Structure Elucidation of Bioactive Marine Natural Products using

Structure Elucidation of Bioactive Marine Natural
Products using Modern Methods of Spectroscopy
(Strukturaufklärung bioaktiver mariner Naturstoffe mit
modernen Methoden der Spektroskopie)
Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf
vorgelegt
von
Mohamed A.A. Ashour
Aus El-Sharkiya, Ägypten
Düsseldorf, 2006
Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf
Eingereicht am : 04.12.2006
Referent : Prof. Dr. Peter Proksch
Koreferent : Dr. Rainer Ebel, Juniorprofessor
Erklärung
Hiermit erkläre ich ehrenwörtlichen, daß ich die vorliegende dissertation
„Strukturaufklärung
bioaktiver
mariner
Naturstoffe
mit
modernen
Methoden
der
Spektroskopie“ selbständig angefretigt und keine anderen als die angegebenen Quellen und
Hilfsmittel benutzt habe. Ich habe diese Dissertation in gleicher oder ähnlicher Form in
Keinem anderen Prüfungsverfahren vorgelegt. Außerdem erkläre ich, daß ich bisher noch
keine weiteren akademischen Grade erworben oder zu erworben versucht habe.
Düsseldorf, 04.12.2006
Mohamed A.A. Ashour
In the name of Allah, the Compassionate, the Merciful.
Recite! (or read!) in the name of your Lord who created (1) Created man from
clots of blood (2) Recite!, your Lord is the most Gracious (3) Who taught by the
pen (4) Taught the man what he knew not (5).
(The first verses of the holy Qur'an that came dwon from the sky )
To the Almighty God “ALLAH” who has granted me all these graces to fulfil
this work and who supported me and blessed me by His power and His mercy in
all my life. To Him I extend my heartfelt thanks.
Acknowledgements
Many institutions and individuals were responsible for the crystallisation of this
humble work, whose associations and encouragement have contributed to the accomplishment
of the present thesis, and I would like to pay tribute to all of them.
I specially wish to express my sincere thanks and gratitude to Prof. Dr. Peter
Proksch, chairman of the Department of Pharmaceutical Biology and Biotechnology,
Heinrich-Heine University, Düsseldorf, for his kindness, admirable supervision, direct
guidance, generous considerations and valuable support during my study in his group.
I would also like to express my deep thanks to my sincere teacher Dr. RuAngelie
Edrada for her guidance, fruitful discussions, constructive advises, NMR courses and
particularly for sharing her expertise in NMR data interpretation and revision of this thesis.
I would also wish to thank J. Prof. Rainer Ebel, of the same department for his direct
guidance, valuable comments and suggestions and specially for sharing his expertise in both
NMR spectroscopy and mass spectrometry.
I am deeply indebted to Dr. Victor Wray (Gesellschaft für Biotechnologische
Forschung, Braunschweig), for the measurement of the NMR spectra, HRMS, and his vital
comments in the structure elucidation of the isolated compounds.
I am grateful to Dr. R. van Soest (Zoological Museum, University of Amsterdam) for
the identification of the sponge materials.
I am thankful to both Dr. Steube of DSMZ (Deutsche Sammlung von
Mikroorganismen
und
Zellkulturen)
and
Prof.
Dr.
W.E.G.
Müller
(Universität
Mainz,Germany) for the cytotoxicity tests.
My deep thanks are also to PD Dr.Wim Wätjen Institut für Toxikologie, HeinrichHeine-Universiät Düsseldorf, Universitätsstr. 1, Geb. 22.21, 40225 Düsseldorf, Germany
I am thankful to Dr. Thomas Schmidt (HHU Düsseldorf) for his kind help in the
measurement of some compounds at the GC machine.
I would also like to express my gratefulness to Dr. Peter Tommes, Dr. U. Matthiesen
(HHU Düsseldorf) for the measurement of EIMS, FABMS and high resolution ms data.
I am very thankful to AnagnosTec [Gesellschaft für Analytische Biochemie und
Diagnostik mbH im Biotechnologiepark, TGZ II , D-14943 Luckenwalde, Berlin] for peptide
sequancing by MALDI-TOF-PSD.
I am thankful to the Egyptian Ministry of Higher Education, Missions Office for
providing the financial support for my study in Germany.
My deep thanks are to my friends, Dr. Mustafa abdelgawwad and Dr. Ihab Elkhayat for their kind friendship, valuable help, encouragement and co-operation during the
first monthes of my stay in Germany.
My deep thanks are also to my new and old colleagues at the Department of
Pharmaceutical Biology and Biotechnology, Düsseldorf, Amal Hassan (Egypt), Arnulf Diesel
(Germany), his wife Ine Inderiani (Indonesia), Clécia Freitas (Brasil). Dr. Yasman
(Indonesia), Dr. Franka (Germany), Dr. Carsten (Germany), Dr. Hefni (Indonesia), Dr. Sabrin
(Egypt), Dr. Gamal (Egypt), Dr. Bärbel (Germany), Dr. Tu (Vietnam), Dr. Yosi (Indonesia),
Dr. Haofu (China), Dr Suwigarn (Thailand), Abdessamad (Morocco), Nadine (Germany),
Mirko (Germany), Frank (Germany), Julia (Germany), Sofia (Sweeden), Idi (Indonesia), Yodi
(Indonesia), Triana (Indonesia), Yao (China) and all the others for their help, friendship and
for the good working atmosphere.
My special thanks to my colleague Annika Putz for his help in the revision of the
German summary in my thesis.
My deep thanks to Mareike, Katrin and Mrs Schlag for their kindness and for always
providing me with the materials and glassware which I needed in my work.
I would also like to express my gratefulness to my supervisors for my Master thesis,
Prof. Dr. Hassan Ammar, Prof. Dr. Taha Khalifa and Ass.Prof. Dr. Samir Al-Donditi and
also to Prof. Dr. Hazem kadry and all staff members and colleagues of the Department of
Pharmacognosy, Faculty of Pharmacy, al-Azhar University (Boys), Nasr City, Cairo for
providing me the basic science and for their motivations, recommendations and for teaching
me very precious knowledge about Pharmacognosy and Phytochemistry offering me such a
kind of opportunity to study in Germany and their continuous encouragement and kind
advises.
My special thanks for my wife Dr. Hasnaa Abouseif for her vast understanding,
everlasting, moral support and contineous encouragement and also for my children Yasmin
Ashour and Ahmed Ashour who could relieve any kind of tirdness by their lovely smiles and
nice atmosphere at home.
Table of contents
I- Introduction………………………………………………….….………….…….………
1
1.1- The significance of the study…………………………...……..……………………
1
1.2 - Natural Products…………………………………………………...………………...
1
1.3- The biological activity of natural products…………...………...…………………...
10
1.4 -The strategies and approaches that infeluence the extraction, isolation
and purification of natural products…………………….…...………………...…
16
1.5-The structural elucidation of the natural products….………...…..……….…..…….
21
1.6 -Marine Natural Products……………………..……....…………...……...………..… 33
1.7- Aim of the work………………………...…….…………….…………...………….. 40
II-Materials and Methods …………………………………….….………….……. ……… 41
2.1- Animal materials………………………………..….……….…………...………….. 41
2.2- Chemicals used……..……………………….…….…..….…..…….…...…………..
48
2.3- Equipments used…………………………….….………….…..………...…………..
49
2.4- Chromatographic methods.…………………….…………..………….…………….. 51
2.5- Procedures of the isolations of the secondary metabolites…..…………………….
57
2.6- Structure elucidation of the isolated compounds………………...………………….. 61
2.7- Bioassays…………………………………………………………………………….. 75
III-Results…………………. …….…………………………….….………….…….………
78
3.1-Isolated compounds from Elysia rufescence……………………….…………………. 78
3.1.1- Compound 1 (kahalalide F)…………….…………….…………………...……….. 79
3.1.2- Compound 2 (kahalalide E)…………….……………...….……………...………..
91
3.1.3- Compound 3 (kahalalide D)……………..…………….……...…………...………. 99
3.1.4- Compound 4 (kahalalide B)…………….…………….…….……..……...……….. 104
3.1.5- Compound 5 (kahalalide C)…………….…………….………....………...………. 110
3.1.6- Compound 6 (N,N-dimethyltryptophane metyhl ester)….….…………………….. 113
3.1.7- Compound 7 (β-sitosterol)…………….…….………….……………...………….. 117
3.2-Isolated compounds from Elysia grandifolia…………….……….……….………….. 121
3.2.1- Compound 8 (kahalalide R)…………….……………..…….….………...……….. 122
3.2.2- Compound 9 (kahalalide S)…………….…………….………..…..……...……….. 133
3.2-Isolated compounds from Pachychalina sp..…………….……….……….………….. 144
3.3.1- Compound 10 (5α,8α-epidioxy-24ξ-methylcholesta-6,22-dien-3β-ol)..…….…..… 145
3.3.2- Compound 11 (5α,8α-epidioxy-24ξ-ethylcholesta-6,22-dien-3β-ol)..……..……… 150
I
3.3.3- Compound 12 (8-hydroxy-4-quinolone)…………… …………….……...……….. 155
3.4-Isolated compounds from Hyrtios erectus……………………....….…………………. 160
3.4.1- Compound 13 (hyrtiosine A)…………….…………………..………...………….. 161
3.4.2- Compound 14 (5-hydroxy-1H-indol-3-carbaldehyde)…………….……..………... 164
3.4.3- Compound 15 (indol-3 carbaldehyde)……………………….………………….… 167
3.4.4- Compound 16 (5-deoxyhyrtiosine A)…………..…..…….…………...…..….…… 170
3.4.5- Compound 17 (isohyrtiosine A)………………….…….…………….……….…… 173
3.4.6- Compound 18 (16-hydroxyscalarolide)….………………….……...…….……….. 176
3.4.7- Compound 19 (scalarolide)…………….…….……….………..……...….……….. 182
3.4.8- Compound 20 (12-O-deacetyl-12-epi-scalarin)….…………….………………….. 187
3.4.9- Compound 21 (7-dehydrocholesterol peroxide)………………....……....…..……. 190
3.5-Isolated compounds from Petrosia nigricans..……………….....….…………………. 194
3.5.1- Compound 22 (24ξ-ethyl-cholesta-5-en-3β-ol)…………….…………….……….. 195
3.5.2- Compound 23 (4α-methyl-5α-cholesta-8-en-3β-ol)…………….………..………... 197
3.5.3- Compound 24 (5α,8α-epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol)………………….. 199
3.5.4- Compound 25 (phenylacetic acid)…………..…..…….…….…………….….…… 202
3.5.5- Compound 26 (p-hydroxyphenylacetic acid)…………..…..…….….……….…… 204
3.5.6- Compound 27 (methyl 2-(4-hydroxyphenyl)acetate)……….…………………….. 207
3.5.7- Compound 28 (ethyl 2-(4-hydroxyphenyl)acetate)………….…………………….. 209
3.5.8- Compound 29 (butyl 2-(4-hydroxyphenyl)acetate)……...….…………………….. 211
3.5.9- Compound 30 (adenosine)…………….…………..……………..………..………. 214
3.5.10- Compound 31 (nicotinamide)…………..……….……...…….………….….…… 217
3.5.11- Compound 32 and 33 (petrocerebrosides 1and 2 )…………..….……… ….…… 219
3.5.12- Compound 34 (nigricine 1)…………….…….……..…..………………..………. 228
3.5.13- Compound 35 (nigricine 2)…………..………….……...…….………….….…… 234
3.5.14- Compound 36 (nigricine 3)…………….…….……..…….……….……..………. 238
3.5.15- Compound 37 (nigricine 4)…………..………….……………………….….…… 242
3.5.16- Compound 38 (nigricinol)…………….……...……..……………..……..………. 245
3.6-Isolated compounds from Callyspongia biru..…….…………....….…………………. 251
3.6.1- Compound 39 (Indole-3-acetic acid)….……………………..……..…….……….. 252
3.6.2- Compound 40 (2`-deoxythymidine)…………….………………………..………... 254
IV-Discussion…………..…………………………………………………………………… 256
4.1- Isolation, characterisation, structure elucidation, and biological evaluation of
II
natural products ………………….………………………………………………….. 257
4.1.1- Isolation of natural products …………...………………………………………..
257
4.1.2- Characterisation of natural products …………...…………………………...…….. 258
4.1.3- Structure elucidation of natural products ……………...………………………….. 261
4.1.4- Biological activity of natural products …………..………………….…………….. 262
4.1.5- Technology of HPLC …………...…………………………………..…………….. 263
4.2- Isolated compounds from genus Elysia ( sacoglossan mollusc) ……...…………...... 267
4.2.1- The responsibility for production of the kahalalides …………..……………....
268
4.2.2- The possible biosynthetic pathway of the kahalalides .…...…………………….. 270
4.2.3- Structure-activity relationship ………………...….……………………………….. 271
4.3- Isolated compounds from Pachychalina sp………………………………….………. 272
4.3.1- Epidioxy sterols …………..………………………………………..…………….... 272
4.4- Isolated compounds from Hyrtios erectus……………...…….…...………….………. 275
4.4.1- Excessive harvesting of the wild-type living organism ………..……………….... 275
4.4.2- Biosynthetic pathway of the scalaran-typ sesterterpenoids .…………………….. 276
4.4.3- The pharmacological activity of scalaran-typ sesterterpenoids …………...….….. 277
4.4.4- Indole derivatives from Hyrtios erectus.……………………….………………….. 277
4.4.5- Biosynthetic pathway of indole derivatives from Hyrtios erectus …………….….. 278
4.5- Isolated compounds from Petrosia nigricans………......…………………….………. 279
4.5.1- Biosynthetic pathway of aromatic acids …………...……………….…………….. 279
4.5.2- Cerebrosides from Petrosia nigricans …………...……………………………….. 280
4.5.3- Biosynthesis of cerebrosides …………...………………………...……………….. 280
4.5.4- New purine derivatives from Petrosia nigricans …………...…………………….. 281
4.5.5- The possible biosynthetic pathway of nigricines 1-4………..…...……………….. 282
4.6- Isolated compounds from Callyspongia biru ..…………...…………………….……. 284
V-Summary…………..……………………………………………………………………
286
VI-References ……..…..…………………………………………………………………… 288
List of abbreviation .…………………………………….………………………………… 302
III
Zusammenfassung
Moderne Techniken und überlegene Werkzeuge haben die Arbeit von Naturstoff-Chemikern
erleichtert und ermöglichen im Vergleich zu älteren Methoden eine beträchtlich schnellere
Entdeckung, Isolierung und Strukturaufklärung von bioaktiven Naturstoffen. Naturstoffe
besitzen oft neue oder ungewöhnliche strukturelle Eigenschaften und / oder viel versprechende
biologische Aktivität. Die Anwendung neuer Methoden reduziert Zeit, Aufwand und Kosten
der Entdeckung bioaktiver Naturstoffdrogen. In dieser Studie wurde oben genanntes Ziel mit
Hilfe von modernen Techniken wie NMR, MS und HPLC Systemen als effiziente Werkzeuge
verfolgt. Das in dieser Studie verwendete biologische Material umfasst sechs marine
Invertebraten, die vier verschiedenen geographischen Standorten entstammen. Insgesamt
wurden 40 Substanzen in reiner Form gewonnen und deren Struktur aufgeklärt. Einige von
diesen zeigten sehr viel versprechende biologische Aktivität und stellen somit potentielle
Kandidaten für die Entwicklung zukünftiger Naturstoffdrogen dar.
1. Elysia rufescens
Der Mollusk Elysia rufescens (Sacoglossa) wurde beim schwarzen Punkt „Kahala“ auf der
Oahu Insel (Hawaii) gesammelt. Aus Extrakten dieser Tiere konnten neben fünf bekannten
Kahalaliden (Kahalalid B, C, D, E und F) β-Sitosterol und ein bislang als FabaceenSekundärmetabolit bekannter N,N-Dimethyltryptophanmethylester gewonnen werden.
2. Elysia grandifolia
Der Mollusk Elysia grandifolia (Sacoglossa) wurde im Golf von Mannar und Palk Bay
(Rameswaram, Indien) gesammelt. Im LCMS-Spektrum des methanolischen Rohextrakts
zeigten sich neben den Peaks der Kahalalide B, C, D, E, F, G, J, K, und O auch zwei neue
Peaks ähnlich den Kahalaliden R und S. Im Fokus des Isolationsprozesses standen diese neuen
Substanzen. Zusätzlich zu diesen konnten noch zwei bereits bekannte Kahalalide isoliert
werden (Kahalalid D und F).
3. Pachychalina sp.
Der unbekannte Schwamm Pachychalina sp. stammte aus Pulau, Baranglompo (Indonesien).
Im methanolischen Rohextrakt fanden sich sowohl zwei 5α, 8α-Epidioxysteroide als auch das
bereits bekannte 8-Hydroxy-4-quinolon, welches als Bestandteil der Tinte des Riesen-Oktopus
Octopus dofleini martini beschrieben wurde.
IV
4. Petrosia nigricans
Der Schwamm Petrosia nigricans wurde in Baranglompo (Indonesien) gesammelt. Der
methanolische Extrakt wurde intensiv untersucht, es konnten insgesamt 17 Substanzen isoliert
werden. Davon waren zehn bereits bekannte Naturstoffe, zwei neue Cerebroside, ein bisIndolderivat und vier neue Oxopurinderivate.
5. Callyspongia biru
Proben des Schwammes Callyspongia biru stammten aus Taka Bako (Indonesien). Aus dem
methanolischen Rohextrakt konnten die fünf bekannten Substanzen Indol-3-carbaldehyd, Indol3-essigsäure, p-Hydroxyphenylessigsäure, p-Hydroxyphenylessigsäuremethylester und 2`Deoxythymidin gewonnen werden.
6. Hyrtios erectus
Individuen des Schwammes Hyrtios erectus wurden bei El-Quesir im Roten Meer (Ägypten)
gesammelt. Im methanolischen Rohextrakt befanden sich neun Substanzen, darunter ein
Epidioxycholesterolderivat, drei Sesterterpene vom Scalaran-Typ, vier bekannte Indolderivate
und ein neues 5-Hydroxyindolderivat.
V
Introduction
I. Introduction
1.1- The significance of the study:
Although effective drugs for many diseases that afflict mankind have been discovered,
many health problems remain untreatable. These problems include various types of cancer;
viral infections such as HIV and viral hepatitis, particularly types related to hepatitis C viral
infections;
severe
fungal
infections,
especially
in
immunocompromised
patients;
cardiovascular diseases, and inflammatory and allergic disorders (Gullo, 1994). Only,
approximately, one third of all diseases can be treated efficiently (Müller et al. 2000).
Therefore the search for novel therapeutic agents continues, and the need still remains to
uncover the initial structural lead that interacts with therapeutic targets. Natural products give
a good chance for the discovering of an effective medication of the remaining untreatable
diseases either, by direct therapeutic effect, after semisynthetic modification or by new
synthesis of chemicals based on the natural product models (Cragg et al. 1997). The discovery
of natural products with therapeutic potential is affected by many factors comprising all steps
of extraction, isolation, characterisation, structural elucidation, and biological evaluations. In
the last few years these methods were highly advanced in order to save time, and economy of
isolation process, discover, and ensure the proper biological activity of natural products. Now,
using this technological evolution, one is sometimes able to extract, isolate, and elucidate the
natural product in only one day (Steinbeck 2004). This work deals with natural products and
covers many aspects in which they are included.
1.2- Natural Products
Natural products have inspired chemists and physicians for millenia. Their rich
structural diversity and complexity has prompted synthetic chemists to produce them in the
laboratory, often with therapeutic applications in mind, and many drugs used today are natural
products or natural product derived. Recent years have seen considerable advances in our
understanding of natural product biosynthesis coupled with improvements in approaches for
natural product isolation, characterization and synthesis; these could be opening the door to a
new era in the investigation of natural products in academia and industry (Clardy & Walsh,
2004). Nature has continuously provided mankind with a broad and structurally diverse array
of pharmacologically active compounds that have proved to be indispensible for the cure of
1
Introduction
deadly diseases or as lead structures for novel pharmaceuticals (Newmann et al, 2000a). In
the present drug discovery programs, compounds derived from natural products account for
more than 40% of the newly registered drugs (Cragg, et al, 1997)
The discovery of antibiotics gave the study of natural products a great boost in
microbiology departments and ensured that natural products remained central to growing
pharmaceutical companies ( Firn & Jones 2003).
1.2.1- Definitions of „Natural products“:
Most scientists define the natural products as chemical substances that are made by
organisms and are not active participants in primary metabolism. In other words they are
chemicals that are usually found in few families or species and which do not seem to serve a
purpose in minute-to-minute activities of the cells. Indeed another term used for natural
product is the term secondary metabolite or secondary product. The term secondary products
or secondary metabolites is widely used now to distinguish this type from the other one „
primary metabolites“. (Firn 2004)
Bennett (1995) mentioned five different definitions for the term secondary metabolism :
•
secondary metabolites are those metabolites which are often produced in a phase
subsequent to growth, have no function in growth (although they have survival function),
are produced by certain restricted taxonomic groups of microorganisms, have unusual
chemical structures, and are often formed as mixtures of closely related members of a
chemical family. (Martin & Demain 1978 )
•
the simplest definition of secondary products is that they are not generally included
in standard metabolic charts (Davies, 1985)
•
A metabolic intermediate or product, found as a differentiation product in restricted
taxonomic groups, not essential to growth and the life of the producing organism, and
biosynthesized from one or more general metabolites by a wider variety of pathways than
available in general metabolism. (Bennett& Bentley, 1989).
•
Secondary metabolites are not essential for growth and tend to be strain specific.They
have a wide range of chemical structures and biological activities. They are derived by
unique biosynthetic pathways from primary metabolites and intermediates (Vining,1992)
2
Introduction
•
Secondary metabolism includes biochemical pathways that are not necessary for
growth or reproduction of an organism, but which can be demonstrated genetically,
physiologically or biochemically (Hunter 1992).
1.2.2- Primary and secondary metabolism :
Primary metabolism refers to the processes producing the carboxylic acids of krebs
cycle, α-amino acids, carbohydrates, fats, proteins and nucleic acids, all are essential for the
survival and well-being of the organism. All organisms possess the same metabolic pathways
by which these compounds are synthesized and utilized. Secondary metabolites on the other
hand, are non essential to life but contribute to the species´fitness for survival (Torssell 1997).
Other descriptions of primary metabolism refer to all biochemical processes for the
normal anabolic and catabolic pathways which result in assimilation, respiration, transport,
and differentiation. Primary metabolism shared by all cells are virtually identical in most
living organisms in order for production of extremly similar molecules giving the same
biological function. The secondary products, having no role in the basic life process, are
produced by pathways derived from primary metabolic routes. Secondary metabolite products
accounting for the plant colours, flavours and smells, are source of fine chemicals, such as
drugs, insectisides, dyes, flavours, and fragrances, and phyto-medicines found in medicinal
plants. The concept of secondary metabolism was first introduced by Kössel,1891,
(Haslam,1986; Seigler,1998; Turner, 1971).
1.2.3- The role of secondary metabolite in the producing organism
„ to every thing there is a season“
In certain scientific circles it is something of a sport to theorize about function, often with
the intent of finding one overriding axiom true for all secondary metabolism. Speculations
range from the notion that they are waste products or laboratory artefacts, to the concept that
they are neutral participants in an evolutionary game, to ideas of chemical weaponry and
signalling, through a number of other creative notions. (Bennett, 1995).
A)
Waste product hypothesis
The role of the secondary metabolites has been rather ambiguous, and initially they were
thought to be just waste materials. The relatively large number and amount of secondary
metabolites observed in nature and the notion that these compounds arose from´´errors´´ in
primary metabolisms in plants, led to the idea that secondary compounds arise and
3
Introduction
accumulate as „waste-products“. However, considering their non-motile nature and the lack of
sophosticated immune system, plants have developed their own defense system against
pathogens and predators, and systems to lure motile creatures, for fertilisation and
dissemination (Luckner, 1990; Seigler, 1998)
B)
The overflow or excess of the primary metbolism hypothesis:
In instance of unbalanced growth, secondary metabolites have been envisioned by some as
shunt metabolites produced in order to reduce abnormal concentration of normal cellular
constituents. The synthesis of enzymes designed to carry out secondary metabolism permits
primary metabolic enzymes to continue to function until such time as circumstances are
propitious for renewed metabolic activity and growth. This could be linked to the depletion of
nutrients such as phosphorous or nitrogen ( Bu´Lock, 1980; Haslam, 1986).
C)
The increased fitness hypothesis:
This hypothesis is based on the fact that many natural products trigger very specific
physiological responses in other organisms and in many cases remarkably bind to its
complementary receptors. In other words, natural products may aid an organism´s survival in
the absence of an immune system. This supports the hypothesis that secondary metabolites
increase the fitness of the individuals that possess them and that those individuals have been
favoured by the
process of natural selection. Secondary metabolites have an important
ecological role in the interaction with the environment, and are like the communication
interface between a plant and its friends and enemies in the environment, (Harborne, 1986;
Rosenthal and Janzen,1979; Swain, 1977; Torssell,1997).
D)
Secondary metabolism is a defense system :
Some scientists refer the production of the secondary metabolits to be a potent chemical
defense system against herbivorous, deterrants, pathogens….etc. The immobility of plants in
diverse and changing physical environment, along with the danger of attack by herbivores and
pathogens, has led to the development of numerous chemical and biochemical adaptations for
protection and defense (Knox and Dodge 1985, Harborne 1988), plants for example can
produce higly toxic compounds or compounds mimicking substances normally produced by a
herbivore, for example growth hormones or phermones.
It may be sufficient for the plant to produce compounds that are unpleasant, odorous,
or distasteful, or that possess digestibility reducing properties, i.e. compounds that decrease
the uptake of nutrients, thus preventing over-browsing of the plant. One example of a
compound with strong antifeedant activity against numerous insects is azadirachtin (1),
produced by the indian neem tree (Azadirachta indica). The tree has been used for centuries
4
Introduction
to protect other plants and clothes from insects. The structure of the active compound is very
complex and not proven until 1985. Due to its potency and selectivity against insects this
compound has been commercialized as antifeedent (Bratt, 2000).
O
O
O
O
O
O
OH
N
O
O
O
OH
O
O
O
O
N
HO
(2 )
(1 )
Indeed many plants do not keep permanent stores of their defense compounds, but
manufacture them in response to predation. One example is the tobacco plant (Nicotiana
sylvestris) that produces nicotine (2), a compound that deters a wide variety of herbivores.
The level of nicotine produced by the plant is regulated by the extent to which it is being
attacked by herbivores and the wild tobacco plant can increase the amount of nicotine
produced by 3-4 times as response to an attack.(Mann, 1994).
E) The screening hypothesis :
This hypothesis was introduced by Richard D. Firn & Clive G. Jones [(Firn and Jones 1996,
1998, 1999, 2000, &2003), (Jones and Firn 1991), (Firn , 2003 and 2004)]
The hypothesis is composed of two parts.
1.
The identification of a fundamental constraint that must be a very significant factor in the
evolution of secondary metabolism.
Potent, specific biological activity is a rare property for a molecule to possess – that is
why large screening programmes are needed to find useful biological activity. The low
frequency of potent, specific biological activity is a consequence of the specificity of
ligand/binding site interactions. Some organisms can increase their fitness by making and
exploiting biological active molecules. However, the low frequency of evolving new
molecules places severe constraints on the evolution of the biochemical pathways leading to
such products.
2.
A proposal as to how secondary metabolism might have evolved with such a constraint
including a prediction as to the metabolic traits that would be expected to minimize the
effects of these constraints
How do organisms generate sufficient chemical diversity to enhance their chances of
finding the rare beneficial chemical? How do organisms retain the capacity to generate so
much chemical diversity when indivdual compounds or pathways become redundant?. The
Screening Hypothesis proposes that certain metabolic traits (matrix pathways, non-enzymic
5
Introduction
transformations, branched pathways, shared pathways and enzymes with a broad substrate
tolerance) would all help increase generation and retention of chemical diversity and
evolution of secondary metabolism.
Some consequences of this hypothesis:
•
One should not expect all naturally made chemicals to have a role in the organisms
that make them. Many will have no role and will never have had any role in the organisms
in which they are found. Many chemicals will simply have been made because the
metabolic machinery cabable of their production has a benefit for the producer. If only one
product that those pathways can produce has a beneficial biological activity, the pathways
will be sustained if the costs of possessing that capacity is sustainable.
•
One should not assume that some biological activity found in a screening trial
conducted by humans has any significance to the role of the chemical in the organism that
produces it. If organisms are producing chemical diversity they must inevitably produce
chemicals with structures that will possess fortuitous biological activity in non-target
organisms.
•
The metabolic traits predicted by the screening hypothesis will sometimes make it
hard to precisely genitically manipulate secondary product pathways. For example, it is
proposed that in order to enhance the production and retention of chemical diversity, many
enzymes involved in secondary product biosynthesis will have low substrate specificity.
Consequently, if a new enzyme is introduced into an organism to cause the production of a
new secondary product, there is high probability that existing enzymes in the transformed
organism will further elaborate the new product to produce more novel diversity.
•
Some of the metabolic traits predicted (for example low substrate specificity) might
be exploited in biotransformation and bioremedation studies.
•
The flux of carbon through secondary metabolite pathways must have been very large
throughout the period of life on earth. The metabolic traits predicted by the screening
hypothesis may have played a part in encouraging the catabolism of this huge amount of
chemical diversity. The world has never been a clean place chemically hence organisms
must have the capacity to survive and thrive in the presence of chemical diversity. Most
synthetic chemicals released into the environment in small amounts will find enzymes that
can transform them.
6
Introduction
1.2.4- The secondary metabolite pathways:
Secondary metabolites are produced through other metabolic pathways than that of primary
metabolites. These pathways are more characteristic for the particular family or genus and are
related to the mechanism of evolution of species. In the fact, the specific constituents in a
certain species have been used to help with systemic determination, groups of secondary
metabolites being used as markers for botanical classification (chemotaxonomy)
(Torssell,1997) .
The differentiation between primary and secondary metabolism is not clear and the
two types are linked together because primary metabolism provides the small molecules that
are the starting materials of the secondary metabolic pathways (Figure 1.1).
Polysaccharides
Glycosides
Nucleic acids
CO2 + H2O (photosynthesis)
hv
HO
Monosaccharides
O
HO
OH
HO
shikimic acid
CH3COCOOH
pyruvic acid
Aromatic amino acids
CH3COSCoA
Acetyl-SCoA
Aliphatic amino acids
OOC-CH2COSCoA
Cinnamic acid derivatives
Other aromatic compounds
Lignan
Peptides
Proteins
Alkaloids
Phenols
HO
O
Malonyl-SCoA
HO
OPP
OH
active isoprene
mevalonic acid
Polyketides
Phenols
fatty acids
Isoprenoids
(Terpenes, Steroids, Carotenoids)
Figure 1.1. Primary metabolites and their links to secomdary metabolism
1.2.5- The chemical diversity of the secondary metbolites:
“Organic chemistry just now is enough to drive one mad. it gives the impression of a
primeval tropical forest, full of the most remarkable things, a monstrous and boundless
thicket, with no way to escape, into which one may well dread to enter. „ (Wöhler in a letter
written to Berzelius in 1835).
7
Introduction
When Wöhler wrote those words he was expressing his despair at the emerging
complexity of the composition of natural products. Yet within a few decades, this complexity
was tamed by the Kekule` theory of structure. Increasingly, confident chemists took up the
challenge of determining the structures of ever more complicated natural products. There are
more than 5oo,ooo secondary chemical products already estimated in plants alone. The
majority of natural products described in plants are made by a relatively small number of key
pathways and the structural diversity is therefore largely a consequence of diversity of product
within these few broad groups of chemical clases of natural products (Firn & Jones,1996).
„It is widely accepted that the mutation of a gene coding for an enzyme will result in various
outcomes“. (Firn 2004)
This concept leads to :
1)
The enzyme activity will be unaltered (the most likely scenario).
2)
The kinetic properties of the enzyme will be changed, usually in a detrimental way.
3)
The enzyme will change its substrate specificity.
4)
Sites necessary for the allosteric control of the enzyme will be altered.
It is not widely believed that a mutation of the gene coding for an enzyme will change the
type of transformation carried out. Thus most biochemical inventivness will arise from
existing catalytic functions being applied to new substrates rather than new types of
transformations being applied to the original substrate. Such biochemical inventivness
requires that multiple copies of a gene must exist if the gene being mutated codes for enzyme
that is involved in primary metabolism, otherwise a loss of an essential part of primary
metabolism would result (Firn & Jones,1996)
Some simple scenarios to illustrate the constraints that might have operated during the
evolution of natural product pathways. (Firn 2004)
B
B
E2
C
E3
E4
D
A
E1
A
E4
C
E4
E2
B´
E3
C´
E1
D´
D
Fig. 1.2 : relaxed substrate specificity facilitates the generation and retention of
chemichal diversity. such predictable characteristics have been found in
"secondary metabolism"-carotenoids, phenylpropanoids, terpenoids, etc..
8
Fig .1 3 single product converted to
multiple products by one enzyme.
Introduction
In (figure 1.2) there is a diagram showing how 3 enzymes (E1-3) convert A to D. Now
if the substrate specificity is relaxed the order in which the substrates are converted in
unimportant pathway, hence the 3 enzymes will generate 6 products not 3. So this helps
generate diversity. Furthermore, if D increases the fitness of the producer then B´,C´ and D´
may still get made even though they play no role currently- this helps retain chemical
diversity.
This phenomenon is evident in the present study where, Kahalalide F, the major
depsipeptide in a saccoglossan molluscs Elysia sp. has antipredatory effect against fish
predators, its biosynthesis was accompanied with the production of other depsipeptides, that
have no antipredatory effects.
Another example, (figure 1.3) illustrates another proposal where one enzyme produces
multiple products instead of the usual single one. Again this helps generate and retain
chemical diversity.
X
X
E1
X
A
X*
A
A
E3
E2
B
B
F ig. 1.4. hypothetical pathw ay. illustrated three enzym es of
very strict substrate spicificity , resulting in production of
only three products.
The third example (figure 1.4) of illustrating this principle is to consider a hypothetical
pathway which has three enzymes leading from a precursor. If the enzymes have a very strict
(narrow) substrate specificity then only three products will be formed.
However if the enzymes have a broad substrate tolerence it is possible that they could
each carry out their own little piece of biochemistry on a selected part of any molecule
irrespective of the whole structure of the molecule. Theoretically, many more structures
could be generated using just these three enzymes as shown in ( figure 1.5).
9
Introduction
X
X
X
X*
A
A
E1
E3
X*
E3
E2
E3
E1
X*
E1
B
B
X
X
A
A
A
A
A
E2
E1
E3
E2
B
X*
X*
X
A
A
A
X*
A
E2
E3
B
A
B
B
Fig. ( 1.5 ) many structures generates from the hypothetical pathway in which
enzymes have a broad substrate tolerance
1.3- The biological activity of natural products.
1.3.1- The history of biological benefits of natural products
Man has used natural products since the down of time as remidies for diseases, spices,
narcotics, dyes, and poison for warfare and hunting. Most of these compounds were used in
their crude forms and active components were mostly not isolated until the nineteenth
century. Morphine (3), first isolated from opium (Papaver somniferum and P. setigrum) in
1803 is a well-known example. It is one of the powerful analgesics known, and also possesses
strong narcotic effects (Mann, 1987).
H
HO
N
O
HO
N
O
HO
N
3
4
10
Introduction
Another example is quinine (4) an anti-malaria agent isolated from the Cinchona tree
as early as in the seventeenth century (Mann 1987). Of course there was a widely held
view in the 19th century that God had created all organisms for man`s benefit. Hence
many considered that the chemicals other organisms contained were for human use. Such
views would have influenced the thoughts of some scientists (Firn and jones 2003, Firn
2004).
Before the 19th century, the only real interest in natural products came from herbalists
and physicians. They were interested in the fact that plants (and sometimes fungi)
contained substances that were thought to be useful in treating patients. For some
considerable time, botanists had been sent from the United Kingdom, and many other
countries, to scout the world for new, exotic plants, and the number of species that were
accessible to herbalists and physicians increased. By the 19th century chemistry had began
to ask questions about the nature of substances and there was a growing scientific interest
in the natural products however this interest was still biased towards their use by humans
rather than their role in the organisms that made them. (Firn 2004)
1.3.2- The importance of biological activity testing of the isolated natural
products:
The fact that microbially-derived antibiotics found by screening make up most of a
$16.5 billion per annum antimicrobial pharmaceutical sales (Nisbet 1992), explains why
so many large antibiotic screening programmes have been conducted during the last half
century (Firn 2004).
This antimicrobial screening of the isolated natural products represents only one of
many lines of biological activity testing that made in order to overcome the most recently
encountered problematic diseases for example, viral hepatitis, AIDS, cancers, autoimmune
diseases, parasites,etc.
The biological activity testing should also expand to comprise most toxicological
studies in order to show the most advantages together with the disadvantages of the given
total extract or the purified natural product. At the end of these biological and
toxicological studies we can decide whether the given product is usefull or not.
This problematic equation (effective, cheap, of wide safty margin, and widely
available natural product drugs) is not easy to be accomplished. For example :
11
Introduction
(i)
400,000 microbial cultures were assayed over a 10 year period and only three
utilisable compounds were found (Fleming et al. 1982, Nelson 1961)
(ii)
21,830 isolates screened in one year to give 2 possible compounds (Woodruff et al
1979).
(iii)
10,000 Microorganisms gave only one clinically effective agent (Woodruff and
MacDaniel 1958).
Those sceptical of such evidence leads man to ask, Is biological activity a rare
property for a natural product to possess? in another word, Is it true that, the
majority of secondary metabolites have no biological benefits?
A meaningful answer requires a definition of the term BIOLOGICAL ACTIVITY.
At a time when natural products were mainly of interest only to herbalists, the father
of modern toxicology, Paracelsus (1493-1541) astutely observed that whether one thought
of a substance as a poison or not depend entirely on the dose that was adminstered. it is
therefore obvious that a discussion of the biological activity of any compound is
meaningless unless some comparative standard is adopted with regards to the dose. The
problem is especially acute when the biological activity is evaluated on the basis of an
adverse effect on an organism, because a sufficient dose of many substances will have an
adverse effect on virtually any organism (Rodricks 1992).
For purposes of this discussion it seems sensible that the only realistic bases for
judging the biological activity of a substance is whether the compound would have a
significant effect at a dose that a recipient organism would receive. A biological activity
that is found when the compound is applied to test organisms at unrealistic concentrations
cannot sensibly be regarded as meaningful. The importance of these considerations can be
illustrated by considering model dose-response curves for three hypothetical substances
C1, C2, and C3 (Fig. 1.6 ). It is appearent that all three substances can be regarded as
showing biological activity in that all saturate the response when applied at high
concentrations (>10-5 M). However, suppose that each substance occures in an organism
at a concentration of 10-8 M. In that case, only compound C1 can be considered to possess
meaningful biological activity. Compounds C2 and C3 show no significant activity at their
endogenous concentrations and would therefore confer no selective advantage to the
organism [Firn and Jones 1996] .
12
Introduction
Response % of maximu
m
100%
75%
C1
C2
C3
50%
25%
0%
10-11
10-9
10-7
Concentration of applied substances
10-5
(Molar)
Fig. 1.6 „the dose makes the poison“. The dose response curves of three
hypothetical substances C1,C2 and C3 illustrate that whether or not a substance
is considered to be biologically active depends on the dose being considered .
(firn &Jones 1996)
The law of mass action explains why substances C2 and C3 are actively bound to the
protein binding sites producing their effect at high concentrations and inactive at low
concentrations.
Chemical + Protein
Ka
Chemical protein complex
Kd
Where:
Ka = rate of association
Kd = rate of dissociation
The rate of association and the rate of dissociation are properties of the particular
protein and molecule combination if the former is larger than the latter, there is a high
chance of the protein having a molecule bound to it at any moment (the ratio of the two
rate constants is used to assess the strength of any binding process). The law of mass
action tells us that the equilibrium will be driven to the right as the concentration of the
molecule increases. Consequently there is a much greater probability of finding an
interaction between any chemical and any protein if you test the chemical at high
concentration than that at low concentration. This relationship is also helpful when
assessing the safety margin of a tested compound.
Also, it is obvious that the wider the range of organisms for which biological activity
of a compound is assessed, the greater the chance that some effect will be found, and also
it must be borne in mind that the selective pressures that operate on any individual within
a population at any one time will be quite specific and only a limited range of
13
Introduction
opportunities will be available where chemical interactions can be beneficially used.[ Firn
and Jones 1996] .
Firn and Jones 1996, mentioned some limitations that affect the biological activity
studies of the secondary metabolites and consequently minimize the chance of detecting
highly biologically active compounds, specially for commercial use, for example:
-
The high selectivity required for a biologically active substance means that many
active compounds will be rejected at an early stage because of lack of specificity.
-
Some active compounds are unstable and are either lost during isolation or are
unsuitable for use .
-
Some compounds are too difficult to synthesise or extract from cultures and are never
developed.
-
Some compounds have already been isolated (and possibly patented before).
-
The type of biological activity found is not meaningful in terms of the particular
usage sought.
-
The screening process is inappropriate.
-
Some forms of biological activity are dependent on the presence of other compounds
(synergists) which are lost during the purification processes before the substance is
bioassayed.
Each of the above reasons could be valid in some circumstances but even taken together
they only partially account for the fact that screening natural products has produced relatively
few biologically active compounds with high selective functions.
Although there are several limitations that minimize the chance of detecting the
biologically active natural product, there are many active natural products that extensively
affect the different forms of our life and can be beneficially used as nutritional supplements ,
dyes, insecticides and also there are many natural products that are already used for treatment
of many diseases that counteract our healthy life.
1.3.3- Approaches to the discovery of biologically active natural products:
In general, drug discovery strategies can be trivially separated into three categories:
1.
Chemically driven, finding biological activities for purified compounds.
2.
Biologically driven, bioassay-guided approach beginning with crude extracts.
3.
Combination of chemically and biologically driven approaches (vide infra).
14
Introduction
For marine-derived drug discovery, strategies may involve one or more of the
following elements:
1. In vivo screens
2. Mechanism-based screens
3. Functional, whole cell, or tissue-based assays
4. On-site assays versus post collection assays
5. „Dereplication,“ via biological profiles or chemical profiles, e.g., thin layer
chromatography (TLC), nuclear magnetic resonance (NMR), and high pressure liquid
chromatography (HPLC), (McConnell et al. 1994)
Beginning in the 1970´s through today, the majority of the academic-based research
efforts have become essentially „biologically driven“ i.e., the object of the search has shifted
to discover natural products with biological activity. The biological activities include
exploring their potential as agrochemicals (Crawley 1988) and pharmaceuticals, as well as
their possible chemical ecological roles (Bakus et al. 1986; Hay and Fenical 1988)
Drug discovery in industry has evolved to the use of specific assays with target receptors
and enzymes involved in the pathogenesis of disease rather than cellular or tissue assays
(Johnson and Hertzberg 1989), and has benefitted immensely from breakthroughs in receptor
technology (Hall 1989; Reuben and wittcoff 1989). These assays reflect new opportunities
due to the recent identification of previously unrecognized biomolecular targets for therapy
(Larson and Fischer 1989). More specifically, this approach for most disease areas is
characterized in industry by:
1. Essentially exclusive reliance on biological activity of crude extracts in numerous targetspecific assays, i.e., enzyme assays and receptor-binding assays, for selection of crude
extracts and bioassay-guided fractionation of the crude extracts (prioritization criteria
emphasize selectivity and potency).
2. High volume, automated screening, i.e., thousands of samples per year for smaller
companies and thousands per week for larger companies.
3. The use of „functional“ or whole-cell assays to confirm activity in a particular disease
state and to further prioritize samples for fractionation.
4. The use of gentically engineered microorganisms, enzymes, and receptors.
Because of low correlation between cytotoxity and antitumor activity, a number of
programs have utilized in vivo tumor models directly for drug discovery (Johnson and
Hertzberg 1989). From 1958 through 1985, NCI used in vivo L1210 and P-388 murine
15
Introduction
leukemia assays as primary screens (Suffness et al. 1989; Boyd et al. 1988) and was
successful primarily in identifying compounds possessing clinical activity against leukemias
and lymphomas. Unfortunately, they were not very successful in finding compounds active
against slow growing tumours in humans. Further, these in vivo assays were expensive, time
consuming, and relatively insensitive (Suffness et al. 1989). In other disease areas it was
shown that activity in in vitro antiviral assays does not translate well to in vivo activity, e.g.,
using Herpes simplex. In contrast, reasonable correlations exist between in vitro and in vivo
antifungal activity, e. g., using Candida albicans.(McConnell et al., 1994)
Chemically and biologically driven approaches can be combined. The combination
means that selecting extracts for chemical fractionation based on the biological activity
profile of the crude extract. However, instead of using a bioassay-guided approach to purify
the compounds responsible for the activity of the extract, NMR and some chromatographic
techniques are used to isolate the chemically most interesting substances. Ideally the
structurally unusual or novel compounds are also responsible for the activity of the extract.
This approach works well when the active compounds are present in high concentration and
the assay turnaround time is longer than a couple of weeks. This approach is indeed
productive with respect to isolating numerous new compounds, at least some of which usually
express some of the activity observed for the crude extract, but is obviously not the best
method to identify the most active compounds if they are present in low concentrations
(McConnell et al., 1994).
1.4- The strategies and approaches that influence the extraction, isolation
and purification of natural products.
Unlike the medicinal chemist, who usually concentrates on a series of compounds of
similar chemical and physical properties and hence is able to master the limited number of
separation techniques applicable to the specific chemotype, the natural product chemist must
be prepared to deal with molecules of the whole spectrum of bioactive metabolites. These can
vary in hydro- and lipophilicity, charge, solubility, and size (McAlpine and Hochlowski1994).
In general, the more hydrophilic metabolites may be candidates for ion exchange
chromatography, reversed phase silica gel chromtography, or size exclusion chromatography
on polysaccharide resins. The more lipophilic
metabolites can be further purified by
chromatography on normal phase silica gel, florisil, alumina, or lipophilic size exclusion
resins such as sephadex LH-20. They may be also candidates for a variety of high-speed
16
Introduction
countercurrent techniques or chromatography on polyresins (McAlpine and Hochlowski
1994). Natural product chemistry is one of the oldest branches of the chemical sciences, its
origin dating back to the first decades of the 19th century, or even earlier. Presently after
almost 200 years of study, this is still vibrant and evolving. What are the reasons for this
continuous and continuing interest? Possible answers would have to include the challenges
offered by the detection, isolation and purification procedures; by the permanently improving
methods of structure elucidation; and by the complexities of the biogenetic pathways leading
to these compounds (Shamma 1989).
The modern highly specific and sensitive screens to detect bioactive molecules have
become available and as researchers have looked at ways to concentrate extracts for primary
screening, novel metabolites have been discovered from fermentations in which they were
produced in levels as low as 1µg/l. (McAlpine and Hochlowski1994). High performance
liquid chromatography (HPLC) including both normal and reversed phases (RP) is now a
well-developed and widely used technique for separation of complex mixtures (Exarchou, et
al, 2005). All parts of the process leading to an elucidated structure have experienced an
immense speed-up in the past fifty years. Separation technology, analytical and spectroscopic
methods have improved steadily and with good fortune, a chemist might be able to go from a
crude extract to a full set of 2D NMR spectra in one day (Steinbeck 2004).
The traditional way of studying natural products includes fractionation of a crude
mixture or extract, separation and isolation of the individual components using liquid
chromatography and structure elucidation using various spectroscopic methods (UV, IR,
NMR, MS). (Exarchou, et al, 2005). It is therefore of great importance to be able to gain
information about extract constituents before investment in the preparative isolation process
(Lambert, et al, 2005).
The chemical components of a biological source are isolated one by one, by
chromatography of the respective extracts, and then their structures are elucidated, such a
complete analytical screening is, unfortunately very time-consuming and material-intensive,
since it is a major effort – and a waste of resources – to isolate all of the compounds in a pure
form, even the known ones. Furthermore, to obtain the required milligram quantities of the
sometimes rare biological source – and of expensive adsorbents and eluents, and thirdly,
unstable compounds will decompose already during the preparative separation and thus
escape the analysis (Bringmann and Lang 2003). A solution of this problem is coupling of the
two steps, the isolation and the structural elucidation, by combining them online. This may
help with the facile acquisition of metabolic profiles, for deciding which of the extracts to be
17
Introduction
analyzed should have the highest priority, for avoiding a tedious isolation of known
compounds, and for checking whether a compound preparatively isolated is genuine natural
product or possibly an artifact (Bringmann and Lang 2003).
In order to discover new bioactive compounds from their biological sources, which
could become new leads or new drugs, extracts should be submitted at the same time to a
chemical screening and to various biological or pharmacological targets. The chemical
screening or metabolite profiling is aimed at distinguishing between already known
compounds (dereplication) and new molecules directly in crude extracts. Thus, the tedious
isolation of known compounds can be avoided and a targeted isolation of constituents
presenting novel or unusual spectroscopic features can be undertaken. (Wolfender, et al 2005)
Metabolite profiling in crude extracts is not an easy task to perform since natural
products display a very important structural diversity. For each compound the order of the
atoms and stereochemical orientations have to be elucidated de novo in a complex manner and
the compounds can not simply be sequenced as it is the case for genes or proteins.
Consequently a single analytical technique does not exist, which is capable of profiling all
secondary metabolites in the biological source. The dereplication procedure strongly relies
mainly on hyphenated techniques coupled to HPLC such as LC-UV-photodiode array
detection (LC/UV-DAD), LC-mass spectroscopy (LC-MS, LC-MS-MS) and LC-NMR which
has been successfully and practically achieved in the last decade. (Wolfender, et al 2005, and
Exarchou, et al, 2005). A common step in the purification and analysis of chemicals of
unknown structure is the separation of the individual components from a chemical or
biological mixture. Chromatographic separation techniques have been coupled with large
number
of
detection
methods,
including
ultraviolet-optical
spectroscopy,
Raman
spectroscopy, mass spectrometry, conductivity measurements and NMR spectroscopy. Each
technique can be characterised in terms of easy implementation, intrinsic sensitivity, structural
information produced, and non destructive nature (Webb , 2005).
The combination of LC-UV and LC-MS information can be helpful in a first step of
dereplication, especially when this information is combined with taxonomical considerations
for cross search in natural product databases. This approach is, however, limited by the
unavailability of general LC-MS and LC-MS-MS databases. For the on-line de novo structure
determination of natural products, LC-NMR (see figures 1.7 and 1.8) plays a key role and
allows the recording of precious complementary on-line structure information when LC-UVMS data are often insufficient for unambiguous peak identification. (Wolfender, et al 2005).
18
Introduction
The online technique of liquid chromatography coupled with solid-phase extraction
and NMR (LC-SPE-NMR) has recently been used to analyze mixtures originating from
natural product extracts, drug metabolites, and pharmaceutical impurities. The growing use of
this technique results largely from the capability of on-line LC-SPE to isolate, enrich and
allow NMR analysis of an individual analyte present in a complex micture (Xu and Alexander
, 2005).
Fig. (1.7) Various LC-NMR modes and their applications in natural product analysis
(Exarchou, et al, 2005)
Fig. (1.8) Schematic diagram of the experimental set-up used for HPLC-NMR coupling; BPSU= Brucker peak
sampler unit; (
) capillary junctions; (-----) electric junctions. (Albert, 2002)
Other isolation strategies are focused on the biological activities of the constituents of
the given biological source through what is called „bioactivity-guided isolation“ , where the
isolation steps will continue only with regard to the bioactve fractions and subfractions of the
19
Introduction
biological mixture or extract until the isolation of the bioactive pure compound (Gunatilaka et
al , 1994)
An example for bioactivity-guided isolation for natural product-based anticancer
agents is demonstrated in figure 1.9, where the bioactive compounds and their analogues have
been subjected to cytotoxicity assays with a view to selecting candidates for further
development as anticancer agents.
Extraction
screening for
bioactivity
Plant material
Hex.Ext.
MEK Ext.
MeOH Ext.
Bioactive Ext.
Bioactivity-guided
fractionation
Confirm bioactivity
Structure elucidation
SAR- Studies
Solvent-solvent
partition
Sephadex LH-20
gel filtration
Si gel and/or
RP CC
Si gel and/or
RP HPLC
Si gel and/or
RP PTLC
Bioactive
compounds
physical and
spectroscopic data
Cytotoxic assay
Analog synthesis
Fig (1.9) , Steps in bioactivity-guided isolation,( Gunatilaka et al , 1994)
With few exceptions, intense and systematic bioassay-guided studies of extract and
compounds from marine organisms by academic, government, and industerial research groups
using clinically relevant assay to discover naturally occuring substances with therapeutic
potential have only been underway for less than 10 years. Because of the tremendous
advances in understanding of the biology of certain diseases and the concomitant explosion of
new assays, researchers are now in a position to explore fully the potential of natural products.
These biological advances highly complement the devlopment of new chemical technology,
i.e., new separation and structure elucidation techniques. ( McConnell et al., 1994).
20
Introduction
1.5- The structural elucidation of the natural products:
Tens of thousands of new compounds have to be synthesized or extracted from natural
sources in order to discover a potential drug. Despite the use of rational drug design
techniques, economic success mainly depends on the number of new candidates available for
activity tests. Consequently, research groups have begun to introduce new automation
procedures such as synthesis robots and combinatorial chemistry. These enhancements on the
production side are only part of the whole task. Additional efforts in the subsequent structure
elucidation process are also vital in order to avoid bottlenecks ( Neudert and Penk 1996). In
natural product drug discovery programs, the major bottleneck has always been structure
elucidation (Jaspars 1999). Conventional approaches to the structure elucidation of organic
compounds are based on the use of
spectroscopic data from different sources. The
spectroscopist´s task is to interpret the spectra and to derive structure proposals. The
efficiency of this process depends mainly on his or her knowledge of structure-spectrum
correlations, acquired in the course of everyday work (Neudert and Penk 1996).
The usual spectroscopic methods that used in the structural elucidation of natural product
chemistry includes UV, IR, NMR, and MS (Exarchou, et al, 2005). Recently, the
3D
structural determination was available through X-ray spectroscopy even if no other additional
spectral informations exist. An X-ray crystal structure determination is the ultimate analysis.
No other analytical technique currently available can deliver such complete and unambiguous
information about the nature of the substance being investigated, but this technique has some
limitations :
- Good single crystals are required, and these are sometimes difficult or impossible to obtain
without considerable effort.
- Decomposition of the compound during crystallisation attempts can be a difficulty with
reactive compounds.
- The analysis is done on a single crystal, which may not be representative of the bulk
material.
- The conformational results apply to the solid state and may be different to the molecular
conformations present in solution, which is where most reactions take place.
The modern and highly advanced technology applied in NMR spectroscopy and mass
spectrometry provide unequivocal structural information for the individually isolated
compounds (Exarchou, et al, 2005).
21
Introduction
1.5.1- Mass spectrometry :
Mass spectrometry (MS) has been appropriately used for analysis of molar masses of
molecules for the past 50 years (Burlingame,1992). MS remains the method of choice for
determining molecular formulas and identifying known substances. Where applicable,
depending largely upon volatility and fragmentation patterns, MS can be a very powerful tool,
as has been demonstrated in the analysis and sequencing of peptides and carbohydrates. In
parallel with development of NMR, the field has benefited greatly from studies of peptides
and proteins where the problem of volatility has been paramount (Hensens 1994).
The application of MS to large biomolecules and synthetic polymers has been limited
due to low volatility and thermal instability of these materials. These problems have been
overcome to a great extent through the development of soft ionization techniques such as
chemical ionization (CI), (Silverstein, et al ,1991, and Cotter, 1980) , secondary-ion mass
spectrometry (SIMS) (Silverstein, et al ,1991, Cotter, 1980 and Bletsos et al, 1991 ), field
desorption (FD) (Silverstein, et al ,1991, and Cotter, 1980), fast atom bombardment (FAB),
(Silverstein, et al ,1991, and Cotter, 1980), electrospray ionization (ESI) (Fenn J. B.2003, and
Ashcroft, A. E., 1997) and matrix asssted laser desorption ionization mass spectrometry
(MALDI), (Karas, et al, 1988, Karas, and Hillenkamp, 1991, and Tanaka, K., 2003).
Mass spectrometry (fig. 1.10) is an analytical technique that can provide both
qualitative (structure) and quantitative (molecular mass or concentration) information on
analyte molecules after their conversion to ions. The molecules of interest are first introduced
into the ionization source of the mass spectrometer, where they are first ionized to acquire
positive or negative charges. The ions then travel through the mass analyser and arrive at
diffferent parts of the detector according to their mass (m)-to-charge(z) ratio (m/z). After the
ions make contact with the detector, usable signals are generated and recorded by a computer
system. The computer displays the signals graphically as a mass spectrum showing the
relative abundance of the signals according to their m/z ratio ( Ho, et al , 2003).
Fig (1.10) a simple Mass spectrometer diagram
22
Introduction
The analyser and detector of the mass spectrometer, and often the ionisation source
too, are maintained under high vacuum to give the ions a reasonable chance of travelling from
one end of the instrument to the other without any hindrance from air molecules. The entire
operation of the mass spectrometer, and often the sample introduction process also, is under
complete data system control on modern mass spectrometers. (Ashcroft, A. E., 1997)
Methods of sample ionization:
The choice of ionization methods depends on the nature of the sample and type of
information required from the analysis. so-called “soft ionization” methods such as field
desorption and electrospray ionization tend to produce mass spectra with little or no
fragmentation content whereas “hard ionization” methods such as electron ionization (EI)
which are also refered to as electron impact ionization tend to produce mass spectra with large
amount of fragments or daughter ion peaks. There are several methods that are used
effectively to provide ionization in the currently available mass spectrometrs. In most
ionization methods there are the possibility of creating both positively and negatively charged
sample ions, depending on the proton affinity of the sample. Before embarking on an analysis
the user must decide whether to detect the positively or negatively charged ions (Ashcroft, A.
E., 1997).
Collision Induced dissociation ( CID):
Collision induced dissociation (CID) sometimes also called collisionally activated
decomposition (CAD) is one of the most common fragmentation procedures that is applied in
biopolymer (e.g. peptides or polysaccharides) sequencing, structural elucidation, and analyte
identification through finger-printing. The precursor ion enters the collision cell containing a
high pressure of energised, chemically inert collision gas (e.g. Ar, He, N2, CO2 etc.) The
precursor ions undergo repeated collisions with the collision gas, building up potential energy
in the molecule, until eventually the fragmentation threshold is reached and product ions are
formed, see figure (1.11). The types of fragmentaion that occur vary considerably with the
type of product ion and amount of energy involved. At lower energies (close to the threshold),
fragmentation reactions are often limited to neutral losses (H2O, MeOH, CO, CO2, MeCN
etc.) depending on the nature of the precursor ion. These neutral loses are often not cosidered
structurally significant, although they can be used to obtain information about functional
groups. At higher energies, retro-synthetic type reactions are often observed. These are much
more structurally significant, and often result in cleavage of the molecule at characteristic
positions.
23
Introduction
Collision
gas
Fragnent
ion
Neutral
lost
Collision
cell
Precursor
ion
fragment ions
(product ions)
Activated
ion
Fragmenting
ion
Activated fragment ion
(continues to fragment)
Fig (1.11). A Schematic diagram of CID fragmentation
If the energy is too high, C-C bond cleavage can occur leading to uncontrolled
fragmentation; this should be avoided. Usually it is best to work at around the fragmentation
threshold, or just above, to maintain most control over the fragmentation processes. Ion-trap
and FT-MS instruments allow for the most control over CID, but also tend to produce less
energetic reactions. Triple quadrupole and Q-TOF instruments tend to produce more energetic
CID with more fragmentation, but less operator control. Ion-trap and FT-MS allow multistage
fragmentation experiments to be conducted through, which is essential for structural
elucidation studies (Ojima et al 2005 and Gates, 2005b).
1.5.2- Nuclear magnetic resonance spectroscopy of Natural Products
(NMR):
Structural elucidation in general involves determining an extended sequence of bond
connectivities. Natural product chemists have to build up a structure from scratch in a logical
manner as they require some biogenetic knowledge and chemical intuition. Indeed they have
to be quite successful in solving structures where initially they had no idea what class of
compound was involved (Rycroft 1988)
Advances in radio frequency and probe technology, in the application of higher
magnetic fields and the ever expanding repertoire of pulse sequences in one-dimentional (1D)
and two-dimensional (2D) NMR and in the biological area of three-dimensional (3D) NMR
and even four-dimensional (4D) NMR, will inevitably be passed down to the structural
organic chemist to allow the resolution of more complex structural problems on increasingly
24
Introduction
smaller sample quantities. This non destructive methodology contrasts sharply with chemical
degradation studies that so typically have dominated natural product structure determination
in the not too distant past (Hensens 1994). The most of the recent advanced NMR techniques
will be disscussed, briefely, through the following structural elucidation strategies:
1.5.2.1- The most applied structral elucidation strategy depends largely on NMR and
MS modern techniques:
1) the establishment of the molecular formula: the determination of the molecular formula
is critical in the structure determination process of natural product. MS remains the
method of choice for determining molecular formulas and identifying known substances
(Hensens 1994). Depending on the particular instrumentation available, the accuracy of
these methods does not always define an emperical formula uniquely but provides a range
of formulas especially in the molecular weight range above 500 Dalton (Da). increasing
number of examples have recently appeared in the litrature where determining emperical
formulas of natural products with molecular wieght 500 Da or more have required an
interplay between MS and NMR methods.
2) Determination of carbon count of the molecule: a proton–decoupled 13C spectrum can in
principle provide a reliable carbon count of the molecule. However, depending on carbon
spin-lattice relaxation times, the
flip angle (pulse width) and the
acquisition time
employed, quaternary carbons can sometimes appear as week signals that may not be
readily distinguished from impurity peaks that are present. In this case, changing the
solvent pH, temperature, and/or parameter selection may be helpful. Demonstrating its
long-range connectivity to an assigned proton usually ensures that a quaternary carbon
belongs to molecule. It should be taken in mind that the No. of
13
C resonances in the
molecule does not immediately infer the correct carbon count and degeneracy in chemical
shift position is often the reason this drowback may be largely overcome by remeasurment
in different solvents. In other cases the degeneracy may be associated with a symmetry
feature of the molecule such as monomeric versus dimeric forms or overlaping of more
than one carbon peaks.
3) Determination of the proton count: The next step in the strategy is the determination of
the proton count, several methods are currently available to determine carbon
multiplicities and consequently the carbon-bound proton count. for example the Jmodulated attached-proton-test (APT), and the more preferred one „distortionless
25
Introduction
enhancement by polarization transfer“ (DEPT). DEPT is more preferred for various
reasons, it has reduced dependence on J, is not very sensitive to misset pulses or in
homogeneities in the rf, has definit sensitivity advantages, require less amount based on
the availabe aparatus. The solvent peak is effectively suppressed in all spectra, which may
thus advantageously detect nonquaternary carbons obscured by the solvent peak. Having
established the No. of carbon-bound protons in the molecule, it remains to determine the
No. of active protons, which is usually less straightforward. Deuterium (2H) exchange
experiments are often used. As far as MS methods are concerned for the determination of
the No. of active protons in a molecule, the formation of trimethylsilyl (TMS) ethers and
esters have enjoyed great popularity. MS data for the derivatives are compared with that
of the corresponding deuterated d9-TMS derivatives.
4) Determination of the No. of possible empirical formulas: With the molecular weight, 1H
and 13C counts in hand, severe restrictions are now placed on the No. of possible empirical
formulas for a given molecule. This is particularly the
case if some knowledge of
elements present has been obtained from either a combustion analysis or by inference
from NMR data. The HRMS data can only be regarded as consistent with the calculated
empirical formula as distinct from rigorously establishing it. This should be kept in mind
because journals such as the Journal of the American Chemical Society and Journal of
Organic Chemistry, for example, set widely varying, acceptable limits in 1991 of ± 3 and
± 13 mmu, respectively for mol.weights up to 500 and ± 6 and ± 16 mmu respectively, for
molecular weights 500 to 1000.
5) Determination of partial structures: This can be established from the 1H-1H connectivity
data, obtained from conventional double irradiation or 2D-COSY-Type experiments.
Included in the development of partial fragments is usually some knowledge of
preliminary
13
C NMR data as well as one-bond 1H-13C correlations. One-bond 1H-13C
techniques, which by definition are limited to nonquaternary carbons, provide little
connectivity data unless used in conjunction with long-range 1H-13C data where such 13C
NMR assignments are essential. The most important information the 2D
1
H-13C
correlation experiment offers is that can provide a clear distinction between methine and
methylene proton positions in crowded regions of a 1H NMR spectrum. This information
cannot always be unambiguously obtained from COSY-type experiments and when
attempted, is solely inferred from the No. and size of the couplings involved, a process
that is not unambiguous and difficult in situations where there is significant overlap.
26
Introduction
5a) H-H connectivities: this may be established from the basic correlation spectroscopy
(COSY) experiment, double-quantum filtered COSY (DQF-COSY), the complementary
relayed COSY (RCOSY), and homonuclear Hartman-Hahn spectroscopy (HOHAHA) or
total correlation spectroscopy (TOCSY) experiments.
5b)
1
H-13C one-bond connectivities: The 2D-heteronuclear correlation (HETCOR)
experiment, which correlates a 13C nucleus with its attached protons or the more preferred
and more sensetive experiment, 1H-detected heteronuclear multiple quantum coherence
experiment, (HMQC) or Hetero nuclear single quantum coherence (HSQC) are applicable
in this stage of the strategy.
6) Determination of the total Structure: this stage of the strategy can be performed by
NMR experiments that are used to bridge isolated spin systems in natural product
compound. For example :
6a) 1H-1H through space correlations: this correlations can be obtained from J-coupled
methods e.g. long range correlations in the COSY experiment (LOCOSY), or from dipolar
coupled methods e.g. 1D- (NOE) or 2D- (NOESY) nuclear overhauser effect or Rotating
frame NOESY, (ROESY) which give indications of distance-dependent „through space“
dipolar interactions. 1D- or 2D NOE experiment can give valuable informations not only
about the sequence and attachment of the separated partial structures but also about the
stereochemical/conformational informations.
6b)1H-13C long range connectivities: powerful as this widely applicable methodology of
13
C-detected and especially 1H-detected 1H-13C long range correlation experiments has
become, it nevertheless has its shortcomings. By implication, this technique depends on
each carbon being strategically located usually 2- or 3-bonds away from a proton in the
molecule. Many NMR experiments may be used in this stage e.g. long range version of
the
HETCOR experiment (LR-HETCOR) or correlation via long range coupling
(COLOC) which are 2D- 13C-detected 13C-1H long range correlation experiments. But the
more applicable and widely used experiment is heteronuclear multiple bond correlation
(HMBC), which inverse 1H-13C long range coupling through 1H-detected 1H-13C long
range correlation experiment. Because of the extreme usefulness of the HMBC
experiment, it is highly recommended that for optimal results, several experiments be run
under different experimental conditions (e.g., solvent or temperature), optimized for
different couplings and that these experimental conditions be reported.
27
Introduction
1.5.2.2- The Computer-Assisted Structural elucidation strategy (CASE):
The
most time-consuming process, that of assembling structures using the
substructure information extracted from the spectra can be performed by computers (Neudert
and Penk 1996). The ideal of computer assisted structure elucidation (CASE) is to generate,
exhaustively and without redundancy, all possible structures that are consistent with a
particular set of spectroscopic data. The aim is to achieve this goal with the minimum amount
of human intervention to overcome the
major bottleneck in the
natural product drug
discovery (Jaspars 1999).
Some approaches without resorting to 2D NMR data, have been tried using 13C NMR
data alone. One example of this type of system is Richert´s Specsolv (Will et al. 1996), which
is a new module of the NMR database SpecInfo. SpecInfo has used data from thousands of
compounds to calculate typical chemical shifts for a carbon with a particular set of neighbours
(a substructure). SpecSolv allows the user to enter the
13
C NMR spectrum of the unknown ,
without having to give the molecular formula, and structures matching these chemical shifts
are returned. For 80% all compounds containing only C,H,N,O,S,P and halogens, the correct
structure is derived. The program relies on a subspectrum search, which is then translated to a
collection of substructures. The substructures are assembled to give the greatest degree of
overlap, and the
13
C chemical shift is calculated for each generated structure. The structure
which gives the correct 13C NMR spectrum is returned to the user as the most likely candidate
structure. With „exotic unknowns“ such as complex natural products no final structure can be
proposed by SpecSolv due to the lack of spectral matches. In addition, two carbons with the
same neighbouring groups, but in different conformations may have very different chemical
shifts, and this may confound the subspectrum search. Although 13C shift based programs are
likely to find great utility in a synthetic laboratory with a high turnover of compounds, it is
unlikely to fulfil the ideal of CASE (Jaspars 1999).
Another approach to CASE, which does incorporate the use of 2D NMR data, is to
compose a new NMR pulse sequence which enables the direct determination of proton spin
systems in a molecule (Eggenberger and Bodenhausen 1989).The generation of proton spin
systems is also possible using a graph theoretical method which determines a C-C
connectivity matrix for protonated carbons by the direct determination of the matrix product
of 1H-1H COSY and 1H-13C COSY (1bond) spectra. These last two methods are useful in
generating spin systems only, but they do not allow the generation of complete structures
without the use of further long range data. This goal can be achieved through the application
28
Introduction
of CASE programs which are able to use routinely available 2D NMR data (e.g. 1H-1H
COSY, HMQC or HSQC, HMBC, NOESY and INADEQUATE) (Jaspars 1999).
The human thought process:
It is important to appreciate firstly, how a spectroscopist will elucidate a structure
from spectroscopic data. The process is summerized in Fig. (1.12). Normally the molecular
formula is derived from a combination of 13C NMR, DEPT and MS data. Using IR, UV and
13
C NMR the functional groups can be proposed, and 1H NMR coupling data or 2D NMR
correlations are used to assemble substructures. These are then combined into „working
structures“ which are possible combination of the substructures. These are then checked for
consistency with the 2D NMR data and MS fragmentations etc. The
13
C chemical shifts of
the surviving structure(s) are then compared with litrature, database or predicted values to
confirm the 2D structure of the molecule. To determine the relative stereochemistry of the
molecule, 1H coupling constant (J) and NOE data are used. The absolute streochemistry can
then be determind by a variety of methods such as optical rotatory dispersion-circular
dichroism (ORD-CD), derivatisation or degradation. It is important, as early as possible, to
know whether the unknown compound has previously been described, a process known as
dereplication, which can be performed using a combination of molecular formula,
substructures and chemical/structural databases (Corely and Dorley 1994).
Dereplicate by MF
Molecular
formula
Unsaturation
Number (UN)
MS,
NMR
Pure
compound
, IR
NMR
UV
Working 2D
Structures
Draw all isomers
Functional
groups
List of
Dereplicate
working 2D
structures by structure
NMR
NMR, MS, IR, UV
Substructures
New 2D
molecular
structure
X-RAY
Very secure
3D molecular
structure
Total
synthesis.
Reasonable 3D
molecular
structure
NMR
ORD
molecular
modeling
Known
molecular
structure
Fig. (1.12 ) Strategy for structure elucidation from spectroscopic data. (Crews et al. 1998)
Once it has been established that the compound in question has not been reported
before, the process of structure elucidation as depicted in Fig (1.12) can begin (Jaspars 1999).
29
Introduction
Two common strategies are employed when elucidating organic structures, one
involving C-C correlations from a 2D INADEQUATE spectrum, the other using C-C
connectivities inferred from C-H data. The INADEQUATE strategy is summarised in Fig.
(1.13a). The main problem of this approach is the inherent insensitivity of the INADEQUATE
experiment, which dictates that a large amount of sample is needed, which may be not
available in some cases, and that it must be (if available) soluble in a small amount of solvents
(Jaspars 1999).
The alternative strategy involves the use of more 2D NMR experiments, but these can
be obtained in a reasonable time using inverse detected techniques on a multimilligram
sample. This strategy is outlined in Fig. ( 1.13b) (Jaspars 1999).
Get 13C NMR spectrum
get multiplicities
Get 1H, 13CNMR spectra
get multiplicities and integrals
Get 2D INADEQUATE
Get 13C-1H correlation spectrum
(e.g. HMQC)
Make C-C map
Get one bond 13C-1H correlations
assign 1H resonances to 13C resonances
Get 13C-1H correlation spectrum
(e.g. HMQC)
Get 1H-1H correlation data (e.g. COSY)
Check assignment of diastereopic
protons using COSY and HMQC
Get one bond 13C-1H correlations
assign 1H resonances
Assemble substructures using
COSY data
Get long range 13C-1H correlation
spectrum (e.g. HMBC)
Bridge heteroatoms
using long range 13C-1H
correlation spectrum (e.g.
HMBC)or using NOE data
combine structures into all
possible working structures
Check all working structures for
consistency with 2D NMR data
Generate 2D structure
2D structure
Fig. (1.13a). A possible structure
elucidation strategy using
C-Ccorrelation data.
Fig.(1.13b). A possible structure
elucidation strategy using H-H
and H-C correlation data
Structure of the CASE Program:
The CASE system is composed of several steps (Fig.1.14) (Jaspars 1999).
1- The first step is the input of the spectra, or „peak-picking“, to convert the data into a
more computer digestible form. This can rely on the skill of a spectroscopist who
30
Introduction
translates the cross peaks of a 2D spectrum into correlations, or ideally on a sophisticated
peak picking program.
2- The next step is to produce a list of possible components (e.g. CH3, CH2-O etc.) present in
the molecule. Generated substructures can be checked during the process of structure
generation (Prospective checking) or after all complete structures have been generated
(retrospective checking). Clearly, prospective checking will be faster, as those
substructures that are not consistent with the 2D NMR data are removed from the
structure generation process. In the case of retrospective checking a combinatorial
explosion occurs for exhaustive structure generation, even for molecules of a moderate
size. These generated components are fed into the most important part of the program, the
structure generator.
Peak picking
routine
Component
generator
generate all
possible
structures
Generate
substructure
Yes
Structure
consistent with
2D NMR data?
Structure
generation/
Checking
routine
Add component
to substructure
Substructure
consistent with
2D NMR data?
Yes
All components
used up?
No
Yes
Possible solution
Prospective checking
Retrospective checking
Fig. (1.14). Components of a CASE program.
3- The next part is „the structure generator“ which will use the components that are
generated by one of the methods mentioned above to generate exhaustive list of all
possible structures without redundancy, and without missing out any plausible structures.
The structure generator is the part of the CASE program that will take the greatest
amount of CPU time, and this is also where the greatest time savings can be made by the
use of efficient algorithms.
31
Introduction
4- The generated structures are checked for consistency with the 2D NMR data. An
innovative feature to determine whether the structure generator is heading in the right
direction is by checking the rate at which 2D NMR constraints are being satisfied. In
general as the generation extends towards the correct structure, the number of constraints
satisfied should increase. As long as this rate of constraint satisfaction is above a
predetermined value, the structure generation continues, if it falls below this level
generation using this particular substructure is discontinued. This is a very powerful way
to direct the structure generation process, and greatly reduces the time taken to achieve a
plausible solution.
5- Determination of stereochemistry: For large molecules the determination of three
dimensional structure is performed by using a combination of molecular modelling and
constraints derived from NOE data as well as coupling constant information (Evans,
1995). The absolute stereochemistry will still need to be determined by the use of
degenerative methods, auxiliary reagents, or optical rotatory dispersion-circular dichroism
(ORD-CD) (Jaspars 1999).
Currently, the modification of CASE systems is continued to overcome as great as
possible, the above encountered problems including the CASE programs, structure generators,
and the database projects, in order to make the CASE more applicable.
Interaction between a spectroscopist and the CASE system will remain important in order
to generate the correct structure rapidly. Therefore CASE will complement the skills of the
spectroscopist, not replace them. The use of CASE system is likely to increase in the near
future, and this will enable the bottleneck so often caused by structure elucidation to be
removed from the natural product drug discovery process (Jaspars 1999).
32
Introduction
1.6- Marine Natural Products
1.6.1- Marine organisms are rich biological sources for bioactive natural product
discovery:
Picture from : Marine Pharmacology: Potentialities in the Treatment of
Infectious Diseases, Osteoporosis and Alzheimer’s Disease. (Bourguetkondracki and Kornprobst, 2005)
Selection of marine organisms as biological materials in this work was largely
attributed to the tremendous level of worldwide interest in marine natural products with
therapeutic potential in industry, academia, and government research labs (McConnell et al.,
1994, Proksch et al 2002). Marine natural products chemistry is essentially a child of the
1970´s that developed rapidly during the 1980´s and matured in the last decade (Faulkner,
2000a). By 1975 there were already three parallel tracks in marine natural products chemistry:
marine toxins, marine biomedicinals and marine chemical ecology. It is the integration of the
three fields of study that has given marine natural products chemistry its unique character and
vigour . Marine organisms have provided a seemingly endless parade of novel structures. New
carbon skeletons were discovered and several functional groups are uniquely or
predominantly marine (Faulkner 2000a).
The first natural products isolated from marine organisms that proved to be valuable
lead structures for the development of new pharmaceuticals were the unusual nucleosides
33
Introduction
spongouridine and spongothymidine from the caribbean sponge [Cryptotethia crypta,
Tethydae, (Bregmann and Feeney 1951)] which served as models for the development of
adenine arabinoside (ARA-A), (Vidarabin, Thilo), for treatment of Herpes simplex infection
and cytosine arabinoside (ARA-C), (Cytarabin, Alexan, Udicil), for the treatment of leukemia
respectively (Ireland et al 1993). The discovery of sizeable quantities of prostaglandins, which
had been discovered as important mediators involved in inflammatory disease, fever and pain
in the gorgonian Plexaura homomalla by Weinheimer and Spraggnis in 1969 is considered
as the take-off point of systematic investigation of marine environments as sources of novel
biologically active componds (Newman, et al., 2000a; Proksch et al., 2002).
1.6.2- Marine natural products:
Marine natural products with their unique structural features and pronounced
biological activities continue to produce lead structures in the search for new drugs from
nature. Invertebrates such as sponges, tunicates, shell-less mollusks and others that are either
sessile or slow moving and mostly lack morphological defense structures have so far provided
the largest number of marine-derived secondary constituents including some of the most
interesting drug candidates (Proksch et al. 2003)
It is clear that marine natural products chemistry has had a major impact over the past
30 years, and man can not predict what will happen in the next few years (Faulkner 2000a).
Faulkner 2000a, predict a great impact of chemical and biological researches including
genetic engineering concerning different forms of marine living forms ranging from marine
invertebrates to the marine-derived microorganisms. Faulkner also expects that, in the near
future we able to transfer biosynthetic genes from one marine organism to another and
imagine the marine natural product chemist of 2025 still involved in structural elucidation,
but considerable effort to the genetic engineering required to produce unique metabolites by
fermentation of genetically modified microbes. This will accomplish the goal of having the
marine organisms provide the inspiration for new compounds while avoiding their excessive
harvesting.
The sponges are the source of the greatest diversity of marine natural products. About
one-third of all marine natural products have been isolated from sponges, which make them
currently the most popular source of novel compounds (Whitehead, 1999). The marine
sponges are considered not only as a very important source of new natural products but also a
34
Introduction
source for bioactive compounds. These compounds are interesting candidates for new drugs,
primarily in the area of cancer, anti-inflammatory and analgesic (Proksch et al, 2002).
1.6.3- The biological evaluation of marine natural products
Marine organisms have provided a large proportion of the bioactive natural products
reported over the last 20 years, but non of these compounds have reached the pharmaceutical
marketplace (Faulkner 2000b).
Now, marine natural products are already available in the market as effective drugs.
Ziconotide (Prialt) which is a 25-aminoacid peptide isolated from the venom of the marine
snail Conus magus is now available in the market as a potent analgesic for severe chronic
pain, its analgesic effect is comparable to the opioid analgesics (e.g. Morphine) but its mode
of action not includes binding to the opioid receptors and its actions are not blocked by opioid
antagonists. Ziconotide has a unique mechanism of action,
binding to N-type calcium
channels on nerves in the spinal cord and blocking their ability to transmit pain signals to the
brain. Unlike the opioid analgesics, it doesn’t cause tolerance or addiction (Hussar 2006).
However, several marine-derived compounds have generated considerable interest
scientifically, commercially, and from public and health point of view, these include
prostaglandins, palytoxin, ciguatoxin. Further, because of their unique and potent biological
activities, several marine-derived compounds have already found use as biological probes or
biochemical tools and are sold commercially, e.g., palytoxin, brevetoxins, ocadaic,
tetrodotoxin, saxitoxin, calyculin A, manoalide, and kainic acid (McConnell et al., 1994).
Several marine derived natural products have a significant biological activity and many of
them, are currently, in different phases of clinical trials as drug candidates. Some of these
bioactive marine-derived natural products will be mentioned below.
Approximately, half of all marine natural products papers report bioactivity data for
new compounds (Faulkner 2000b). Significant number of marine-drived natural products
have been entered into antitumor preclinical or clinical trials since the early 1980s (Newmann
and Cragg 2004).
Table 1.2 Status of marine-derived natural products in clinical and preclinical
anticancer trials*
Name
Source
Status
comment
Didemnin B
Trididemnum solidum
Phase II
Dropped middle 90s (very toxic)
Dolastatin 10
Dolabella auricularia
Phase I/II
35
Introduction
Giroline
Pseudaxinyssa cantharella Phase I
Discontinued (hypertension)
Bengamide
derivative
Jaspis sp.
Phase I
Licensed to Novartis, Met-AP1
inhibitor, withdrawn 2002
Cryptophycins
(also arenastatin)
Nostoc sp.& Dysidea
arenaria
Phase I
Licensed to Lilly, but withdrawn 2002.
Bryostatin 1
Bugula neritina
Phase II
TZT-1027
Synthetic dolastatin
Phase II
Cematodin
Synthetic dolastatin 15
Phase I/II
ILX 651,
synthetadin
Synthetic dolastatin 15
Phase I/II
also known as auristatin PE and
soblidotin
Ecteinascidin 743 Ecteinascidia turbinata
Phase
II/III
Aplidine
Aplidium albicans
Phase II
Dehydrodidemnin B, made by total
synthesis
E7389
Lissodendoryx sp.
Phase I
Synthetic halichondrin B derivative
Discodermolide
Discodermia dissoluta
Phase I
Kahalalide F
Elysia rufescens / Bryopsis Phase II
sp.
ES-285
(spisulosine)
Spisula polynyma
Phase I
Rho-GTP inhibitor
HTI-286
(hemiasterlin
derivative)
Cymbastella sp.
Phase II
Synthetic derivative, licensed to Wyeth.
KRN-7000
Agelas mauritianus
Phase I
Squalamine
Squalus acanthias
Phase II
also has antiangiogenic activity.
Æ-941(Neovastat) shark
Phase
II/III
Defined mixture of <500 kDa from
cartilage, also has antiangiogenic
activity.
NVP-LAQ824
Synthetic
Phase I
Derived from Psammaplin trichostatin,
and trapoxin structures.
LU103793
Dolabella auricularia
Phase II
Semisynthetic pseudopterosin
Laulimalide
Cacospongia mycofijiensis preclinical synthesized by a variety of investigators
Curacin A
Lyngbya majuscula
Vitilevuamide
preclinical
Didemnum cucliferum &
Polysyncraton lithostrotum
Diazonamide
Diazona angulata
preclinical synthesized and new structure
elucidated.
Eleutherobin
Eleutherobia sp.
preclinical synthesized and also derivatized, can be
produced by aquaculture
Sarcodictyin
synthetic
derivatives.
Sarcodictyon roseum
preclinical
Licensed to PharmaMar, (isolated also
from E. grandifolia together with very
similar
analogues
as
bioactive
depsipeptides in the present work.)
preclinical synthesized
36
Introduction
Peloruside A
Mycale hentscheli
preclinical
Salicylhalimides
A
Haliclona sp.
preclinical first marine Vo-ATPase inhibitor,
synthesized.
Thiocoraline
Micromonospora marina
preclinical DNA polymerase α inhibitor
Ascididemnin
preclinical Reductive DNA-cleavage agents
Variolins
Kirkpatrickia variolosa
preclinical Cdk inhibitors
Dictyodendrins
Dictyodendrilla
verongiformis
preclinical Telomerase inhibitors
(*Reported from Proksch et al.2002 and Newmann and Cragg 2004)
There are other marine-derived natural products showing vast pharmacological
activities, many of them are currently evaluated as drug candidates and entered clinical trials.
Table (1.3) shows other marine-derived natural products that are, currently, in clinical trials
for non antitumour therapeutic activities.
Table 1.3. Status of marine-derived natural products in clinical and preclinical
trials* other than antitumors.
Name
Source
Status (disease)
comment
GTS-21 (aka
DMBX)
Amphiporeus lactifloreus Phase I
(Alzheimer´s /
schizophrinia)
Manoalide
Luffariaella variabilis
Phase II
(antipsoriatic)
discontinued (formulation
problems)
IPL-576,092 (aka
HMR-4011A)
Petrosia contignata
Phase II
(antiasthmatic)
licensed to Aventis
IPL-512,602
derivative of IPL-576092 Phase II
(antiasthmatic)
licensed to Aventis
IPL-550,260
derivative of IPL-576092 Phase I
(antiasthmatic)
licensed to Aventis
Ziconotide (aka
Prialt)
Conus magus
Phase III
(neuropathic pain)
Licensed by Elan to Warner
Lambert (it is on the market
now)
CGX-1160
Conus geographus
Phase I (pain)
Contulakin G
CGX-1007
Conus geographus
Phase I (pain &
epilepsy)
Conantokin G; discontinued
AMM336
Conus catus
Preclinical (pain)
ω-conotoxin CVID
χ-conotoxin
Conus sp.
Preclinical (pain)
Conotoxin MR1A/B
CGX-1063
Thr10-contulakin G
Preclinical (pain)
Modified toxin
ACV1
Conus victoriae
Preclinical (pain)
Α-conotoxin Vc1.1
Methopterosin
Pseudopterogorgia
elisabethae
Phase I
(inflamation /
wound)
Semisynthetic pseudopterosin
derivative
Modification of a worm toxin :
contignasterol 8 IZP-94,005
(*Reported from Proksch et al.2002 and Newmann and Cragg 2004)
37
Introduction
Although during 2000 no new marine natural product was approved for patient care by
the U.S. Food and Drug Administration (FDA), during 2000 preclinical pharmacologic
research with marine chemicals continued to proceed at a very active pace, involving both
natural product chemists and pharmacologists from foreign countries and the United States
(Mayer and Hamann 2004)
In order to reflect the worldwide interest towards the marine derived natural products
as drug candidates, Mayer and Hamann 2004 reported a review for preclinical trials involving
pharmacological and toxicological studies for some bioactive marine natural products that are
published during only one year (2000), 35 marine natural products reported as antibacterial,
anticoagulant, antifungal, antimalarial, antiplatelet, antituberculosis, antiviral and 20 marine
natural products having antiinflammatory, immunosuppressant, cardiovascular activity, and
compounds affecting nervous systems. Some of this report is presented in Tables (1.4 and 1.5
respectively.)
Table
1.4.
Marine
compounds
with
antibacterial,
anticoagulant,
antifungal,
antimalarial, antiplatelet, antituberculosis, and antiviral activities, that are reported in
one year (2000) as drug leads in preclinical trials.
Drug class
Compound/organism
Chemistry
Pharmacologic activity
Molecular
mechanism of
action (MMOA)
Antibacterial
Acetylenic acids/sponge
Fatty acid
Gram-positive and
Undetermined
nigative inhibition
Antibacterial
Discorhabdin R /sponge
Alkaloid
Gram-positive and
Undetermined
nigative inhibition
Anticoagulant
Anticoagulant
Fucoidan/alga
Proteoglycan/alga
Sulfated
Inhibition of
No effect on P-
polysaccharide
microvascular thrombus
and L-selectin
Polysaccharide
Anticoagulant
Inhibition of
thrombin and
potentiation of
antithrombin III
Anticoagulant
Sulfated D-galactan/alga
Sulfated
Anticoagulant
Polysaccharide
Inhibition of
thrombin and
factor Xa
Antifungal
Lyngbyabellin B/bacterium
Depsipeptide
C. albicans inhibition
undetermined
Antifungal
Naamine D/sponge
Alkaloid
C. neoformans inhibition
Nitric oxide
38
Introduction
inhibition
Antimalarial
Ascosalipyrrolidin 1A/fungus Polyketide
P. falciparum inhibition
p561ck tyrosine
Antimalarial
Homofascaplysin A &
Sesterterpene
P. falciparum inhibition
Undetermined
Alkaloid
In vivo P. berghei
Undetermined
fascaplysin/sponge
Antimalarial
Manzamine A/sponge
inhibition
Antiplatelet
Eryloside F/sponge
Sterol
Platelet aggregation
Thrombin
glycoside
inhibition
receptor
antagonist
Anti-
Axisonitrile-3/sponge
Sesquiterpene
M. tuberculosis inhibition Undetermined
Elisapterosin B/soft coral
Diterpene
M. tuberculosis inhibition Undetermined
Cyclodidemniserinol
Polyketide
In vitro HIV infection
HIV-1 integrase
inhibition
inhibition
tuberculosis
Antituberculosis
Antiviral
trisulfate/ascidian
Antiviral
Dragmacidin F/sponge
Alkaloid
In vitro HSV-1 and HIV- Undetermined
1 inhibition
Antiviral
Lobohedleolide, 17-
Diterpene
dimethylamino/soft coral
Antiviral
Mololipids /sponge
In vitro HIV infection
Undetermined
inhibition
Alkaloid
In vitro HIV-1 infection
Undetermined
inhibition
Table.1-5. Marine compounds with anti-inflammatory and immunosuppressant effects and
others affecting cardiovascular and nervous system that reported in one year (2000) as drug
candidates in preclinical trials.
Drug class
Compound/organism
chemistry
Pharmacologic activity
Molecular mechanism
of action (MMOA)
Anti-
Cavernolide /coral
Terpene
inflammatory
Anti-
Contignasterol/ sponge
sterol
inflammatory
In vitroTNF-α, NO and
sPLA2, iNOS and COX2
PGE2 inhibition
inhibition
In vivo allergen-induced
Undetermined
plasma protein exudation
inhibition
Anti-
Cyclolinteinone/sponge
Sesterterpene In vitro NO and PGE2
inflammatory
inhibition
NF-kB binding, iNOS
and COX2 expresion
inhibition
Anti-
Oxepinamide A/fungus
Alkaloid
inflammatory
Antiinflammatory
In vivo neurogenic
Undetermined
inflammation assay
Sterol/alga
Sterol
In vivo inflammation
glycoside
assay
39
Undetermined
Introduction
Cardiovascular
Docosahexanoic acid/fish
Fatty acid
In vivo vascular reactivity Undetermined
assays and in vitro
biochemical assays
Immuno-
Theonellapeptolides/
suppressant
sponge
Nervous system
Conantokin G, T/snail
peptide
In vitro mixed
Undetermined
lymphocyte reaction assay
Peptide
In vivo parkinson´s
Alteration of striatal
disease model
efferent neurons
function
Nervous system
Conantokin-G/snail
Peptide
In vivo block of
NMDA receptor
dopamine-enhancing drug antagonism
methamphetamine
Nervous system
Nervous system
Nervous system
Conantokin-G/snail
Conantokin-G/snail
Conantokin-R/snail
Peptide
Peptide
Peptide
In vitro neuronal and
Competitive antagonist
oocyte whole-cell
of NR2B NMDA
electrophysiology
receptors
In vivo and in vitro
Decrease Ca++ response
neuroprotective assays
to NMDA
In vitro NMDA receptor
NR2 NMDA receptor
antagonist and in vivo
selectivity
anticonvulsant assays
Nervous system
Conantokin-R/snail
Peptide
In vitro binding assays
Disulfide loop not
essential for receptor
and cation binding.
1.7- Aim of the work:
The present work is focused on some of the modern methods that currently included in
the recent discovery of natural products with therapeutic potentials. This study deals with the
modern technical applications ranging from the use of various types of HPLC, the use of
hyphenated analytical techniques during the isolation procedures (e.g. HPLC/UV, HPLC/MS,
HPLC/MS/MS, GC/MS), new methods of chemical modification and different techniques in
molecular weight determination during the structural elucidation, different methods of NMR
spectral analysis and bioactivity guided isolation.
In the present study, the isolation of some new natural product is guided by
dereplication procedure using hyphenated techniques coupled to HPLC such as LC-mass
spectroscopy (LC-MS, LC-MS-MS). Some soft bodied marine organisms are included in this
study as they are attractive biological materials that, attract worlwide interest of natural
product chemists and pharmacologists.
40
Material and Methods
II. Materials and Methods
2.1 Animal materials
All biological materials included in this work are marine animals, and collected from four
different geographical zones and including 2 saccoglussan mollusks, Elysia rufescens which
was collected from the
black point Kahala, in the
Pacific Ocean (Hawaii), and Elysia
grandifolia which was collected from the Indian Ocean (India), and 4 sponges, unidentified
Pachychalina sp., Petrosia nigricans, and Callyspongia biru which were collected from the
Pacific Ocean (Indonesia), in addition to Hyrtios erectus which was collected from the Red Sea
(Egypt).
2.1.1 Sacoglossan mollusks from the genus Elysia :
Genus Elysia belongs to the herbivorous marine mollusks, sacoglossans, with the
ability to sequester from their algal diet functioning chloroplasts, which then may participate in
the biosynthesis of secondary metabolites. (Hamann and Scheuer 1993)
2.1.1.1. Elysia rufescens
Elysia rufescens Pease, 1871
Order: Sacoglossa
Superfamily: Elysioidae
Family: Elysiidae
Elysia rufescens : "Length: 11 mm up to 60 mm; parapodial height: 3 mm. Parapodia is low but
erect, with two or three undulations; rhinophores are long, slender and directed anteriorly. The
pericardial prominence is a white sac at the anterior end of the parapodia. The color is dark red
maculated with spots of creamy green which impart a reticulated appearance to the animal. The
parapodial margine is orange, while the rhinophores are dark red in color tipped with purple".
(Kay, E.A. 1979; Pease, W.H. 1871; Rudman, W.B., 1999). Elysia rufescens was collected and
provided by D. Horgen (University of Hawaii at Manoa) from Black point, Kahala, Oahu
Island in Hawaii, dried and sent as coarse powder to our Laboratory in April 2003.
2.1.1.2- Elysia grandifolia
Elysia grandifolia Kelaart, 1858
Order: Sacoglossan
Superfamily: Elysioidea
Family: Elysiidae
41
Material and Methods
The mollusc E. grandifolia (17 molluscs ; 77 g wet wt) which were observed to be feeding on
the algae Bryopsis plumosa (Hudson), were collected by snorkeling from the Gulf of Mannar
and Palk Bay, Rameswaram, India (9°15' N; 79°15'E) at 1 to 2 meter depth in May, 2003,
immediately preserved in methanol and kept frozen until extraction work. The animal was 10
cm in length and had a translucent green color with a large parapodial margin. The parodial
margin had a very thin, black line, and a submarginal yellow or orange band.
Rhinophores
Parapodial undulations
Rhinophore
Parapodia
Figure ( 2.1) Elysia sp
Fig. ( 2.2 ) Elysia rufescens 60mm,
on Bryopsis pennata (algal diet)
Fig. ( 2.3 ) Elysia rufescens
Figure ( 2.4 ) Elysia ornata
Fig. ( 2.5 ) Elysia grandifolia
42
Material and Methods
There was no white line between the orange and yellow bands (distinct. from E.
ornata). The tip of the rhinophores had a similar color as its orange bands. The body is covered
with numerous black and white dots. It had a long renopericardial ridge with 7 pairs of dorsal
vessels as described by Eliot (1906). The figure of the teeth showed blunt tips and rather broad,
denticulate blades as reported by O’Donoughe (1932) for specimens from the Gulf of Mannar.
The sacoglossan was identified by Dr. Kathe Rose Jensen, National Natural History Museum,
Copenhagen. Voucher specimens (EG-TMMP-3) were deposited in the Centre for Marine
Diversity, University of Kerala, India. Jensen (1992) reported that E. grandifolia feeds on
Bryopsis in both the Caribbean and the Indo West Pacific. (Jensen, 1992 & Rudman, 1999). It
is very similar in colour to Elysia ornata. It is possibly the same species, Rudmann 2001
thought that, there are probably good grounds for considering it distinct. It grows to a very
large size, 10 cm or more. He suspect that Elysia ornata and E. grandifolia are part of a group
of similarly coloured species which need closer examination. (Rudman, 2001 & Kelaart, 1858).
2.1.2- The sponges
"Sponge" is the common name for the Poriferae, who are members of the phylum Porifera. It is
named for the pores with which every sponge is covered, 'porifera' meaning 'pore bearer'.
Sponges come in an incredible variety of colours and an amazing array of shapes. They
probably achieved their greatest diversity during the Cretaceous period and between 5000 and
10000 species live today. They are the simplest form of the multicellular organisms and all are
aquatic, benthic organisms. Most are marine, with only 150 fresh water species known today.
They are known to be present in all seas and in several lakes.
Sponges are divided into three distinct groups, as follows:
Hexactinellida, or glass sponges. These are characterized by siliceous spicules consisting of
six rays intersecting at right angles.
Demospongia. This is by far the most diverse sponge group, although they are not well
represented in the fossil record, as they do not possess skeletons that would easily fossilize.
Demosponge skeletons are composed of 'spongin' fibres and/or siliceous spicules.
Demosponges take on a variety of growth forms from encrusting sheets living beneath stones to
branching stalks upright in the water column. They tend to be large and only exhibit the more
complex 'leucon' grade of organization.
43
Material and Methods
Calcarea. Members of the group Calcarea, the calcareous sponges, are the only sponges that
possess spicules composed of calcium carbonate.
Morphology:
Sponges have a cellular grade of organization, lack symmetry, and unlike all other
marine invertebrates, they do not possess any structures that can be considered true tissues or
organs. Sponges do not have stomachs , kidneys. They do not have nerve or muscle cells. The
body of a sponge is hollow, and it is composed of a simple aggregation of cells, between which
there is little nervous coordination. However, these cells perform a variety of bodily functions
and appear to be more independent of each other than are the cells of other animals. This
gelatinous matrix is supported by an internal skeleton of spicules of silica, calcium carbonate,
or fibrous protein known as 'spongin'. The sponge's body encloses a vast network of chambers
and canals that connect to the open pores on their surface. The porous nature of the sponges
makes them ideally suited for habitation by opportunistic crustaceans, various worms, etc. in
addition to these macro-organisms, bacteria, fungi, blue-green algae and dinoflagellates are also
observed in many species. Many of the most common types of cells are described below.
Pinacocytes
These cells are the "skin cells" of sponges. They line the exterior of the sponge body
wall. They are thin, leathery and tightly packed together.
Choanocytes
These distinctive cells line the interior body walls of sponges. These cells have a central
flagellum that is surrounded by a collar of microvilli. It is their striking resemblance to the
single-celled protists called choanoflagellates that make many scientists believe that
choanoflagellates are the sister group to the animals Choanocytes. Their flagella beat to create
the active pumping of water through the sponge, while the collars of the choanocytes are the
primary areas that nutrients are absorbed into the sponge. Furthermore, in some sponges the
choanoflagellates develop into gametes.
Mesenchyme
Between the two layers is a thin space called mesenchyme or mesohyl. The
mesenchyme consists of a proteinaceous matrix, some cells, and spicules.
Archaeocytes
Archaeocytes are very important to the functioning of a sponge. These cells are
totipotent, which means that they can change into all other types of sponge cells. Archaeocytes
44
Material and Methods
ingest and digest food caught by the choanocyte collars and transport nutrients to the other cells
of the sponge. In some sponges, archaeocytes develop into gametes.
Sclerocytes
The secretion of spicules is carried out by sclerocytes. Other cells, called spongocytes,
secrete the spongin skeletat fibres when those are present.
Myocytes and Porocytes
Poriferans do not have any muscle cells, so their movement is rather limited. However,
some poriferan cells can contract in a similar fashion as muscle cells. Myocytes and porocytes
which surround canal openings and pores can contract to regulate flow through the sponge.
Taxonomic classification
The Porifera are sometimes placed into the Subkingdom Parazoa of Animalia, while all the
other animals are in the Metazoa. As described above, they are divided into three distinct
groups, the Hexactinellida (glass sponges), the Demospongia, and the Calcarea (calcareous
sponges). The classification of sponges once relied on the characters of the spicules and the
fibres, since the outer shape and the colour vary with the habitat. However, the classification is
now more complex. The following extract, from the systematics page of the excellent Berkeley
University description of Sponges [http.//www.ucmp.berkeley.edu/porifera/porifera.html.]
explains the development of the current taxonomic classification of sponges.
At one time, a diagnostic feature of the Porifera was the presence of spicules. As a
result, certain fossil groups whose organization was consistent with that of living sponges were
not placed within the phylum Porifera. In particular, groups with a solid calcareous skeleton
such as the Archaeocyatha, chaetetids, sphinctozoans, stromatoporoids, and receptaculids were
problematic. A great deal of insight into the phylogenetic affinities of these groups was gained
with the discovery of more than 15 extant species of sponges having a solid calcareous
skeleton. These species are diverse in form, and would be classified with the chaetetids,
sphinctozoans and stromatoporoids if found as fossils. However, with the living material in
hand, histological, cytological, and larval characteristics can be observed. This information
suggests that these 15 species can readily be placed within the Calcarea and the Demospongia.
This radically changes our view of poriferan phylogeny.
It is widely accepted among poriferan biologists that the Calcarea and the Demospongia
are more closely related to each other than either is to the Hexactinellida. With the discovery of
living chaetetids, stromatoporoids, and sphinctozoans, a fourth class was erected for these socalled sclerosponges. However, the Sclerospongia is not a natural monophyletic grouping and
is thus being abandoned. The abundant fossil chaetetids, stromatoporoids, and sphinctozoans
are probably part of the classes Demospongia and Calcarea, though some uncertainty still
45
Material and Methods
remains. The Archaeocyatha pose a special case. No living representative of this group has
been discovered. Their organization is consistent with that of living sponges. The one
phylogenetic analysis (carried out by Reitner) that included archaeocyaths with other sponges,
grouped them as sisters to the demosponges. Therefore, although the taxonomic term
Archaeocyatha is often accorded phylum status it is likely a sub-clade of the phylum Porifera,
thereby violating the ranking system. (www.bbm.me.uk/portsdown/PH_321_sponge.htm,
Pechenik 2000, Barnes 1986, Goerge and Goerge, 1979, Gerwick and Bernart 1992, Pearse et
al. 1987, and Hooper and van Soest 2002)
2.1.2.1- Pachychalina sp.
The sponge Pachychalina sp. (Phylum: Porifera, Class: Demospongia, order:
Haploscleridea,
suborder:
Haplosclerina
family:
Niphatidae)
is
columnar,
clavate,
subcylindrical growth forms, having a well-developed aquiferous system with large canals
rendering the texture highly compresible. Numerous small rounded auiferous openings not
clearly differentiated into oscules or pores. The whole skeleton is rather confused, with the
choanosomal skeleton irregular, diffuse, and occuring as a radially plumose network of
primary, ramified, multispicular fibers, irregularly connected by no proper secondary
multispicular fibers and no visible spongin. The outer surface is light rose in colour, the inner
side is white to faint yellow, the growing branches are finger-like branches. It was collected in
Pulau Baranglompo (Indonesia) at depth of 27 ft. (25. 07. 1997). A voucher specimen has been
deposited in the Zoological Museum Amsterdam under registration number ZMA POR17545
2.1.2.2- Petrosia nigricans
The sponge Petrosia nigricans Lindgren, 1897 (Phylum: Porifera, class: Demospongia,
order: Haplosclerida, suborder: Petrosina, family: Pterosiidae) is a massive sponge, irrigularly
globular, with a large base that produces several fused lobes. Aquiferous system with a terminal
deep aquiferous cavity connected with a unique volcano-shaped osculumat the end of the
lobes. Surface smooth, fine, compact, covered by a fine ectosomal layer, hispid to the touch.
Texture hard, firm. The collected specimens are brown, hard, rag-like, the outer surface is
rough sometimes with long fistulae, the inner side is white to faint yellow. The specimen
shows small krabs introduced their heads into the osculum of the sponges. It was collected in
Pulau Baranglompo (Indonesia) at depth of 30 ft. (25. 07. 1997). A voucher specimens has
been deposited in the Zoological Museum Amsterdam under registration numbers ZMA
POR17546 and ZMA POR17713
46
Material and Methods
Fig (2.6) Microscopic
view of a Poriferan
wall
(www.ucmp.berkeley.ed
u/porifera/pororg.html)
Figure (2.8), Callyspongia biru
Figure ( 2.7 ) Pachychalina sp.
Figure (2.10), Hyrtios erectus
Figure (2.9), Petrosia nigricans
47
Material and Methods
2.1.2.3- Callyspongia biru
The sponge Callyspongia biru. (Phylum: Porifera, Class: Demospongia, order:
Haploscleridea, suborder: Haplosclerina family: Callyspongiidae) is lobate repent encrusting to
massive or short anastomosed upright tubes with apical oscule. Surface smooth. Single
ectosomal non-hispid, layer and regular size of single, rounded to polygonal mesh.
Choanosomal a well- developed network with rectangular empty mesh, primary choanosomal
fibers multispicular, non-fasciculated, non ramified, spongin sheath well-defined. The
specimens are blue, soft , finger-like branched sponge. the inner side is white to faint yellow.
It was collected in Taka Bako (Indonesia) at depth of 30 ft. (31. 07. 1997). A voucher specimen
has been deposited in the Zoological Museum Amsterdam under registration number ZMA
POR18290
2.1.2.4- Hyrtios erectus
The sponge Hyrtios erectus. Keller (Phylum Porifera, class Demospongia, order
Dictyoceratida, family Thorectidae) is cake shaped, with protruding lobes forming large erect
rounded digits, occasionally branched, with a rounded apex. The consistency is very spongy
and difficult to tear. It is very distinctive black, was collected from a depth of 15-20 m from El
Quseir, 120 km south of Hurghada, Egypt, in the Red Sea, on January 22, 2005. The color in
life is deep greenish black, the interior slightly lighter black to brown. The texture is
incompressible, and the sponge easily snapped in half as the sponge fibers are packed solidly
with sand grains. The surface is regularly conulose and quite spiky to the touch. black, hard,
rag-like. A voucher specimen has been deposited in the Zoological Museum Amsterdam under
registration number ZMA POR18348.
2.2 Chemicals used
2.2.1. General laboratory chemicals
Agar-Agar
Merck
Anisaldehyde (4-methoxybenzaldehyde)
Merck
(-)-2- butanol
Merck
Dimethylsulfoxide
Merck
Formaldehyde
Merck
Hexamethyldisilazane
Merck
Chlorotrimethylsilane
Merck
Hydrochloric acid
Merck
Potassium hydroxide
Merck
48
Material and Methods
Pyridine
Merck
Concentrated sulphuric acid
Merck
Trifloroacetic acid (TFA)
Merck
Concentrated amonia solution
Merck
D-Glucose
Sigma
L-Glucose
Sigma
D-Galactose
Sigma
L-Galactose
Sigma
Acetic anhydride .
Merck
Ortho-phosphoric acid 85% (p.a.)
Merck
N-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide.
Merck
Aminoacids standards.
Merck
Sodium bicarbonate
Merck
2.2.2. Solvents
Acetone
Acetonitrile
Dichloromethane
Ethanol
Ethyl acetate
Hexane
Methanol
Butanol
The solvents were purchased from the institute of chemistry, university of Düsseldorf. They
were distilled before using and special grade were used for spectroscopic measurements.
2.3. Equipments used
Balances
: Mettler 200
: Mettler AT 250
: Mettler PE 1600
: Sartorious RC210P
Centrifuge
: Kendro D-37520 osterde
Fraction collector
: ISCO Cygnet
49
Material and Methods
Freeze dryer
: LYOVAC GT2; Pump TRIVAC D10E
Hot plate
: Camag
Syringe
: Hamilton 1701 RSN
Mill
: Molinex 354
Magnetic stirrer
: Variomag Multipoint HP
Mixer
: Braun
PH-Electrode
: Inolab
: Behrotest PH 10-Set
Rotary evaporator
: Büchi Rotavap RE 111; Buchi Rotavap R -200
Drying Ovens
: Heraeus T 5050
Sonicator
: Bandelin Sonorex RK 102
UV Lamp
: Camag (254 and 366)
Vacuum Exicator
: Solvent speed vac SPD 111V
2.3.1. Hyphenated HPLC equipments
I. Dionex Analytical HPLC (LC/UV) :
HPLC program
: Chromeleon Ver 6.3
Pump
: Dionex P580A LPG
Detector
: Dionex, Photoiode Array Detector UVD 340S
Autosampler
: ASI-100T
Column Thermostat:
: STH 585
Mobile Solvents:
Methanol LiChrosolv HPLC
Merck
ortho-phosphoric acid 0.15 %, pH 2.0 (prepared from
ortho-phosphoric acid 85% p.a.)
Merck
Nanopure water
Barnstead
II. Semi-preparative HPLC ( LC/UV) :
Pump:
: Gynkotek, M40
HPLC program:
: Gynkosoft (V. 5.4)
Detector:
: Gynkotek, Photoiode Array Detector UVD 340
Autosampler:
: Gynkotek Autosampler GINA 50
Printer:
: NEC P60
Mobile Solvents:
50
Material and Methods
Methanol LiChrosolv HPLC
Merck
Nanopure water
Barnstead
III. Analytical HPLC (LC /UV/ MS hyphenated system) :
MS spectrometer
: Finnigan LC Q-DECA
HPLC system (Pump,
Detector and autosampler)
: Agilent 1100 series
Column Knauer (125mm L, 2 mm ID), prepacked with Eurosphere- 100 C-18 (5 µm)
and with integrated pre-column.
Mobile Solvents:
Acetonitrile or Methanol LiChrosolv HPLC
Merck
ortho-phosphoric acid 0.15 %, pH 2.0 (prepared from
ortho-phosphoric acid 85% p.a.)
Merck
Nanopure water
Barnstead
LC /ESI-MS was carried out using a Finnigan QDECA-7000 mass spectrometry connected to a
UV detector. The sample is dissolved in methanol and injected to HPLC/ESI-MS
hyphenated system. HPLC was run on a Eurospher C-18 reversed phase column. The mass
spectra were generated on a dual octopole ion trap mass spectrometer operated in positive
and negative ion modes and fitted with an atmospheric pressure electrospray-ionization
sample introduction device. Fragmentation experiments were performed by automatic MS
technique.
2.4. Chromatographic methods
2.4.1. Thin layer chromatography (TLC)
Pre-coated TLC Alu Plates ( Silica gel 60 F254, Layer thickness 0.2 mm)
Merck
Pre-coated TLC glass Plates ( RP-18, F254 S, Layer thickness 0.25 mm)
Merck
Preparative TLC glass plate ( Silica gel G 60,
Merck
spread on a clean glass sheets and activated in oven at 120 °C 5 Hours)
The compounds were monitored by the UV absorbance at 254 and 366 nm or by spraying with
Anisaldehyde reagent , Methanol-sulphouric acid, Dragendorf´s spray reagents.
Anisaldehyde /H2SO4 spray reagent (DAB10)
Anisaldehyde
:5 parts
51
Material and Methods
Glacial Acetic Acid
: 100 Parts
Methanol
: 85 parts
Conc. H2SO4
: 5 parts (added slowly)
The solution was stored in amber-coloured bottles and kept refrigerated until use. TLC
was used for the fractions and for the pure compounds to identify the fractions and determine
the purity of the isolated compounds. After spraying, the TLC plates were heated at 110 °C in
order to monitor the UV-undetected sample components.
2.4.2. Vacuum liquid chromatography
Vacuum liquid chromatography (VLC) is a useful method as rapid
and initial
fractionation procedure for a crude extracts or large amounts of fractions. The apparatus
consists of
a sintered
glass büchner
filter funnels with different lengthes and internal
diameters suitable for different sample quantities. Fractions are collected in Erlenmeyer
flasks. Silica gel 60 was packed to a hard cake at a height of 5-10 cm under applied vacuum.
The sample used was mixed with a small amount of silica gel using a volatile solvent. The
resulting sample mixture was then packed onto the top of the column. Step gradient elution
with non-polar solvent (hexane) then increasing the amount of the polar solvent (EtOAc,
MeOH) is added to each successive fraction. The flow is produced by vacuum and the
column is allowed to run dry after each fraction was collected.
2.4.3. Column chromatography
Fractions obtained from VLC were subjected to series of chromatographic columns using
different stationary phases and solvent systems according to the chemical nature and
quantity of the fraction components.
The following separation systems were used :
a) Normal phase chromatography uses a polar stationary phase, typically silica gel in
conjunction with a non-polar mobile phase
(n-Hexane, EtOAc, CH2Cl2,..etc). thus
hydrophobic compounds elute more quickly than do hydrophilic compounds.
b) Reversed phase (RP) chromatography uses a non polar stationary phase and a polar mobile
pase (water, methanol). The stationary phase consists of silica packed with n-alkyl chains
covalently bound. For instance, C-18 signifies an octadecyl ligand in the silica matrix. The
more hydrophobic the ligand on the matrix, the greater the tendency of the stationary
phase to elute the hydrophilic components of the sample and retain the hydrophobic ones.
52
Material and Methods
c) Size exclusion chromatography involves separations based on molecular size of the sample
components. The stationary phase consists of porous beads. The larger compounds will be
excluded from the interior of the bead and thus will elute first. The smaller compounds
will be allowed to enter the beads and elute according to their ability to exit from the small
sized pores they were internalised through.
2.4.4. Semi-preparative HPLC
Semi-preparative HPLC ( Merck-HPLC apparatus, using MERCK HITACHI pump L7100 and MERCK HITACHI UV Detector L-7400, Hitachi,Ltd. Tokyo Japan,) was used for
the purification of relatively pure compounds obtained from fractions eluted by column
chromatography or in some cases used to separate very closely related compounds (see figure
2.11). Each injection was in concentration of 3 mg of the dried fraction dissolved in 1 ml of
solvent system. The injection volume up to 1 ml was injected into the column and the flow rate
was 5 ml/min. The eluted peaks were detected by online UV detector involved in the apparatus.
821 ms030701 #4
mAU
A
600
400
egme3-3
UV_VIS_1
WVL:235 nm
9 - 31,390
Kah F
Ret. Time : 31,77
Kah R
Ret. Time : 32,21
Kah S
Ret. Time : 30,10
8 - 30,403
200
0
min
-222
27,75
29,00
30,00
31,00
32,00
33,00
34,00
35,00
36,51
Kah S
Ret. Time : 30,10
Kah R
Ret. Time : 32,21
Kah F
Ret. Time : 31,77
350 ms030707 #6
mAU
egab2e
B
300
250
UV_VIS_1
WVL:235 nm
400 ms030707 #13
mAU
egKah F prep
UV_VIS_1
WVL:235 nm
180 ms030707 #8
mAU
egab4b
UV_VIS_1
WVL:235 nm
9 - 32,212
6 - 31,775
10 - 30,098
150
300
125
C
200
200
D
100
150
75
100
50
100
50
0
25
67 -- 1,211
1,266
1,342
-0,922
1,485
1,089
1,022
123458-9---0,537
0,665
-50
0,0
7 - 47,621
-0,919
1,239
1,020
12345- --0,339
1,109
min
10,0
20,0
30,0
40,0
50,0
60,0 -50
0,0
56 - 1,207
7 - 1,268
1,333
1,087
-0,908
1,499
12348---0,613
1,012
min
10,0
20,0
30,0
40,0
50,0
60,0
-20
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
Figure (2.11) HPLC chromatogram A, shows sub-fraction of E. grandifolia–methanol extract containing three
closely related peptides , their retention times are very close to each others. Chromatograms B, C, and D shows the
pure compounds Kahalalide S, F, and R respectively after successful separation using preparative HPLC.
53
Material and Methods
For separation of the above mentioned peptides (kahalalide S, F, and R), the separation
was carried out by using Methanol : H2O gradient elution; [ solvent A: nanopure H2O 100%,
solvent B: MeOH 100%, the gradient commenced with 50% solvent B to 100% in 20 min then
the mobile phase was stabilised at 100% MeOH for 10 min, then MeOH-percentage was
decreased until 50 % for an additional 5 min], at a flow rate of 5 ml/min through a C-18
reversed phase column to yield three well-separated peaks of Kahalalide S (8 mg), F (103mg),
and R (20 mg) at retention times 17, 19, and 20 min. respectively. The semi-preparative RPcolumns were (300 x 8mm) pre-filled with Eurospher-100 , C-18 reversed phase silica gel beds.
2.4.5. Analytical HPLC
Analytical HPLC was used to identify the UV-absorbable compounds in the isolated
fractions or to evaluate the pure componds. The plotting of the peaks was guided by UV-VIS
photodiode array detector operating in four different wave lengthes 235, 254, 280 and 340 nm.
The solvent gradients used started with 10:90 [MeOH 100 % : nanopure H2O (adjusted to pH 2
with o-phosphoric acid)] , the commencing methanol ratio was equilibrated for 5 min at 10 % ,
then gradiently increased up to 100% after 35 min., then washing with 100 % MeOH for
additional 10 min. In some cases, special programs were applied using the same solvent
systems but in different ratios according to the chemical nature and the relative retention times
of the compounds being analysed. The flow rate of the mobile phase was at 1 ml / min. The
analytical HPLC column was a reversed phase column, thus eluting the polar compounds
firstly, then the relatively nonpolar compounds.
Role of ortho-phosphoric acid in the analytical HPLC process:
o-phosphoric acid gives a good compound-separation, allowing the UV-detector to
impart sharp peaks, but it has some disadvantages that, in some cases of poly-nitrogenous
compounds (e.g. Purine derivatives, nucleotides and other similar small biologically active
metabolites), the presence of acid makes the nitrogenous compounds highly protonated (in
other word, highly polar according to the affinity of that compounds to accept protons), which
in turn elute the compounds very rapidly and in some cases can not be detected with the UVdetector). This problem prompted some analytical chemists (e.g. Nordström 2004) to derivatise
such basic compounds to be more hydrophobic and consequently improve their retentions on
reversed phase materials and enhance their ESI response. The following example will explain
the effect of ortho-phosphoric acid in masking of the peaks for the nitroginous compounds
during HPLC analysis.
54
Material and Methods
Detection and isolation of new purine derivatives in sub-fractions of EtOAc-Fraction of
Petrosia nigricans:
200 ms040710 #3
mAU
tm7-et-18
UV_VIS_1
WVL:235 nm
3 - 1,271
4 - 1,325
150
6 - 5,425
100
12 - 20,167
50
5 - 3,451
7 - 10,3249 - 15,642
11 - 17,971
8 - 14,035
10 - 16,278
13
- 29,999
14
- 30,811
15 - 46,934
16 - 51,396
- 0,930
1 2- 0,197
-20
0,0
mi n
10,0
20,0
30,0
40,0
50,0
60,0
Fig (2.12) HPLC chromatogram of EtOAc subfraction (Et-18) of Petrosia nigricans, pH 2
nanopure water was maintained by phosphoric acid.
From the experiment (figure2.13), it is noted that, in case of acidified water, low
concentrated sample showed nothing and the test purine peak was completely undetected, while
in more concentrated sample the corresponding peak was broad with low resolution, and short
retention time. On the other hand, the chromatograms that plotted during the measurment in
which the neutral nanopure water was applied, showed a purine peak with high resolution, good
peak-sharpness, longer retention times, and more descriptive. It is noted also the resulting UVspectra were varied according to pH value of the mobile phase, and the compounds of the same
chromophoric functions give the same UV spectra.
55
Material and Methods
110
ms050503 #15
et18h
9.25
mAU
4.000
% No spectra library hits found!
UV_VIS_1
WVL:235 nm
300
ms050506 #15
mAU
et18h
10 - 16,822
17 - 34,093
250
A
19 - 36,233
22
23- -37,678
-38,213
38,651
21-24
-36,715
37,165
20
2.500
80
10 - 29,507
1.250
18 - 35,397
60
- 33,737
1516- 32,882
344.7
150
16.90
No spectra library hits found!
228.1
B
200
12- -30,426
30,899
530.3
551.711
Peak #10
%
70,0
209.0
281.3
nm
-500
200
40
UV_VIS_
WVL:235 nm
400
14 - 32,109
595
100
13 - 31,750
3 - 1,088
4 - 1,117
8 - 24,155
9 - 25,234
20
5 - 1,210
6
7 -- 1,258
1,318
0,950
12 -- 0,758
0
nm
-10,0
200
50
300
400
500
11 - 47,527
15,412
89- -14,673
67 -0,778
1,237
1,360
-0,459
1,108
0,597
12345
---Peak
1 - 0,046
595
min
-10
0,0
10,0
20,0
30,0
200 ms050503 #13
mAU
40,0
50,0
et18f
C
150
min
-50
0,0
UV_VIS_1
WVL:235 nm
9 - 4,528
70,0
60,0
10,0
30,0
1.200 ms050506 #13
mAU
Peak #9
4.68
No spectra library hits found!
%
40,0
50,0
et18f
60,0
UV_VIS_1
WVL:235 nm
8 - 14,308
1.000
800
289.7
70,0
D
210.4
25,0
100
20,0
Peak #8
%
14.27
No spectra library hits found!
228.3
281.2
242.5
600
560.3
50
12 - 15,506
7 - 1,214
8 - 1,367
6 -0,598
1,125
12345--0,188
1,057
-0,515
0,964
400
nm
-10,0
200
400
595
16 - 47,606
11 - 12,692
10 - 9,838
0
nm
-10,0
200
200
15 - 30,910
13 - 24,164 14 - 30,573
300
9 - 15,406
10 - 16,195
7 - 13,800
-0,781
1,264
1,346
0,910
1,117
123456---0,478
400
12 - 36,770
11 - 32,482
500
595
13 - 47,608
min
-20
0,0
10,0
20,0
30,0
70,0 ms050503 #12
mAU
40,0
50,0
et18e
7 - 5,034
10,0
30,0
12 - 13,230
70,0
315.6
F
300
25,0
37,5
200
40,0
50,0
et18e
Peak #7 5.07
% No spectra library hits found!
214.2
20,0
400 ms050506 #12
mAU
UV_VIS_1
WVL:235 nm
70,0
E
50,0
min
-200
0,0
60,0
Peak #12
%
227.0
202.7
60,0
UV_VIS_1
WVL:235 nm
13.32
No spectra library hits found!
300.5
13 - 30,907
25,0
11 - 24,172
400
15 - 47,523
595
100
14 - 36,729
300
400
8 - 10,394
67
1,129
1,204
1,337
89-----10
1,272
- 2,394
0,930
1,012
0,860
0,457
1234-5-Peak
1 - 0,063
0,5921 - 0,063
0,375
123---Peak
-10,0
0,0
nm
-10,0
200
10 - 18,680
6 - 1,341
0,0
-10,0
200
12 - 30,569
9 - 13,491
5 - 1,210
4 - 1,118
12,5
nm
min
10,0
20,0
30,0
40,0
50,0
-50
0,0
60,0
14 - 36,762
13 - 32,479
11 - 10,578
500
595
15 - 47,571
min
10,0
20,0
30,0
40,0
50,0
60,0
Figure (2.13) shows three HPLC chromatograms A, C & E of three pure new purine
derivatives isolated from Petrosia nigricans subfraction Et-18, measured in HPLC using
standard mobile phase that composed of methanol 100 % and acidified water pH 2 using
phosphoric acid. The peak resolution in HPLC chromatograms was improved when the
same samples were chromatographed again using the same mobile phase and the same
gradient program under the same condition, but the difference was only the use of neutral
water instead of pH 2 water, where chromatograms B, D & F, respectively were obtained.
56
Material and Methods
2.4.6. Flash chromatography
Flash chromatography was used for rapid isolation of compounds. A silica gel 60 GF254
pre-packed column was used and the sample was dissolved in a small volume of the initial
solvent used. The resulting mixture was then packed onto the top of the column using special
syringe. The flow rate was maintained by an air pump and 5 ml fractions were collected and
monitored by TLC.
2.4.6. Gas Chromatography :
Gas chromatography (GC) was used to determine the nature of the absolute stereochemistry of
sugar units (D or L forms). The sugar fraction of the test sample was injected in the GC
after hydrolysis and derivatisation using a special program. The samples were analysed by
comparison of the retention times with those observed for the authentic samples.
2.5. Procedures of the isolations of the secondary metabolites:
2.5.1. Isolation of secondary metabolites from Elysia rufescens
total methanol
extract
solvent partition
n-Hexane : methanol
Methanol extract
n-Hexane extract
Sephadex column
methanol 100 %
Fraction A
prep.
HPLC
Fraction B
Fraction C
RP-18, column
MeOH:H2O
80:20
Kahalalide F
120 mg
N,N-dimethyl-tryptophan
methyl ester
4.6 mg
ß-sitosterol
Prep HPLC
Kahalalide
B
5mg
Kahalalide
D
3.1 mg
57
Kahalalide
E
4 mg
Kahalalide
C
1 mg
Material and Methods
2.5.2. Isolation of secondary metabolites from Elysia grandifolia
total methanol
extract
solvent partition
n-Hexane : methanol
(90%)
Methanol extract
n-Hexane extract
Dereplication procedure using LC/MS
new compounds were detected, and
cosequently were targeted for isolation
Sephadex column
methanol 100 %
Fraction 2-4
Fraction 5-6
Fraction 13-14
Fraction 7-12
Kahalalide F
50 mg
mixture of known
kahalalides 10mg
LC/MS dereplication
procedure detect a mixture
of 2 new kahalalides plus
kahalalide F
Kahalalide D
3.4 mg
prep. HPLC
Kahalalide S
8mg
Kahalalide F
53 mg
Kahalalide R
20 mg
2.5.3. Isolation of secondary metabolites from Pachychalina sp.
total methanol
extract
solvent partition
n-Hexane : methanol
(90%)
n-Hexane extract
Methanol fraction
solvent partition
n-butanol : dist. H2O
butanol fraction
aqueous fraction
silica gel column
DCM:MeOH 9:1
n.hexane fraction
No. 6
preparative HPLC
1) silica gel column
DCM:MeOH 9:1
2) preparative HPLC
8-hydroxyquinolin-4-one
3.8 mg
58
sterol peroxide 1
5 mg
sterol peroxid 2
4.5 mg
Material and Methods
2.5.4. Isolation of secondary metabolites from Callyspongia biru
total methanol
extract
solvent partition
EtOAc:H 2O
EtOAc fraction
Aqueous fraction
VLC
solvent partition
n-butanol : dist. H 2O
subfractions, Et.A------------Et.Q
butanol fraction
subfraction Et.j
silica gel column
DCM:MeOH,
gradient elution
aqueous fraction
1) silica gel column
DCM:MeOH 9:1
2) preparative HPLC
subfractions, But.A------------But.V
bioassay guided isolation
using brine shrimp assay
and antimicrobial activity
testing
indol-3
-acetic acid
2.5mg
2-deoxy
thymidine
10mg
only subfraction (But.V) shows
brine shrimp ++ve assay,
and mild antimicrobial activity
4-hydroxyphenylacetic acid
1.7 mg
alkylpyridinium
compound
30mg
indol-3
-carbaldehyde
1.5 mg
4-hydroxyphenylacetic acid methylester
1.3 mg
2.5.5. Isolation of secondary metabolites from Hyrtios erectus
M e th a n o l e x tra c t
s o lv e n t p a rtitio n
D C M : H 2O
H 2 O e x trc t
-c o n c e n tra tio n
-m e th a n o l e x tra c tio n
ppt
(m a rin e s a lts )
D C M e x tra c t
1 0 f ra c tio n s
h b a - --- -h b j
M e th a n o l
e x tra c t
V LC
1 2 fra c tio n s
hbj
hbf
S i g e l c o lu m n
f ra c tio n s 4 -1 0
- s ilic a g e l c o lu m n
-p re p H P L C
H y r tio s in e
A
5 mg
5 -d eo x y H y r tio s in e
A
2 mg
5 - (O H )-in d o l-3 c a r b a ld e h y d e
2 .5 m g
in d o l- 3 c a r b a ld e h y d e
2 .3 m g
I s o h y r t io s in e
A
3 mg
h b ff
h b fc
1 6 -h y d r o x y s c a la r o lid e
3 .5 m g
1 2 -d e a c e t y l-1 2 e p i -s c a la r in
1 .5 m g
s c a la r o lid e
5m g
59
S ter o l
p e r o x id e
4m g
Material and Methods
2.5.6. Isolation of secondary metabolites from Petrosia nigricans
total methanol
extract
solvent partition
n-Hexane:Methanol
Methanol fraction
n-Hexane fraction
VLC
VLC
TM7 Butanol
fraction
Methanol
Si gel column
CH2Cl2:MeOH
n-Hexane
solvent partition
BuOH: dist. H2O
26 fractions
TM7 Butanol
fraction
fraction 17
A
TM7
EtOAc
fraction
silica gel column
n-Hexane:EtOAc
gradient elution
H2O fraction
Si gel column
CH2Cl2:MeOH
24-ethyl
cholesterol
2g
Adenosine
23 mg
sterolperoxide
25 mg
steroid
15 mg
B
1) Si gel columnn
2) Flash chromatography
3)Si gel column
4)preparative TLC
Petrocerebroside 1
Petrocerebroside 2
35 mg
TM7 EtOAc
fraction
Si gel column
CH2Cl2:MeOH
et.6,7&8
Prep. HPLC
et.11
Prep.
HPLC
Nigricinol
3.25 mg
et.13,14
Prep.
HPLC
Prep.
HPLC
p-hydroxy
phenylacetic
acid 5.2 mg
et.17&18
Prep.
HPLC
Nicotinamide
2.9 mg
phenylacetic
acid
6.0 mg
p-hydroxy
phenylacetic
acid ethylester
3.2 mg
et.16
Nigricine 1
2.3 mg
p-hydroxy
phenylacetic
acid methyl
ester 4.4 mg
Nigricine 2
6.1 mg
Nigricine 3
7.5 mg
Nigricine 4
1.1 mg
p-hydroxy
phenylacetic
acid butylester
2.5 mg
60
Material and Methods
2.6. Structure elucidation of the isolated compounds
2.6.1. Mass spectrometry (MS)
2.6.1.1. Low resolution MS:
Low resolution mass spectra were measured by ESI, EI, MALDI, and FAB-MS on a
Finnigan MAT 8430 mass spectrometer. Measurements were done in Institute für
Pharmazeutische Biologie and Institute für Anorganische und Strukturchemie, Heinrich-Heine
University, Düsseldorf.
2.6.1.2. HRMS (High resolution mass spectra):
High resolution mass spectra
were determined on a micromass Q-Tof 2 mass
spectrometer.
2.6.1.3. LC/ESI-MS (liquid chromatography/Electrospray ionization mass spectrometry):
LC/ESI-MS was carried out using a Finnigan LC QDECA mass spectrometer connected
to a UV detector. The samples were dissolved in methanol and injected into HPLC/ESI-MS setup. HPLC was run on a Nucleosil C-18 reversed-phase column. Measurements were done at
Institute für Pharmazeutische Biologie, Heinrich-Heine University, Düsseldorf. Standard
mobile phase system was applied, which was composed of 10:90 Acetonitrile:nanopure H2O
(0.1% HCOOH) – 100 % acetonitrile in 35 minutes in gradient elution system.
Electrospray ionization Mass spectrometer ( ESI-MS) :
Electrospray ionization (ESI) is a gentle method of the atmospheric pressure ionization
(API) technique and is well-suited to the analysis of polar molecules ranging from less than
100 Da to more than 100,000 Da in molecular weight. ESI –MS has been shown to be a
successful and popular tool for the analysis of noncovalently bound protein macromolecules,
(Ashcroft, A. E., 2005).
Capillary, 3-4 kV
Droplets
containing
ions
Droplet
evaporating
Ions evaporating
from the surface
of the droplets
Fig (2.14 ) The electrospray ionization (ESI) process
61
Material and Methods
The mechanism of electrospray ionization spectrometer
The sample is dissolved in a polar, volatile solvent and pumped through a narrow,
stainless steel capillary (75-150 µm i.d.) at a flow rate of between 1µl/min and 1 mL/min. A
high voltage of 3-4 Kv is applied to the tip of the capillary, which is situated within the
ionization source of the mass spectrometer, and as a consequence of this strong electric field,
the sample emerging from the tip is dispersed into an aerosol of highly charged droplets, a
process that is aided by a co-axially introduced nebulising gas flowing around the outside of
the capillary. This gas, usually nitrogen, helps to direct the spray emerging from the capillary
tip towards the mass spectrometer. The charged droplets diminsh in size by solvent evaporation
(see figure 2.14), assisted by a warm flow of nitrogen known as the drying gas which passes
across the front of the ionization source. Eventually charged sample ions, free from solvent,
are released from the droplets, some of which pass through a sampling cone or orifice into an
intermediate vacuum region, and from there through a small aperture into the analyser of the
mass spectrometer, which is held under high vacuum. The lens voltages are optimized
individually for each sample (Ashcroft, A. E., 2005).
The ESI-MS was modified by John Fenn, 1987. Fenn´s modifications include the
replacement of the simple orifice into the vacuum system by glass capillary tube (see fig. 2.15).
That glass tube was metallized at each end to provide an electrical contact. The inlet end of the
capillary would be maintained at the required potential below ground so the ions entering the
tube would be in a potential well. The fairly high-velocity flow of bath gas through the glass
(dielectric) tube into the vacuum system could drag the ions out of that potential well up to
whatever potential might be desired at the metallized exit end of that tube. Indeed, the gas flow
could raise the ions to an exit potential of several tens of kV, sufficient to inject the ions into
the analyzer. At the same time all exposed external parts and surfaces of the instrument could
be maintained at ground potential, thereby posing no hazard to an operator. Thus, Fenn`s
modification brought the proteins and biomacromolecules to fly, or as John Fenn himself said,
to give “wings to molecular elephants“ (Fenn 2003).
Fig. (2.15) Fenn´s modification of ESI-MS
62
Material and Methods
2.6.1.4. MALDI-TOF-PSD-MS
Post source decay mass spectrum
was
obtained
using
Matrix-assisted
laser
desorption/ionization technique coupled to Time-of-Flight mass analyser. This PSD-MS is
the
method of choice for sequence determination of the amino acids of isolated
depsipeptides (or peptides). MALDI-TOF-PSD spectra were obtained on a Perspective
Biosystems Voyager-DE PRO MALDI-TOF mass spectrometer and Bruker Esquire 2000
LC-MS system equipped with an electrospray source. Sample application for MALDI-TOFPSD MS was carried out directly on sample plates with a mixture of 1µL of matrix
(saturated 2,5- dihydroxybenzoic acid in 50 % acetonitrile, 0.3%TFA) and 1µL of a 50%
MeOH solution containing about 0.2µg of the sample.
The analysis was measured through AnagnosTec (Gesellschaft für Analytische
Biochemie und Diagnostik mbH im Biotechnologiepark, TGZ II , D-14943 Luckenwalde,
Berlin). Post source decomposition mass spectrum was recorded as peaks corresponding to
molecular weight [M+], [M+] - 1 mole amino acid, [M+] - 2 moles, [M+] - 3 moles, and so on.,
where the decomposition was predominantly at the amide (peptide) bonds (i.e. retrobiosynthetic decomposition). The data collection was assisted automatically by specific
computer program, plotted and interpretted using a computer program. This method has an
advantage, that the
cyclic peptide can be measured directly without linearisation or
decyclisation and it also needs no terminal NH2 group.
Matrix-assisted laser desorption ionization (MALDI)
The use of Laser desorption/ionisation-mass spectrometry (LDI-MS) was limited to
compounds that could be vaporised without being decomposed, since the laser light is absorbed
directly into the analyte, the molecular bonds may be broken owing to the increased internal
energy. In other words, there is an increased risk that measurement would occur in the
decomposed state. This method was usually inadequate for compounds exceeding 1000 Dalton
(Da) and ionization of compounds having a molecular weight exceeding 10000 Da was
considered by chemists at that time to be impossible (Tanaka, 2003).
In 1987, Koichi Tanaka, proposed the application of suitable matrix (glycerin) together
with a good laser light absorpable material, UFMP, (Ultrafine metal powder). The mixing of
this combination with the sample allowed to provide an efficiently enough heating and release
of solid sample from its crystalline state to dissolve in the liquid, thereby assisting ionization
(see figure 2.16). Thus, he was able to measure an ion cluster having a mass number exceeding
100, 000 Da (Tanaka, 2003).
63
Material and Methods
Fig (2.16) Desorption/ionization of macromolecules by using the
UFMP-glycerin mixed matrix.
The mechanism of MALDI spectrometer:
The mechanism of MALDI is believed to consist of three basic steps ( Gates, P., 2005a):
(i)
Formation of a solid solution: it is essential for the matrix to be in access thus leading
to the analyte molecules being completely separated from each other. This eases the
formation of the homogenous solid solution required to produce a stable desorption of
the analyte.
(ii)
Matrix extraction: the laser beam is focused onto the surface of the matrix-analyte
solid solution. The chromophore of the matrix couples with the laser frequency causing
rapid vibrational excitation, bringing about localised disintegration of the
solid
soltuion. The clusters ejected from the surface consists of analyte molecules surrounded
by matrix and salt ions. The matrix molecules evaporate away from the clusters to
leave the free analyte in the gas-phase.
(iii)
Analyte Ionization : The photo-excited matrix molecules are stabilised through proton
transfer to the analyte. Cation attatchment to the analyte is also encouraged during this
process. It is in this way that the characteristic [M+X]+ ( X= H, Na, K etc.) analyte
ions are formed. This ionization reactions take place in the desorbed matrix-analyte
could just above the surface. The ions are then extracted into the mass spectrometer for
analysis (see figure 2.17).
64
Material and Methods
Fig. (2.17) Schematic diagram of matrix-assisted laser desorption ionization mechanism
Matrix-assisted laser desorption/ionisation - time-of-flight mass spectrometer (MALDITOF MS):
It is a relatively new mass spectrometer in which matrix assisted laser
desorption/ionization procedure was applied together with the efficient mass detector, Time-ofFlight (TOF) detector which is available in two modes, linear and refractory modes (Fig 2.18).
Fig. ( 2.18) basic components of a linear (upper) and reflecting (lower )
TOF mass spectrometer.
65
Material and Methods
Matrix-assisted laser desorption/ionisation-time-of-flight in source decompostion mass
spectrometer (MALDI-TOF-ISD MS):
A recent extension of the MALDI in source design dramatically increased the ion
resolution and mass accuracy. The technique was dubbed “time lag focusing” (TLF), “delayed
extraction” (DE) or “pulsed ion extraction” (PIE). After desorption/ionization, the ions are
kept in the ion source under field-free conditions for a short period of time, like 100-400 ns,
before they are extracted with a high electrical field and accelerated towards the detector.
During the brief time between ion desorption and the extraction pulse, analyte ions can
undergo prompt fragmentaion within the ion source (in source decompostion, ISD). Many
reports of this phenomenon were published in which linear TOF instruments were incorporated
to provide a protein sequencing. Because the fragmentation occurs in the source, product ions
experience some of the effects of pulsed ion extraction, but due to the higher laser power
required to induce fragmentation it is very difficult to obtain high resolution and mass accuracy
for product ions in linear mode. Suckau and Cornett 1998, presented results from ISD
measurements made using an instrument with ion reflector, which provided isotopic resolution
for product ions up to several thousand Da. (Suckau and Cornett 1998).
Matrix-assisted laser desorption/ionisation-time-of-flight post source decay mass
spectrometer (MALDI-TOF-PSD MS):
PSD analysis is an extention of MALDI/MS that allows one to observe and identify
structurally informative fragment ions from decay taking place in the field free region after
leaving the ion source (Spengler, 1997). Mass spectrometric analysis of product ions from post
source decay of precursor ions that were formed by matrix-assisted laser desorption ionization
(MALDI-PSD) has evolved into a powerful method for primary structure analysis of
biopolymers. Especially in the field of peptide sequencing, MALDI-PSD has been widely
applied, mainly because of its high sesitivity for prepared sample amounts in the range 30-100
fmol and because of its high tolerance of sample impurities and sample inhomogenity
(Spengler, 1997).
It was first concluded that ions formed by MALDI must be extremely stable and
internally cool and that MALDI is therefore a very soft ionization technique (i.e. provide highly
stable molecular ion or provide no fragment ions ), the main reason for this assumption was
that these large biopolymers could be desorbed and ionized intact (which was impossible with
66
Material and Methods
other ionization techniques) and these molecular ions had obviously survived hundreds of
microseconds during their flight through the instrument until detection (Spengler, 1997).
More recently, fragment ions, which are produced after leaving the ion source during
the flight in the field-free region were used for peptide sequencing, (see figure 2.19). These
ions are called “Post-source decay” or PSD ions and can be observed in instruments equipped
with an ion reflector. In contrast to classical Edman sequencing, PSD-sequence determinations
are possible even from complex mixtures like proteolytic digests if a gated electrostatic ion
selector is used (Suckau and Cornett 1998).
Mechanism of MALDI-PSD:
With reflector TOF-MS, it is in theory possible to obtain structural information on a
selected quasimolecular ion by mass analysis of daughter ions issued from in-flight
fragmentation of the parent ion. Intact molecular ions leaving the ion source and having
acquired sufficient internal energy during the desorption process (photoactivation, low energy
collisions,) can release this energy by undergoing fragmentation while traveling the first fieldfree drift path of the instrument called post-source-decay or (PSD). The fragment ions have the
same velocity as their precursor ions but have different energy as a function of their mass.
Fragment ions are then discriminated as a function of their kinetic energy (thus their mass) by
the time dispersions induced by the electrostatic reflector. Large fragment ions (with higher
keinetic energy) will penetrate deeper into the reflectorn than smaller fragment ions and will
appear at later time on the resulting reflectron time-of-flight spectrum (Chaurand et al ,1999).
An important feature of MALDI-PSD instrument is their MS/MS capability, allowing one to
preselect a certain precursor ion in a mixture of multiple components. Precursor selection is
done by electrostatic “beam blanking” or “ion gating”.
All ions passing the beam blanking device are deflected off the ion detector except a
certain mass window which is transmitted without deflection. Deflection is performed by a fast
high voltage drop applied to the device which typically built of small plates, wires or strips.
Positioning of the ion gate within the flight path is always a compromise between positiondependent dispersion of precursor ions of different masses and high transmission for (low
energy) PSD ions already formed at the position of the ion gate (Spengler, 1997).
67
Material and Methods
(M+H)+
Small PSD
fragments
Stable ions
(precursor)
Large PSD
fragments
Detector
U1
L2
U2
d1
DE
d2
Ion gate
Sample
neutral fragment
+ve fragment
first field free drift region
Ua
L1
Fig.(2.19) Principle of PSD analysis in MALDI/MS, using two-stage gridded ion reflector. Mass analysis of these
ions is done in a series of consecutive steps by lowering the potentials U1 (decelerating voltage) and U2 (reflecting
voltage) of the reflector grid. Total instrument lengths can vary between 1m and several meters.
2.6.1.5. GC/MS (hyphenated Gas chromatography /Mass spectrometry):
GC/MS is a combination of two microanalytical techniques: a separation technique, GC,
and an identification technique, MS. GC/MS combination overcomes certain deficiencies or
limitations caused by using each technique individually and gives a two-diminsional
identification consisting of both GC retention time and a mass spectrum for each component of
the mixture. This combination has several advantages. First, it can separate the components of a
complex mixture so that mass spectra of individual compounds can be obtained for qualitative
68
Material and Methods
purposes, second , it can provide a quantitative information on these same compounds. GC/MS
can provide complete mass spectrum for as little as 1 pmol of an analyte. It gives direct
evidence for the molecular weight and the characteristic fragmentation pattern or chemical
fingerprint that can be used as the bases for identification. GC/MS is limited to the analysis of
those compounds that can be made volatile without thermal decomposition.
GC/MS consists essentially of three components: the gas chromatograph, the mass
spectrometer and the data system. The sample was dissolved in a volatile solvent (methanol, nhexane or EtOAc) and injected manually (10µl) in the GC/MS set-up working in the EI-MS
mode. The sample which eluted from the GC-column, was divided -in the same time- into two
parts, one of them passed through GC-detector (electron capture detector, ECD), and another
part passed through the EI-MS detector involved in the system. GC chromatograms were
obtained as quantitative peak intensities plotted versus retention times. EI-MS of each peak
was recorded depending on the retention times of each compond.
2.6.2. Nuclear magnetic resonance spectroscopy (NMR)
NMR measurements were done at the Institut für Anorganische Chemie und
Makromolekulare Chemie of Heinrich-Heine University, Düsseldorf. ID and 2D, 1H and 13CNMR spectra were recorded at 300º K on Bruker DPX 300, ARX 400, 500 or GBF 600 NMR
spectrometers. All 1D and 2D spectra were obtained using the standard Bruker software. The
samples were dissolved in deuterated solvents ( DMSO-d6, CDCl3, CD3OD, Pyridine-d5), the
choice of the solvent depends mostly on the solubility of the compound. Residual solvent
signals of (CD3OD at 3.3 ppm ,1H, and 49.0 ppm,13C), (CDCl3 at 7.26 ppm and 77.0 ppm),
(DMSO-d6 at 2.49 ppm and 39.5 ppm) were considered as internal reference signal for
calibration. The observed chemical shift values (δ) were given in ppm. and the coupling
constant (J) in Hz.
2.6.3. The optical activity
Optically active compounds are compounds which have at least one chiral carbon.
Optically active compounds can rotate the plane polarized light. Optical rotation was
determined on a Perkin-Elmer-241 MC Polarimeter. The substance was stored in a 0.5 ml
cuvette with 0.1 dm length. the angle of rotation was measured at a wavelength of 546 and 579
nm of a mercury vapour lamp at room temperature (25ºC). The specific optical rotation was
calculated using the equation:
69
Material and Methods
[α] 20
D =
[α] 579 x 3.199
[α] 579
[α] 546
4.199
Where [α]20D
= the specific rotation at the wavelength of the sodium D-line, 589 nm, at a
temperature of 20ºC.
[α]579 and [α]546 = the optical rotation at the wavelength 579 and 546 nm respectively,
calculated using the formula:
100 x α
[ α] λ =
I xC
Where:
α = the measured angle of the rotation in degrees
I = the length in dm of the polarimeter tube,
C = the concentration of the substance expressed in g/100 ml of the solution.
2.6.4 Special procedures:
2.6.4.1 LC/MS dereplication procedure for targeting new kahalalides of Elysia grandifolia
Total methanol extract of E. grandifolia was subjected to ESI-MS analysis using
LC/MS set-up available in the Institut für Pharmazeutische Biologie, Heinrich-Heine
University, Düsseldorf. ESI-MS spectrum (Fig 2.20 & 2.21 ) detects many positive molecular
ion peaks, 879.8, 914.9, 596.7, 1478.8, 1494.3, and 1240.0, of known kahalalides, kahalalide B,
kahalalide C, kahalalide D, kahalalide F, kahalalide G and kahalalide J, respectively, that have
been found and were previously reported from E. rufescens extract.
Indeed , it shows two minor new kahalalides (based on mol. wt.) close to the major one
(kahalalide F). The corresponding molecular ion peaks of both peptides appeard in both sides
left and right to the corresponding peak of the major peptide. The mol.wt. of the first one,
kahalalide S (M+1= 1536.6), was 58 mass units more than kahalalide F and slightly more polar,
and the other, kahalalide R (M+1= 1520.5), was 16 mass unit (which equal to one oxygen
atom) less than the first new one, and slightly less polar than kahalalide F (Fig. 2.11)
Both new kahalalides were targeted for isolation based on the obtained data including
the molecular weight and the HPLC retention times, where the total methanol extract was
chromatographed by gel filtration, to separate as pure as possible the targeted peptides.
Complete isolation and purification was carried out by semi-preparative HPLC.
70
Material and Methods
Figure (2.20) ESI-MS survey for the methanol extract of Elysia grandifolia detect 2 new
depsipeptides, kah.S [m/z 1536.5 (M+H)]+ and kah. R [m/z 1520.5 (M+H)]+ in addition
to the known kah. F [m/z 1478.8 (M+H)]+.
71
Material and Methods
B
G
J
C
K
D
O
E
Fig ( 2.21 ): ESI-MS survey for the methanol extract of Elysia grandifolia detect many of
known depsipeptides. (Kahalalides B, C, D, E, G, J, K, O).
2.6.4.2. Determination of amino acid diastereoisomers of isolated kahalalides :
Marfey reagent N-(5-flouro-2,4-dinitrophenyl)-L-alanine amide (FDAA) was used to
prepare diastereoisomers of amino acid derivatives. This diastereoisomers can be very useful in
quantitative determination of D- and L- amino acids in protein hydrolysates by HPLC analysis.
The L- amino acid derivatives were eluted more rapidly than the D-isomers due to the strong
intramolecular hydrogen bonding (more hydrophobic) in the latter diastereoisomers (Marfey,
72
Material and Methods
1984). Marfey´s procedure was applied for the isolated kahalalides after complete acid
hydrolysis.
Each peptide (0.1 mg) was hydrolysed by heating in a sealed vial with 6N HCl (2 ml)
until complete hydrolysis and liberation of free amino acids. The hydrolysate was cooled, dried
and dissolved in 0.5 ml of 1 % acetone solution of N-(5-fluoro-2,4-dinitrophenyl)Lalaninamide [FDAA], followed by 20 µmol of NaHCO3, the contents were mixed and heated
over a hot plate at 40 °C for 1 hour with frequent mixing, and after cooling, a 20 µmol HCl was
added, and the mixture was filled to a volume of 1 ml by addition of MeOH. Amino acid
standards were individually derivatised in the
same manner using 2.5 µmol amino acid
standard to react with 3.6 µmol FDAA to obtain the amino acid-DAA derivative.
The obtained derivatives were centrifuged and the supernatants were injected in the
HPLC/MS spectrometer, the elution time was 1 hour for each peptide-hydrolysate-DAA
derivative. Standard amino acid-DAA derivatives were used in order to determine the
molecular weight (MW) and the retention time (RT) of each amino acid unit. After comparison
of the MW and RT of the hydrolysate derivatives with that of derivatised standards,
diastereoisomers were detected in each peptide hydrolysate. The test was repeated again using
the same procedure by co-chromatography (peak enrichment technique) to confirm this
determination. The derivatised standards were added separately to the derivatised hydrolysate
of each kahalalide and run again under the same condition in HPLC-MS spectrometer, to show
what diastereoisomer-standard peak will be enriched or completely overlap the unknown peak
of the hydrolysate derivatives. Complete overlapping means complete stereosimilarity between
standard amino acid and the corresponding amino acid in the hydrolysate.
2 N HCl / 130 °C / 20 Hours
Free aminoacids
Kahalalide
Free aminoacids
1 % acetone solution of FDAA
1M NaHCO 3 / 40 °C /1 Hour
Aminoacid-DAA derivative
(A.A.Diastereoisomer-DAA deriv.)
H 2N
R
NH2
H 2N
R
NH
O
L-aminoacid
-
N+
F
O
-
- HF
R
-
O
O N+
O
L-aminoacid- DAA deriv.
O
OH
ON+
O
H
N
O
OH
NH
O
OH
O
O
N+
O
NH 2
N-(5-Fluoro- 2,4-dinitrophenyl )L-alaninamide
H 2N
D-aminoacid
O
R
HPLC ret. time (longer)
due to
strong intramolecular
hydrogen bonding
12-membered ring
N
H
O
H
9-membered ring
NH
O-
O
O- N+
O
N+
O
D-aminoacid- DAA deriv.
73
Material and Methods
The following reaction products were detected in the new kahalalide hydrolysates ( kahalalide
R and kahalalide S) using LC/MS analysis:
O
H2N
H2N
NH
HO
C O
H2C CH
H2C HN
C OH
O
O N+
O-
O
N+
O-
F
O N+
O-
N-(2,4-dinitro-5-fluorophenyl)-L-alaninamide
H2N
O
O
H2C
HO
CH CH3
C
O
O
O
O
N+
HN
O
N+
O-
O-
O
N+
N+
HN
H2N
NH
CH
H2C
CH
N+
O-
C15H21N5O7
Exact Mass: 383,14
Mol. Wt.: 383,36
DAA- Isoleucine
CH OH
C CH
HO
HN
C
NH
OHO N+
O
N+
O-
C14H20N6O7
Exact Mass: 384,14
Mol. Wt.: 384,34
DAA-ornithine
C18H19N5O7
Exact Mass: 417,13
Mol. Wt.: 417,37
DAA-phe.ala.
C13H17N5O8
Exact Mass: 371,11
Mol. Wt.: 371,30
O-
DAA-threonine
NH2
H
N
O
O
N+
O-
O
NH2
O
NH
N+
O
C14H19N5O7
Exact Mass: 369,13
Mol. Wt.: 369,33
O-
NH2
O
DAA- valine
O-
O-
H3C
O
O N+
O-
OH
O
O
NH
NH
-
N+
C O
CH CH
HN
H3C
H3C
NH
DAA- glutamic acid
(FDAA)
NH2
HO
C14H17N5O9
Exact Mass: 399,10
Mol. Wt.: 399,31
C9H9FN4O5
Exact Mass: 272,06
Mol. Wt.: 272,19
CH3
O
O
O
O
NH2
C OH
N
O N+
NH
O
N+
O- C14H17N5O7
Exact Mass: 367,11
Mol. Wt.: 367,31
DAA-proline
O-
2.6.4.3. Acid hydrolysis and GC analysis :
Petrosia cerebrosides (2.0 mg) were hydrolysed by treatment with 3N HCl and stirred at
80 °C for about 5h. The solution was concentrated using N2. The residue was re-dissolved in ()-2-butanol (0.5 mL) and one drop of triflouroacetic acid. The solution was transferred to an
ampoule, sealed and heated at 130 °C in an oven overnight until complete butanolysis. The
solution was concentrated to dryness in a vacuum evaporator and then connected to the freeze
dryer
overnight
until
complete
dryness.
The
residue
was
then
treated
with
hexamethyldisilazane-chlorotrimethylsilane-pyridine (0.1 mL, 1:1:5) for 30 min at room
temperature. The solution was then centrifuged and the supernatant (1µL) was analysed by GC
on HP-5 column. The injection port and detector temperatures were 200 °C and 220 °C,
respectively. The temperature gradient was programmed as linear increase from 135 °C to 200
°C at 1 °C/min. Four peaks of hydrolysate were detected at 37.21, 39.91, 41.98, 42.95 minutes,
which was in agreement with the four peaks obtained for authentic D-galactose at 37.49,
40.15, 42.43, 43.32 minutes treated simultaneously in the same manner, [Leontein, et al.1977
and Gerrit, et al., 1977].
74
Material and Methods
2.7. Bioassays
2.7.1. Antimicrobial activity
Microorganisms
The crude extracts and the pure compounds were tested for activity against the following
standard strains: gram positive bacteria Bacillus subtilis DSM2109, Staphylococcus aureus
ATCC 25923 and gram negative bacteria Escherichia coli DSM10290; the yeast
Sacharomyces cerevisiae DSM1333 and two fungal strains Cladosporium herbarum
DSM62121 and C. cucumerinum DSM62122.
Culture preparation
The agar diffusion assay was performed according to the Bauer-Kirby-Test (DIN 58940,
Bauer et al, 1966). Prior to testing, a few colonies (3 to 10) of the organism to be tested, were
subcultured in 4 ml of tryptose-soy broth (Sigma, FRG) and incubated for 2 to 5 h to produce a
bacterial suspension of moderate cloudiness. The suspension was diluted with sterile saline
solution to a density visually equivalent to that of a BaSO4 standards. The standards were
prepared by adding 0.5 ml of 1 % BaCL2 to 99.5 ml of 1% H2SO4 (0.36 N). The prepared
bacterial broth is inoculated onto Müller-Hinton-Agar plates (Difco, USA) and dispersed by
means of sterile beads.
Agar diffusion assay
For screening, aliquots of the test solution were applied to sterile filter-paper discs (5
mm diameter, Oxoid Ltd) using a final disc loading concentration of 500 µg for the crude
extract and 50 and 100 µg for the pure compounds. The impregnated discs were placed on agar
plates previously seeded with the selected test organisms, along with discs containing solvent
blanks. The plates were incubated at 37°C for 24 hr. and anti-microbial activity was recorded as
the clear zones of inhibition surrounding the discs. The diameter was measured in mm.
2.7.2 Bioactivity guided isolation using brine shrimp lethality test:
This technique is an in vivo lethality test involving the whole body of a tiny crustacean,
the brine shrimp, Artemia salina Leach. It has been previously utilised in various bioassay
systems including the analysis of pesticide residues, mycotoxins, stream pollutants,
anaesthetics, dinoflagellate toxins, morphine-like compounds, toxic oil dispersants,
carcinogenic phorbol esters and toxicants in marine environments (Mayer, et al., 1982).
75
Material and Methods
This test takes into account the basic premise that pharmacology is simply toxicology at a
lower dose, and that toxic substances might indeed elicit, at a lower nontoxic dose, interesting
pharmacological effects. The procedure determines LC50 values in µg/mL of active compounds
and extracts in the brine medium.
Sample preparation
Crude extract, butanol fraction, EtOAc fraction, and all butanol sub-fractions of
Callyspongia biru were subjected to the brine shrimp lethality test. The test samples were
dissolved in a suitable organic solvent and the appropriate amount was transferred to 10 mL
sample vials. Bioassay was done on 1.0 & 0.5 mg test samples. The samples were then dried
under nitrogen and redissolved in 20 µl DMSO. Negative control vials contain the same
amount of DMSO were also prepared.
Hatching the eggs
Brine shrimp eggs (Dohse, Aquaristik GmbH, Bonn, Germany) were hatched in a small
tank filled with artificial sea water which was prepared with a commercial salt mixture (33 g,
Sera Sea-Salt, Aquaristik GmbH, Bonn, Germany) and distilled water (1000 mL). The eggs
hatched and became ready for the bioassay after 48 hours. Twenty nauplii were taken out by
pipette (counted macroscopically in the stem of the pipette against a lighted background) and
transferred into each test sample vial. Artificial sea water was added to render the volume 5 mL
and the vials were maintained under illumination. Survivors were counted, with the aid of a
magnifying glass after 24 hours and the mortality at each dose and control were determined.
The LC50 values were determined using the probit analysis method. The LD50 was derived from
the best fit line obtained by linear regression analysis.
Results
The most active fraction was butanol subfraction (But V) the last fraction eluted from
the silica gel column of the butanol fraction of the sponge. Where it shows 80 % and 100 %
lethality effects at concentrations of 0.5 mg and 1.0 mg respectively. Therefore the isolation
was continued for this fraction where alkylpyridinium compound was isolated from this
fraction.
76
Material and Methods
2.7.4. Cytotoxicity test
Cytotoxicity Assay
Antiproliferative activity was examined against several cell lines, which included MCF-7,
PC12, HeLa, L1578Y and H4IIE, and was determined by an MTT assay as described earlier
(Kreuter et al 1992, Mosmann 1983).
Cell cultures
L5178Y mouse lymphoma cells were grown in Eagle’s minimal essential medium supplement
with 10% horse serum in roller tube culture. H4IIE-cell line was grown in a DMEM-medium
with 10% fetal bovine serum, while HELA, PC12, and MCF-7 cells were grown in RPMI 1640
medium supplemented with non-essential amino acids, sodium pyruvate, 10 µg/mL insulin and
10% fetal bovine serum. The cell culture media contained 100 units/mL penicillin and
100 µg/mL streptomycin and were changed twice per week. The cells were maintained in a
humidified atmosphere at 37°C with 5% CO2.
Determination of cytotoxicity (MTT colorimetric assay )
MCF-7 cells, PC12, HeLa, L1578Y, and H4IIE were plated on 96-multiwell plates with
50.000 cells/well. The cells were allowed to attach for 24 h and then treated with different
concentrations of kahalalides for 24 h. After this treatment the medium was changed and the
cells were incubated for 3 h under cell culture conditions with 20 µg/ml MTT (3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). The conversion of the tetrazolium salt
MTT to a colored formazan by mitochondrial dehydrogenases was determined as a marker of
cell viability according to Mosman 1983 (Kreuter et al 1992, Mosmann 1983). After this
incubation the cells were fixed on the plate with an aqueous solution containing 1%
formaldehyde and 1% calcium chloride and then lysed with 95% isopropanol-5% formic acid.
The concentration of reduced MTT as a marker for cell viability was measured photometrically
at 560 nm.
77
Results
III. Results
3-1 Natural Products from Elysia rufescens:
Sacoglossan molluscs of the genus Elysia have been intensively investigated for their
biologically active natural products which include diterpenoids (Paul and Van Alstyne 1988),
polypropionates (Gavagnin, et al 1994, Cueto et al 2005), and depsipeptides (Hamann and
Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al 2000). Elysia-derived
natural products show significant biological and pharmacological activities. The diterpenoids
were reported to be active against anti-HIV opportunistic infections as well as against Herpes
simplex type II (HSV II) (Paul and Van Alstyne 1988). The polypropionates exhibited
ichthyotoxicity (Gavagnin, et al 1994, Cueto et al 2005), while the depsipeptides were shown
to have fish deterrent activity and are used as a chemical defence by the sacoglossan (Becerro
et al 2001). The peptide derivatives which carry the trivial name kahalalides, were further
described to have anti-malarial, anti-cancer, anti-psoriatic, anti-tuberculosis, and anti-fungal
activities (Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al
2000, Bourel-Bonnet et al. 2005, El-Sayed et al 2000). The name kahalalide, was derived
from the mollusc’s collection site, at Black Point near the shores of Kahala District of the
Oahu Island in Hawaii. Kahalalides have been isolated from the sacoglossans E. rufescens
(Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997), E. ornata (Horgen et al
2000) (Plakobranchidae) as well as from their green algal diet, Bryopsis sp. (Bryopsidaceae)
(Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al 2000). The
green algal diet, Bryopsis sp., has been found to yield kahalalides A, B, F, G, K, P and Q.
(Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al 2000, Kan et
al 1999, Dmitrenok et al 2006). The kahalalide peptides consist of 3 to 13 amino acid units,
ranging from a C31 tripeptide to a C75 tridecapeptide, with cyclic and linear components, the
latter terminating in a saturated fatty acid moiety. Ten of these derivatives are cyclic
depsipeptides, kahalalides A to F, K, O, P and Q, while three analogues, kahalalides G, H, and
J are linear peptides. Kahalalide F and its linear analogue kahalalide G are the only congeners
that feature the atypical amino acid, Z-dehydroaminobutyric acid (Z-Dhb).
Due to their biological importance, kahalalides A, B, and F have been chemically
synthesized. (López-Maciá et al 2001a, López-Maciá et al 2001b, Bourel-Bonnet et al.
2005). To date, kahalalide F is the only derivative reported to have significant activity
towards solid tumor cell lines, including human colon and lung cancers, and against some of
78
Results
pathogenic microorganisms that cause opportunistic infections in HIV/AIDS patients
(Hamann and Scheuer 1993). Kahalalide F displays both in vitro and in vivo anti-tumor
activity in various solid tumor models, which include colon, breast, non-small cell lung, and in
particular prostate carcinoma. In vitro anti-proliferative studies showed activity among certain
prostate cancer cell lines (PC-3, DU-145, T-10, DHM, and RB), but no activity was found
against the hormone-sensitive LnCAP (prostate cancer) cell line. In vivo models also
confirmed selectivity and sensitivity of the prostate tumor xenograft derived from hormoneindependent prostate cancer cell lines, PC-3 and DU-145. Furthermore, in vitro evaluation
exhibited that this activity is selective but not restricted to prostate tumor cells (RademakerLakhai et al 2005). It has also been revealed that kahalalide F induces cell death via oncosis
preferentially in the tumor cell (Suarez et al 2003). Kahalalide F has attracted most attention
and has been the subject of a patent application. Its mode of action and preclinical toxicity has
also been studied (García-Rocha et al 1996, Sewell et al 2005, Janmaat et al 2005, Suarez et
al 2003) and the compound is currently in phase II clinical trial as a potential anticancer
agent (Brown et al 2002, Ciruelos et al 2002, Rademaker-Lakhai et al 2005).
Chemical investigantion of methanol extract of Elysia rufescens led to the isolation of
five known kahalalides in addition to β-sitosterol and N,N-dimethyltryptophan methyl ester
3.1.1- Kahalalide F (1, Known compound)
Kahalalide F was isolated as a white amorphous powder, with [α]D of – 5° (c 0.35
MeOH). It has UV absorbance at λmax 203 nm. FAB-MS showed pseudomolecular ion peak
m/z 1478 [M+1]+ and 1500 [M+Na]+, suggesting the molecular formula C75H124N14O16. The
molecular weight of 1 was also confirmed by ESI-MS, and MALDI-TOF-MS. The 1HNMR
spectrum of kahalalide F revealed 14 deshielded amide NH resonances in the lower field
region, ranging from 6.76 ppm to 9.69 ppm, suggesting the peptidic nature of the compound.
One NH proton, a sharp singlet at δ 9.69, indicated an α,β-unsaturated amino acid, a broad 2H
singlet at δ 7.69 was shown by a cosy experiment to be the terminal NH2 of ornithine.
Compound 1 was ninhydrin-positive, thus supporting the presence of the free amino group of
ornithine. The remaining 11 NH protons were doublets; those that are part of the ring are
sharp, while the NH protons of the linear portion of the molecule are splitted and broad due to
several conformations of the molecule. COSY spectrum showed 14 different spin systems,
that were also confirmed by TOCSY spectrum, 12 spin systems possessing deshielded amide
79
Results
NH resonances at δ 6.76 Val-1 NH, δ 9.69 Dhb NH, δ 8.79 Phe NH, δ 7.62 Val-2 NH, δ 8.82
Ile-1 NH, δ 8.56 Thr-1 NH, δ 7.90 Ile-2 NH, δ 7.95 Orn NH, δ 8.10 Val-3 NH, δ 7.57 Val-4
NH, δ 7.82 Thr-2 NH, δ 7.88 Val-5 NH (Abbreviations used for amino acids and the
designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in: J. Biol. Chem. 1972, 247, 977-983). The other 2 spin systems show no
correlations to amide NH protons, they were assigned to proline and to the aliphatic acid, 5methylhexanoic acid. Each NH proton showed COSY correlation to the next α –proton, then
to the β-proton, then to γ- and δ-proton resonances. The COSY spectrum revealed the
presence of one proline spin system, [δ 4.36 (dd, J= 9.1, 6.6 Hz, H-α), 2.03,1.97 (m,m, 2H-β),
1.86 (m, 2H- γ) and δ 3.76, 3.52 (m,m, 2H- δ)]. The presence of 5-methylhexanoic acid (5MeHex) was indicated by the 2D NMR data, sequential COSY correlations were observed
between the α-methylene signals at δ 2.13 and the subsequent methylenes at δ 1.47 (m, 2H-3),
1.11 (m, 2H-4) followed by aliphatic methine proton at δ 1.47 (m, H-5) and two methyl
groups at δ 0.82 (d, J = 7.1 Hz ) and 0.82 (d, J = 7.0 Hz ). As shown by the broad NH signals
for the linear part of compound 1, a second regio-isomer of the aliphatic acid was detected
from the COSY spectrum as exhibited by the signals at δ 2.2 (m, 2H-2), δ 1.6 (m, 2H-3), δ
1.14 (m, 2H-4), δ 1.35 (m, H-5) and two methyl groups at δ 0.80 (d, J = 6.9 Hz, CH3 -6 ) and
0.80 (d, J = 7.0 Hz, CH3-7). TOCSY experiment showed an uncommon spin system which
was assigned as dehydroaminobutyric acid (Dhb) at 9.69 (s, NH), 6.34 (q, J =7.0Hz, 1H),
1.26 (d, J =7.5Hz, CH3). The ROESY correlation between NH and CH3 protons indicated a Z
stereochemistry of this uncommon amino acid. This uncommon amino acid was reported as a
constituent of peptides isolated from the terrestrial blue-green alga (Moore et al 1989), and
from an herbivorous marine mollusk (Pettit et al 1989). The ester linkage of the ring part of
the compound was confirmed by HMBC correlation between the β-Proton (shifted downfield
at δ 4.96) of threonine and the carbonyl at δ 164.4 of valine-1.
13
C NMR and DEPT spectra displayed signals for 75 carbons including 14 carbonyls
at δ 169.5 , 164.4, 171.3, 172.9, 170.0, 169.7, 170.6, 173.0, 172.6, 171.3, 169.8, 169.0, 172.2,
and 173.8 of val-1, Dhb, Phe, Val-2, Ile-1, Thr-1, Ile-2, Orn, Pro, Val-3, Val-4, Thr-2, Val-5,
and 5-MeHex, respectively. In addition to 6 sp2 methines and 2 sp2 quaternary carbones, 18
methyl signals, 12 methylenes and 22 sp3 methines including 12 α-methines and two
oxygenated β methines one of them is more deshielded at δ 69.98 for Thr-1 indicating the
ester linkage to Val-1, and another at 67.4 ppm for Thr-2 as mentioned in table 3.1.2.
HMBC and ROESY experiments established the connectivity of the amino acids.
The 5-MeHex—Val-5—Thr-2—Val-4—Val-3 connectivity could be established by HMBC
80
Results
correlations between NH signals of Val-5, Thr-2, Val-4 and Val-3 at δ 7.88, 7.82, 7.57 and
8.10 respectively, to the vicinal carbonyls of 5-MeHex, Val-5, Thr-2, and Val-4 at δ 173.8,
172.2, 169.0, and 169.8, respectively. Connections of the amino acids Thr-1—Ile-2—Orn—
Pro were determined through HMBC correlations of the NH signals at 8.56, 7.90, 7.95 ppm to
the vicinal carbonyls at 170.6, 173.0, 172.6 ppm, respectively. The connectivity of Pro to Val3 was established through the HMBC correlation between δ-proton of Pro at δ 3.52 and the
carbonyl of Val-3 at δ 171.3, and through the ROESY correlation between δ-protons of Pro at
δ 3.76, 3.52 and the α-proton of Val-3 at δ 4.26. The cyclo [Val-1—Dhb—Phe—Val-2—Ile1—Thr-1] linkage was deduced through HMBC correlations of NH signals at 6.76, 9.69, 8.79,
7.62, 8.82 ppm to the vicinal carbonyls 164.4, 171.3, 172.9, 170.0, 169.7 ppm respectively,
while the connectivity of Val-1 to Thr-1 was confirmed by an HMBC correlation between the
β-proton at δ 4.96 of Thr-1 and the carboxyl at δ 169.5 of Val-1, and also the ROESY
correlation between the β-proton at δ 4.96 of Thr-1 and the α-proton of Val-1 at 3.86 ppm.
The deduced data of compound 1 as obtained by NMR were further confirmed by
ESI-MS/MS experiment, MALDI-TOF-PSD MS (matrix –assisted laser desorption/ionisation
– time-of-flight – post source decay mass spectrometry) and by Marfey´s analysis. The
reflectron mode MALDI-TOF mass spectrum performed with delayed extraction (DE)
showed a positive ion signal at m/z 1499.92 which was identified as sodium-ion associated
monoisotopic peak [M+Na]+ of compound 1, and showed also a positive ion signal at m/z
1515.9, [M+K]+ and at m/z 1478.5, [M+1]+.
MALDI-TOF-PSD spectrum confirmed the sequence of the depsipeptide, kahalalide
F, as shown in table (3.1.3). Similar results were obtained from tandem ESI-MS/MS
spectrum, where the positive protonated fragment ion peaks were evident at m/z 1266.7,
1165.7, 1066.6, 967.8, 836.0, 756.8 , 642.7 and 443.5 corresponding to [M+1]+-[5-MeHexVal-5], [M+1]+ -[ 5-MeHex- Val-5- Thr-2], [M+1]+ -[ 5-MeHex- Val-5- Thr-2- Val-4],
[M+1]+-[ 5-MeHex- Val-5- Thr-2- Val-4- Val-3], [(5-MeHex- Val-5- Thr-2- Val-4- Val-3Pro- Orn-Ile-2)+1]+, [cyclo (Val-1- Dhb- Phe- Val-2- Ile-1- Thr-1) + Ile-2+1]+, [cyclo (Val-1Dhb- Phe- Val-2- Ile-1- Thr-1) +1]+ and [(Dhb- Phe- Val-2- Ile) +1]+, respectively.
The stereochemistry of the amino acids were determined using Marfey analysis
(Marfey, 1984), and the experiment resulted in the presence of D-Pro, L-Orn , D-aIle, LaThr, D-aThr, D-Val, L-Val, and L-Phe. The NMR data, amino acid sequences, and
stereochemistry of compound 1 were identical to those of kahalalide F (Hamann and Scheuer
1993).
81
Results
3.1.1- Kahalalide F (1, Known compound)
2.74
40.1
7.69
H2N
L- Orn.
1.67
24.4
1.48
29.6
0.77
11.6
D- pro
2.03, 1.97
29.6
val-3
0.86
19.6 H3C
N
H
172.6
4.49
52.9
4.36
60.2
186
25.4
1.94
30.5
N
H
O
7.90
4.26
57.6
O
H3C
0.58
18.8
169.8
3C
O
164.4
3.86
60.3
0.80
181 H3C
7.57
HO
19.7
4.26
58.9
6.34
131.3
7.82
0.84 H3C
18.4
NH
H
N
4.42
56.3
171.3
O
CH3 18.6
val-2
217
32.4
CH3
0.62
18.9
O
2.93
36.8
L- Phe.
7.28
129.0
C75H124N14O16
Exact Mass: 1476,93197
Mol. Wt.: 1477,87126
7.2
127.6
0.82
22.5
CH3
1.47
24.0
1.47
28.1
1.11
39.0
CH3
0.82
22.5
Kf
UV_VIS_1
WVL:235 nm
70,0
5 - 31,780
100
4.46
58.6
8.79
172.9
7.88
2.13
36.3
0.77
NH
7.28
127.0
173.8
1.02
14.6
7.62
9.69
1.26
12.5
D-Ile
CH3
170.0
O
NH
O
120 test #1
mAU
4.31
57.5
4.23
59.6
H3C
val-5
130.3
1.73
38.8
HN
138.2
1.96
30.7
1.31
26.8
169.7
1.07
17.3 CH3
172.2
H3C
CH3
8.82
CH3
NH
O
0.84
19.6
4.96
71.1
169.5
Z-Dhb
0.77
11.7
O
O
HN
169.0
3.97
67.4
4.88
L-Thr 0.98
1.39
30.8
O
HN
val-4
H3C
6.76
4.28
59.1
1.98
31.3
D-Thr
4.53
57.4
val- 1O
0.62
14.6
O
HN 8.10
0.80
195 H
170.6
HN 8.56
171.3
H3C
14.6
4.37
57.3
173.0
O
CH3 1.03
N
48.0
3.76, 3.52
0.86
18.8
1.69
38.0
1.30
26.6
O
7.95
D-Ile
H3C
80
50,0
Peak #5 31.87
%
202.8
60
25,0
40
3 - 1,041
20
0
4 - 1,220
12--Peak
0,4961 - 0,046
-20
0,0
6 - 47,005
min
10,0
20,0
30,0
40,0
50,0
-10,0
200
nm
400
595
60,0
Fig. ( 3.1.1 ): chemical structure with NMR values (up), HPLC
chromatogram (down left and UV spectrum (down right) of compound 1,
kahalalide F
Yield : 120 mg
82
Results
723
FAB-MS
H2N
868
O
512
H3C
N
H
HN
H3C
H3C
O
CH3
H3C
HN
O
O
HN
HO
O
CH3
NH
212
H3C
H3C
CH3
NH
H
N
CH3
CH3
O
O
[M+1]+
[M]+
[M+Na]+
O
H3 C
1265
CH3
HN
O
NH
1065
313
642
O
H3 C
O
836
O
N
H
O
HN
O
412
CH3
N
H3 C
1066
755
H3 C
610
967
NH
CH3
O
CH3
[M+1]+
ESI-MS/MS
ESI-MS
Fig.( 3.1.2 ) : FAB-MS , ESI-MS and ESI-MS/MS of compound 1
C=O
reg i o n
P h e-p h e n y l
& Dhb
o l efen i c
C = C r e g io n
β - T h r-2
α - C r e g io n
β- T h r-1
Fig. (3.1.3) : 13C-NMR and DEPT spectra of compound 1
83
Results
Fig. (3.1.4) : different reigons of 1HNMR spectrum of compound 1
β/me
Thr-1
γ Hs, & CH3
β/me
Thr-2
α/βVal -1
β Hs
α Hs
α /β
Phe
β-Phe
δ/γ Orn
Pro
Thr-1
NHs
β/α
Thr-1
Fig. (3.1.5) : Total COSY (left), part of COSY (right) spectra of compound 1
84
Results
m e - D h b
α P he
α V a l- 2
α O rn
α V a l-3
α T h r -2
α V a l-4
m e -Ile - 1
m e - V a l-1
m e - V a l-2
β V a l- 2
γ − P ro
α
5 M e H ex
β P h e
α T h r-1
α Il e -1
α I le -2
α V a l -5
α P ro
β I le - 1
δ − P ro
δ O rn
α V a l- 1
β T hr-2
β T h r-1
D h b -C H
P h e .p h e n y l
Fig. (3.1.6) : Total HMQC spectrum of compound 1
Fig.(3.1.7) : NH-detected spin systems (left), Proline and 5-MeHex (right) spin
systems from partial TOCSY spectrum of compound 1
Fig.(3.1.8) : NH-detected ROESY correlations (left), upfield part of ROESY
spectrum (right) of compound 1
85
Results
H 2N
H3 C
O
H 3C
N
H
CH 3
H
N
H
O
N
HN
HN
O
H 3C
O
CH3
H 3C
H3 C
O
O
HN
O
CH 3
NH
NH
H
N
NH
H
HN
CH 3
HN
O
HO
CH 3
O
H 3C
O
H 3C
C-C HMBC Correlations
O
CH3
H
CH 3
C=C
HMBC Correlations
O
O
O
H3 C
H3 C
H 3C
NH
CH 3
O
H
CH3
CO
HMBC Corre lations
Fig.(3.1.9) : Total HMBC spectrum showing different H-detected HMBC
correlations (up), NH-detected correlations to C=O (down left), and aliphatic
protons–detected HMBC correlations to C=O (down right) of compound 1.
Table (3.1.1) Marfey´s analysis results of kahalalide F hydrolysates :
Amino acidDAA deriv.
MW
(+ve mode)
MW
(-ve mode)
Ret.time in
minutes
D.Ile
L.Phe
L. Val
D. Val
D.Pro
L.Thr
D.Thr
384.0
418.0
370.0
370.0
368.1
372.0
372.0
L.
Orn*
385.1
382.4
416.5
368.3
368.3
366.3
370.3
370.3
383.5
24.53
22.98
15.93
16.60
13.44
20.97
22.77
18.68
&
14.14
86
Results
* It was detected that L-ornithine isomer has two different retention times, because ornithine
contains α- and δ- reactive amino groups which could both react with FDAA producing two
ESI-MS-detectable products as shown in figure 3.1.12.
Table (3.1.2) : 1H and 13C NMR data of compound 1 in DMSO-d6
Amino No.
acid
1
Val-1
2
3
4
5
13
1
C,PP
M
169.5
60.3
30.8
14.6
18.8
H,PPM
multiplicity
(NH) 6.76
3.86
1.39
0.62
0.58
(d, J=9.0Hz)
( t,J= 9.0 Hz )
(m)
(d, J=7.0Hz)
(d, J=6.0Hz)
Amino
acid
Pro
Dhb
1
2
3
4
164.4
130.3
131.3
12.50
(NH) 9.69
6.34
1.26
(s)
(q, J=7.0Hz)
(d,J=7.5Hz)
Val-3
Phe.
1
2
3
4
5,5`
6,6`
7
1
2
3
4
5
1
2
3
4
5
6
171.3
56.3
36.8
138.2
129.0
127.0
127.6
172.9
58.6
32.4
18.9
18.6
170.0
57.5
38.8
26.8
14.6
11.7
(NH) 8.79
4.42
2.93
7.28
7.28
7.2
(NH) 7.62
4.46
2.17
0.62
0.77
(NH) 8.82
4.31
1.73
1.31
1.02
0.77
(d, J=5.5Hz)
(q, J=6.5Hz)
(m)
(m)
(m)
(m)
(d, J=8.5Hz)
(m)
(m)
(d, J=7.0Hz)
(d, J=6.5Hz)
(d, J=10.0Hz)
(m)
(m)
(m)
(t, J= 7.6 Hz)
(d)
Val-4
Thr-1
1
2
3
4
169.7
57.4
71.1
17.3
(NH) 8.56
4.53
4.96
1.07
(d, J=8.0Hz)
(t, J=7.8Hz)
(m)
(d,J=6.5Hz)
5-Me-Hex.
1st conf
Ile-2
1
2
3
4
5
6
1
2
3
4
5
-
170.6
57.3
38.0
26.6
14.8
11.6
173.0
52.9
29.6
24.4
40.1
-
(NH) 7.90
4.37
1.69
1.30
1.03
0.77
(NH) 7.95
4.49
1.48
1.67
2.74
(NH2),7.69
(d, J=8.2.0Hz)
(m)
(m)
(m)
(t, J= 6.5 Hz)
(d)
(d, J=8.50Hz)
(m)
(m)
(m)
(m)
b.singlet
5-Me-Hex.
Val-2
Ile-1
Orn.
Thr-2
Val-5
2st conf.
87
No.
13
C,PPM
1
H,PPM
multiplicity
(dd,J= 9.1,
6.6 Hz )
(m,m)
(m)
(m,m)
(d, J=8.5Hz)
(m)
(m)
(m)
(m)
(d, J=8.5Hz)
(m)
(m)
(m)
(m)
1
172.6
-
2
3
4
5
1
2
3
4
5
1
2
3
4
5
60.2
29.6
25.4
48.0
171.3
57.6
30.5
19.6
18.8
169.8
59.1
31.3
19.5
18.1
4.36
2.03,1.97
1.86
3.76,3.52
(NH) 8.10
4.26
1.94
0.86
0.86
(NH) 7.57
4.28
1.98
0.80
0.80
1
2
3
4
1
169.0
58.9
67.4
19.7
172.2
2
3
4
5
59.6
30.7
19.6
18.4
1
2
3
4
5
6
7
1
2
3
4
5
6
7
173.8
36.3
24.0
39.0
28.1
22.5
22.5
174.1
33.9
32.8
38.8
29.5
19.5
11.5
(NH) 7.82
4.26
3.97
0.98
(OH) ,4.88
(NH) 7.88
2ndconf. (
7.85)
4.23
1.96
0.84
0.84
2.13
1.47
1.11
1.47
0.82
0.82
2.2
1.6
1.14
1.35
0.80
0.80
(d, J=8.0Hz)
(m)
(m)
(d,J=6.5Hz)
(d,J=5.0Hz)
(d, J=7.5Hz)
(d, J=7.5Hz)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
Results
R
T :
5 .0 0
- 4 5 .0 0
N
2
S
M
n
4
n
4
M
2 2 .7 7
1 0 0
9 5
D-Valine
9 0
L-Phenylalanine
8 5
R
e la t i v e
A b s o r b a n c e
8 0
D-Proline
7 5
7 0
L
.7
p
a
m
0
m
0
o
:
8 E 5
e c tr u m
x i m
u m
= 2 1 0 .0 0 .0
= 3 9 9 .5 0 .5
P D A
h a m m
e d 2 9 7
L-Valine
6 5
6 0
D-Threonine
5 5
5 0
2 0 .9 7
4 5
2 4 .5 3
1 8 .8 8
4 0
D-isoleucine
L-Threonine
3 5
3 0
1 6 .6 0
2 5
1 5 .9 3
2 0
L-Ornithine
1 4 .1 4
1 5
1 0
1 3 .4 4
5
7 .2 9
5
8 .6 5
2 8 .2 4
1 2 .4 9
1 0
1 5
2 0
T i m
2 5
e ( m
3 4 .4 7
3 0
3 6 .2 0
3 7 .6 0
3 5
3 8 .8 1
4 4 .7 6
4 0
4 5
i n )
D-Isomer enriched
D-isoleucine
L-Isomer enriched
L-Phenylalanine
D-Valine
L-Isomer enriched
L-Valine
D-Proline
L-Isomer enriched
D-Threonine
L-Threonine
L-Isomer enriched
L-Isomer enriched
L-Ornithine
L-Ornithine
Fig (3.1.10): Marfey´s analysis results of kahalalide F-amino acids (up), ESI-MS
chromatogram showing base peak search for each individual amino acid
(chromatograms right) and their peak enriched analysis (opposite chromatograms
left) for D-Ile, L-Phe, L-Val& D-Val, D-Pro, D- Thr &L-Thr, and L-Orn.
88
Results
L-Thr
L-Threonine
enriched
D-Thr
L-Thr
L-Proline
enriched
D-Threonine
L-Threonine
D-Thr
L-Thr
D-Thr
D-Proline
Kah F hyd.
D-Proline
L-Pro
D-Pro
D-Pro
Fig (3.1.11): Compound 1–hydrolysate ESI-MS (+ve) : comparison of D-Thr and DPro [right] against enriched L isomers [left], the retention time was not always
constant. Therefore, the peak enrichment technique was applied to unambiguously
confirm the stereochemistry of each amino acid.
2x L .O rn d erivatives
D . O rn d eriv atives
S tan d ard D & L O rn
T w o O rn derivativ es resulted from L .O rnith ine
S ta n d ard L .O rn
Fig (3.1.12) : Standard L.Orn vs DL.Orn. , showing two possible products for each
stereoisomer.
89
Results
RT: 5.00 - 45.00
22.77
100
95
90
D-Valine
85
L-Phenylalanine
80
75
L-Valine
70
D-Proline
Relative Absorbance
65
60
D-isoleucine
D-Threonine
55
20.97
24.53
50
45
18.88
L-Threonine
40
35
16.60
30
25
L-Ornithine
20
15.93
14.14
15
10
13.44
5
7.29
5
8.65
10
34.47 36.2
28.24
12.49
15
20
25
Time (min)
30
Fig (3.1.13) : relative peak area of D-Val
(right) to the L-Val (left) of compound 1
[ratio = 3:2]
35
Fig.(3.1.14 ) The stereochemical profile of
Amino acid units of compound 1
Table (3.1.3): The calculated mass fragments from MALDI-TOF-PSD-MS of
compound 1 and sequence determination.
90
Results
3.1.2- Kahalalide E (2, Known compound)
21.5
0.83
1.25
D-Ala-1
21.5
22.0
0.85
0.85
1.50
D-Ala-2
H
N
27.0
O
1.14
2.65,
2.46
27.0
9-Me-3Decol
27.4
1.15
33.1
5.1
28.5
H
N
170.8
8.26
170.4
7.05
171.6
H
N
4.27
1.82, 1.99
171.4
25.2
8.12
1.89, 2.0
28.1
O
L-Pro
Leu-1
1.37
124
46.3
4.28
1.45
7.1
3.47, 3.66
N
58.9
24.0
26.8
25.0
N
H
111
58.0
O
50.2
3.11, 3.08
7.30
4.41
O
53.8
109.2
121.5
L-Trp
38.9
O
4.38
136.3
Leu-2
1.58
7.08
O
71.5
7.5
128.1
N
H
17.1
117.5
6.95
H
N
171.4
1.25
40.0
O
118.5
4.18
7.9
50.5
170.5
1.40
1.18
171.9
21.0
0.83
28.6
47.7
3.99
8.17
1.10
1.15 29.0
16.4
O
0.84
0.79
10.8
22.0
22.1
C45H69N7O8
Exact Mass: 835,521
Mol. Wt.: 836,071
600 ms04052
mAU
rp2,7
UV_VI
WVL:280 nm
Peak #1133.26
60,0
%
No spectra library hits found!
220.8
9 - 33,231
40,0
250
20,0
281.1
288.5
5
1,163
6
1,254
8----Peak
-0,619
1,421
7
1,346
1,037
123
4
1,121
1 - 0,071
-50
0,0
min
10,0
20,0
30,0
40,0
50,0
0,0
200
60,0
nm
250
300
350
400
450
500
550
595
Fig (3.1.15): chemical structure of compound 2 with δC and δH values (up), HPLCchromatogram (down left) and UV-spectrum (down right).
Yield : 4 mg
ESI-MS
(M +1) +
(2M+H 2 O) +
(M + Na )+
MALDI-TOF-MS
(M +H) +
ESI-MS
(M-1) +
(M +K) +
(M +HCOOH) +
(2M +H 3 PO4) +
(2M+HCOOH) +
Fig.( 3.1.16 ) : MALDI-TOF-MS and ESI-MS of compound 2
91
Results
Kahalalide E was isolated as a white amorphous powder, with [α]D of + 5° (c 0.25
MeOH). It has UV absorbance at λmax 221, 281, 288 nm. MALDI-TOF-MS showed
pseudomolecular ion peak at m/z 858.57 [M+Na]+ and 874.55 [M+K]+ and 836.65[M+1]+
suggesting the molecular formula C45H69N7O8. The molecular weight of 2 was also confirmed
by ESI-MS, and MALDI-TOF-PSD-MS. The 1HNMR spectrum of 2 showed 5 deshielded
amide-NH resonances in the lower field region, at 7.08, 7.90, 8.12, 8.17 and 8.26 ppm,
suggesting the peptidic nature of the compound. In addition to the most downfield NH proton
which is a sharp singlet at δ 10.8, an ABCD aromatic spin system as resembled by resonances
at 7.50, 6.95, 7.05 and 7.3 ppm was observed and suggested the presence of tryptophane, Trp.
COSY spectrum together with TOCSY experiment indicated 5 spin systems commencing
from the above mentioned five NH-resonances, in addition to the characteristic indole spin
system of Trp. TOCSY spectrum showed also the presence of characteristic proline spin
system (Hα 4.28, Hβ 1.89 & 2.00, Hγ 1.83 & 1.99 and Hδ 3.47 & 3.66 ppm) and β-hydroxy
aliphatic acid, 9-methyl-3-hydroxydecanoic acid (9-Me-3-Decol) [(H-2) at δ 2.65 & 2.46, (H3) at δ 5.1, (H-4) at δ 1.40, (H-5) at δ 1.15, (H-6) at δ 1.14, (H-7) at δ 1.15, (H-8) at δ 1.10,
(H-9) at δ 1.15 and two methyls at δ 0.85 and 0.85 ppm].
13
C NMR spectra displayed signals for 45 carbons including 7 carbonyls at δ 170.8,
171.6, 171.4, 170.4, 171.4, 171.9, 170.5 of Trp, Leu-1, Pro, Leu-2, Ala-1, Ala-2 and 9-Me-3Decol, respectively, in addition to 8 aromatic carbons, 6 α-CH, one downfield aliphatic
methine carbone at 71.5 (C-3 of 9-Me-3-Decol), 3 highfield methines,12 CH2 and 8 methyls
as mentioned in table 3.1.5.
HMBC and NOESY experiments established the connectivity of the individual spin
systems. H-2 of indole at 7.1 ppm showed HMBC correlation to C-3, C-3a, C-7 and C-7a at δ
109.2, 128.1 111.0 and 136.3 respectively. The connectivity of Trp to Leu-1 was evident
through HMBC correlation between Trp-NH at δ 8.26 to the vicinal carbonyl of Leu-1 at
171.6 ppm. The sequence for Ala-1-Ala-2-9-Me-3-Decol was established also by HMBC
correlations of NH resonances at δ 7.90 and 8.17 to vicinal carbonyls at δ 171.9 and 170.5,
respectively. Connection of 9-Me-3-Decol to Trp was established through HMBC correlation
of Hβ-9-Me-3-Decol to carboxyl at 170.8. Although there are no evident HMBC correlations
between Leu-2-NH and the vicinal carbonyl of Ala-1, due to peak broadness, the connection
of Leu-2 to Ala-1 was determined by the HMBC correlation at δ 4.41 (Hα, Leu-2) to Ala-1
carbonyl at 171.4. HMBC experiment showed a correlation between Hα Pro at δ 4.28 and
Leu-2 carbonyl at 170.4 ppm. The connectivity of Leu-1 to Pro was established through the
NOE between Leu-1 NH at 8.12 and Hα-Pro 4.28 ppm. Also, the connection of the amino
92
Results
acids of Pro- Leu-2 was confirmed by NOE between δ-protons Pro at 3.47, 3.66 ppm and Hα
of Leu-2 at δ 4.41. NOESY experiment showed also a through space correlation between Ala1 NH at 7.9 ppm to Ala-2 NH at δ 8.17, while Ala-2 NH showed a through space correlation
to Hβ of 9-Me-3-Decol at 2.65 and 2.46. In addition, further NOEs were observed between the
downfield Hβ of 9-Me-3-Decol at 5.1 ppm and Hα of Trp 4.38 ppm. The NMR data of
compound 2 were identical to those of kahalalide E (Hamann et al 1996).
D ow n field region
Indole
C H -7
NH
A la-2 N H
Trp-N H
A la-1 N H
C H -2
Leu 2 N H
2x M e- A liphatic acid
C H -6
C H -4
M e-A la-2
C H -5
2x M e
Leu -1 N H
M e-A la-1
Leu -2
M e Leu -1
M e Leu -1
H γ Leu -2
H
α - region
H β Leu -2
H
β
A liphatic acid
Trp
Leu -1
H
Pro
δ-
H
Por
H
β
α
H β Leu -1
H γ Leu -1
A liphatic acid
H
Trp
β-
Por
H
γ-
Por
Leu -2
A la-1
A la-2
H -9 A liphatic acid
Fig (3.1.17): 1H NMR spectrum.
C H -2
Ala-1 NH
Indole
NH
Ala-2 NH
Leu -1 NH
C H -7
C H -4
C H -6
C H -5
Trp-NH
Leu -2 NH
β
γ
β
γ
β
β
Trp
Ala-1
Ala-2
Trp
α
α
Leu -1
Leu -2
α
α
α
ABCD
spin system
Indole
NH/ C
H -2
Fig (3.1.18): parts of TOCSY Spectrum of Compound 2 showing NH-detected spin
system .
93
Results
HMBC correlations to indole aromatic carbons
HMBC correlations to C=O
C-2
HβTrp
C-3a
C-3
Indole carbons
C-2
C-3a
NH Ala-2β/
C=OFatty acid
H-3 Fatty acid/
C=O-Trp
NH Ala-1/
C=O Ala-2
NH Trp/
C=O Leu-2
H-2 /C=O
Ala-1
H-2 Pro/
C=O Leu-2
H-3/CO
Pro
C=O
2x H /C=O
H-2 Ala-2/ Trp Fattyβacid
C=O Ala-2
CH3/CO
Ala-1
CH3/CO
Ala-2
Fig (3.1.19): Total HMBC spectrum of compound 2 (up), different parts (down)
showing important HMBC correlations
94
Results
NH Trp /
H-2 Leu-1
NH Ala-1/
H-2 Ala-1 & H-2Ala-2
NH Ala-1/
NH Ala-2
NH Leu-1/
H-2 Pro
Fig (3.1.20) : NH-detected NOESY correlations.
Deduced data of compound 2 obtained by NMR were confirmed by ESI-MS/MS
experiment and MALDI-TOF-PSD MS.
MALDI-TOF – PSD spectrum confirmed the sequence of a depsipeptide, kahalalide
E, as shown in table (3.1.4). Similar results were obtained from tandem ESI-MS/MS
spectrum, where the positive protonated fragment ion peaks were evident at m/z 808.4, 765.2,
650.1, 599.1, 581.3, 537.1, 464.7, 440.0, 415.1, 395.1, 326.9, 308.9, 282.1 corresponding to
[M+1-CO]+, [M-Ala] +, [M+1-Trp]+, [M+1-(Leu+pro+CO)]+, [M+1-(Ala+9-Me-3-Decol)]+,
[M+1-(Leu+Trp)]+,
[M+1-(Trp+9-Me-3-Decol)]+,
[9-Me-3-Decol+Ala+Ala+Leu+1]+,
[Trp+9-Me-3-Decol+Ala+1-(CO)]+, [Ala+Leu+Pro+Leu+1]+, [9-Me-3-Decol+Ala+Ala+1]+,
[Ala+Leu+Pro+CO+1]+, and [Ala+Leu+Pro+1]+ respectively.
Fig (3.1.21) ESI-MS/MS spectrum of compound 2.
95
Results
[M+1] + = [ 9-Me-3-Decol+Trp+Leu+Pro+Leu+Ala+Ala+1]+ = 836.41
-[Trp]
-m/e 186
-[Ala]
-m/e 71
- [Pro+Leu+CO]
-CO
- m/e 237
-28
O
H
N
O
O
O
O
O
O
N
H
N
O
O
N
H
[Leu+Pro+Leu+Ala+
Ala+9-Me-3-Decol+1]+
m/e 650
O
H
N
O
N
H O
H O
H
N
N
O
N
H
HN
-[Leu+Ala-CO]
- m/e 156
[Ala+9-Me-3-Decol+Trp+1-CO]+
H
N
H
N
O
O
O
O
N
H
N
O
O
NH
m/e 415
N
H
O
[Leu+Pro+Leu+
Ala+Ala+1]+
m/e 650
N
O
m/e 537
NH
O
N
H
H
N
O
O
[Ala+9-Me-3-Decol+Trp
+....+Leu+Ala+1-CO]+
N
[M+1-CO]
(m/e 808.4 )
O
O
O
+
-m/e 184
H
N
H
N
N
H
N
H
H
N
O
-[9-Me-3-Decol]
- m/e113
- [Leu]
O
O
H
N
m/e 599.1
H
N
N
H
O
O
[Pro+Leu+Ala+
Ala+9-Me-3-Decol+1]+
- [Pro]
- m/e 97
O
O
O
O
H
N
HN
O
N
H
N
O
N
H
- m/e113
- [Leu]
H
N
O
O
H
N
O
O
m/e 440
O
N
H
[9-Me-3-Decol+Trp+Leu+Pro+Leu+Ala+1]+
m/e 765
-[9-Me-3-Decol]
-m/e 184
HN
H
N
HN
HN
O
O
H
N
O
O
N
H
N
O
[Trp+Leu+Pro+Leu+Ala+1]+
m/e 581
-[Trp]
-m/e 186
H
N
HN
- m/e113
- [Leu]
O
O
O
N
H
N
O
HN
O
N
O
O
m/e 282
m/e 395
[Leu+Pro+Leu+Ala+1]+
[Pro+Leu+Ala+1]+
Fig (3.1.22) Possible fragmentation pattern and amino acids-sequencing of
compound 2 by interpretation of Tandem ESI-MS spectrum.
96
O
N
H
O
m/e 327
[Ala+Ala+9-Me-3-Decol+1]+
[Leu+Ala+
Ala+9-Me-3-Decol+1]+
O
O
O
H
N
H
N
Results
Table (3.1.4): The calculated mass fragments from MALDI-TOF-PSD-MS of
compound 2
X = 9-Me-3-hydroxy Decanoic acid [9-Me-3-Decol]
97
Results
Table (3.1.5) : 1H and 13C NMR data of compound 2 in DMSO-d6
Amino acid
Carbon
Tryptophan
1
2
3
4
5
6
7
8
9
10
11
1
2
3
4
5
6
1
2
3
4
5
1
2
3
4
5
6
1
2
3
1
2
3
1
2
3
4
5
6
7
8
9
10
11
Leucine-1
Proline
Leucine-2
Alanine-1
Alanine-2
9-Me-3-Decol
13
C NMR
ppm, Mult
170.8, s
53.8, d
26.8, t
109.2, s
124.0, d
136.3, s
128.1, s
117.5, d
118.5, d
121.5, d
111.0, d
171.6, s
50.2, d
24.0, t
25.0, d
22.0, q
22.1, q
171.4, s
58.9, d
28.1, t
25.2, t
46.3, t
170.4, s
58.0, d
38.9, t
28.6, d
21.0, q
21.5, q
171.4, s
47.7, d
16.4, q
171.9, s
50.5, d
17.1, q
170.5, s
40.0, t
71.5, d
33.1, t
28.5, t
27.4, t
27.0, t
29.0, t
27.0, d
22.1, q
22.1, q
1H NMR, ppm
Multiplicity , J= Hz
NH,8:26
4.38
3.11; 3.08
d, J=7.7
q, J=7.4
m; dd, J=14.7, 8.1
7.1; NH,10.80
s; s
7.50
6.95
7.05
7.30
NH, 8.12
4.27
1.37
1.45
0.84
0.79
d, J=8.1
dt, J=7.0, 0.7
dt, J=7.0, 1.1
d, J=8.1
br d
m
m
m
m
d, J= 5.9
4.28
2.00; 1.89
1.99; 1.83
3.66; 3.47
NH, 7.08
4.41
1.58
1.25
0.83
0.83
NH, 7.90
4.18
1.18
NH, 8.17
3.99
1.25
m
m; m
m; m
m; m
br d
m
m; m
m
m
m
d, J= 7.7
p, J= 7.3
d, J= 7.0
d, J= 5.6
p, J= 6.3
d, J= 7.4
2.65; 2.46
5.10
1.40
1.15
1.14
1.15
1.10
1.50
0.85
0.85
dd, J= 14.7, 5.6; dd, J= 14.7, 7.0
p, 6.3
m
m; m
m
m
m
m
d, J= 6.7
d, J= 6.7
98
Results
3.1.3- Kahalalide D (3, Known compound)
7 .3 4
7 .1 9
7 .0 5
6 .9 7
7 .4 8
L -P r o
1 .4 1 , 1 6 3
1 0 .9 1
H
N
2 .7 2 ,
3 .7 0
O
1 .6 0 , 1 .9 0
L -A r g
O
N
4 .0 7
NH
HN
4 .5 5
1 .6 8 ,1 .5 8
3 .1 4 , 2 .9 8
8 .8 5 N H
4 .1
3 .0 3
N
H
7 .6 3
1 .3 9
D -T r p
O
O
2 .5 2 , 2 .6 1
NH2
O
5 .0 5
1 .6 7 , 1 5 7
1 .2 5
7 -M e -3 -O c to l
C 31H 45N 7O 5
E x a c t M a ss: 5 9 5 ,3 4 8
M o l. W t.: 5 9 5 ,7 3 3
1 .1 5
H 3C
0 .8 4
1 .5 2
CH3
0 .8 4
160 ms030627 #2
mAU
Egme1-13
UV_VIS_3
WVL:280 nm
60,0
Peak #7 100%
%
219.4
125
37,5
100
75
203.1
25,0
50
12,5
280.6
25
1
-20
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
10,0
200
nm
250
300
350
400
450
500
550
595
Fig (3.1.23 ): chemical structure of compound 3 showing δH values (up) and HPLC
chromatogram (down left) and UV spectrum (down right).
Yield : 3.1 mg
ESI-MS/MS
M+1
2M+1
M+HCOOH-1
2 M+3HCOOH-1
2 M+2HCOOH-1
Fig.(3.1.24) : ESI-MS and ESI-MS/ MS spectra of compound 3
99
Results
H
N
O
O
N
NH
HN
NH
H
N
O
O
N
O
NH
HN
NH
N
H
(16 %)
H3C
-NH2
NH2
CH3
O
O
C31H43N6O5•
Exact Mass: 579,329
Mol. Wt.: 579,71
O
H3C
-CH3N2
CH3
N
H
O
O
H
N
O
O
N
+
HN
[Mol. wt. +H] = m/z 595
(80%)
NH
O
O
-NHCNHNH2
-(Arg+H2O)
NH
O
C30H41N5O5•
Exact Mass: 551,311
Mol. Wt.: 551,677
(100 %)
H3C
-[Arg + Prol]
CH3
[- (Trp +Fatty acid)+H]
H
N
O
NH
H
N
O
N
(18 %)
O
NH
O
O
CH3
O
(13 %)
H3C
NH
HN
O
N
NH
C25H32N3O3• O
Exact Mass: 422,244
Mol. Wt.: 422,54
H3C
N
O
CH3
N
H
O
NH2
C30H40N4O5
Exact Mass: 536,3
Mol. Wt.: 536,662
+H
-
( 54 % )
N
O
C11H21N5O2•+
Exact Mass: 255,169
Mol. Wt.: 255,316
HN
O
H
N
C20H25N2O2•
Exact Mass: 325,192 O
Mol. Wt.: 325,425
OH
NH
O
H3C
C20H27N2O3•
Exact Mass: 343,202
Mol. Wt.: 343,44
O
NH
(43%)
H3C
CH3
CH3
( 3 %)
Fig (3.1.25):Possible fragmentation pattern and sequence determination from ESIMS/MS spectrum of compound 3
Kahalalide D was isolated as a white amorphous powder, with [α]D of - 40° (c 0.25
MeOH). It has UV absorbance at λmax 203, 219, 280 nm. Positive ESI-MS showed
pseudomolecular ion peak m/z 596.7 [M+1]+ and 1191.0 [2 M+1]+ and negative ESI-MS
showed pseudomolecular ion peak at m/z 641.0 [M+HCOOH-1]- and 1327.8 [2M+3HCOOH1]- suggesting the molecular formula C31H45N7O5. The molecular weight and the sequence
100
Results
determination of 3 was confirmed by ESI-MS/MS fragmentation pattern as shown in figure
3.1.25. The 1HNMR spectrum of 3 showed characteristic proton resonances that belongs to
the simplest known kahalalide, which is kahalalide D. Compound 3 is made up of three amino
acids consisting of tryptophane (Trp), proline (Pro), and arginine (Arg) plus one aliphatic acid
[7-Me-3-Hydroxy-Octanoic acid]. 1HNMR showed 2 deshielded amide-NH resonances in the
lower field region, at 6.78 (d, J = 5.68 Hz, Arg-NH) and 8.85(d, J = 6.9 Hz, Trp-NH) in
addition to downfield proton resonance at 7.63 (br t, J = 5.63 Hz, Arg-NH) and a
characteristic indole protons of Trp resonating at 6.97 (t, J = 7.4 Hz, Trp-CH-10), 7.05 (t, J =
7.3 Hz, Trp-CH-9), 7.34 (d, J = 8.2 Hz, Trp-CH-9), 7.48 (t, J = 8.2 Hz, Trp-CH-11), 7.19 (d, J
= 2.3 Hz, Trp-CH-5) and a downfield resonance at 10.91 (d, J = 1.2 Hz) for an indol-NH.
TOCSY experiment and COSY spectrum showed the rest of arginine-proton resonances as
mentioned in table 3.1.6. 1HNMR showed a characteristic proline spin system at δ 4.07 (dd, J
= 8.6, 4.3 Hz, H-2), 1.60, 1.90 (m,m, H-3),1.41, 1.63 (m,m, H-4), 3.70, 2.72 (m,m, H-5)
which was also confirmed by COSY and TOCSY experiments. The aliphatic acid of 3 was
assigned to be 7Me-3-Octol from the COSY and TOCSY spectra of compound 3 at δ 2.52,
2.61 (dd, J = 15.2, 4.95 Hz, H-2a and dd, J = m, H-2b), 1.67,157 (m,m, H-4), 1.25 (m, H-5),
1.15 (m, H-6), 1.52 (m, H-7), the presence of downfield proton resonance at δ 5.05 (m)
indicated the presence of an oxygenated methine group of the fatty acid (H-3) where its
position was established by direct COSY correlations to H-2. As are most of fatty acid
moieties of known depsipeptides, compound 3-fatty acid is an iso-acid showing a two methylproton resonances at δ 0.84 and 0.84 (d, J 6.3 Hz, H-8 and H-9). The presence of a guanidine
group was established through ESI-MS/MS fragmentation where the presence of molecular
ion fragments at 579.3 [M - NH2, (16 %)], 551.3 [M - NHCHNH2, (100 %)] and 536.5 [M –
guanidine (14 %)] confirmed the presence of guanidine group. The connection of a different
spin systems was confirmed by ROESY and ESI-MS/MS fragmentation data. ROESY
spectrum showed a NOEs between (NH-Trp) at δ 8.85 ppm to Hα of 7Me-3-Octol and Hα-Pro
at δ 2.52, 2.61 and 4.07ppm, respectively. H-3 of the fatty acid at δ 5.05 showed an NOE to
Arg NH at δ 6.78, while Arg-NH at 6.78 showed an NOE to proline-Hα at 4.07, and
additional correlations to Trp-NH and Hα of the fatty acid moiety. ROESY, COSY and
TOCSY correlations as well as 1HNMR data are identical to those of kahalalide D (Hamann
et al 1996).
101
Results
Fig (3.1.26): 1H NMR spectrum of compound 3
N H T rp
N H In d o le
N -6 H
A rg
N -6 H /
H -4 A rg
H -5 a P ro
N H A rg
H -3
A rg
a& b
H -2 F a tty a c id
H -3 a T rp ,
H -3 b T rp
H -2 P ro &
H -2 A rg
c o rre la tio n s
to H -2 P ro
H -2 T rp
N H A rg /
H -3 F a tty a c id
Fig (3.1.27): Total COSY spectrum (up) showing different spin
systems and NH-detected ROESY correlations (down) of compound 3
102
Results
7 -M e -3 -O c to l
( F a tty a c id )
P r o lin e
S p in s y s te m
Fig (3.1.28): Total TOCSY spectrum (up), and part of TOCSY spectrum
showing proline and Fatty acid spin systems of compound 3 (down).
Table (3.1.6): NMR data of compound 3 in DMSO-d6
Amino acid
Carbon
Tryptophan
Proline
Arginine
9-Me-3-Decol
(F.A)
1
2
3
5
1H NMR,
ppm
(NH) 8.85
4.55
3.14; 2.98
7.19
Multiplicity , J=
Hz
d, J=6.9
q, J=7.4
m; dd, J=14.7, 7.1
d, J=1.9
8
9
10
11
2
3
4
5
1
(NH) 10.91
7.34
7.05
6.97
7.48
4.07
1.60; 1.90
1.41; 1.63
3.70; 2.72
NH, 6.78
d, J=1.3
d, J=8.2
t, J=7.6
t, J=7.3
d, J=8.2
m
m; m
m; m
m; m
d, J=5.68
2
3
4
5
6
2
4.1
1.68;158
1.39
1.03
7.63
2.52; 2.61
3
4
5
6
7
8
9
5.05
1.57;167
1.25
1.15
1.52
0.84
0.84
m
m
m
m
br t, J= 5.6
dd, J= 14.7, 5.6; dd,
J= 14.7, 7.0
p, 6.3
m; m
m; m
m
m
d, J= 6.3
d, J= 6.3
103
ROESY correlations
Pro H-2; F.A H-2
Trp H-3, H-5
Trp H-5
indole NH, Trp H-2, H-8, H11; Pro H-5
Trp H-5;
Arg NH, Trp NH
Arg H-3a &b, NH-6; Pro H-2;
Trp H-2; F.a H-3
F.A H-3; Trp NH;
Arg NH
Results
3.1.4- Kahalalide B (4, Known compound)
126.5
7.18
17.2
0.75
127.5
7.16
129.0
7.21
18.5
0.78
1.50
38.0
11.5
0.81
137.2
23.0
1.10
8.32
H
41.2
2.11
8.03
H
N
N
173.5
4.29
56.0
O
2.78,
2.83
7.00
129.8
168.1
4.4
50.4
171.7
2.91,
2.93
38.5
O
1.35
27.1
4.8
53.2
N
H
7.63
173.1
8.9
H
N
11.0
0.75
38.0
1.28
23.5 1.51,
1.40
4.10
50.8
O
O
35.5
3.35
O
47.2
127.3
5.4
HO
O
6.68
115.0
157.7
O
167.1
3.61,
3.91
42.0
HO
67.0
H
4.2
59.1
170.8
N
H
4.38
60.8
171.5
4.45
7.37
H
N
3.51,
3.96
12.0
1.25
N
H
7.2
47.0
23.0
2.0,
1.8
2.10, 28.5
2.05
O
O
C 45H 63N 7O 11
Exact Mass: 877,459
Mol. Wt.: 878,022
300
ms050512 #7
mAU
rp2-4e
250
UV_VIS_1
WVL:235 nm
70,0
Peak #8
30.81
No spectra library hits found!
%
8
204.9
50,0
200
7
150
25,0
100
9
50
277.2
10
0
562.4
123456
min
-50
0,0
10,0
20,0
30,0
40,0
50,0
60,0
nm
-10,0
200
300
400
595
Fig (29) : compound 4 (Kahalalide B)
Yield : 5 mg
Kahalalide B was isolated as a white amorphous powder, with [α]20D of + 35° (c 0.25
MeOH). It has UV absorbance at λmax 205, 228sh, 277 nm. ESI-MS showed positive
pseudomolecular ion peak m/z 878.8 [M+1]+ and 895.6 [M+H2O]+, 1774.9 [2M+H2O]+ and
1755.8[2M+1]+ and negative pseudomolecular ion peak m/z 913.1 [M+HCOOH-1]-, 975
[M+H3PO4-1]-, suggesting the molecular formula C45H63N7O11. The 1HNMR spectrum of 4
showed 6 deshielded amide-NH resonances in the lower field region, at δ 8.90 (d, J= 0.9 Hz),
8.32 (d, J= 5.4 Hz), 8.03 (d, J= 9.6 Hz), 7.63 (d, J= 9.4 Hz), 7.37 (t, J= 6.2 Hz), and 7.20 (d,
J= 7.3 Hz) of Leu, Tyr, Ser, Phe, Gly, and Thr, respectively, suggesting the peptidic nature of
the compound. COSY spectrum together with TOCSY experiment indicated 6 spin systems
104
Results
commencing from the above mentioned 6 NH resonances as mentioned in table 3.1.8, in
addition to the characteristic proton resonances of p-disubstituted benzene ring (the rest of
Tyrosine protons) at δ 6.68 (d, J= 8.2 Hz) and 7.0 (d, J=8.2 Hz) and proton resonance cluster
at δ 7.16-7.21 ( 5 aromatic proton of phenylalanine). COSY and TOCSY spectra revealed the
presence of two more spin systems which were assigned to proline and 5-methyl hexanoic
acid (see table 3.1.8). As in the case of kahalalide F, kahalalide B showed a second regioisomer of 5MeHex. 13C chemical shifts were extracted from HMQC and HMBC spectra as
mentioned in table 3.1.8.
HMBC and ROESY experiments established the connectivity of the individual spin
systems. The NHs of Thr, Gly, Ser, Phe, and Leu at δ 7.20, 7.37, 4.4, 7.63 and 8.90,
respectively showed a ROESY correlation to Pro H-2, Thr H-2, Phe NH, Ser H-2 and Phe H2, at δ 4.38, 4.2,7.63, 4.4 and 4.8, respectively, thus the connections of the amino acids ThrGly-Ser-Phe-Leu were determined. The connectivity of Leu to Pro was established by
ROESY through NOEs between Leu H-2 at 4.1 ppm and Pro H-5 at 3.51 ppm. Furthermore,
the sequence [5MeHex-Tyr-Ser] was established through NOEs between Tyr NH and Tyr H2 at 8.32 and 4.29, respectively to H-2 of 5MeHex and Ser NH at δ 2.11, and 8.03,
respectively.
Again Ser-Gly connectivity through the ester bond between the terminal C-3 of Ser
and the carboxyl of Gly was confirmed by HMBC correlation of Ser H-3 at δ 3.35 to the
carboxyl at 167.1 ppm. The above deduced connectivities of the different amino acids were
confirmed through additional HMBC correlations. NHs of Leu, Tyr, Ser, Phe, Gly, and Thr
showed HMBC correlations to vicinal carbonyls at 173.1, 173.5, 171.7, 168.1, 170.8, 171.5 of
Phe, 5MeHex, Tyr, Ser, Thr, and Pro respectively. ROESY, COSY, TOCSY and HMBC as
well as 1HNMR and
13
C NMR data are identical to those of kahalalide B (Hamann et al
1996).
105
Results
Fig (3.1.30): 1H NMR spectrum, NHs and aromatic region (up), and α-protons and
higher field region (down)
Fig ( 3.1.31 ) : Part of COSY spectrum of compound 4, showing Lower field part
106
Results
Thr
Tyr
Ser
L eu
Phe
G ly
Phenyl
D is u b s titu te d
p h e n y l (T y r)
Figure (3.1.32): Parts of TOCSY spectrum , NH-detected spin systems (up).
Phenyl
L eu
Tyr
Ser
T h r D is u b s titu te d
p h e n y l (T y r)
P h e G ly
to H - 3 F .A
to H -2 F .A
to H -2 ty r
to H -2 S e r
to H -2 P h e
N H -T h r/ H -2 P ro
to H -2 T h r
fig (3.1.33): Parts of ROESY spectrum of compound 4, NH/NH correlation (upleft), higher field region (up-right) and NH/ aliphatic proton correlations (down)
107
Results
T hr
P henyl
D is u b s titu te d
p h e n y l (T y r)
T yr
L eu
S er
P he
G ly
C 7- T yr
P he C = O
5 M eH ex
C = O
S er
T yr C = O
C = O
P ro C = O
T hr C = O
Fig (3.1.34): Part of HMBC spectrum of compound 4 showing NHdetected correlations to vicinal carbonyls.
Phe
C 9 H 9 NO ••
Exact M ass: 147,068
Ser
C 3 H 4 NO 2 •••
Exact M ass: 86,024 O
Leu
H
5M eH ex
C 7 H 13 O •
Exact M ass: 113,097
C 6 H 11 N O ••
Exact M ass: 113,084
H
N
N
H
H
N
N
O
O
O
O
tyr
H
C 9H 9N O 2
E xact M ass: 163,063
H
N
H
N
H
O
HO
N
HO
O
••
G ly
C 2 H 3 N O 2 ••
Exact M ass: 73,016
O
O
Pro
C 5 H 7 N O ••
Exact M ass: 97,053
T hr
C 4 H 7 N O 2 ••
Exact M ass: 101,048
Fig (3.1.35): ESI-MS/MS of compound 4
Table (3.1.7 ) ESI-MS/MS Fragment ions of compound 4 and sequence determination
Ion composition
m/z
M-[C2H5OH (Thr side chain)]+1
M-[Fatty acid] +1
M-[Fatty acid + C2H5OH+ H2O] +1
M-[Fatty acid + Tyr] +1
M-[Fatty acid + Tyr + H2O] +1
M-[Fatty acid + Tyr +2 H2O] +1
Tyr + Ser + Phe + Leu –[H2O] + 1
832.1
765.1
702.6
603.1
585.2
567.1
492.0
Tyr + Ser + Phe + Leu –[H2O + C=O] + 1
Ser + Phe + Leu –[H2] + 1
Pro + Thr + Gly +1
464.1
345.0
256.0
108
Results
Table (3.1.8) : 1H and 13C NMR data of compound 4 in DMSO-d6
13
Carbon
C NMR
ppm, Mult
Gly
1
2
167.1 s
42.0 t
(NH) 7.37
3.61 &3.91
Thr
1
2
3
OH
4
1
2
3
4
5
170.8 s
59.1 d
67.5 d
(NH) 7.20
4.2
4.45
5.4
1.25
1
2
3
4
5
6
1
2
3
4
5,5´
6,6´
7
1
2
3
1
2
3
172.5
50.8 d
23.5 t
38.0 d
11.0 q
11.5 q
173.1 s
53.2 d
38.5 t
137.2 s
129.0 d
127.5 d
126.5 d
168.1 s
50.4 d
47.2 t
171.7 s
56.0 d
35.5 t
4
5,5´
6,6´
7
1
2
3
4
5
6
7
127.3 s
129.8 d
115.0 d
157.7 s
173.5 s
41.2 t
27.1 t
23.0 t
38.0 d
11.5 q
12.0 q
Amino acid
Pro
Leu
Phe
Ser
Tyr
5-MeHex
12.0q
171.5 s
60.8 d
28.5 t
23.0 t
47.0 t
Multiplicity, J= Hz
1H NMR,
ppm
4.38
2.10, 2.05
1.80, 2.00
3.51, 3.96
(NH) 8.90
4.1
1.4 , 1.51
1.28
0.75
0.81
(NH) 7.63
4.8
2.91, 2.93
7.21
7.16
7.18
(NH) 8.03
4.4
3.35
(NH) 8.32
4.29
2.78 , 2.83
t, J= 6.2
dd, J= 17.4, 5.4;
dd J=17.6, 5.9
d, J= 7.3
dd, J= 8.8, 1.0
m
d, J= 9.2
d, J= 7.3
m
m,m
m,m
dd, J= 16.7, 8.8;
dd, J= 6.5, 9.5
d, J= 0.9
m
m,m
m
d, J= 7.1
d, J= 7.1
d, J= 9.4
dd, J= 15.5, 8.8
m;m
m
m
m
d, J= 9.6
m
m
d, J= 5.4
dd, J= 13.3,7.3
dd, J= 14.1, 8.5;
dd J=14.8, 7.2
7.0 0
6.68
d, J=8.2
d, J= 8.2
2.11
1.35
1.10
1.5
0.75
0.78
t, J= 7.2Hz
m
m
m
d, J= 6.9
d, J=6.9 H-7
109
Results
3.1.5- Kahalalide C (5, known compound)
Phe
Thr
OH
Tyr
O
H
N
B u ta n o ic
ac id
O
HN
NH
N
H
HN
O
N
H
O
O
O
H
N
O
NH2
A rg
NH
O
Ile
Tyr
OH
C 47H 63N 9O 10
E x a c t M a ss: 9 1 3 ,4 7
M o l. W t.: 9 1 4 ,0 5 7
E S I -M S /M S o f K a h C
Fig(3.1.36):Compound 5 (kahalalide C).
Yield : 1 mg
Kahalalide C was isolated as a white amorphous powder, with [α]D of + 40° (c 0.11
MeOH). It has UV absorbance at λmax 230, 278 nm. ESI-MS showed pseudomolecular ion
peak m/z 914 [M+1]+ and 937 [M+Na]+, 953 [M+K]+ and 971[M+K+H2O]+ and
110
Results
pseudomolecular ion peak m/z 913.1 [M-1]-, 1827 [2M-1]-. MALDI-TOF-MS showed
positive pseudomolecular ion peak at m/z 914 [M+1]+ and 937 [M+Na]+, suggesting the
molecular formula C47H63N9O10. The indivdual amino acids and the sequence were
established through interpretation of both ESI-MS/MS [see figure 3.1.36 and table 3.1.9] and
MALDI-TOF-PSD [see figure 3.1.38 and table 3.1.10]. This kahalalide was previously
isolated from the same mollusc (E. rufescens) which was collected from the same
geographical zone (Hamann et al 1996).
Table (3.1.9) ESI-MS/MS Fragment ions of compound 5 and sequence determination
Ion composition
m/z
M-[OH] +1
M-[C=O] +1
M-[C2H5OH (Thr side chain)]+1
M-[guanidine] +1
M-[Ile] +1
M-[Ile + H2O] +1
M-[Tyr ] + 1
M-[ Phe + Fatty acid] + 1
M-[ Phe + Fatty acid + OH] + 1
M-[ Phe + Fatty acid + OH + OH ] + 1
M-[Ile + Tyr + H2O] + 1
M-[Ile + Tyr + H2O + NH3] + 1
[Arg + Tyr + Thr + Phe - H2O] +1
[Tyr + Thr + Phe + Fatty acid - H2O] +1
Tyr + Thr + Phe + Fatty acid – [C=O + OH]
[Tyr + Thr + Phe + Fatty acid – (C=O+H2O)] +1
Arg + Tyr + Ile –[C=O]
[Arg + Tyr + Thr –(NH2 + OH)] +1
[Arg + Tyr ] +1
[Arg + Tyr –( OH)] +1 //also // [Thr + Phe + Fatty acid – (OH)] + 1
897
886.4
869.4
855.5
801.3
783.2
752.3
697.3
680.3
662.4
620.2
603.3
549.4
463.9
436.0
434.2
404.1
386.0
320.7
302.9
Fig (3.1.37): MALDI-TOF-MS of compound 5
111
Results
Fig (3.1.38): MALDI-TOF-PSD-MS of compound 5
Table (3.1.10) MALDI-TOF-PSD
Fragment ions of compound 5 and sequence
determination
Ion composition
m/z
M-[C2H5OH (Thr side chain)]+1
M-[guanidine] +1
M-[Ile] +1
M-[Ile + H2O] +1
M-[Tyr ] + 1
M-[Tyr + OH] + 1
M-[ Phe + Fatty acid] + 1
M-[ Phe + Fatty acid + OH] + 1
M-[ Phe + Fatty acid + OH + OH ] + 1
M-[Ile + Tyr + H2O + NH3] + 1
M-[Arg + Tyr + ( C=O)] + 1
[Ile + Tyr + Arg + Tyr - ( C=O)] + 1
[Arg + Tyr + Thr + Phe - H2O] +1
[Tyr + Thr + Phe + Fatty acid - H2O] +1
Tyr + Thr + Phe + Fatty acid – [C=O + OH]
[Tyr + Thr + Phe + Fatty acid – (C=O+H2O)] +1
Arg + Tyr + Ile –[C=O]
[Arg + Tyr + Thr –(NH2 + OH)] +1
[Tyr + Thr + Phe – (C=O + OH + NH)] +1
[Arg + Tyr ] +1
[Thr + Phe + Fatty acid ]+ 1
[Arg + Tyr –( NH3)] +1
[Arg + Tyr –(OH)] +1 //also // [Thr + Phe + Fatty acid – (OH)] + 1
[Thr + Phe + Fatty acid – H2O]+ 1
Arg + Tyr – [C=O]
Thr + Tyr – [OH]
Phe + Fatty acid
Arg + NH
Tyr-[C=O]
Phe – [C=O]
Ile – [H2] +1
Phe – [NH + C=O]
Ile – [C=O]
Arg-[Guanidine + C=O]
869.8
855.4
801.7
783.2
751.8
734.7
697.3
680.5
662.7
603.3
567.6
567.6
549.4
463.9
436.6
434.2
404.1
386.4
350.6
320.7
319.6
304.0
302.9
301.3
292.2
247.3
218.7
183.7
136.3
120.2
112.2
104.0
86.1
70.1
112
Results
3.1.6 – N,N-dimethyl Tryptophane metyhl ester (6, new marine natural product)
O
51.0
3.18
N
3.68
78.7
109.2
127.1
C14H18N2O2
Exact Mass: 246,137
Mol. Wt.: 246,305
123.8
7.15
7.05
120.8
136.0
111.3
7.32
300 ms040608 #5
mAU
48.6
3.16
O
23.2
3.2
7.60
118.4
6.98
118.1
167.2
rp1c3
N
H
UV_VIS_3
WVL:280 nm
Peak #711.86
60,0
%
No spectra library hits found!
218.9
6 - 11,673
40,0
200
20,0
100
279.5
286.5
1,212
345 -- 1,041
1,138
1,283
12
1,109
-50
0,0
7 - 27,706
8 - 51,602
min
10,0
20,0
30,0
40,0
50,0
60,0
10,0
200
nm
250
300
350
400
450
500
Fig (3.1.39):Compound 6, structure with 1H and 13C- chemical shift values (up),
HPLC chromatogram (down left), UV-spectrum (down right).
Yield : 4.6 mg
Fig (3.1.40): ESI-MS (left), ESI-MS/MS of compound 6 (right).
113
550
595
Results
Compound 6 [N,N-dimethyl-tryptophan methyl ester] was isolated as a brownish
white amorphous powder, with [α]D of + 20° (c 0.25 MeOH). It has UV absorbance of typical
indole compounds at λmax 218, 279(sh), 286(sh) nm. ESI-MS showed positive
pseudomolecular ion peak m/z 247 [M+1]+ and 493 [2M+1]+, 515 [2M+Na]+ and negative
pseudomolecular ion peak m/z 245.4 [M-1]-, 291.6 [M + HCOO-]-, 537.8 [2M + HCOO-]-,
suggesting the molecular formula C14H18N2O2. 1H NMR spectrum showed characteristic
proton resonances of tryptophan at δ 3.68 (1H, dd, J=10.1,3.8 Hz, H-2), 3.20 (2H, m, H-3),
7.15 (1H, d, J=1.2 Hz, H-5), 7.32(1H, d, J=8.2 Hz, H-8),7.05(1H, t, J=7.3 Hz, H-9 ), 6.98(1H,
t, J=7.0 Hz, H-10 ), 7.6(1H, d, J=8.2 Hz, H-11), 10.86 (1H, s, NH), in addition to one
methoxy group at δ 3.16 (3H, s) and two N-methyls at δ 3.14 (6H, s). 13C NMR showed the
presence of one carboxyl at δ 167.2 (s. C-1), the presence of methoxy carbon and two Nmethyls were confirmed by resonances at δc 48.6 ppm and 51.0 ppm respectively. The α- and
β-carbon resonances of Trp were evident at 78.7 and 23.2 ppm respectively. COSY spectrum
established the connection of H-2 to H-3 and demonstrated the characteristic ABCD spin
system of indole moiety. The exchangeable NH was confirmed by measuring of the 1HNMR
in deuterated methanol. The connection of one methyl to carboxyl and the substitution of the
two amino protons with two methyls were confirmed through HMBC correlations between δH
at 3.16 and δc 167.2 ppm and between δH at 3.18 and δc 78.7 ppm respectively as shown in
table 3.1.11 and figure 3.1.45. These NMR data and UV-spectrum were identical with those
of the terrestrial natural product [N,N-dimethyl-tryptophan methyl ester] which has been
previously isolated from the Australian fabaceous plant Pultenaea altissima (Fitzgerald
7.7
7.6
7.5
7.4
7.0
4.3251
8.2304
Integral
1.0504
7.1
1578.24
1587.70
1578.24
1587.70
1834.24
1837.39
1843.69
1847.48
2041.05
2058.07
3479.92
3487.48
3494.42
3520.90
2063.12
7.2
O-Me
N(CH3)2
10
1.0515
9
1.0341
7.3
(ppm)
3527.84
3535.40
3479.92
3575.13
3487.48
3576.39
3494.42
3520.90
3527.84
3535.40
3575.13
3576.39
1.0000
5
1.1212
8
1.0882
Integral
11
3655.83
3655.83
3664.03
3664.03
3758.61
3793.92
3802.12
3758.61
3793.92
3802.12
5431.40
1963). To the best of our knowledge this is the first report of 6 as a marine natural product.
3.2
6.9
3.1
(ppm)
3
11.0
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
4.3251
8.2304
1.5246
1.8147
1.0504
1.0515
1.0341
1.0000
1.1212
1.0882
2
0.9997
Integral
Indole NH
3.0
2.5
2.0
1.5
(ppm)
Figure (3.1.41): 1H NMR spectrum of compound 6 meassured in DMSO-d6
114
1.0
0.5
0.0
N
13C
200
CH
2
O
5
HN
7
190
C
1
6
8
9
180
170
(ppm)
160
150
N H /H -5
10.0
NMR spectrum
5
140
130
11
6 9 10
7
120
110
8
100
8.0
115
90
80
6.0
70
7.0285
4.0
60
α-CH
50
4.0
Fig (3.1.44 ): COSY spectrum of compound 6.
3.8
3.0
40
3.6
(ppm)
2.0
O-Me
Figure (3.1.43): C NMR and DEPT spectra of compound 6
13
(ppm)
30
20
(ppm)
C H /C H 2
4.0
A B C D s p in s y s te m
6.0
8.0
10.0
10
7.0285
8.2663
2.2621
1.1717
Integral
2
23.2161
5.0
8.2663
5
48.5712
6.0
2.2621
1.1717
0.9932
10
50.9883
78.6834
7.0
109.2482
111.2993
118.1846
118.4349
120.8520
8.0
1.0958
0.9294
9
123.8469
9.0
1.0108
1.0000
11
8
127.0536
Figure (3.1.42 ): 1H NMR spectrum of compound 6 measured in CD3OD
10.0
136.0285
167.2096
Integral
1646.30
1640.62
1944.85
(ppm)
1.0
3
CH2
O
4
11
10
DEPT
N-(Me)2
CH2
4
0
1640.62
1646.30
1706.83
1711.87
1716.29
1935.71
1941.07
1950.21
1706.83
1711.87
1716.29
1935.71
1941.07
1944.85
1950.21
3505.09
3506.04
3512.03
3512.98
3513.92
3519.91
3520.86
3534.10
3535.36
3542.30
3543.24
3549.23
3550.49
3583.91
3652.95
3653.90
3660.84
3801.76
3808.69
3809.64
3810.59
Results
N(CH3)2
O-Me
3
3.4
0.0
Results
2
3
(ppm)
40
O
N
C
2
O
3
80
HN
120
160
(ppm)
7.2
6.4
5.6
4.8
4.0
3.2
Fig (3.1.45) HMBC of compound 6 [Methanol-d4]
Table (3.1.11): NMR data of 6 (500 MHz, DMSO-d6),
Carbon
•
13
C NMR,
ppm
1
2
3
4
5
167.2 s
78.7 d
23.2 t
109.2 s
123.8 d
6
7
8
9
10
11
OMe
N-(CH3)2
127.1 s
136.0 s
111.3 d
120.8 d
118.1 d
118.4 d
48.6 q
51.0 q, q
1
H NMR*, ppm
(Multiplicity, J=
Hz)
1
H NMR, ppm
(Multiplicity, J= Hz)
HMBC
H -------C
3.86 (dd, J=9.1; 5.4)
3.41 (m)
3.68 (dd, J=10.1; 3.8)
3.20 (m)
C-1, C-4, C-3, N(CH3)2
C-1, C-2, C-4, C-5, C-6, N(CH3)2
7.16 (s)
7.15 (d, J=1.2)
(NH) 10.86 (s)
C-4, C-6, C-7
C-6
7.32 (d, J=7.9)
7.08 (dt, J=1.0,6.9)
7.03 (dt, J=1.0, 6.9)
7.61 (dd, J=7.9, 1.0)
3.28 (s)
3.28 (s , s)
7.32 (d, J=8.2)
7.05 (t, J=7.3)
6.98 (t, J=7.0)
7.6 (d, J=8.2)
3.16 (s)
3.18 (s , s)
C-6, C-10
C-7, C-11
C-6, C-8
C-4, C-7, C-9
meassured in (CD3OD)
116
C-2, C-3, N-Me
Results
3.1.7- β-sitosterol(7, Known compound):
0.91
11.8
23.0
0.93
33.9
18.8
36.2
12.3
1.10
21.1
19.4
39.7
42.3
31.6
36.5
71.8
HO
0.81
19.6
28.2
0.83
24.3
50.1
140.7
42.2
56.0
28.9
26.3
56.7
37.2
3.52
19.0
46.1
0.68
31.9
31.9
121.7
C29H50O
Exact Mass: 414,386
Mol. Wt.: 414,707
5.35
Fig (3.1.46): compound 7
Yield : 15 mg
Compound 7 [β-sitosterol] was isolated from the ether fraction of the total exract as a
white amorphous powder, with [α]D of - 37° (c 0.35 CHCl3). EI-MS showed molecular ion
peak m/z 414 [M]+ and fragment ions at 386, 368, 271, 255, 69, 55, 43 suggesting the
molecular formula C29H50O. 1H NMR spectrum showed resonances for six methyl groups at δ
0.68 (3H, s, Me-18), 1.10 (3H, s, Me-19), 0.93 (3H, d, J=6.2 Hz, Me-21), 0.81 (3H, d, J=7.25
Hz, Me-27), 0.83 (3H, d, J=7.57 Hz, Me-26) and 0.86 (3H, t, J=7.11 Hz, Me-29). The
were indicative for ∆5-6 mono
resonances at δ 5.35 (1H, m, H-6), 3.52 (1H, m, H-3)
hydroxylated steroidal nucleus (Itoh et al , 1983).13C NMR showed six methyl signals at δ
11.8, 19.4, 18.8, 19.6, 19.0 and 12.3 for the methyl groups 18, 19, 21, 26, 27 and 29,
respectively. 13C NMR spectrum showed one quaternary olefenic carbon resonating at δ 140.7
and one olefenic CH at 121.7 and oxygenated methine at 71.8, in addition to two quaternary
aliphatic carbons, 7 aliphatic methines, and 11 aliphatic methylenes (see table 3.1.12). The
above NMR data were identical with those of β-sitosterol, which was previously isolated from
many other natural sources (Greca et al , 1990).
314
-- - - - - - -- - - - - - - -- - - - - - -- - - - - - - -- - - - - - -- - - - - - -- - - - - - - - --
386
43
69
143
271
231
HO
28
386
368
303
69
55
Fig (3.1.47):EI-MS spectrum of compound 7
117
Results
29
19
28
24
22
21
18
26
20
25
23
18
27
12
17
11
13
16
14
15
19
9
1
8
10
3
7
HO
5
6
6
4
3
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
(ppm)
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
1
Fig (3.1.48): HNMR spectrum of compound 7 ( CDCl3)
10
13
6
19
11
18
29
5
145
140
135
130
125
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
(ppm)
3
9
14
29
24
17
8
20
25
21
26 27
28
26
24
22
21
20
25
23
18
12
11
27
17
13
16
14
15
23
19
9
1
8
10
3
12
7
HO
22
28
15
5
6
4
1
140
130
120
110
100
90
80
70
60
50
40
(ppm)
Fig (3.1.49): 13CNMR and DEPT spectra of compound 7 (CDCl3)
118
7
2
16
30
20
10
Results
Table (3.1.12): 1H, 13C-NMR data of compound 7 in (CDCl3 , 500, MHz)
13
1
No.
C (Multiplicity)
H (Multiplicity, Hz)
1
37.2 t
*
2
31.6 t
*
3
71.8 d
3.52 (m)
4
42.2 t
*
5
140.7 s
6
121.7 d
5.35 (m)
7
31.9 t
*
8
31.9 d
*
9
50.1 d
*
10
36.5 s
11
21.1 t
*
12
39.7 t
*
13
42.3 s
*
14
56.7 d
*
15
24.3 t
*
16
28.2 t
*
17
56.0 d
*
18
12.3 q
0.68 ( s)
19
20.4 q
1.10 ( s)
20
36.2 d
*
21
18.8 q
0.93 (d, J=6.2 Hz)
22
33.9 t
*
23
26.3 t
*
24
46.1 d
*
25
28.9 d
*
26
19.6 q
0.83 (d, J=7.5 Hz)
27
19.0 q
0.81 (d, J=7.3 Hz)
28
23.0 t
0.82 (d, 6.6 Hz)
29
11.8 q
0.86 (t, J=7.1 Hz)
* not confirmed δH, because they are present in the highly overlapping multiplet signals of aliphatic methines
and methylenes in the higher field region between 1.00 and 2.28 ppm.
Bioactivity :
Compound 1 (kahalalide F) was reported as the most active kahalalide. Kahalalide F
was reported to have a significant biological activity against solid tumour cell lines, human
colon and lung cancers and some pathogenic microorganisms that cause the opportunistic
infections of HIV/AIDS. Its mode of action and preclinical toxicity have also been studied. It
is currently in Phase I and II clinical trials. Kahalalide F displays both in vitro and in vivo
antitumor activity in various solid tumor models, including colon, breast, non–small cell lung,
and in particular prostate cancer. In vitro antiproliferative studies showed activity among
certain prostate cancer cell lines (PC-3, DU-145, T-10, DHM, and RB), but no activity was
found against the hormone-sensitive LnCAP. In vivo models also confirmed selectivity and
sensitivity of the prostate tumor xenograft derived from hormone-independent prostate cancer
cell lines, PC-3 and DU-145. Further in vitro evaluation showed selective activity but not
119
Results
restricted to prostate tumor cells. Suares et al, 2003 revealed that kahalalide F induces cell
death via oncosis preferentially in tumor cells. (Hamann et al 1993, Hamann et al 1996,
García-Rocha et al 1996, Sewell et al 2005, Janmaat et al 2005, Suarez et al 2003, Brown et al
2002, Ciruelos, et al 2002, Rademaker-Lakhai et al 2005). Kahalalide A was reported to have
antimalarial and antituberculosis activity (Hamann et al, 1996; Copp 2003 and El-Sayed et al
2000). Furthermore kahalalide E was reported to have selective anti-viral activity against
Herpes simplex (Hamann et al, 1996).
The cytotoxic activity studies of 1 against three different cell lines (L5178Y, Hela, and
PC12) and showed ED50 values of 1 as 6.3µg/ml, 6.7µg/ml and >10 µg/ml respectively. The
cytotoxicity assays were summerized in Figure (3.1.50). Compounds 1 (kahalalide F), 3
(kahalalide D) and 4 (kahalalide B) showed mild antibacterial activity against B. subtilis and
S. cereviisae. Kahalalide B showed significant cytotoxicity (99 % inhibition ) compared to
kahalalide D (65.2 % inhibition), kahalalide E (94.8 %), while compound 6 showed no
significant activity against L5178Y cancer cell line at concentration of 10µg/ml each, see
figure (3.1.51).
Cell proliferation %
120%
0µg/ml
1µg/ml
3µg/ml
10µg/ml
100%
80%
60%
40%
20%
0%
L5178Y
Hela
PC12
6
com
p.
10µ
g/m
l
hE
ml
ka
10µ
g/
hB
ml
ka
10µ
g/
ml
ka
10µ
g/
g/m
l
0.0
µ
hD
100
80
60
40
20
0
con
trol
%
Cell growth
Fig (3.1.50): In vitro cytotoxicity assay of kahalalide F against three different cancer
cell lines .
Fig (3.1.51): In vitro cytotoxicity assay of compound 2 (kah E), 3 (kah D), 4 (kah B)
and 6 ( N,N-dimethyl tryptophan methyl ester) against L5178Y cells .
120
Results
3-2 Natural Products from Elysia grandifolia:
Although many natural products were isolated from the genus Elysia this is the first
report for isolated natural products from E. grandifolia. Our interest with this sacoglossan
mollusc came up by the strong biological activity of its crude MeOH extract. Organic extracts
from the specialist herbivore E. grndifolia and its dietary green alga Bryopsis plumosa
exhibited antimicrobial, cytotoxicity, feeding deterrence and ichthyotoxicity (Padmakumar
1998, Bhosale et al , 1999).
Dereplication procdure using LC-MS was applied for targeting new natural products
from E. grandifolia. Inspection of the LC-MS chromatograms suggested the presence of
kahalalide derivatives giving molecular ion peaks corresponding to kahalalides B, C, D, E, F,
G, J, K, O (see figures 2-15 and 2-16). The presence of two unidentified peaks at m/z 1520.2
and 1536.0 aroused our interest to do further chemical work on the mollusc extract.
Chemical investigation of Indian sacoglossan mollusc E. grandifolia Kelaart 1858,
yielded two known analogues, kahalalide F (53.0 mg) and D (3.4 mg) along with two other
new kahalalide derivatives, which we designated as kahalalide R (20 mg) and S (3.4 mg). The
structural elucidation of the new kahalalides R and S will be discussed in detail.
121
Results
3.2.1 Kahalalide R (8, New natural product)
H 2N
2 .7 4
1 1 .8 5
0 .8 2
3 8 .3
H 3C
2 8 .5
1 .5 2
7 .72
1 .6 9 ,1.8 2
3 0 .9 2
1 4 .6
0 .8 4
3 0 .2
1 .7 3
1 .0 2 ,1 .2 1
2 6 .0
CH3
4 .4 9
O
1 7 2 .0
2 .0 1 ,1 .9 8
2 9 .7
1 .7 8 ,1 8 9
2 7 .2 4
2 2 .8 5
0 .8 4
H 3C
HN
7 .8 7
1 9 .2
0 .6 0
O
HN
4 .3 8
5 9 .5
3 1 .6
1 .8 8
H 3C
1 3 0 .4
H
1 .2 8
12 .51
1 7 1 .2
3 0 .7
1 .9 5
4 .3 7
5 1 .8 9
H 3C
9 .6 9
8 .7 8
NH
NH
1 7 1 .3
CH3
O
CH3
2 .1 8
3 1 .5
4 .4 5
5 5 .4 3
1 7 2 .6
CH3
5 5 .6 5
0 .8 0
19 .5
O
2 .9 2 ,2 .9 4
3 6 .1
1 3 7 .0 2
HN
H 3C
H
N
18 .5
0 .6 2
7.6 1
O
O
7 .8 6
CH3
5 9 .3
4 .1 3
1 70 .03
4 .4 3
13 0 .1 9
O
7 .2 8
1 2 8 .5
1 7 0 .8 8
0 .8 6
2 2 .9
1 6 9 .4 5
7 .2 9
1 2 9 .5
5 6 .0 3
4 .4 1
2 .75
HO
8 .8 2
CH3
1 6 3 .5
HN
1 9 .5
0 .8 2
3 1 .5
1 .9 4
O
6 .3 6
7 .8 9
2 2 .2
0 .8 4
H 3C
HN
1 .0 8
17 .3
6 .7 3
0 .8 1
1 8 .6
O
1 6 8 .7
O
3 .8 5
6 0 .2
1 7 1 .2
0.8 3
1 9 .6
6 9 .9 8
4 .9 6
1 6 9 .7
H 3C
8 .5 5
5 6 .3 7
4 .5 0
1 .3 8
3 0 .1
1 6 .5
0 .6 2
O
1 7 1 .3
O
H 3C
8 .1 1
H 3C
4.3 4
HN
O
1 9 .3
0 .8 2
N
H
N
4 .26
5 5 .9 5
H 3C
5 7 .2 7
1 7 1 .5
O
1 72 .6
1 9 .0
0 .8 3
5 1 .1 4
7 .9 3
5 5 .6 5
4 .3 7
4 7 .0
3 .7 8 ,3 .5 2
3 1 .0
1 .9 4
N
H
1 .59 , 1 .5
3 8 .5
2 8 .0
7 .2 5
1 2 6 .7 5
7 .9 1
NH
O
1 7 2 .5
2 .1 1
O
2 3 .6
1 .5 1
0 .8 0
1 8 .0
3 5.1
1.2 5
22 .0
1 .2 8
22 .6
1.5 2
3 0 .6
1 .1 0 , 1 .1 5
2 2 .3
0 .8 1
1 8 .1
C 7 7 H 12 6 N 1 4 O 1 7
E x a c t M a s s: 1 5 1 8 ,9 4 3
M o l. W t.: 1 5 1 9 ,9 0 8
Fig ( 3.2.1 ): Compound 8, kahalalide R.
180 ms030707 #8
mAU
egab4b
UV_VIS_1
WVL:235 nm
60,0
9 - 32,212
150
50,0
Peak #9 50%
%
100%
-50%
993.00
995.52
203.5
37,5
100
25,0
50
12,5
10 - 32,976
5
6
1,268
7 -- 1,207
1,333
1,087
-0,908
1,499
12348---0,613
1,012
-20
0,0
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
500
Fig (3.2.2): HPLC chromatogram and UV spectrum of compound 8.
Yield : 20 mg
122
595
Results
742
837
856
H2 N
H 3C
CH3
O
953
H 3C
1052
N
H
O
O
N
H3C
HN
HN
O
O
CH 3
H3C
O
HN
H3 C
H3 C
O
O
H 3C
O
1151
N
H
O
CH 3
C H3
HN
O
NH
H
HN
629
H 3C
NH
H
N
O
CH 3
CH3
O
O
H3C
1250
HN
H 3C
HO
NH
O
1378.8
O
Fig ( 3.2.3 ) : ESI-MS (up) and ESI-MS/MS (down) of compound 8
Kahalalide R (compound 8) was obtained as a white amorphous powder. The reflector
mode MALDI-TOF mass spectrum of 8 performed with delayed extraction showed positive
monoisotopic ion peaks at m/z 1520.2, [M+H]+, 1542.2 [M+Na]+, and 1557.2 [M+K]+. In (+)ESI-MS, a pseudomolecular ion was detected at m/z 1520.8 [M+H]+ that was compatible with
the molecular formula C77H127N14O17 as established by HRESIMS. The 1H and 13C NMR data
of 8 were comparable to those of kahalalide F (compound 1), but had a higher molecular
weight of 42 mass units. Inspection of the 1H and
13
C NMR spectra of 8 revealed that
kahalalide R shared very similar structural features with kahalalide F. As in 1, the region
between 6.50 and 9.70 ppm of the 1H spectrum of 8 accounted for a similar number of 14
deshielded amide NH resonances. Eleven of those are doublets, one is a singlet at δ 9.69 for
the α,β-unsaturated amino acid Z-Dhb, and a broad 2H singlet at δ 7.69 for the terminal NH2
of Orn. Like kahalalide F, compound 8 was also ninhydrin-positive, thus supporting the
presence of a free amino group found in Orn. The aromatic region also disclosed the presence
of Phe, the only aromatic amino acid in the structure of the new analogue as well as in the
known congener (kahalalide F). An apparent difference observed between the 1H spectrum of
123
Results
8 and 1, was the absence of a second methyl triplet at δ 1.02 that suggested the loss of one Ile
unit in the structure of the new derivative. Major differences to kahalalide F were more
obvious from the
13
C NMR and DEPT spectra of 8. The
13
C NMR spectrum displayed 77
carbons signals instead of 75 carbons as in kahalalide F. An additional carbonyl signal was
observed between 163.5 and 174.1 ppm. Again, the DEPT spectrum also revealed the
presence of 12 regular amino acid residues in the new analogue 8, as indicated by the 12 αmethine carbon signals between 51.0 and 60.0 ppm. The DEPT spectrum showed 15 instead
of 12 methylene carbons and a loss of an oxygenated methine carbon signal in the 65 to 75
ppm region, which further indicated a deficit of one Thr unit that was replaced by another
amino acid compared to kahalalide F. Similar to kahalalide F, the sp2-carbon region of the
13
C NMR spectrum of 8 showed evidence for the occurrence of Phe and Dhb. The 13C NMR
and DEPT spectral data were in agreement with the molecular formula as determined by
HRMS.
The COSY spectrum of 8 revealed 14 spin systems, 12 of which commenced with a
deshielded amide NH doublet while two other spin systems showed no correlations to any
amide NH proton and were then allotted to Pro and a saturated fatty acid. The COSY
spectrum indicated the presence of 7-methyloctanoic acid (7-Me-Oct). Kahalalide R contained
a linear n-alkyl side chain that showed an iso- type methyl branching at the terminus of the
alkyl chain similar to that found in kahalalide F, which could be detected in the COSY spectra
of both 8 and 1. Sequential correlations of the iso-fatty acid (7-Me-Oct) observed from the αmethylene signal at δ 2.11 to the subsequent methylenes at δ 1.51 (H2-3), 1.28 (H2-4), 1.25
(H2-5), and 1.10/1.15 (H2-6), which then terminated with an aliphatic methine proton at δ 1.52
(H-7) and two methyl functions at δ 0.80 and 0.81. In the 13C and DEPT spectra of the latter,
characteristic signals appeared for the terminal carbon atoms, i.e., Cω, δ C-8/9 22.5 (2 × q); Cω-1,
δC-7 27.3 (d); and Cω-2, δC-6 38.3 (t) which were unequivocally assigned by HMQC (see Table
3.2.2).
A TOCSY experiment corroborated the assignments obtained from the COSY
spectrum. The TOCSY spectrum unambiguously resolved the amine- and α-proton resonances
of each of the different 13 amino acid residues in the structure of the new depsipeptide
congener 8. Kahalalide R (8) was thus shown to contain six units of Val, one unit each of Phe,
aIle (allo-Ile), aThr (allo-Thr), Orn, Pro, Glu, also the unusual Dhb, and 7-Me-Oct. In the
new analogue, Val and Glu units replaced Thr and Ile previously found in kahalalide F. The
TOCSY spectrum allowed the overlapping 12 methyl doublets (J = 6.5 Hz) that belong to the
six Val units, which occur between 0.60 and 0.85 ppm, to be explicitly assigned to their
124
Results
respective spin systems. The presence of Glu also further explains the additional carbonyl
signal observed in the
13
C NMR spectrum while the extra two methylene carbons could be
accounted for by the replacement of the 7-methyl-hexanoic acid (7-Me-Hex) in kahalalide F
with 7-methyl-octanoic acid (7-Me-Oct) in compound 8. Again, two structural regio-isomers
of the fatty acid moiety could be identified in the TOCSY spectrum of 8.
HMBC and ROESY experiments established the connectivity and sequence of the
amino acids in the peptide structure of 8 (Figure 3.2.4). The sequence 7-Me-Oct–Glu–Val-6–
Val-5–Val-4, which was elucidated as fragment I, was established through the HMBC
correlations of NH signals at δ 7.93, 7.86, 7.89, and 8.11 for Glu, Val-6, Val-5, and Val-4,
respectively, to each of their neighboring vicinal (2J) carbonyls of 7-Me-Oct, Glu, Val-6, and
Val-5 resonating at δ 172.5, 170.9, and 171.2, respectively. Connectivities for fragment II,
Pro–Orn–aIle– aThr–Val-3–Val-2–Phe–Z-Dhb–Val-1, were similarly determined through
HMBC correlations of the NH signals at δ 7.93, 7.87, 8.55, 8.82, 7.61, 8.78, 9.69, 6.73,
respectively, to their neighboring vicinal carbonyls at δ 172.6, 171.5, 171.3, 168.7, 170.0,
172.6, 171.3, and 163.5, respectively. The cyclization of Val-1 to aThr was confirmed by an
HMBC cross peak between the carboxyl signal at δ 169.7 for Val-1 and the β-proton of aThr
at δ 4.96, which arises from a characteristic low field acylation shift. This ring closure was
corroborated by the ROESY correlation of the β-proton of aThr with the α-proton of Val-1 at
δ 3.85. The connectivity of fragment I with II was established through the HMBC correlation
of the δ-proton of Pro at δ 3.52 with the carbonyl of Val-4 at 172.6 ppm. This was in
agreement with the ROESY cross peak between the δ-proton of Pro at 3.78 ppm and the αproton of Val-4 at 4.26 ppm.
L- Orn
H2N
D-aIle
O
O
N
H
D- Pro
N
H
O
D-aThr
N
HN
Val-1
Val-4
O
O
HN
HN
O
Z-Dhb
Val-5
O
O
O
O
HN
NH
H
N
Val-2
O
L- Phe
O
NH
HO
Val-3
O
NH
NH
Val-6
O
HN
D-Glu
O
Fragment II
O
7-Me-Oct
Fragment I
Figure 3.2.4. Key HMBC correlations of kahalalide R (8)
125
Results
The ROESY spectrum of compound 8 also indicated the Z stereochemistry of
dehydroaminobutyric acid as shown by the NOE effect of the sharp NH singlet at δ 9.69 on
the methyl doublet at δ1.28 (J = 6.93 Hz). Together with kahalalides F and G, the new
analogues kahalalides R (8) and S (9), are the only derivatives that contain the amino acid ZDhb. This uncommon amino acid, Z-Dhb, was reported as a constituent of peptides isolated
from terrestrial blue-green algae (Moore et al 1989), and from an herbivorous marine mollusc
(Pettit et al 1989).
L- Orn.
H2 N
H 3C
D- Ile
CH3
O
D- pro
N
H
N
H
O
val-3
H 3C
O
D-Thr
N
H3 C
HN
H 3C
O
HN
O
O
HN
O
Z- Dhb
HN
HO
O
O
C H3
phenyl
Thr-2
Val -5
Ile-2
va l-2
CH3
L- Phe.
Dhb
H 3C
Phe
NH
H3 C
Ile -1
CH 3
M eHex
9.6
H 2N
9.2
8.8
8.4
8.0
7.6
7.2
6.8
6.4
6.0
5.6
Ile
H3 C
Thr-2
H-3
Thr-2
OH
Thr-1
H-3
Dhb
Val -1
Val -4
ThrVal -3 Val -2
CH 3
O
Kahalalide F
Orn-NH2
Orn
O
O
H 3C
val-5
NH
H
N
NH
CH3
NH
CH 3
HN
C H3
va l-4
L-Thr
CH3
D-Ile
O
H 3C
H 3C
H 3C
O
val- 1O
5.2
(ppm)
4.8
4.4
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
CH 3
O
N
H
Pro
H 3C
N
H
O
O
N
HN
Val -1
O
H3 C
H3 C
O
HN
O
O
C H3
H3 C
H 3C
O
HN
Thr
O
O
Val -6
C H3
O
H 3C
NH
H
HN
NH
Val -2
CH 3
O
Glu
Phe
ThrVal -4
Val -3
0.9
19
1
NH
O
Dhb
Phe
HO
Kahalalide R
OrnIle
C H3
H
N
O
HN
H3 C
phenyl
Val -6
Val -5
GluOrn-NH2
Val -3
CH3
O
Dhb
H 3C
H 3C
HN
10.0
0.8 1.0 0.8
77 08 99
0 5 6
9.5
9.0
Val -2
0.9 5.3 3.5 1.0
83 72 22 13
9 9 6 7
8.5
8.0
7.5
Dhb
Val -1
1.1
44
9
0.9
36
6
7.0
Thr
H-3
0.7
92
1
6.5
1.0
64
0
6.0
5.5
5.0
3.3 1.0 3.3 4.0 1.0 1.0 1.1 1.3
49 70 73 00 59 74 60 54
4 3 1 4 6 3 8 4
4.5
4.0
3.5
2.0 2.4
48 07
7 2
3.0
3.3 1.8 1.6 4.7 2.2 2.6 3.2 1.7 3.8 5.6 2.2 3.5 3.4 10.
92 57 31 91 22 67 65 73 80 90 12 91 97 00
0 2 5 8 6 3 3 0 4 7 7 0 9 0
2.5
2.0
1.5
1.0
0.5
O
7-MeOct
13C
Region of
aromatic carbons
NMR of kahalalide R
Region of carbonyls
170
160
Thr
C-3
150
140
130
120
110
100
90
80
70
Higher field region
Region of
α-carbons
60
50
40
30
20
10
(ppm)
13C
NMR of kahalalide F
Thr -1
C-3
Thr -2
C-3
Fig ( 3.2.5 ): Comparison between 1H NMR of kahalalide F and R (up) and between 13C NMR of
kahalalide R and F (down).
126
Results
Ile
Glu
Val -6
Val -5
Orn
Orn-NH2
phenyl
Dhb
Phe
Thr
Val -3
0.91
91
9.8
0.871.00
70 85
9.6
9.4
9.2
9.0
8.8
0.89
96
8.6
Val -2
Val -4
0.98
39
8.4
8.2
5.37
29
8.0
3.52
26
7.8
1.01
37
7.6
Dhb
Val -1
1.14
49
7.4
0.93
66
7.2
7.0
6.8
0.79
21
6.6
6.4
6.2
(ppm)
7-MeOct
H-2 2nd conf.
Hα
Val -2
H-3
Glu
Orn Pro
Ile
Val -2
Val -6
Val -5
Thr
H-3
1 .0
6 4
0
5 .0
3 .3
4 9
4
1 .0
7 0
3
4 .5
Thr
Val -4
Val -3
3 .3
7 3
1
4 .0
0 0
4
Val -1
1 .0
5 9
6
4 .0
1 .0
7 4
3
Phe
H-3
Pro
H-5
1 .1
6 0
8
1 .3
5 4
4
2 .0
4 8
7
3 .5
2xMe 7-MeOct
1xMeVal -2
2xMe Val -3
2x MeVal -4
2xMeVal -5
2xMeVal -6
1xMe Ile
H β, H γ
7-MeOct
H-2 1st conf.
Glu
H-4
Orn
H-5
Val -1
H-3
2 .4
0 7
2
3 .0
2x MeVal -1
1x Me Val -2
DhbThr-Me
Me
3 .3
9 2
0
2 .5
1 .8
5 7
2
1 .6
3 1
5
4 .7
9 1
8
2 .2
2 2
6
2 .0
2 .6
6 7
3
3 .2
6 5
3
1 .7
7 3
0
3 .8
8 0
4
1 .5
5 .6
9 0
7
2 .2
1 2
7
3 .5
9 1
0
1 .0
3 .4
9 7
9
1 0 .
0 0
0
0 .5
Fig ( 3.2.6 ): 1H NMR spectrum of 8, NHs and aromatic region (up) and higher field region
of α,β,γ and methyl proton signals(down).
(ppm)
7-Me Oct
2.0
Proline
spin system
NH-detected spin system
4.0
6.0
8.0
(ppm)
8.0
6.0
4.0
2.0
Fig ( 3.2.7): Total TOCSY spectrum of compound 8 ( Kahalalide R).
127
Results
Orn
GluVal -5
Val -6 Ile
Dhb
Phe
Thr
Val -3
Me
Orn-
Val -4
Me
Phenyl
Val -1 Dhb
Val -2
Me
0.8
β
β
(ppm)
Me
Me
Me
1.6
β
β
2.4
δ
β
3.2
α
α
4.0
α
α
α
α
4.8
β
(ppm)
9.6
8.8
8.0
7.2
6.4
Fig ( 3.2.8): NH-detected TOCSY correlations of compound 8 ( Kahalalide R).
(ppm)
48
Orn
Pro C-5
Val -6
52
Thr Glu
Phe
Val -4
56
Val -2
Pro
Ile
Val -1
60
Val -5
Val -3
64
Thr
C-3
(ppm)
68
4.8
4.4
4.0
3.6
Fig (3.2.9 ): Part of HMQC spectrum of compound 8 showing H----C direct correlations
of α-carbons.
128
Results
Orn
GluVal Val - Ile
y
Phe
Val -3
Phenyl
Orn-
Val -
Thr
Dhb
Val -1
Val -2
(ppm)
Me-Val -1,
Val-2
H2N
H3C
O
N
H
29.7
H3C
O
N
H3C
HN
O
O
HN
H
HN
O
H3C
HN
CH3
H3C
H3C
H3C
O
O
O
2.4
59.3
NH
NH
H
N
O
36.1
CH3
1.6
Hβ-Val-
O
H3C
O
HN
MeDhb Me
CH3
N
H
O
0.8
CH3
CH3
3.2
O
O
H3C
HN
H3C
Hα- Val-1
4.0
HO
NH
O
Hα of most
amino acids
O
4.8
Hβ-Thr
(ppm)
8.8
8.4
8.0
7.6
7.2
6.8
6.4
Fig (3.2.10): significant NOEs deduced from the ROESY spectra of compound 8.
NHs
Orn
H2N
Hα
(ppm)
Ile
H3C
CH3
O
Pro
H3C
Val -4
N
H
N
H3C
CH3
Dhb
H3C
HN
O
H 3C
H 3C
Val -5
Thr
O
H3 C
O
80
NH
CH3
Val -2
H
N
CH3
O
HMBC correlations to Aromatic carbons
120
C=C
NH
O
Val -3
CH3
Phe
HN
HO
H 3C
HN
O
NH
CH3
O
H3C
O
HN
H
HN
Val -6
O
O
H3 C
O
40
O
Val -1
O
HN
N
H
O
Glu
O
7-MeOct
C=O
NH-detected HMBC correlations
to vicinal carbonyls
Hα-----C=O
(ppm)
Fig (3.2.11): Total HMBC spectrum of compound 8
129
8.0
6.0
4.0
160
2.0
Results
The deduced data obtained by 2D NMR (Figure 3.2.4) that determine the amino acid
sequence of kahalalide R (8) were further confirmed by mass spectrometric methods
involving both ESI (Spengler 1999) and MALDI–TOF–PSD ( Giorgiani et al 2004, Loo and
Loo 1997, Puapaiboon et al 1999) experiments. Similar results were obtained from the (+)
ESI-MS/MS spectrum, where fragment ion peaks were observed at m/z 1251.6, 1151.6,
1052.6, 952.9, 742.7 and 629.5 corresponding to [M–(7-Me-Oct–Glu)]+, [M–(7-Me-Oct–Glu–
Val-6)]+,
[M–(7Me-Oct–Glu–Val-6–Val-5)]+,
[M–(7-Me-Oct–Glu–Val-6–Val-5–Val-4)]+,
[M–(7-Me-Oct–Glu–Val-6–Val-5–Val-4–Pro–Orn)]+ and [M–(7-Me-Oct–Glu–Val-6–Val-5–
Val-4–Pro–Orn–aIle)]+, respectively. However, this MS/MS fragmentation of 8 was limited to
confirmation of the linear peptide side chain. In contrast, the MALDI–TOF–PSD spectrum
established unequivocally the sequence of the depsipeptide, kahalalide R, as shown in Table
3.2.1. The amino acid sequence of the cyclic component was indicated by ion composition
peaks at m/z 628.4 [Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3]+, 330.2 [Val-1–Dhb–Phe]+ or
[Dhb – Phe – Val-2]+, 247.1 [Val-2–Phe]+, and 183.1 [Val-1–Dhb]+. The terminal functional
units 7-Me-Oct–Glu were also shown by peaks at m/z 270.1 [7-Me-Oct–Glu]+ and 142.1 [7Me-Oct]+. The stereochemistry of the amino acids was determined by Marfey analysis
(Marfey 1984), which identified one mole unit each of D-Glu, D-Pro, L-Orn , D-aIle, D-aThr,
and L-Phe. For the six Val units, Marfey analysis suggested the presence of both D and L
isomers in compound 8. The stereochemistry of the individual Val units could not, however,
be unambiguously determined as there are three moles each of Val in both the cyclic and
linear fragments. This is not suprising as the stereochemistry of Val-3 and Val-4 in kahalalide
F has been long debated. The overall structure of kahalalide F was initially elucidated by
Scheuer’s group in 1993 (Hamann and Scheuer 1993), where Val-3 and Val-4 were
respectively assigned the L and D stereochemistry (Goetz et al 1999). The originally proposed
structure (Goetz et al 1999) was then synthesized by the groups of Albericio and Giralt
(López-Maciá et al 2001a), and showed differences in chromatographic and spectroscopic
behavior between the synthesized and the natural peptide. They indicated that the
stereochemistry of Val-3 and Val-4 should be reversed. Later, Rinehart’s group (Bonnard et al
2003) confirmed this indication and proved that the stereochemistry of Val-3 and Val-4 plays
an important role in the activity of kahalalide F since the depsipeptide with L-Val-3 and DVal-4 in its structure was not active while the molecule with D-Val-3 and L-Val-4 possesses
the activity.
130
Results
Table 3.2.1 Important MALDI-TOF-PSD fragment ions of Kahalalide R
Ion composition
m/z
M–[7-Me-Oct– Glu]+
1250.6
+
M–[7-Me-Oct–Glu–Val-6]
1150.7
M–[7-Me-Oct–Glu–Val-6–Val-5]
+
1051.6
M–[7-Me-Oct–Glu–Val-6–Val-5–Val-4]
+
952.6
M–[7-Me-Oct –Glu–Val-6–Val-5–Val-4–Pro]
+
855.5
M–[7-Me-Oct–Glu–Val-6–Val-5–Val-4–Pro–Orn]
+
741.4
Cyclo[Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3]
+
[Val-1–Dhb–Phe] or [Dhb–Phe–Val-2]
[Val-2–Phe]
628.4
+
330.2
+
[Val-1–Dhb]
[Pro–Orn]
+
247.1
+
183.1
+
212.1
[7-Me-Oct-Glu]
[7-Me-Oct]
+
270.1
+
142.1
RT : 5.00 - 45.00
D-Val
22.26
100
95
90
L-Val
85
80
NL:
3.06E5
Spectr
Maxim
nm=21
400.0
nm=39
400.5
Moham
L-Phe
22.46
75
70
Re
lati65
ve 60
Ab
sor55
ba
nc 50
e
45
D-Pro
D-Glu
20.47
D-Thr
40
24.00
35
L-Orn
30
25
16.24
20
D-Ile
17.04
13.87
15
10
13.20
5
7.25
0
5
8.30
10
27.58
12.27
15
20
25
T ime (min)
33.78
30
34.32
35
37.15
38.38
40
44.32
45
Fig (3.2.12): Marfey analysis result and sterochemical profile of Kahalalide R
131
Results
Table 3.2.2. 1H and 13C NMR Data of Kahalalide R in DMSO-d6.
13
Amino
No.
acid
1
Val-1
2
3
4
5
1
(Z)2
Dhb
3
4
C
(ppm)
169.7
60.2
30.1
16.5
19.2
163.5
130.4
130.2
12.5
1
2
3
4
5,5’
6,6’
7
1
2
3
4
5
1
2
3
4
5
171.3
55.65
36.1
137.0
128.5
129.5
126.7
172.6
55.4
31.5
19.5
18.5
170.0
59.3
31.5
18.6
19.5
1
2
3
4
1
2
3
4
5
6
1
2
3
4
5
168.7
56.4
70.0
17.3
171.3
57.2
30.2
14.6
26.0
11.8
171.5
51.1
30.9
28.5
38.3
Phe
Val-2
Val-3
aThr
aIle
Orn
1
H (ppm)
mult.
(NH) 6.73
3.85
1.38
0.62
0.60
(NH) 9.69
(d, J=8.5 Hz)
( t, J= 9.0 Hz )
(m)
(d, J=6.3 Hz)
(d, J=6.3 Hz)
(s)
6.36 (q, J=7.0 Hz)
1.28 (d, J=7.5 Hz)
(NH) 8.78 (d, J=5.5 Hz)
4.43 (q, J=6.5 Hz)
2.94, 2.92 (m, m)
7.28
7.29
7.25
7.61 (NH)
4.45
2.18
0.80
0.62
(NH) 8.82
4.13
1.94
0.81
0.82
(m)
(m)
(m)
(d, J=8.5 Hz)
(m)
(m)
(d, J=7.0 Hz)
(d, J=6.5 Hz)
(d, J=8.5 Hz)
(m)
(m)
(d, J=7.0 Hz)
(d, J=6.5 Hz)
(NH) 8.55
4.50
4.96
1.08
(NH) 7.87
4.34
1.73
1.21
1.02
0.82
(NH) 7.93
4.49
1.69, 1.82
1.52
2.74
(NH2) 7.72
(d, J=8.0Hz)
(t, J=7.8 Hz)
(m)
(d, J=6.5Hz)
(d, J=8.2 Hz)
(m)
(m)
(m)
(t, J=6.5 Hz)
(d, J=6.5 Hz)
(d, J=8.5 Hz)
(m)
(m, m)
(m)
(m)
(bs)
13
Amino No
acid
.
1
Pro
2
3
4
5
Val-4 1
2
3
4
5
Val-5 1
2
3
4
5
Val-6
Glu
1
2
3
4
5
1
2
3
4
5
7-Me- 1
2
Oct
3
4
5
6
7
8
9
C
(ppm)
172.6
55.6
29.7
27.2
47.0
172.6
55.9
31.0
22.8
19.0
171.2
59.5
31.6
19.3
19.6
171.2
51.89
30.7
22.2
22.9
170.9
2nd regioisomer
56.0
28.0
38.5
169.4
172.5
35.1
23.6
29.5
29.5
38.3
27.3
22.5
22.5
*Resonance is underneath the methyl signals of Val and aIle.
132
1
H (ppm)
mult.
4.37
2.01, 1.98
1.78, 1.89
3.78, 3.52
(NH) 8.11
4.26
1.94
0.84
0.83
(NH) 7.89
4.38
1.88
0.82
0.83
(m)
(m, m)
(m, m)
(m, m)
(d, J=8.5 Hz)
(m)
(m)
(m)
(m)
(d, J=8.5 Hz)
(m)
(m)
(m)
(m)
(NH) 7.86
4.37
1.95
0.84
0.86
(NH) 7.93
(NH) 7.91
(d, J=8.5 Hz)
(m)
(m)
(m)
(m)
(d, J=7.5 Hz)
(d, J=7.5 Hz)
4.41
1.59, 1.50
2.75
(OH) 7.71
(m)
(m)
(m, m)
(bs)
2.11
1.51
1.28
1.25
1.15, 1.10
1.52
0.80
0.81
(m)
(m)
(m)
(m)
(m)
(m)
*
*
Results
3.2.2- Kahalalide S (9, New natural product)
2 .7 4
3 8 .3
H 2N
7 .7 2
O rn
1 1 .8 5
0 .8 2
H 3C
1 .0 2 , 1 . 2 1
2 6 .0
1 .7 3
1 .5
2 8 .5 1 .6 9 ,1 .8 2
3 0 .9 2
5 1 .1 4 4 . 4 9
O
P ro
1 9 .4
0 .8 4
H 3C
V a l- 4
1 9 .0
0 .8 3
H 3C
1 .7 8 ,1 8 9
2 4 .8
O
5 5 .6 5
4 .3 7
2 9 .7
4 7.2
N
3 1 .0 3 .7 8 ,3 .5 2
1 .9 4
1 7 0 .2
O
O
V a l -5
1 7 1 .3
1 .8 8
0 .83
1 9 .6 H 3 C
D hb
7 .8 8
HN
O
1 7 1 .2
2 2 .2
0 .8 4
H 3C
V a l- 6
4 .3 2
5 9 .5
3 1 .6
3 0 .7
1 .9 5
7 .8 9
HN
O
6 .3 6
H
0 .8 1
1 8 .9
H 3C
HN
8 .8 2
O
G lu
2 .1 8
3 2 .5
V a l- 2
CH3
0 .8 0
1 9 .5
O
O
2 . 9 2 ,2 . 9 4
3 6 .1
1 3 7 .0
V a l- 3
1 8 .5
0 .6 2
CH3
7 .6 1
NH
1 7 0 .0
8 .7 8
1 3 1 .0 9 .6 9
4 .4 5
H
NH
4 .4 3
N
5 5 .4 3
5 5 .6 5
1 3 0 .1
1 7 1 .3
1 7 2.8
7 .2 8
1 2 8 .5
Phe
O
56 .0 3
4 .2 1
1 9 .1
0 .8 2
CH3
5 9 .3
4 .1 3
7 .2 9
1 2 9 .5
1 70 .8 8
1 . 5 9 , 1 .5
2 8 .0
7 .7 2 1 6 9 . 4 5
3
8
.
5
HO
2 . 7 5 , 3 .2 9
1 .9 4
3 1 .5
1 6 3 .0
1 2 .5
1 .2 8 C H 3
4 .2 4
5 1 .8 9
H 3C
0 .8 6
2 2 .9
Thr
1 9 .2
0 .6 0
O
H 3 C 1 .3 8
O
3 0 .1
1 6 7 .9
1 6 .5
3 .8 5
1 .0 8 C H 3
0 .62
1 7 .3
6 0 .2
H 3C
O
H N 6 .7 3
H N 8 .1 1
1 9 .3
0 .8 2
H 3C
O
H N 8 .5 5
5 7 .1
4 .5 0
6 9 .9 8
1 6 8 .5
4 .9 6
V a l- 1
4 .2 6
5 9 .5
I le
5 7 .2 7
N 4 .3 4
H
7 .8 7
1 7 1 .3
1 7 1 .5
N 7 .9 3
1 7 2 .0 H
2 . 0 1 , 1 .9 8
1 4 .6
0 .8 4
CH3
3 0 .2
7 .2 5
1 2 6 .7 5
7 .9 2
NH
O
O
4 .2 2
OH
1 7 2 .5
2 3 .6
2 .1 1 1 .6 1 ,1 .5 2
3 5 .4
1 .2 3
2 2.6
3 .4 1
6 7 .5
0 .8 0
1 8 .0
1 .7 1
3 0 .6
1 .12
2 2 .3
0 .8 1
1 8 .1
7 - M e - 5 -O c t o l
C 77H 126N 14O 18
E x a c t M a s s : 1 5 3 4 ,9 3 7
M o l. W t.: 1 5 3 5 ,9 0 7
Fig (3.2.13): compound 9, Kahalalide S
18
egab2
UV_VI
W V L :2 3
mA
60,0
7 -
50,0
15
Peak #7 50%
%
100%
-50%
992.76
994.78
203.0
37,5
10
25,0
5
12,5
8 9 -
345 -6 12 --
0,0
mi
0,
10,
20,
30,
40,
50,
60,
-10,0
200
nm
300
400
500
595
Fig (3.2.14): HPLC chromatogram and the corresponding UV spectrum of
compound 9.
Yield: 8 mg
133
Results
Fig ( 3.2.15 ): ESI-MS of compound 9.
H2N
Ile
H3C
O
N
H
N
H
O
O
H 3C
N
Val-4
H 3C
O
O
O
HN
O
NH
O
CH3
Val -6
Val -5
Orn Ile
Glu
CH3
Val-2
H
N
NH
H
HN
Val-3
CH3
HN
CH3
H3C
Dhb
H3C
H3C
O
H 3C
O
HN
H3C
Val-5
Thr
O
HN
Val-1
CH 3
O
O
Dhb
O
H3C
Val-6
Phe
HN
H 3C
Phe
HO
NH
Val -3
OH
O
Glu
7-Me-5Octol
H-5
Kahalalide S
CH 3
Orn
Pro
phenyl
Orn-NH2
Val -1
Val -2
Thr Val -4
Thr
H-3
Dhb
O
7-Me-5-Octol
L- Orn.
H2N
D-Ile
H3C
CH3
O
D- pro
N
H
N
H
O
val-3
H3C
D-Thr
HN
HN
O
CH3
Z-Dhb
HN
O
NH
CH3
NH
H3C
N
H
N
H
H3 C
N
H3C
HN
Val-1
Val-4
O
CH3
H3 C
H3C
Thr
O
O
H3 C
O
O
O
HN
NH
H
O
CH3
O
H 3C
HO
7.6
7.2
6.8
6.4
6.0
5.6
5.2
4.8
4.4
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
H
N
CH 3
Dhb
Val-2
CH3
Phe
O
Val -3
Phe
HN
Kahalalide R
Glu Orn-NH2
CH 3
NH
phenyl
Val -6
Val -5
Orn Ile
Val-3
O
Glu
8.0
(ppm)
HN
O
Dhb
HN
H 3C
8.4
O
O
H3 C
8.8
CH 3
O
Val-6
9.2
CH3
Ile
H 3C
Orn
Pro
H3C
9.6
CH3
H2N
Val -4
Thr Val -3 Val -2
Thr-2
H-3
Thr-2
OH
Thr-1
H-3
Dhb
Val -1
Ile -1
NH
H3C
MeHex
Val-5
CH3
L- Phe.
O
HN
Phe
val-2
Kahalalide F
Orn-NH2
Dhb
CH3
O
O
O
H3C
val-5
NH
H
N
val-4
HO
Orn
CH3
HN
O
HN
O
L-Thr
CH3
D-Ile
O
H3 C
H3 C
H3C
O
val- 1O
H3 C
O
H3 C
phenyl
Thr-2
Val -5
Ile-2
O
N
NH
0.9
19
1
O
O
7-MeOct
10.0
Thr Val -4
0.8 1.0 0.8
77 08 99
0 5 6
9.5
9.0
8.5
Val -2
0.9 5.3 3.5 1.0
83 72 22 13
9 9 6 7
8.0
7.5
1.1
44
9
7.0
Dhb
Val -1
Thr
H-3
0.9
36
6
1.0
64
0
0.7
92
1
6.5
6.0
5.5
5.0
3.3 1.0 3.3 4.0 1.0 1.0 1.1 1.3
49 70 73 00 59 74 60 54
4 3 1 4 6 3 8 4
4.5
4.0
3.5
2.0 2.4
48 07
7 2
3.0
3.3 1.8 1.6 4.7 2.2 2.6 3.2 1.7 3.8 5.6 2.2 3.5 3.4 10.
92 57 31 91 22 67 65 73 80 90 12 91 97 00
0 2 5 8 6 3 3 0 4 7 7 0 9 0
2.5
2.0
Fig (3.2.16): comparison of 1H NMR spectra of Kahalalides F, R and S.
134
1.5
1.0
0.5
Results
13C
Region of
aromatic carbons
NMR of kahalalide R
Region of carbonyls
170
160
150
Thr
C-3
140
130
120
110
100
90
80
70
Higher field region
Region of
α-carbons
60
50
40
30
20
10
(ppm)
13C
Thr -1
C-3
NMR of kahalalide F
Thr -2
C-3
DEPT of kahalalide S
13C
C-3
Thr
NMR of kahalalide S
C-5
7Me-5-Octol
Fig (3.2.17): comparison of 13C NMR spectra of Kahalalides F, R and S.
Kahalalide S (9) was obtained as a white amorphous powder. The reflector mode
MALDI-TOF mass spectrum of 9 performed with delayed extraction showed positive
monoisotopic ion peaks at m/z 1536.0, [M+H]+, 1558.0 [M+Na]+, and 1574.0 [M+K]+. In
(+)-ESI-MS, a pseudomolecular ion was detected at m/z 1535.9 [M+H]+ that was compatible
with the molecular formula C77H127N14O18 as established by HRESIMS
Kahalalide S differs from kahalalide R by 16 mass unit (one oxygen atom) in the fatty
acid, 7-methyl-5-hydroxyoctanoic acid (7Me-5-Octol). It consists of all amino acid residues
found
in
kahalalide
R.
It
is
also
the
fourth
kahalalide
containing
unusual
dehydroaminobutyric acid (Dhb), table 3.2.3 shows the structural differences between the four
largest kahalalides (F, G, R and S) .
135
Results
In comparison with kahalalide R, 9 has 16 mass units more, which implied the
occurrence of an additional oxygen atom in the molecule. Inspection of the 1H and 13C NMR
spectra of 9 revealed that kahalalide S shared very similar structural features with 8 (Table
3.2.5). The amino acid residues of depsipeptide 9 were identical to those of 8. The only
obvious difference was exhibited in the
13
C NMR and DEPT spectra of 9 where an extra
oxygenated methine carbon was observed at δ 67.5 and the subsequent loss of one methylene
carbon in the 20 to 25 ppm region. COSY and TOCSY spectra of 9 revealed changes
occurring in the fatty acid residue. Instead of 7-Me-Oct as in 8, the fatty acid residue in 9 was
substituted by 5-hydroxy-7-methyloctanoic acid (7-Me-5-Octol). Sequential COSY
correlations were observed between the α-methylene signal at 2.11 ppm to the subsequent
methylene signals at δ 1.61, 1.52 (CH2-3), 1.25 (CH2-4), then to the oxygenated methine at δH5
3.41 and further to the methylene proton at δ 1.12 (CH2-6), which terminates with an
isopropyl unit at δ 1.71 (CH-7), 0.80 (CH3-8) and 0.81 (CH3-9) Table 3.2.5.
HMBC and ROESY spectra of 9 also gave similar results to 8, indicating an identical
amino acid sequence to 8. The ROESY spectra of 9 also implied the occurrence of Z-Dhb in
the depsipeptide. The connectivity of 7-Me-5-Octol to Glu was also afforded by the HMBC
correlation of the Glu–NH resonance at δ 7.92 with the carbonyl signal at δ 172.5, which was
assigned to C-1 in 7-Me-5-Octol.
The amino acid sequence of depsipeptide 9 was further confirmed by ESI-MS/MS
fragmentation and MALDI–TOF–PSD (Table 3.2.4) experiments, which again confirmed the
2D NMR results. The terminal unit, 7-Me-5-Octol–Glu, was evident from the peak at m/z
286.2 in the MALDI–TOF–PSD spectrum. Amino acid analysis using Marfey´s method
revealed the prevalence of a similar stereochemistry for each of the amino acids as found in 8.
The same problem was encountered in 9 as in 8 regarding the assignment of the
stereochemistry of each of the six Val units.
Except for the presence of glutamic acid (Glu) in kahalalides R (8) and S (9), the new
derivatives have a similar set of amino acids to those of kahalalide F (1). The new analogues
further differ from kahalalide F with regard to the occurrence of octanoic acid instead of
hexanoic acid. Kahalalide S (9) was found to contain an identical sequence of amino acids
along with the unusual amino acid, Z-Dhb, as in kahalalide R (8) and differed only in the
terminal fatty acid function, which was 7-Me-5-Octol instead of 7-Me-Oct. (see tables 3.2.3
and 3.2.5 )
136
Results
Table ( 3.2.3 ) Structural differences between the four largest kahalalides, (F, G, R and
S) .
Kah Mol.wt
Mol.formula
Glu
0
Ileu
2
Aminoacids
Orn Phe Pro
1
1
1
The
2
Val
5
F
1477.9
C75H124N14O16
Dhb
1
G
1495.9
C75H126N14O17
1
0
2
1
1
1
2
5
P
1519.9
C77H126N14O17
1
1
1
1
1
1
1
6
Q
1535.9
C77H126N14O18
1
1
1
1
1
1
1
6
Orn
Glu
Val -6
Ile Val -5
Dhb
Fatty acid Structure
form
5-MeHex cyclic
peptide
5-MeHex linear
peptide
7-MeOct cyclic
peptide
7-Me-5cyclic
Octol
peptide
phenyl
Dhb
Orn-NH2
Phe
Thr
Val -4
Val -1
Val -2
Integral
Val -3
9.8
9.6
9.4
9.2
9.0
Hα
8.8
8.6
Ile
Thr
H-3
5.0
4.8
4.6
7-Me-5-Octol
OH
Thr
Val -4
4.2
8.0
(ppm)
7.8
7.6
4.0
Val -2
H-3
7-Me-5-Octol
H-5
Val -1
Val -3
4.4
8.2
7-Me-5-Octol
H-2 2nd conf.
Glu
Orn Pro
Val -2
Val -6
Val -5
8.4
3.8
Phe
H-3
Pro
H-5
3.6
3.4
3.2
3.0
7.4
7.2
7.0
2xMe 7-MeOct
1xMeVal -2
2xMe Val -3
2x MeVal -4
2xMeVal -5
2xMeVal -6
2xMe Ile
7-Me-5-Octol
H-2 1st conf.
DhbMe
6.6
6.4
Thr-Me
6
1x Me Val -2
Hβ, Hγ
Glu
H-4
Orn
H-5
2.8
(ppm)
6.8
2x MeVal -1
Val -1
H-3
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
1
Fig (3.2.18 ): H NMR spectrum of compound 9, NH and aromatic region (up), high
field region (down).
Orn
Dhb
Glu Val -5
Val -6 Ile
Phe
Val -3
Thr
Me
Val -4
Me
Orn-NH 2
Phenyl
Val -2
Me
Me
Val -1
Me
β
β
Dhb
Me
β
δ
β
α
α
α
α
α
α
β
Fig (3.2.19): Part of TOCSY of compound 9 showing NH-detected correlations.
137
Results
(ppm)
20
C-5
Pro
40
α- carbons
60
β- carbon
Thr
C-5
7-M e-5-Octol
80
100
Phenyl CHs
120
=CH-Dhb
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.2.20): Total HMQC of compound 9
Orn
Val -6
IleVal -5
Glu
Val -3
Phe
Thr
Val -4
phenyl
Val -2
(ppm)
0.8
1.6
2.4
3.2
4.0
4.8
(ppm)
8.8
8.4
8.0
7.6
7.2
Fig (3.2.21): Part of the ROESY spectrum of compound 9.
138
Results
NHs
H 2N
Hα
H3C
CH3
O
N
H
N
H
O
O
H3C
N
H3C
HN
H 3C
O
H 3C
O
HN
O
O
HN
O
NH
C H3
H
N
NH
H
HN
HMBC correlations to Aromatic carbons
C H3
O
C H3
O
O
O
H3C
C H3
HN
CH 3
H 3C
H 3C
H 3C
O
O
C=C
HN
NH-detected HMBC correlations
to vicinal carbonyls
Hα-----C=O
H 3C
HO
C=O
NH
OH
O
O
Fig (3.2.22 ): Total HMBC correlations of compound 9
The sequence of kahalalide S was confirmed using MALDI-TOF-PSD modus, and
LC-MS/MS spectra. The reflector mode MALDI-TOF mass spectrum performed with delayed
extraction (DE) showed a positive ion signal at m/z 1558.0, identified as sodium-ion
associated monoisotopic peak [M+Na]+ of kahalalide S, and showed also a positive ion signal
at m/z 1574.0, [M+K]+ and at m/z 1536.0, [M+1]+. MALDI-TOF– PSD spectrum revealed and
confirmed the sequence of a depsipeptide, kahalalide S, as shown in table (3.2.4). Similar
results were obtained from LC-MS/MS spectrum, where the positive protonated fragment ion
peaks could be seen at m/z 1379.9, 1250.6, 1151.6, 1052.6, 952.9, 742.7 and 629.5
corresponding to M-[ 7Me-5-Octol], M-[ 7Me-5-Octol - Glu], M-[ 7Me-5-Octol - Glu- Val6], M-[ 7Me-5-Octol - Glu- Val-6- Val-5], M-[ 7Me-5-Octol - Glu- Val-6- Val-5- Val-4], M-[
7Me-5-Octol - Glu- Val-6- Val-5- Val-4- Pro- Orn] and M-[ 7Me-5-Octol - Glu- Val-6- Val5- Val-4- Pro- Orn- Ileu] respectively, See figure (3.2.23).
Amino acid analysis using Marfey´s method revealed the presence of D-Glu, D-pro, L-Orn,
D-aIle, D-athr, L-Phe , D-Val & L-Val,
139
Results
742
855
H2N
681
CH3
O
953
N
H
N
H
Pro
583
N
HN
Val-1
Val-4
O
O
HN
O
D hb
Val-5
H3C
Val-3
CH3
NH
CH3
H
N
NH
H
HN
Val-2
CH3
O
1151
H 3C
CH3
H 3C
H3C
O
HN
HN
O
627
Thr
O
H3C
O
H3 C
909
O
O
H3 C
1052
Ile
H3 C
Orn
CH 3
O
O
O
1250
Val-6
Phe
H3 C
HN
H3 C
HO
Glu
NH
OH
O
O
1379
7-Me-5 -Octol
Fig (3.2.23): ESI-MS/MS of compound 9
Table 3.2.4. Important MALDI-TOF-PSD fragment ions of Kahalalide S
Ion composition
m/z
M–[7-Me-5-Octol–Glu] +
1250.6
M–[7-Me-5-Octol–Glu–Val-6] +
1150.7
M–[7-Me-5-Octol–Glu–Val-6–Val-5]
+
1051.6
M–[7-Me-5-Octol–Glu–Val-6–Val-5–Val-4] +
952.6
M–[7-Me-5-Octol–Glu–Val-6–Val-5–Val-4–Pro] +
855.5
Ile + Cyclo[Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3] +
741.4
Cyclo[Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3] +
628.4
[Val-1–Dhb–Phe] + or [Dhb–Phe–Val-2] +
330.2
[Val-2–Phe] +
247.1
[Val-1–Dhb] +
183.1
[Pro–Orn] +
212.1
[7-Me-5-Octol–Glu] +
286.2
140
Results
Table 3.2.5 1H and 13C NMR data of Kahalalide S in DMSO-d6.
Amino
No.
acid
Val-1
(Z)Dhb
Phe
Val-2
Val-3
aThr
aIle
Orn
13
C
(ppm)
1
H (ppm)
13
C
Amino
No.
(ppm)
acid
mult.
1
2
3
4
5
1
2
3
4
167.9
60.2
30.1
16.5
19.2
163.0
131.0
130.1
12.5
(NH) 6.73
3.85
1.38
0.62
0.60
(NH) 9.69
1
2
3
4
5,5’
6,6’
7
1
2
3
4
5
1
2
3
4
5
171.3
55.6
36.1
137.0
128.5
129.5
126.7
172.8
55.4
32.5
19.5
18.5
170.0
59.3
31.5
18.9
19.1
(NH) 8.78 (d, J=5.5 Hz)
4.43 (q, J=6.5 Hz)
2.94 (m)
(d, J=9.0 Hz) Pro
( t, J=9.0 Hz )
(m)
(d, J=7.0 Hz)
(d, J=6.0 Hz)
(s)
Val-4
6.36 (q, J=7.0 Hz)
1.28 (d, J=7.5 Hz)
7.28
7.29
7.25
(NH) 7.61
4.45
2.18
0.62
0.80
(NH) 8.82
4.13
1.94
0.81
0.82
(m)
(m)
(m)
(d, J=8.5 Hz)
(m)
(m)
(d, J=7.0 Hz)
(d, J=6.5 Hz)
(d, J=8.5 Hz)
(m)
(m)
(d, J=7.0 Hz)
(d, J=6.5 Hz)
Val-5
Val-6
Glu
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
172.0
55.6
29.7
24.8
47.2
170.2
59.5
31.0
19.4
19.0
171.3
59.5
31.6
19.3
19.6
1
2
3
4
5
1
171.2
51.9
30.7
22.2
22.9
170.9
2nd
regioisomer
56.0
28.0
38.5
169.4
172.5
35.4
23.6
22.6
67.5
2
3
4
5
(NH) 8.55 (d, J=8.0 Hz) 7-Me- 1
4.50 (t, J=7.8 Hz) 5-Octol 2
3
4.96 (m)
4
1.08 (d, J=6.5 Hz)
5
(NH) 7.87 (d, J=8.2.Hz)
4.34 ( m )
6
1.73 (m)
7
1.21 (m)
8
1.02 (t, J=6.5 Hz)
9
0.82 (d, J=6.5 Hz)
(NH) 7.93 (d, J=8.5 Hz)
4.49 (m)
1.69, 1.82 (m, m)
1.5 (m)
2.74 (m)
(NH2) 7.72 (bs)
*Resonance is underneath the methyl signals of Val and aIle.
1
2
3
4
1
2
3
4
5
6
1
2
3
4
5
168.5
57.1
70.0
17.3
171.3
57.27
30.2
14.6
26.0
11.9
171.5
51.1
30.9
28.5
38.3
141
38.3
27.3
18.0
18.1
1
H (ppm)
mult.
4.37
2.01, 1.98
1.78, 1.89
3.78, 3.52
(NH) 8.11
4.26
1.94
0.84
0.83
(NH) 7.88
4.32
1.88
0.82
0.83
(m)
(m, m)
(m, m)
(m, m)
(d, J=8.5 Hz)
(m)
(m)
(m)
(m)
(d, J=8.5 Hz)
(m)
(m)
(m)
(m)
(NH) 7.89
4.24
1.95
0.84
0.86
(NH) 7.92
(NH) 7.91
(d, J=8.5 Hz)
(m)
(m)
(m)
(m)
(d, J=7.5 Hz)
(d, J=7.5 Hz)
4.21
1.59, 1.50
2.75, 3.29
(OH) 7.72
(m)
(m)
(m)
(bs)
2.11
1.61,1.52
1.25
3.41
(OH) 4.22
1.12
1.71
0.80
0.81
(m)
(m)
(m)
(m)
(d)
(m)
(m)
*
*
Results
R T : 5 .0 0
- 4 5 .0 0
2 2 .7 1
1 0 0
9 5
D - V a l in e
1 8 .8 6
9 0
L - V a l in e
8 5
8 0
L - P h e - a la n i n e
7 5
D - P r o l in e
7 0
2 0 .9 3
2 2 .9 8
Relative Absorbance
6 5
6 0
D - G lu ta m ic a ci d
5 5
5 0
D -T h re o n i n e
4 5
2 4 .5 1
4 0
D - I so le u c in e
1 7 .4 5
3 5
1 4 .1 5
3 0
L - O r n it h in e
2 5
2 0
1 3 .4 3
1 5
1 0
5
7 .2 7
5
1 0 .9 9
2 8 .2 3
1 2 .4 9
1 0
1 5
2 0
2 5
T im e (m in )
3 4 .4 5
3 6 .4 7
3 0
3 7 .6 3
3 8 .4 8
3 5
4 3 .7 7
4 0
4
Fig (3.2.24) Marfey analysis results showing the stereochemical profile of
amino acids of compound 9.
Table 3.2.6 Marfey´s analysis results of kahalalide F, R and S hydrolysates :
Amino acid-DAA
deriv.
Mol. wt (+ve mode)
Mol. wt
(-ve mode)
Ret.time in minutes
L.
aIle
384.0
382.4
D.
aIle
384.0
382.4
L.
Phe
418.0
416.5
L.
Val
370.0
368.3
D. Val L.
Pro
370.0 368.1
368.3 366.3
D.
Pro
368.1
366.3
L.
aThr
372.0
370.3
D.
aThr
372.0
370.3
L.
Glu
400.0
398.3
D.
Glu
400.0
398.3
L.
Orn*
385.1
383.5
D.
Orn*
385.1
383.5
23.48
24.53
22.98
20.97
22.77
18.68
15.93
16.60
16.61
17.45
13.44
13.54
&
16.40
-
-
14.14
+
-
18.02
&
Occcurence in
Kah. F
Occcurence in
Kah. R
Occcurence in
Kah. S
-
+
-
+
-
+
+
+
+
-
+
+
+
+
+
+
-
+
-
+
-
+
+
-
+
+
+
-
+
-
+
-
+
+
-
*it was detected that each Ornithine isomer has two different retention times, because ornithine contains α- and δreactive amino groups which called be react with FDAA producing ESI-MS detectable products.
Bioactivity :
Crude aqueous methanol extract obtained from E. grandifolia was reported to exhibit
significant antifungal activity against Aspergillus japonica, A. fresenii and A. niger (Bhosale
et al , 1999). Although many natural products were isolated from genus Elysia, this is the first
report for isolated natural products from E. grandifolia.
The known derivatives, kahalalides B, D, E, F, together with the new congeners,
kahalalides R and S, were assayed for their cytoxicity toward L1578Y, HELA, PC12, H4IIE,
and MCF7 cancer cell lines. Kahalalides F and R were found to be comparably cytotoxic
toward MCF7 cells with IC50-values of 0.22 ± 0.05 µmol/L and 0.14 ± 0.04 µmol/L,
respectively. Kahalalide S and E were less cytotoxic in MCF7 cells with IC50-values of 3.55
± 0.7 µmol/L and 4.5 ± 0.49 µmol/L, respectively (Figure 3.2.25). Kahalalide R was cytotoxic
towards the mouse lymphoma L1578Y cell line at an IC50 of 4.28 ± 0.03 nmol/mL, which is
142
Results
almost identical to that of kahalalide F with an IC50 of 4.26 ± 0.04 nmol/mL. The kahalalides
including kahalalides F and R were found to be inactive toward HELA, H4IIE and PC12
cancer cell lines. This implied the cytotoxic selectivity and specificity of kahalalides F and R.
Statistics
Data are given as mean ± S.E.M. of 3 independent experiments. The significance of changes
in the test responses was assessed using a one-way ANOVA followed by LSD test (Analyseit, Leeds, UK), differences were considered significant at p<0.05.
cell viability
(absorption 560 nm)
0,4
0,3
0,2
0,1
*
0
DMSO
Kah B
Kah D
Kah E
Kah F
*
Kah R
Kah S
Fig (3.2.25): Cytotoxicity of Kah B, D, E, F, R and S in MCF7 cells
MCF7 cells were incubated with different kahalalides (1 µmol/L) for 24 h, and then
MTT reduction as a marker of cell viability was measured. Results are expressed as
absorption of reduced MTT (560 nm) ± S.E.M. (n=3), * p < 0.05 vs. control (DMSO).
Antimicrobial activity
In an agar diffusion assay, kahalalide R at a disc loading concentration of 5 µg,
showed strong antifungal activity against the plant pathogens Cladosporium herbarum and C.
cucumerinum with inhibition zones of 16 and 24 mm, respectively. These results were almost
identical to kahalalide F, which exhibited its fungicidal activity with inhibition zones of 17
and 24 mm, respectively, at the same concentration as the latter compound. Using the same
concentration, the fungicidal activity of kahalalides F and R were also comparable to that of
nystatin showing inhibition zones of 19 and 39 mm, respectively. However, kahalalides F and
R did not show a broad spectrum of antibiotic activity as the derivatives did not exhibit any
antibacterial activity. Kahalalide S exhibited neither antibacterial nor antifungal activities.
143
Results
3.3-Natural products from Pachychalina sp :
From some of the unidentified marine poriferian Pachychalina sp. from the South
China Sea, 15 steroids with five different nuclei including 7-en-sterol, 8-en-sterols,
Anorsterols, 5-en-sterols and sterols with 4-Me cholestanol nuclei and glycerin-3-heptacosyl
ether (Zeng et al 1996, Zeng 2000), in addition to methyl-p-hydroxyphenylacetate, thymine,
uracil, thimidine and 2´deoxyuridine (Xiao et al 1997) have been previously reported.
An unidentified Indonesian Pachychalina sp. was chemically investigated in the
present study and three compounds were isolated which included two 5α,8α- epidioxysterols
(compounds 10 and 11) and 8-hydroxy-4-quinolone (compound 12).
400 ms 0 309 26 # 3
mAU
T M 6- he x6
U V_VIS _1
W VL :23 5 nm
C om p ou nd s 1 0 & 1 1
giv in g t he s am e R f valu e
0 n T LC ( R f = 0.5 6)
7 - 38 ,0 06
Pla s tic is e r
300
200
Co mp o u n d 10
100
Co mp o u n d 11
0
8 - 38 ,4 33
10
6 0 ,0
10, 0
Peak #8
%
2 0 5 .4
H e xa n e : EtA c O
7
:
3
20, 0
30, 0
40, 0
3 8 .5 1
50, 0
min
60, 0
60,0
P eak #9
%
203.9
40,0
4 0 ,0
2 0 ,0
39.35
Co mp o u n d -11
Co mp o u n d -10
-1 0 ,0
10 & 1 1
9 - 39 ,2 23
5----0,
1,69
42843
46
2
124
36
0,
1,
99
20
30
07
- 50
0,0
11
20,0
200
400
nm
5 9 5 -10,0
200
400
nm
595
Fig ( 3.3.1 ): HPLC chromatogram showing the mixture of compounds 10 & 11 before
separation (up-left), TLC of both compound showing the
same Rf- value and complete
overlapping before separation (up-right), UV-spectrum of compound 10 (down left) and UVspectrum of compound 11 (down right).
Compounds 10 and 11 are 5α,8α-epidioxysterols. The difference in molecular weight is
only 14 mass units which seems to be an additional CH2 unit in the side chain as explained
below. Both compounds show the same Rf value on TLC and the same UV-spectrum. HPLC
chromatogram shows slight differences in the relative retention times which were
consequently used as the basis for the isolation using semi-preparative HPLC.
144
Results
3.3.1- 5α,8α-epidioxy-24ξ-methylcholesta-6,22-dien-3β-ol (10, known compound)
0.98
20.89
0.91
18.16
5.12
135.39
0.81
12.82
1.68
1.5
34.67
36.94
30.09
3.96
66.44
HO
51.06
82.14
36.91
2.12,1.85
132.39
5.08
56.12
23.38
1.21
1.52
43.04
1.25
39.32
44.54
0.88
18.01
2.03 39.8
1.85
1.27
33.17
1.47
0.81
20.14
28.90
51.68
1.60
79.41
130.73
135.39
6.49
6.25
O
19.63
0.83
20.65
C28H44O3
Exact Mass: 428,329
Mol. Wt.: 428,647
O
tm6hex-6-2
100 ms031006 #3
mAU
UV_VIS_
WVL:235 nm
70,0
10 - 38,407
Peak #11 38.30
%
80
202.6
60
40
25,0
20
0
563.1
68
1,309
1,409
1,342
9-----0,658
1,444
57
1,175
4
1,147
123
0,966
1,083
-20
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
500
595
Fig (3.3.3) HPLC chromatogram and UV-spectrum of compound 10.
Yield : 5mg
Fig (3.3.4) EI-MS spectrum of compound 10
145
Results
Compound 10 was isolated as crystalline white needles, with [α]D of - 5° (c 0.35
CHCl3). Compound 10 has UV absorption at λmax 203 nm. EI-MS showed molecular ion peak
at m/z 428 [M]+ and fragment ions at m/z 396 [M-(O2)]+, 378 [M-(O2 + H2O)]+, 363 [M-(O2 +
H2O + CH3)]+, 271 [M-(O2 + side chain)]+, and 253 [M-(O2 + side chain + H2O)]+ suggesting
the molecular formula C29H46O3. 1H NMR spectrum showed resonances for six methyl groups
at δ 0.81 (3H, s, Me-18), 0.88 (3H, s, Me-19), 0.98 (3H, d, J=6.31 Hz, Me-21), 0.81 (3H, d,
J=6.3 Hz, Me-26), 0.83 (3H, d, J=6.93 Hz, Me-27), 0.91 (3H, d, J=6.63 Hz, Me-28). The
resonances at δ 3.96 (1H, m, H-3), 6.25 (1H, d, J=8.83 Hz, H-6) and 6.49 (1H, d, J=8.83 Hz,
H-7) suggested a ∆6, mono hydroxylated 5α,8α-epidioxysteroidal compound (Gauvin et al ,
2000). This was confirmed by the presence of an ion fragment at m/z 396 [M-O2] through the
loss of O2 from the
molecular ion, presumably by a retro Diels-Alder fragmentation
(Gunatilaka et al 1981), and by
13
C NMR signals at 82.14 and 79.41 of C-5 and C-8,
respectively (Yaoita et al 1998 and Yue et al 2001). The presence of two olefenic protons at δ
5.08 (1H, ddd, J=15.2, 8.83, 2.5 Hz) and 5.12 (1H, ddd, J=15.6, 8.81, 2.3Hz) was indicative
to ∆22 unsaturation and assigned to H-23 and H-22 respectively, which were also confirmed
by
13
C NMR resonances at 135.39 and 132.39 ppm for C-22 and C-23, respectively. The β-
configuration of hydroxy group at position 3, δH 3.96 (1H, m, H-3) and δC 66.44 ppm (d, C-3)
was suggested by comparison with the published data of 3α- and 3β- hydroxy steroids (Eggert
et al 1976, Wright et al 1978, and Gauvin et al, 2000 ). Although the NMR data of the
position 24 were compared with those given by (Gunatilaka et al 1981) the sterochemistry of
this position, is still undetected because the chemical shifts of the side chain carbons were not
similar enough to those (meassured in C6D6) given by (Gunatilaka et al 1981) for 24(R) or
24(S). Therefore, it preferred not to assign the stereochemistry at C-24 for compound 10. The
position of the double bonds and also the methyl groups were confirmed with 2D-NMR
experiments of the compound. The NMR data assigned compound 10 as 5α,8α-epidioxy-24ξmethylcholesta-6,22-dien-3β-ol, that was previously isolated from some marine sponges (like
Axinella cannabina, Tethya aurantia, and Raphidostila incisa (Gunatilaka et al 1981).
146
Results
Table (3.3.1): 1H, 13C-NMR data of compound 10 in (CDCl3, 500, MHz)
No.
13
C (Multiplicity)
1
H (Multiplicity, Hz)
HMBC correlation
H-------C
1
34.67 t
1.68 (m)
2
30.09 t
1.5 (m)
3
66.44 d
3.96 (m)
C-5
4
36.91 t
1.85 (m), 2.12 (ddd J=13.87, 5.04, 1.89 Hz)
C-3, C-5
5
82.14 s
-
6
135.39 d
6.25 (d, J=8.83 Hz)
C-4, C-5, C-8
7
130.73 d
6.49 (d, J=8.83 Hz)
C-5, C-8
8
79.41 s
-
9
51.06 d
1.52 (t)
10
36.94 s
-
11
23.38 t
1.21 (m)
12
39.32 t
1.27 (m)
13
44.54 s
-
14
51.68 d
1.6 (m)
15
20.65 t
16
28.90 t
17
56.12 d
1.25(m)
18
12.82q
0.81 ( s)
C-12, C-13, C-14 ,C-17
19
18.01q
0.88 ( s)
C-10, C-9, C-5
20
39.8 d
2.03(m)
21
20.89
0.98 (d, J=6.31 Hz)
C-20
22
135.39 d
5.08 (ddd, J=15.2, 8.83, 2.5 Hz)
C-20, C-24
23
132.39 d
5.12 (ddd, J=15.6, 8.81, 2.3 Hz)
C-20, C-24
24
43.04 d
1.85 (m)
25
33.17 d
1.47 (m)
26
20.14 q
0.81 (d, J=6.3 Hz)
C-25, C-24
27
19.63 q
0.83 (d, J=6.93 Hz)
C-25
28
18.16 q
0.91 (d, J=6.62 Hz)
C-25, C-24
147
C-8, C-10
C-8, C-13
C-13, C-8
Results
H-22,
H-23
H-7
H-6
Me-18
Me-19
6.5
6.4
6.3
5.20
(ppm)
(ppm)
Me-28
Me-21
H-7
H-6
H-22,
H-23
H-20
H-12
H-4
H-3
7.0
6.5
6.0
5.5
5.0
4.5
4.0
(ppm)
3.5
3.0
2.5
2.0
1.5
1.0
23
6
21
22
28
15
22
18
23
12
7
11
19
13
14
10
3
5
4
HO
9
24
17
25
27
6
17
8
8
12 10
4
3
26
5
16
15
9
1
2
20
14
24
19,21
11
2
26 27
28
20
16
13
7
1
18
25
O
O
0
130
120
110
100
90
80
70
60
50
Fig (3.3.6): 13C NMR and DEPT spectra of compound 10.
148
40
30
20
10
12.8274
18.0178
18.1623
19.6356
20.1460
20.6564
20.8971
23.3816
28.8994
30.0935
33.1750
34.6677
36.9114
36.9403
39.3188
39.8003
43.0455
44.5381
51.0575
51.6834
56.1227
66.4458
79.4074
82.1423
130.7340
132.3903
135.3948
Fig (3.3.5): 1H NMR of compound 10.
Results
7 6
22&23
3
4a
(ppm)
1.00
27/25
3/2
3/4b
23/24
22/20
21/20
3/4a
2.00
3.00
4.00
5.00
6.00
7.00
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.3.8): COSY spectrum of of compound 10
7
6
22&23
4a
3
(ppm)
20
6/4
22/20
40
21/20
23/24
60
2/3
6/5&8
9&14/8
80
2/5
7/5&8
1/5
19/5
100
120
(ppm)
7.00
6.00
5.00
4.00
3.00
Fig (3.3.9): HMBC spectrum of of compound 10
149
2.00
1.00
Results
3.3.2- 5α,8α-epidioxy-24ξ-ethylcholesta-6,22-dien-3β-ol (11, known compound)
1.03
21.01
5.14
137.46
0.81
12.84
0.80
18.16
1.19, 1.41
25.35
2.03 51.06
39.96
1.52
39.40
44.5
28.24
0.88
18.16
56.1
51.67
3.96
66.46
35.32
HO
82.14
31.85
1.47
18.54
0.85
23.38
34.67
30.11
0.80
19.00
39.32
51.16
1.53
129.87
5.04
79.41
130.74
135.38
6.51
6.25
36.91
2.12,1.84
C29H46O3
Exact Mass: 442,345
Mol. Wt.: 442,674
O
O
Fig(3.3.10): compound 11.
tm6hex-6-3
120 ms031006 #4
mAU
100
UV_VIS_1
WVL:235 nm
70,0
Peak #8 39.12
%
8 - 39,181
204.8
75
50
25,0
25
0
--1,151
1,358
1,482
1,430
123456
-7-0,367
- 1,078
1,284
-40
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
500
Fig (3.3.11) HPLC chromatogram and UV-spectrum of compound 11.
Yield : 4.5 mg
Fig (3.3.12) EI-MS spectrum of compound 11
150
595
Results
Compound 11 was also isolated as crystalline white needles, with [α]D of - 10° (c 0.3
CHCl3). Compound 11 has UV absorption at λmax 204 nm. EI-MS showed molecular ion peak
at m/z 442 [M]+ and fragment ions at m/z 410 [M-O2]+ , 398 [M-(C2H3O)]+, 396 [M-(O2 +
CH2)]+, 377 [M-(O2 + H2O + CH3)]+, 363 [M-(O2 + H2O + C2H5)]+, 271 [M-(O2 + side
chain)]+ and 253 [M-(O2 + side chain + H2O)]+. suggesting the molecular formula C29H46O3.
1
H NMR spectrum showed resonances for again six methyl groups at δ 0.81 (3H, s, Me-18),
0.89 (3H, s, Me-19), 1.03 (3H, d, J=6.41 Hz, Me-21), 0.80 (3H, d, J=6.92 Hz, Me-27), 0.85
(3H, d, J=6.91 Hz, Me-26), 0.80 (3H, t, J=6.3 Hz, Me-29) as found in compound 10. The
resonances at δ 3.96 (1H, m, H-3), 6.25 (1H, d, J=8.83 Hz, H-6) and 6.51 (1H, d, J=8.83 Hz,
H-7) suggested a ∆6, mono hydroxylated 5α,8α-epidioxysteroidal compound (Gauvin et al
2000). By comparison between the EIMS spectra of compounds 10 and 11 especially for the
most abundant fragment (M-O2, rel. abundance 100%) for both compounds, compound 11 is
14 mass units larger than 10 which indicated the presence of one additional methylene group
in 11. This CH2 group is assigned at position 28 as evident from its COSY spectrum. The
differences in the NMR data between 10 and 11 are evident only for the side chain as
mentioned in table 3.3.2, while the NMR data of the tetracyclic carbon skeleton are
superimposible. Compound 11 formed two stereoisomers due to the
stereochemistry at
position 24 as evident from 1HNMR spectrum where the triplet signal at δ 0.80 of CH3-29
showed another set of resonances at the same position with only 1.2 Hz difference in the
chemical shift value. Therefore compound 11 was assigned as a racemic mixture of 24(R)
and 24(S) at a ratio of 1:1. This was also confirmed by the corresponding δC values of the side
chain which showed signals with small intensities compared to those of the tetracyclic part.
The NMR data assigned compound 11 as [5α,8α-epidioxy-24ξ-ethylcholesta-6,22-dien-3β-ol],
which was also previously isolated from some marine sponges like Luffariella sp. (Gauvin et
al 2000) and Raphidostila incisa (Gunatilaka et al 1981).
151
Results
Table (3.3.2): 1H, 13C-NMR data of compound 11 in (CDCl3 , 500, MHz)
No.
13
C (Multiplicity)
1
H (Multiplicity, Hz)
HMBC correlation
H-------C
1
34.67 t
2
30.11 t
1.53 (m)
3
66.46 d
3.96 (m)
C-5
4
36.91 t
1.85 (m), 2.12 (ddd J=13.87, 5.04, 1.89 Hz)
C-3, C-5
5
82.14 s
-
6
135.38 d
6.25 (d, J=8.83 Hz)
C-4, C-5, C-8
7
130.74 d
6.51 (d, J=8.83 Hz)
C-5, C-8
8
79.41 s
-
9
51.16 d
10
35.32 s
11
28.24 t
12
39.40 t
13
44.5 s
14
51.67 d
15
23.38 t
16
39.32 t
17
56.10 d
18
12.84 q
0.81 ( s)
C-12, C-13, C-14 ,C-17
19
18.16 q
0.89 ( s)
C-10, C-9, C-5
20
51.06 d
2.03(m)
21
21.01 q
1.03 (d, J=6.31 Hz)
C-20
22
137.46 d
5.14 (ddd, J=15.2, 8.83, 2.3 Hz)
C-20, C-24
23
129.87 d
5.04 (ddd, J=15.6, 8.81, 2.3 Hz)
C-20, C-24
24
39.96 d
1.52 (m)
25
31.85 d
1.47 (m)
26
19.00 q
0.80 (d, J=6.93 Hz)
C-25, C-24
27
18.54 q
0.85 (d, J=6.93 Hz)
C-25
28
25.35 t
1.19, 1.41 (m,m)
29
18.16 q
0.80 (m)
-
-
152
C-28
7.0
170
160
150
6
6.5
6.0
6
22
140
130
82.1423
79.4074
66.4651
56.1035
51.6738
51.1538
51.0575
51.0382
44.5285
39.9640
39.3188
38.7796
36.9114
35.3225
34.6677
33.4736
32.3854
31.8461
31.7884
30.1032
28.9861
28.8802
28.2350
25.3557
23.3912
21.0897
20.9068
20.6949
18.9904
18.9230
18.5475
18.2393
18.1623
12.8370
7
130.7436
129.8769
137.6481
135.3851
Results
19
22
6.4
(ppm)
5.5
120
23
5.1
5.0
110
18
(ppm)
5.0
20
3
1
4.5
Fig (3.3.13): H NMR spectrum of compound 11.
(
100
4.0
7
(
90
153
3.5
)
80
Fig (3.3.14): 13C NMR spectrum of compound 11.
3.0
3
70
60
2.5
9
13
5
50
4
40
21
4a
)
2.0
1.5
13
30
1.0
2
15
19
8
18
23
20
10
N
E
P
Results
21
7 6
22 23
3
(ppm)
21/20
23/24
1.00
3/2
3/4b
3/4a
22/20
2.00
3.00
4.00
23/22
5.00
6/7
6.00
7.00
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.3.15): COSY spectrum of compound 11.
7
6
29/28
27/25
4/3
7/
5&8
6/
5&8
19/5
Fig (3.3.16): HMBC spectrum of compound 11.
154
Results
3.3.3- 8-hydroxy-4-Quinolone (12, known compound)
O
7.58
6.33
181.5
128
109.1
140
7.93
N
H
115
115.2
158
132
7.2
125
7.03
OH
C 9H 7N O 2
Exact M ass: 161,04
1.400
but-a2
ms031002 #9
mAU
UV_VIS_
WVL:235 nm
6 0 ,0
Peak #12
%
1 2 .9 7
50%
100%
-5 0 %
9 9 9 .2 9
9 9 9 .9 4
9 9 7 .6 0
13 - 12,687
2 3 2 .5
5 0 ,0
1.000
3 7 ,5
2 5 ,0
500
2 0 0 .5
3 2 3 .2
1 2 ,5
56
1,273
4
1,196
8
1,460
1,351
7
1,393
9
-0,971
1,152
10
11
-1,509
-12
1,551
1,802
- 4,229
123
-----0,647
-200
0,0
14 - 37,007
16- -47,329
48,089
15
0 ,0
min
10,0
20,0
30,0
40,0
50,0
60,0
nm
-1 0 ,0
200
300
400
500
Fig (3.3.17): HPLC chromatogram and UV-spectrum of compound 12.
Yield : 3.8 mg
[M + H ]+
[2 M + H ]+
[M -H ]+
[2 M -H ]+
Fig (3.3.18): ESI-MS of compound 12.
155
595
Results
Compound 12 [8-hydroxy-4-quinolone] was isolated as a brownish yellow
amorphous powder. Compound 12 has UV absorption at λmax (MeOH) 200, 232, 220, 323
and 335 nm. Positive ESI-MS showed molecular ion peak at m/z 162 [M+H]+ and negative
molecular ion peak at m/z 160 [M-H]- suggesting the molecular formula C9H7NO2. 1H NMR
spectrum showed resonances for five aromatic protons for two spin systems, 1st spin system
composed of two resonances at δ 7.93 (1H, d, J= 6.94 Hz) and 6.33 (1H, d, J= 6.94 Hz), the
2nd spin system composed of three proton resonances at δ 7.58 (1H, dd, J= 7.90, 0.94 Hz), 7.2
(1H, t, J= 7.88 Hz) and 7.03(1H, dd, J= 7.90, 0.94 Hz ). COSY spectrum confirmed the
above spin systems. These data sugested the presence of two aromatic rings, the odd
numbered MW suggested the occurence of one nitrogen atom while the presence of three
nitrogens or more was excluded. The disappearence of the exchangeable protons (NH and
OH) was expected because deuterated methanol was used. By searching in the commercially
available computer program „Dictionary of Natural Product (DNP)“ it was suggested that
compound 12 is 8-hydroxy-4-quinolone which was isolated previously (Siuda 1974). The
NMR data were identical to those of 8-hydroxy-4-quinolone which was previously isolated
from the ink glands of the marine giant octopus, Octopus dofleini martini (Siuda 1974). The
deduced data of compound 12 were confirmed by HMQC and HMBC experiments. The
presence of tautomerisation was evident from the presence of four aromatic spin systems
(two spin systems for each tautomer) as evident from the 1H NMR and COSY spectra of the
same sample in deuterated DMSO as shown in figures (3.3.19 and 3.3.20).
O
OH
5
3
1
8a
N
H
7
8
11.0
OH
10.0
9.5
7'
8'
8a'
9.0
8.5
3109.80
1.7937
3556.84
3549.27
0.4699
3585.53
7.0
(ppm)
0.8894
3593.41
3601.29
3775.95
3881.56
3767.75
7.5
0.6298
2.3893
2.3632
2.1454
3890.39
8.0
1.0000
3942.09
OH
2.4551
10.5
1'
N
3949.03
11.5
6'
2'
1.4769
1.1817
12.0
5'
3'
2.0813
2
4a'
4'
6
3102.86
4a
4
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Fig (3.3.19):1H NMR spectrum of compound 12 (measurred in DMSO)
156
Results
2’
2
5’
67
3’
6’
7’
5
3
(ppm)
4.8
5.6
2/3
6.4
6’/7’
2’/3’
7.2
5/6
O
OH
4a
4
5
3
6
2
1
8.0
2'
5'
6'
1'
N
OH
7.2
8.0
4a'
3'
7
8
8a
N
H
(ppm)
4'
8'
8a'
7'
OH
6.4
5.6
8.8
4.8
4a
4
3158.97
3166.22
3529.40
3530.35
3537.29
3538.23
3597.50
3605.70
3613.58
3821.65
3822.60
3829.85
5
3
6
2
1
2
8
8a
N
H
7
OH
5
8.0
10.5
3830.80
3960.06
O
3953.12
Fig (3.3.20):COSY spectrum of compound 12 (DMSO-d6)
7.5
10.0
9.5
3
6 7
9.0
7.0
8.5
8.0
7.5
6.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
Fig (3.3.21):1H NMR spectrum of compound 12 (methanol-d4)
157
2.5
2.0
1.5
Results
O
4a
4
2
5
3
2
1
8
8a
N
H
6
3
5
6
7
7
(ppm)
OH
6.4
6.8
7.2
7.6
8.0
8.4
(ppm)
8.4
8.0
7.6
7.2
6.8
6.4
Fig (3.3.22):COSY spectrum of compound 12 ( showing H/H correlations)
2 5
6
7
3
(ppm)
20
40
60
O
80
4
4a
3
5
6
100
2
1
N
H
8a
8
7
120
OH
140
(ppm)
8.00
7.00
6.00
5.00
4.00
3.00
2.00
Fig (3.3.23):HMQC spectrum of compound 12 ( showing H/C direct correlations)
158
Results
2
5
3
6 7
(ppm)
7/5
2/3
120
3/4a
6/4a
2/8a
7/8a
5/8a
O
3/2
6/8
140
4
4a
5
3
2
6
1
N
H
8
8a
8.0
160
OH
2/4
(ppm)
7
180
7.2
6.4
5.6
Fig (3.3.24):HMBC spectrum of compound 12 ( showing H/C long range correlations)
Bioactivity:
Many of the reported 5α, 8α- epidioxy sterols showed significant biological activity
including antimycobacterial activity (Cantrell et al 1999), inhibitive activity against the
human T-cell leukemia/lymphotropic virus type I (HTLV-I), cytotoxic activity against the
human breast cancer cell line (MCF7 WT) (Gauvin et al 2000), and antitumor activity against
different tumour cell lines (Bok et al 1999). 8-Hydroxy-4-quinolone (compound 12) was
reported to be one of the ink components that is ejected by the giant octopus Octopus dofleini
martini which was reported to have antipredatory activity (Siuda 1974). Some other
quinolone analogues (e.g. 3,8-dihydroxyquinoline) showed mild cytotoxic activity against the
growth of several human tumour cell lines( Moon et al 1996).
Cytotoxic activity of compounds 10 and 11 showed growth inhibitiory effect of 57.2
% and 38.6 %, respectively at a concentration of 10 µg/mL each against L5178Y cancer cells.
Compound 12 showed neither significant antimicrobial activity nor cytotoxic activity in our
available test systems.
159
Results
3.4-Natural products from Hyrtios erectus
The secondary metabolites from the genus Hyrtios especially Hertios erectus (also
called H. erecta) have been investigated extensively [Kobayashi et al 1990; Kobayashi et al
1994a; Miyaoka et al 2000; Pettit et al 1998a; Pettit et al 1998b; Pettit et al 1998c; Youssef
2005; Youssef et al 2005, Youssef et al 2002]. Previous chemical investigations of different
Hyrtios sp. and their associated microorganisms have revealed the presence of numerous
structurally
unique
natural
products
including
scalarane
sesterterpenoids,
acyclic
triterpenoids, indole alkaloids, macrolides and usual- and unusual steroids. Many of these
compounds possess important biological activities. The most important metabolites of the
genus Hyrtios discovered to date include the powerful anticancer agents spongistatins (Pettit
et al 1993; Pettit et al 1994). Which in turn prompted the same authers to exhaustively
recollect the same sponge Hyrtios erecta (600 kg wet wt.) for further chemical investigations.
The later chemical investigations resulted in the isolation of antineoplastic sesterstatins in
very minute quantities (Pettit et al 1998b, Pettit et al 1998c, and Pettit et al 2005).
However, in the present study, 210 g. dry weight of Hyrtios erectus collected from the
Red Sea – Egypt were chemically investigated and nine compounds were isolated. The
isolated compounds consisted of 8 known compounds and one new 5-hydroxy-1H-indole
derivative.
160
Results
3.4.1- Hyrtiosine A (13, Known compound):
O
4.71
67.1
195.3
7.63
107.5
HO
OH
114.0
155.6
128.0
6.76
113.6
132
113.3
7.28
8.1
133.4
N
H
C10H9NO3
Exact Mass: 191,06
Mol. Wt.: 191,18
400 ms050811 #3
mAU
ih-mixa
UV_VIS_1
WVL:235 nm
70,0
1 - 12,104
300
Peak #1 12.24
%
213.6
200
25,0
251.6
302.9
100
2 - 47,192
0
-50
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
595
Fig (3.4.1):HPLC chromatogram and UV-spectrum of compound 13.
Yield : 5 mg
[M-(CH2OH + OH)]+
[M-(CH2OH + OH)]+
[M-(COCH2OH)]+
[M+H]+
[M-CH2OH]+
[M-H2O]+
Fig (3.4.2):(+ve) ESI-MS spectrum of compound 13 and its MS/MS spectrum.
161
Results
Compound 13, Hyrtiosine A, [ 5-Hydroxy-3-(hydroxyacetyl)-1H-indole] was isolated
as brownish transluscent amorphous powder, with slightly offensive characteristic odour.
Compound 13 shows positive pseudomolecular ion peak at m/z 192 [M+1]+ and negative
pseudomolecular ion peak at m/z 190 [M-1]- suggesting the molecular formula C10H9NO3.
The UV (MeOH) absorption maxima of 13 at λmax 213, 251, 271 and 302 nm indicative of a
3-acyl indole chromophore (Kobayashi et al 1990). The 1H NMR (CD3OD) showed 5 proton
resonances at δ 8.1 (1H, s ; H-2), 7.63 (1H, d, J = 2.21 Hz; H-4), 6.76 (1H, dd, J = 2.21,8.83
Hz; H-6), 7.28 (1H, d, J = 8.83 Hz; H-7), 4.71 (2H, s ; H-9). The
13
C NMR resonances
were obtained indirectly from the long range correlations (HMBC spectrum), see table (3.4.1).
The HMBC spectrum showed long range correlations between CH2-9 and the carbonyl C-8 at
195.3 ppm and C-3 at 114.0 ppm. Indeed, the following HMBC correlations were observed
(H/C): H-2/C-3, H-2/C-3a, H-2/C-7a, H-4/C-5, H-4/C-7a, H-4/C-6, H-4/C-3, H-6/C-7a, H4/C-5, H-7/C-3a, H-7/C-5. The NMR data and the UV absorption maxima identified 13 as
Hyrtiosine A that was previously isolated from the same sponge (Kobayashi et al 1990) and
from Dragmacidon sp (Pedpradab et al 2004).
Table (3-4.1): NMR data of compound 13 (Methanol-d4, 500 MHz).
No.
13
C, (δ =
ppm)
2
133.4
3
114.0
3a
128
4
107.5
5
155.6
6
1
H (δ = ppm), couppling
HMBC correlations
constant (J= Hz)
HÆ C
δ 8.1 (1H, s )
3, 3a, 7a
7.63 (1H, d, J = 2.21)
5, 7a, 6, 3
113.6
6.76 (1H, dd, J = 2.21& 8.83)
5, 7a
7
113.3
7.28 (1H, d, J = 8.83)
5, 3a
7a
132
8
195.3
9
67.1
4.71 (2H, s )
8, 3
162
2341.49
3378.39
3380.91
3387.22
3389.43
3628.08
3636.91
3818.19
3820.39
4047.70
Results
OH
8
3378.39
3380.91
3387.22
3389.43
3628.08
3636.91
3818.19
3820.39
O
4
HO
3a
5
2
2
4
1.0521
0.9974
7.26
(ppm)
10.8
7
6
10.2
9.6
9.0
8.4
7.8
1
N
H
7.2
1.8726
1.0521
0.9974
1.0122
11.4
7a
(ppm)
Integral
12.0
7
6.78
1.0000
1.0000
6
(ppm)
9
3
6.6
6.0
5.4
4.8
4.2
3.6
3.0
2.4
1.8
1.2
0.6
Fig (3.4.3):1H NMR spectrum of compound 13 (CD3OD).
2
4
9
7
6
(ppm)
80
H-2/C-3
H-6/C-4
H-2/C-3a
H-6/C-6
direct correlation
120
H-9/C-2
Correlations to C-3a
Correlations to C-7a
Correlations to C-5
160
H-2/C-7a
H-9/C-8
200
(ppm)
8.0
7.2
6.4
5.6
4.8
Fig (3.4.4): HMBC spectrum of compound 13 (CD3OD).
163
4.0
3.2
Results
3.4.2- 5-Hydroxy-1H-indol-3-carbaldehyde (14, Known compound):
O
H
9.83
186.3
7.55
106.5
HO
119.1
154.3
126.5
6.80
113.6
132.2
113.3
7.30
7.98
139.0
N
H
C9H7NO2
Exact Mass: 161,05
Mol. Wt.: 161,16
ih-mixb
180 ms050811 #4
mAU
UV_VIS_1
WVL:235 nm
70,0
1 - 13,623
150
Peak #1 13.67
%
211.9
252.3
271.8
100
50
2 - 47,171
-20
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
400
Fig (3.4.5):HPLC chromatogram and UV-spectrum of compound 13.
Yield : 2.5 mg
[M-(CHO)]+
[M-(CHO)]+
[M+H]+
Fig (3.4.6):(+ve) ESI-MS spectrum of compound 14 and its MS/MS spectrum.
164
595
Results
Compound 14, [5-Hydroxy-3-formyl-1H-indole] was isolated as a brownish
transluscent amorphous powder with characteristic odour. Compound 14 shows a positive
pseudomolecular ion peak at m/z 162 [M+1]+ and a negative pseudomolecular ion peak at m/z
160 [M-1]- suggesting the molecular formula C9H7NO2. The UV (MeOH) absorption maxima
of 14 at λmax 211, 252, 271 and 302 nm were indicative of a 3-acyl indole chromophore
(Kobayashi et al 1990). The 1H NMR (CD3OD) showed 5 proton resonances at δ 7.98 (1H, s ;
H-2), 7.55 (1H, d, J = 2.52 Hz; H-4), 6.80 (1H, dd, J = 2.52, 8.51 Hz; H-6), 7.3 (1H, d, J =
8.51 Hz; H-7) and 9.83 (1H, s ; H-8). The
13
C NMR resonances were obtained indirectly
from HMBC spectrum, see table 3.4.2 . The following proton-detected long range correlations
were observed (H/C): H8/C-3, H-8/C-3a, H-2/C-3, H-2/C-3a, H-2/C-7a, H-4/C-6, H-4/C-7a,
H-6/C-7a, H-7/C-3a, H-7/C-5. The NMR data confirmed 14 as 5-hydroxy-3-formyl indole
which has been previously isolated from the same sponge (Kobayashi et al 1990) and from
Dragmacidon sp (Pedpradab et al 2004).
Table (3.4.2): NMR data of compound 14 (Methanol-d4, 500 MHz).
No.
13
C, (δ =
ppm)
2
139.0
3
119.1
3a
126.5
4
106.5
5
154.3
6
1
H (δ = ppm), couppling
HMBC correlations
constant (J= Hz)
HÆ C
δ 7.98 (1H, s )
3, 3a, 7a
7.63 (1H, d, J = 2.52 Hz)
7a, 6
113.6
6.8 (1H, dd, J = 2.52& 8.51 Hz)
7a
7
113.3
7.3 (1H, d, J = 8.51 Hz)
5, 3a
7a
132.2
8
186.3
9.83 (1H, s )
3, 3a
165
Results
4
8
2
6
7
O
8
4
HO
5
3a
3
2
9.6
9.2
8.8
8.4
8.0
7.6
7.2
7a
6
7
1
N
H
1.2330
1.3622
1.1309
10.0
6
7
1.1022
1.0000
4
6.8
6.4
6.0
5.6
5.2
4.8
4.4
4.0
3.6
3.2
Fig (3.4.7):1H NMR spectrum of compound 14 (CD3OD).
8
2
4
7
6
(ppm)
100
H-4/C-6
H-8/C-3
H-2/C-3
H-2/C-3a
H-8/C-3a
120
H-7/C-3a
H-2/C-7a
H-4/C-7a
H-6/C-7a
140
H-2/C-2
direct
O
H-7/C-5
8
4
HO
5
3
3a
2
H-8/C-8
direct
6
9.6
8.8
180
1
7a
N
H
7
(ppm)
160
8.0
7.2
Fig (3.4.8): HMBC spectrum of compound 14 (CD3OD).
166
6.4
Results
3.4.3- Indol-3 carbaldehyde (15, Known compound, first isolation from genus Hyrtios):
O
H
9.95
185.2
120.0
8.07
121.0
7.1
119.1
126.5
8.3
139.5
121.5
7.30
138.5
7.6
114.1
N
H
12.13
C 9H 7N O
Exact M ass: 145,05
M ol. W t.: 145,16
140 ms050811 #5
mAU
ih-mixc
UV_VIS_1
WVL:235 nm
1 - 19,113
100
Peak #1
19.10
0,0
%
207.5
75
243.7
298.0
50
2 - 47,277
25
-20
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
0,0
200
nm
300
400
500
595
Fig (3.4.9):HPLC chromatogram and UV-spectrum of compound 13.
Yield : 2.3 mg
Fig (3.4.10):ESI-MS spectrum of compound 15 [direct injection, +ve mode, (M+H)+].
167
Results
Compound 15, [indole-3-carbaldehyde] was isolated as a yellowish white amorphous
powder, with slightly offensive characteristic oder. Compound 15 shows positive
pseudomolecular ion peak at m/z 146 [M+1]+ suggesting the molecular formula C9H7NO.
The UV (MeOH) absorption maxima of 15 at λmax 206, 243, 255, and 298 nm were
indicative of a 3-acyl indole chromophore. The 1H NMR (DMSO-d6) showed 7 proton
resonances which include the aromatic ABCD spin system at δ 8.07 (1H, d, J = 7.4 Hz ; H-4),
7.10 (1H, dt, J = 1.21, 7.4 Hz; H-5), 7.30 (1H, dt, J = 1.31, 7.4 Hz; H-6) and 7.60 (1H, d, J =
7.4 Hz; H-7) in addition to 3 proton resonances at δ 8.30 (1H, s ; H-2) and the aldehydic
proton at 9.95 (1H, s; H-8) and the most downfield broad singlet at δ 12.13 (1H, s ; H-1) see
table (3.4.3). The HMBC spectrum showed long rang correlation between H-2/C-3, H-2/C-3a,
H-2/C-7, H-2/C-7a, H-2/C-8, H-6/C-7, H-6/C-7a, H-4/C-6, H-4/C-7a, H-4/C-3a, H-4/C-3, H5/C-7, H-5/C-3a, H-7/C-3a, H-7/C-5. The NMR data were identical to those of indole 3carbaldehyde ( Aldrich, 1990, Hiort, 2002) but this is the first isolation of 15 from genus
Hyrtios.
Table (3.4.3): NMR data of compound 15 (DMSO-d6).
13
No.
1
C, (δ = ppm),
multiplicity
H (δ = ppm), couppling
HMBC correlations
constant (J= Hz)
HÆ C
1
-
12.13 (1H, s )
2
139.5
8.30 (1H, s )
3
119.1, s
-
3a
126.5 , s
-
4
120.0, d
8.07 (1H, d, J = 7.4 Hz ; H-4)
3, 3a, 7, 7a, 6
5
121.0
7.10 (1H, dt, J = 1.21, 7.4 Hz)
3a, 7
6
121.5
7.30 (1H, dt, J = 1.31, 7.4 Hz)
4, 7, 7a
7
114.1
7.60 (1H, d, J = 7.4 Hz; H-7)
5, 3a
7a
138.5
-
8
185.2
9.95 (1H, s )
3, 3a, 7, 7a, 8
3, 3a
168
3594.36
3595.62
3601.61
3602.55
3609.17
3610.44
3616.43
3618.00
3624.62
3625.57
3631.56
3632.82
Results
O
5,6
8
4
3a
5
8
3
2
7a
7
1
N
H
7
4
5,6
2.1817
6
Integral
2
7.30
7.25
7.20
7.15
(ppm)
13.0
12.0
11.0
10.0
9.0
8.0
2.1817
1.0425
1.0000
0.9870
0.9239
0.5789
Integral
1
7.0
(
6.0
5.0
4.0
3.0
2.0
)
Fig (3.4.11):1H NMR spectrum of compound 15 (DMSO-d6).
2
4
7
5,6
(ppm)
H-8/C-3
120
H-8/C-3a
H-2/C-7a
H-6/C-7a
140
H-2/C-7a
H/C-2
direct correlation
160
180
H-2/C-8
(ppm)
9.6
8.8
8.0
Fig (3.4.12):HMBC spectrum of compound 15 (DMSO-d6).
169
7.2
1.0
Results
3.4.4. 5-Deoxyhyrtiosine A (16, Known compound, first isolation from genus Hyrtios):
O
4.73
66.3
196.2
7. 44
122.0
OH
114.4
7.19
124.2
126.3
7.22
123.2
138.1
113.0
8.23
8.19
133.6
N
H
C10H9NO2
Exact Mass: 175,06
Mol. Wt.: 175,18
350 ms050811 #7
mAU
ih-mixe
UV_VIS_1
WVL:235 nm
70,0
1 - 17,006
Peak #1
%
208.9
17.05
200
241.3
299.3
100
2 - 18,542
3 - 47,191
nm
-10,0
200
min
-50
0,0
10,0
20,0
30,0
40,0
50,0
60,0
300
400
500
595
Fig (3.4.13):HPLC chromatogram and UV-spectrum of compound 16.
Yield : 2 mg
Fig (3.4.14): (-ve) ESI-MS spectrum of compound 16 and its ESI-MS/MS spectrum.
Fig (3.4.15):ESI-MS spectrum of compound 16 (direct injection).
170
Results
Compound 16, 5-deoxyhyrtiosine A, [3-(hydroxyacetyl)-1H-indole] was isolated as
colourless amorphous powder. Compound 16 shows positive pseudomolecular ion peak at m/z
176 [M+1]+ and negative pseudomolecular ion peak at m/z 174 [M-1]- suggesting the
molecular formula C10H9NO2, which seems to be smaller than hyrtiosine A (compound 13) by
only 16 mass units (one oxygene atom). The UV (MeOH) absorption maxima of 16 at λmax
209, 241, 261 and 299 nm were indicative of a 3-acyl indole chromophore. The 1H NMR
(CD3OD) showed 5 aromatic proton resonances (instead of 4 aromatic protons as in hyrtiosine
A) which included an aromatic ABCD spin system at δ 8.23 (1H, dd, J = 6.94, 1.26 Hz; H-4),
7.19 (1H, dt, J = 6.94, 1.26 Hz; H-5), 7.22 (1H, dt, J = 6.94, 1.26 Hz; H-6) and 7.44 (1H, dd,
J = 6.94, 1.26 Hz; H-7) and one proton resonance at δ 8.19 (1H, s ; H-2) in addition to the
most upfield singlet at δ 4.73 (1H, s ; H-9), see table (3.4.4). The 1HNMR confirmed the loss
of a 5-hydroxyl group (compared to Hyrtiosine A). HMBC spectrum showed long range
correlations between H-2/C-3, H-2/C-3a, H-2/C-7a, H-6/C-7, H-6/C-4, H-6/C-7a, H-4/C-6,
H-5/C-7, H-5/C-3a, H-4/C-7a, H-4/C-3a, H-7/C-5, H-9/C-3, H-9/C-8, see table (3.4.4) and
figure (3.4.17). The NMR data and the UV absorption maxima were identical to those of 3(hydroxyacetyl)-1H-indole which has been previously isolated from the Bermudian sponge
Tedania ignis and their associated bacterium, Micrococcus sp. and the red alga ( Dillman and
Cardellina 1991, Stierle 1988, Bernart and Gerwick 1990, respectively), but this is the first
6
3597.19
3602.86
3604.44
3610.11
3611.37
3614.21
3617.05
3618.31
2367.02
7
4
3622.09
3597.19
3623.98
3602.86
3604.44
3610.11
3611.37
3617.05
3618.31
3622.09
3614.21
3720.14
3623.98
2
3721.08
3720.14
3721.08
3728.02
4097.83
4106.65
4107.91
4112.96
3728.02
4097.83
4106.65
4107.91
4112.96
4114.85
4114.85
isolation of 16 from genus Hyrtios.
5
7.5
10.5
10.0
9.5
9.0
8.5
7.3
7.2
8.0
7.5
1.9171
0.8800
0.7790
0.8807
11.0
7.4
(ppm)
Integral
11.5
2.2592
Integral
8.15
0.8800
8.20
(ppm)
2.2592
8.25
0.7790
0.8807
Integral
9
7.0
6.5
6.0
5.5
5.0
Fig (3.4.16):1H NMR spectrum of compound 16 (CD3OD)..
171
4.5
4.0
3.5
3.0
2.5
Results
2
7
4
6
5
(ppm)
O
OH
104
8
4
5
3a
9
3
2/3
6
7a
7
112
1
N
H
4/6
120
6/4
7/5
4/3a
5/7
6/7
2
5/3a
2/3a
128
4/7a
136
6/7a
2/7a
144
(ppm)
8.40
8.20
8.00
7.80
7.60
7.40
7.20
7.00
Fig (3.4.17): HMBC spectrum of compound 16(CD3OD).
Table (3.4.4): NMR data of compound 16 (Methanol-d4).
No.
13
C, (δ =
ppm)
2
133.6
3
114.4
3a
126.3
4
1
H (δ = ppm), couppling
HMBC correlations
constant (J= Hz)
HÆ C
δ 8.19 (1H, s )
3, 3a, 7a
122.0
8.23 (1H, dd, J = 6.94, 1.26 Hz)
6, 3a, 7a
5
123.2
7.19 (1H, dt, J = 6.94, 1.26 Hz)
7, 3a
6
124.2
7.22 (1H, dt, J = 6.94, 1.26 Hz)
4, 7a, 7
7
113.0
7.44 (1H, dd, J = 6.94, 1.26 Hz)
5
7a
138.1
8
196.2
9
66.3
4.73(2H, s )
8, 3
172
Results
3.4.5- Isohyrtiosine A (17, New compound):
O
O
168.0
7.44
106.3
HO
3.81
51.3
107.5
153.7
128.4
6.74
113.6
132.7
113.4
7.24
7.91
133.4
N
H
C10H9NO3
Exact Mass: 191,06
Mol. Wt.: 191,18
400 ms050811 #8
mAU
ih-mixf
UV_VIS_1
WVL:235 nm
1 - 17,284
70,0
300
Peak #1
%
213.5
200
17.38
241.5
285.9
100
2 - 47,280
min
-50
0,0
10,0
20,0
30,0
40,0
50,0
60,0
nm
-10,0
200
400
Fig (3.4.18):HPLC chromatogram and UV-spectrum of compound 17.
Yield : 3.0 mg
160
O
O
132
HO
N
H
m /z
Fig (3.4.19): EI-MS of compound 17
173
191
595
Results
Compound 17, isohyrtiosine A, [5-hydroxy-1H-indole-3-carboxylic acid methyl ester]
was isolated as a transluscent needle crystals. Compound 17 shows positive pseudomolecular
ion peak at m/z 192 [M+1]+ and negative pseudomolecular ion peak at m/z 190 [M-1]+ and EIMS of 17 showed molecular ion peak of m/z 191 [M]+ suggesting the molecular formula
C10H9NO3. The UV (MeOH) spectrum of 13 showed an absorption maxima 213, 241 and
286 nm, (slightly deviated from those of 13 „Hyrtiosine A“). The 1H NMR (CD3OD) showed
5 proton resonances at δ 7.91 (1H, s ; H-2), 7.44 (1H, d, J = 2.21 Hz; H-4), 6.74 (1H, dd, J =
2.21,8.83 Hz; H-6), 7.24 (1H, d, J = 8.83 Hz; H-7), 3.81(2H, s ; H-9). Although the
molecular weight is identical to that of hyrtiosine A, the
13
C NMR spectrum shows some
differences in resonances from those of hyrtiosine A, including the presence of a carboxyl
resonance at 168.01 ppm instead of the keto-carbonyl at 195.3 ppm and the methoxy carbon
at 51.27 ppm replacing the OCH2 at 67.1 ppm which were confirmed by its DEPT spectrum.
The other 13C NMR resonances seems to be similar to those of hyrtiosine A (compound 13),
see table (3.4.5). The attachment of the methoxy group to the carbonyl is also confirmed by
HMBC correlation between CH3-9 and C-8 at 168.1 ppm. Other HMBC correlations were
presented in table 3.4.5 and explained in figure 3.4.22. The NMR data and the UV absorption
maxima identified 17 as isohyrtiosine A (5-Hydroxy-1H-indole-3-carboxylic acid methyl
ester). To the best of our knowledge this is the first report of 17 as a natural product.
9
6,7
5
8
00
190
180
170
2
160
150
7a
3a
140
130
4
3
120
110
100
90
80
70
60
50
40
30
20
10
(ppm)
O
4
HO
5
3a
9
O
8
3
7a
7
195
185
175
165
4
2
2
6
6,7
9
1
N
H
155
145
135
125
115
105
95
85
75
65
55
45
Fig (3.4.20):13C NMR (up) and DEPT (down) spectra of compound 17
174
35
25
15
5
2
3364
3367
1925.9
7
3373
3375
3364.8
3367.3
3373.6
3375.8
3619.2
3628.0
3720.1
3722.3
4
3619
3628
3720
3722
3928
3928.5
Results
9
O
6
O
8
4
7.80
7.70
7.60
7.50
7.40
7.30
5
3a
7.20
7.10
7.00
6.90
6.80
2
6.70
6
(ppm)
7a
9.0
8.0
1
N
H
3.1135
1.0635
1.1139
0.9918
1.0000
Integral
7
10.0
9
3
1.0635
1.1139
0.9918
Integral
1.0000
HO
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Fig (3.4.21):1H NMR spectrum of compound 17
2
4
7
6
(ppm)
H-2/C-3
H-4/C-3
H-2/C-3a
H-6/C-4
H-4/C-6
120
H-7/C-3a
H-2/C-7a
H-6/C-7a
H-4/C-7a
H-6/C-5
140
O
H-4/C-5 H-7/C-5
4
HO
5
3a
6
7a
7
8.0
9
3
160
2
H-2/C-8
(ppm)
O
8
7.6
7.2
6.8
1
N
H
180
6.4
Fig (3.4.22): HMBC spectrum of compound 17
Table (3.4.5): NMR data of cpompound 17 (Methanol-d4).
No.
2
3
3a
4
5
6
7
7a
8
9
13
C, (δ = ppm)
133.41
107.5
128.4
106.27
153.7
113.6
113.4
132.7
168.01
51.27
1
H (δ = ppm), couppling constant (J=
HMBC correlations (HÆ C)
Hz)
δ 7.91 (1H, s )
3, 3a, 7, 8
7.44 (1H, d, J = 2.21 Hz)
3, 5, 7a, 6
6.74 (1H, dd, J = 2.21& 8.82 Hz)
7.24 (1H, d, J = 8.82 Hz)
4, 5, 7a
5, 3a
3.81 (3H, s )
8
175
Results
3.4.6- 16-Hydroxyscalarolide (18, Known compound):
O
O
175.6
HO
1.88, 1.49
25.64
1.79, 0.83
0.84
15.92
57.95
75.54
3.6
0.9
17.4
1.18
16.72
162.25
42.74
4.5
67.9
54.25
28.15
39.6
1.60, 1.44
18.5
37.4
H
37.0
56.67
33.2
H
0.77
21.27
0.81
33.27
0.84
18.1
OH
2.21, 1.7
H
1.15
0.88
1.09,1.38
42.03
4.81, 5.00
70.71
136.93
41.6
1.81, 0.92
1.57, 1.41
C25H38O4
Exact Mass: 402,28
Mol. Wt.: 402,57
350 ms051011 #3
mAU
ihbffa2
300
UV_VIS_1
WVL:235 nm
70,0
Peak #10
%
33.43
No spectra library hits found!
6 - 33,371
50,0
216.7
200
25,0
100
7 - 34,354
0
-50
0,0
590
8 - 44,083
3 - 1,215
45 - 1,358
1,420
1 2- 0,317
- 1,108
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
Fig (3.4.23):HPLC chromatogram and UV-spectrum of compound 18.
Yield : 3.5 mg
[M+H-(2H 2 O)] +
[M+H-(H 2 O)] +
[M+H] +
[M+Na]+
Fig (3.4.24):FAB-MS spectrum of compound 18.
176
595
Results
Compound 18 [16-hydroxyscalarolide] was isolated as a colourless solid with [α]D of
- 23° (c 0.18 CHCl3). Compound 18 has UV absorption at λmax 216 nm and a molecular
formula of C25H38O4, as established from its FAB MS and
13
C NMR data. FABMS of the
compound 18 showed pseudomolecular ion peak m/z 425 [M+ Na]+ and pseudomolecular ion
peak m/z 403 [M+ H]+ suggesting the molecular formula C25H38O4.
1
H NMR spectrum (Table 3.4.6) displayed resonances for 38 protons including five
singlets assigned to five methyl groups at δ 0.81, 0.84, 0.9, 0.84, and 1.18, eight methylenes,
and five sp3 methines. The 13C NMR spectrum revealed signals for 25 carbons including five
methyls, eight methylenes, two downfield sp3 methines, three upfield sp3 methines, and four
sp3 quaternary carbons and three sp2 quaternary carbons. Analysis of the 1H,1H-COSY led to
the assembly of the following structural fragments: C-1 to C-3; C-5 to C-7; C-9 to C-12 with
a hydroxyl group at C-12; and C-14 to C-16 with an hydroxyl group at C-16. The proton
signals at 4.81(H-24a) and 5.00 (H-24b) (J24a/b = 17.97 Hz) together with the carbon
resonances at 70.71 (CH2, C-24), 162.25 (qC, C-17), 136.93 (qC, C-18), and 175.6 (qC, C-25)
were representative of an α,β-unsaturated butenolide moiety (Youssef et al 2005, Pettit et al
1998b, Miyaoka et al 2000). This was supported by interpretation of the HMBC spectrum
(Table 3.4.6), which indicated that C-16 and C-24 were connected to the sp2 quaternary
carbon C-17, with these situated, along with C-18 and C-25, in the α,β-unsaturated-γ-lactone
ring (Youssef et al 2005, Pettit et al 1998b, Miyaoka et al 2000). Connectivities of the five
ring systems of 18, as well as the assignments of all quaternary carbons, were supported
unequivocally by HMBC data. In addition, the placements of the OHs at C-12 and C-16, were
secured from HMBC correlations of H-11/C-12, H-16/C-17, and H-16/C-18. Moreover,
COSY coss-peaks of H-12/H-11a, H-12/H-11b, H-16/H-15a, and H-16/H-15b supported these
assignments. The partial structures and the above α,β-unsaturated-γ-lactone (C-17, C-18, C-24
and C-25) were found to be connected through quaternary carbons based on HMBC data; also
observed were the cross-peak: Me-20/C-3, C-5, C-19; Me-19/C-3, C-5, C-20; Me-22/C-1, C5, C-9; Me-21/C-7, C-9, C-14; Me-23/C-12, C-14, C-18, and H-16/C-14, C-17, C-18, so that
the tetracarbocyclic scalarane skeleton could be constructed. The planar structure of 18 was
thus determined. All trans junctions of the tetracyclic rings (A, B, C, and D) were
demonstrated from
13
C NMR chemical shifts of methyl groups (δC 15.92, 16.7, 17.4, 21.27,
and 33.27), (Miyaoka et al 2000). The NMR spectra of 18 were closely related to those of
known scalarane sesterterpenoids [(scalarolide, seterstatins 1-7, and 16-hydroxyscalarolide),
(Walker et al 1980, Pettit et al 1998b, Pettit et al 1998c and Pettit et al 2005, Youssef et al
2005, Miyaoka et al 2000)]. Compound 18 seems to be identical with the 16177
Results
hydroxyscalarolide see the comparative NMR data, table 3.4.6. The planar structure of 18 was
supported by COSY, and HMBC spectra. The equatorial geometry of OH at C-12 was
established from the large coupling constant of H-12 (J = 11.04 Hz), (Youssef et al 2005,
Pettit et al 1998b). Similarly, the β-configuration of the hydroxyl at C-16 was established
from the coupling constant (J = 9.45 Hz) (Youssef et al 2005, Pettit et al 1998b). From the
above discussion, compound 18 was assigned as 16-hydroxyscalarolide and its spectral data is
identical with those of 16-hydroxyscalarolide isolated from the Okinawan sponge H. erectus
(Miyaoka et al 2000).
Table ( 3.4.6 ): NMR data of compound 18 (CDCl3, 500 MHz)
No.
16-Hydroxyscalarolide
(Miyaoka et al 2000)
13
1
C (mult.)
H [mult., J (Hz)]
1
39.7 (CH2)
2
18.2 (CH2)
3
42.0 (CH2)
4
5
6
33.3 (C)
56.7 (CH)
18.6 (CH2)
7
41.7 (CH2)
8
9
10
11
37.0 (C)
58.0 (CH)
37.4 (C)
25.7 (CH2)
12
75.5(CH)
12-OH
13
14
15
42.8 (C)
54.3 (CH)
28.2 (CH2)
16
17
18
19
67.9 (CH)
162.3 (C)
136.93 (C)
21.3 (CH3)
20
0.78 (1H, m), 1.72
(1H, br d, 13.0)
1.42 (1H, m),1.62
(1H, m)
1.09 (1H, m), 1.38
(1H, m)
0.75 (1H, m)
1.40 (1H, m), 1.58
(1H, m)
0.92 (1H, m), 1.80
(1H, td, 3.3, 12.4)
0.88 (1H, m)
1.49 (1H, dd, 2.1,
13.2), 1.88 (1H, m)
3.64 (1H, dd, 4.5,
10.9)
5.75 , s
1.14 (1H, m)
1.66 (1H, dd, 2.1,
12.4), 2.23 (1H, dd,
6.7, 12.5)
4.50 (1H, m)
compound 18
13
C (mult.)
1
H [mult., J (Hz)]
HMBC
39.6, CH2
1.79, m, 0.83, m
C-10
18.5, CH2
1.60, m, 1.44, m
C-4, C-10
42.0, CH2
1.09, m, 1.38, ddd
(13.5, 13.5, 4.0)
C-4
33.2, qC
56.67, CH
18.1, CH2
0.77, m
1.57, m, 1.41, m
C-4
41.6, CH2
1.81, m, 0.92, m
C-8
0.88, m
C-10, C-12
1.49, ddd (11.5, 4.2,
1.8) 1.88, m
3.60, dd (11.5, 4.4)
C-12
37.0, qC
58.0, CH
37.4, qC
25.7, CH2
75.5, CH
C-13
5.75, s
42.8, qC
54.25, CH
28.2, CH2
1.15, m
1.7, m; 2.21, dd (11.7,
7.1)
C-8, C-13, C-16
C-16
4.50, dd (9.45, 6.93)
C-17, C-18
0.81 (3H, s)
67.9, CH
162.25, qC
136.93, qC
21.27, CH3
0.81, s
33.3 (CH3)
0.84 (3H, s)
33.27, CH3
0.84, s
21
17.4 (CH3)
0.90 (3H, s)
17.4, CH3
0.9, s
22
23
15.9 (CH3)
16.7 (CH3)
0.84 (3H, s)
1.19 (3H, s)
15.92, CH3
16.72, CH3
0.84, s
1.18, s
24
70.7 (CH2)
4.81 (1H, d, 17.8),
4.99 (1H, d, 17.8)
70.7, CH2
4.81, dd (17.87, 1.27)
5.0, d (17.97)
C-4,C-3, C-5, C20
C-4, C-3, C-5, C19
C-8, C-14, C-9,
C-7
C-1, C-10, C-9
C-12, C-13, C18,C-14
C-17, C-18, C-25
25
175.6 (C)
175.6, qC
178
0
190
180
4
19
25
170
160
22
2
10
3
5
H
5.6
9
H
150
5.2
HO
11
21
8
17
140
4.8
23
12
13
1
14
15
130
120
12
4.4
4.0
3.6
110
100
179
3.2
90
2.8
O
16
6
80
Fig(3.4.26): 13C NMR and DEPT spectra of compound 18
2.4
2.0
12 16
70
1.6
60
1.2
7
24
3,7
20
50
0.8
9,5,14
20
10,8
1
40
30
18.5553
18.1982
17.4039
16.7189
15.9245
16
21
21.2735
24a
23
25.6532
12-OH
25
28.1527
O
3.2823
20
OH
6.0462
O
33.2685
6.0
8
H
2.9414
H
16
2.7876
15
42.7494
42.0352
41.6562
39.6741
37.4150
37.0142
5
14
5.2650
1
7.7053
19
H
13
2.9784
4
21
1.0471
12
57.9508
56.6755
54.2488
6.4
10
23
67.8908
11
1.0031
3
9
0.9415
2
0.9426
HO
70.7110
6.8
0.9500
0.9110
22
75.5425
7.2
136.9386
7.6
162.2550
175.5981
Integral
405.48
415.88
420.61
450.56
592.74
894.77
897.60
900.76
907.06
909.90
913.68
922.51
925.03
934.49
1104.10
1111.04
1116.71
1123.02
1812.82
1817.23
1823.53
1827.95
2245.67
2252.29
2255.13
2262.07
2398.58
2416.86
2482.44
2487.48
2505.45
2903.63
Results
22,
20
18
24
17
19
6
7
24b
15a
(ppm)
0.4
0.0
Fig(3.4.25): 1H NMR spectrum of compound 18
25
O
18
24
17
19
23,21,22
H
OH
4
15
11
2,6
13
18
20
10
Results
24b
24a
12-OH
16
12
15a
(ppm)
1.00
12/11b
16/15b
12/11a
2.00
16/15a
3.00
4.00
5.00
6.00
7.00
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig(3.4.27): H-H COSY spectrum of compound 18
23
15a
20,22
21
19
14
(ppm
Methyls
direct correlations
20
H-14/C-8
14
direct correlation
40
H-23/C-13
H-23/C-14
H-21/C-14
H-15/C-16
H-14/C-16
H-11a / 12-C
H-22/C-9
60
H-21/C-9
H-23/C-12
80
O
O
25
HO
23
12
11
22
13
21
9
14
16
15
10
3
H
5
4
19
H
8
10
17
1
2
24
18
OH
H
7
6
12
20
H-23/C-18
14
(ppm)
2.0
1.6
1.2
0.8
Fig(3.4.28): Part of HMBC spectrum of compound 18 showing H/C direct and H/C long
correlations of the methyl groups.
180
Results
24b
24a
16
(ppm)
40
60
H-24a/C-24
direct correlation
80
O
O
25
HO
23
11
22
13
21
9
17
14
16
15
1
2
10
3
H
5
4
H
8
100
24
18
12
120
OH
H
7
6
H-24b, H-24a/C-18
19
140
20
160
H-24b, H-24a/C-17
(ppm)
5.6
5.2
4.8
4.4
Fig(3.4.29): Part of HMBC spectrum of compound 18 showing H/C direct and H/C long
correlations of the methylene group (CH2-24).
181
Results
3.4.7- Scalarolide (19, Known compound):
O
175.9
5.94
HO
25.7
0.84
15.9
57.98
1.18
16.7
75.65
3.6
0.89
17.4
4.81, 5.00
72.1
135.8
162.1
42.2
25.2 2.39
55.2
17.2
39.7
18.5
37.4
H
56.65
42.0
33.2
21.27
0.81
O
H
37.2
H
41.7
18.2
C25H38O3
Exact Mass: 386,28
33.2
0.84
450 ms051004 #7
mAU
ihbfcf
UV_VIS_1
WVL:235 nm
60,0
8 - 35,654
Peak #8
%
220.1
35.70
40,0
300
200
20,0
9 - 36,180
100
342.2
4
5 -- 1,150
1,184
--0,939
1,267
1,080
1236-7--0,572
1,448
min
-50
0,0
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
400
Fig ( 3.4.30 ):HPLC chromatogram and UV-spectrum of compound 19.
Yield : 5 mg
[M+H-(H2O)]+
[M+H]+
[M+Na]+
Fig (3.4.31):FAB-MS spectrum of compound 19.
182
595
Results
Compound 19 [scalarolide] was isolated as a brownish white amorphous solid with
[α]D of + 23° (c 0.2 CHCl3). Compound 19 has a UV absorption at λmax 220 nm and a
molecular formula of C25H38O3, as established from its FAB MS and 13C NMR data. FAB MS
of compound 19 showed pseudomolecular ion peak m/z 409 [M+ Na]+ and pseudomolecular
ion peak m/z 387 [M+ H]+ suggesting the molecular formula C25H38O3.
1
H NMR spectrum (Table 3.4.7) displayed resonances for five methyls at δ 0.81, 0.85,
0.89, 0.85, and 1.13, one downfield sp3 methine at δ 3.67 (dd, J=11.0, 4.4 Hz) and downfield
methylene at δ 4.69 (1H, d, J=16.8 Hz) and 4.68 (1H, d, J=17.3 Hz) and one OH broad
singlet at δ 5.94. The
13
C NMR spectrum revealed signals for 25 carbons including five
methyls, eight upfield methylenes, one downfield methylene at δ 72.1 three upfield sp3
methines, one downfield sp3 methine (oxygenated carbon) at δ 75.6, and four sp3 quaternary
carbons and three sp2 quaternary carbons. The presence of five singlet methyl signals at δ 0.81
(3H), 0.85 (6H), 0.89 (3H), and 1.13 (3H) suggested the pentacyclic scalarin derivative
(Walker et al 1980), the presence of α,β-unsaturated-γ-lactone was evident from the
13
C
chemical shift of carbons resonating at δ 72.1, 162.1, 135.8 and 175.9 corresponding to C-24,
C-17, C-18, and C-25 respectively which also confirmed by HMBC correlations between the
methylene protons at δ 4.69 (2H, d, J=16.8 Hz; d, J=17.3 Hz) and three downfield sp2
quaternary carbons resonating at δ 162.1, 135.8 and 175.9. The HMBC correlation (3J
coupling) between the methyl proton at δ 1.13 (3H, s, CH3-23 ) and the sp2 Quaternary
carbon resonating at δ 135.8 and an oxygenated sp3 methine carbon at 75.5 (C-12) further
indicaterd that the compound 19 seems to be very similar to compound 18 except the loss of
one oxygenated methine carbon (C-16) and consequently the presence of one upfield methine
carbon more than those present in compound 18. Compound 19 is smaller than 18 by 16 mass
unit which could be demonstrated by the loss of an oxygen atom from position C-16 as
demonstrated above. The 1H and 13C NMR data of Compound 19 was found to be identical
with those of scalarolide that isolated before (Walker et al 1980) from the sponge Spongia
idia .
183
Results
Table ( 3.4.7 ): NMR data of compound 19 (CDCl3, 500 MHz)
scalarolide
compound 19
(Walker et al 1980)
No.
13
C (mult.)
1
H [mult., J (Hz)]
13
C (mult.)
1
39.6 (CH2)
39.7 (CH2)
2
18.5 (CH2)
18.5 (CH2)
3
42.0 (CH2)
42.0 (CH2)
4
33.2 (C)
33.2 (C)
5
56.6 (CH)
56.6 (CH)
6
18.1 (CH2)
18.2 (CH2)
7
41.7 (CH2)
41.7 (CH2)
8
37.2 (C)
37.2 (C)
9
57.9 (CH)
57.9 (CH)
10
37.3 (C)
37.4 (C)
11
25.7 (CH2)
25.7 (CH2)
12
75.5(CH)
3.64 (1H, dd, 4.5, 10.9)
75.6 (CH)
5.75 , s
12-OH
1
H [mult., J (Hz)]
3.64 (1H, dd, 4.5, 10.9)
HMBC
C-13
5.75 , s
13
42.1 (C)
42.2 (C)
14
55.1 (CH)
55.2 (CH)
15
17.1 (CH2)
17.2 (CH2)
16
25.2 (CH2) 2.39 (1H, m)
25.2 (CH2)
17
162.0 (C)
162.1 (C)
18
135.8 (C)
135.8 (C)
19
21.2 (CH3) 0.81 (3H, s)
20
2.39 (1H, m)
C-18
21.2 (CH3)
0.81 (3H, s)
C-4,C-3, C-5, C-20
33.2 (CH3) 0.85 (3H, s)
33.2 (CH3)
0.85 (3H, s)
C-4, C-3, C-5, C-19
21
16.3 (CH3) 0.89 (3H, s)
16.4 (CH3)
0.89 (3H, s)
C-8, C-14, C-9, C-7
22
15.9 (CH3) 0.85 (3H, s)
15.9 (CH3)
0.85 (3H, s)
C-1, C-10, C-9
23
16.6 (CH3) 1.13 (3H, s)
16.7 (CH3)
1.13 (3H, s)
C-12, C-13, C-18,C-14
24
72.0 (CH2) 4.69 m
72.1 (CH2)
4.68 (d, J=17.3 Hz),
4.69 (d, J=16.8 Hz)
25
175.9 (C)
175.9 (C)
184
C-17, C-18, C-25
1826.06
1830.47
1837.09
1840.87
2321.65
2338.99
2349.08
2365.16
Results
2Me- 22, 20
Me-19
Me-23
Me-21
CH2-24
CH-12
12-OH
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
1
O
10
3
5
4
19
H
OH
180
3
5
4
19
170
160
H
H
8
7
6
180
170
Comp. 19
C-24
25.7188
25.2742
21.2808
18.5626
18.2419
17.2144
16.7699
16.4347
15.9756
33.2539
150
140
15.9
16.7
17.4
18.1
18.5
21.2
25.6
28.1
C-16
20
C-11
C-18
C-17
160
33.2
C-12
H
130
120
110
100
90
80
70
60
50
40
30
20
10
C-11,
C-16
C-12
C-24
C-25
37.0
37.4
39.6
41.6
42.0
42.7
54.2
16
15
10
20
C-17
14
1
2
Comp. 18
6
21
9
17
56.6
22
24
13
57.9
16
15
H
7
C-25
0
14
8
H
11
O
18
12
17
136
21
9
162
175
22
1
2
13
23
67.8
18
12
25
OH
24
70.7
23
75.5
25
11
42.1809
42.0498
41.7073
39.7032
37.4004
37.2255
O
O
HO
57.9873
56.6537
55.1816
75.6518
72.1320
135.8164
162.1457
175.9844
Fig (3.4.32) H NMR spectrum of compound 19
C-18
150
140
130
120
Fig (3.4.33):comparison between
110
13
100
90
(ppm)
80
70
60
50
40
30
20
C NMR spectra of compounds 18 and 19.
185
10
Results
(ppm)
H-23/C-13
40
O
25
OH
23
12
11
22
21
9
H-23/C-14
24
18
13
17
14
16
H-24/C-24
direct correlation
80
15
1
2
O
10
3
H
5
4
H
8
7
6
H-23/C-12
H
Comp. 19
120
19
20
H-24/C-18
H-23/C-18
H-24/C-17
H-12/C-25
W-correlation
H-24/C-25
(ppm)
7.00
6.00
5.00
4.00
3.00
160
2.00
Fig (3.4.34):HMBC spectrum of compounds 19.
186
1.00
Results
3.4.8- 12-O-deacetyl-12-epi-scalarin (20, Known compound):
HO
O
HO
26.1
0.85
16.6
0.93
9.1
80.5
3.57
136.3
6.86
23.6
18.0
H
37.4
39.9
H
O
127.3
52.7
39.9
2.38, 2.32
56.5
41.5
33.3
21.3
0.83
220 ms051011 #4
mAU
168.0
58.8
2.55
37.4
0.86
16.8
58.7
98.6
5.67
18.5
H
33.3
0.81
42.00
C25H38O4
Exact Mass: 402,28
Mol. Wt.: 402,57
ihbffa3
UV_VIS_1
WVL:235 nm
10 - 34,369
150
60,0
Peak #10
%
221.9
34.41
40,0
100
20,0
50
- 15,906
8 9- 15,028
5 - 1,213
67 -- 1,344
1,421
0,635
4-- -0,371
1,099
1,047
123
-20
0,0
11 - 44,101
12 - 46,836
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
500
Fig (3.4.35):HPLC chromatogram and UV-spectrum of compound 20.
Yield : 1.5 mg
Fig (3.4.36):FAB-MS spectrum of compound 20.
187
595
Results
Compound 20 [12-O-deacetyl-12-epi-scalarin] was isolated as a white solid with
[α]D of - 15° (c 0.1 CHCl3). Compound 20 has a UV absorption at λmax 221 nm and a
molecular formula of C25H38O4, as established from its EIMS and 13C NMR data. EIMS of the
compound 20 showed molecular ion peak m/z 402 [M]+, m/z 384 [M-H2O]+, m/z 356 [MCH2O2]+ , and m/z 191 [rings A+B+ 4(CH3)]+suggesting the molecular formula C25H38O4.
The 1H and 13C NMR spectra measured in CDCl3 (Table 3.4.8) suggested the scaralane-type
sesterterpenoid. 1H NMR spectrum displayed five singlets assigned to five methyl groups at δ
0.81, 0.83, 0.85, 0.86 and 0.93. One olefinic proton at δ 6.86 (1H, q, 3.1 Hz, H-16), 2
oxygenated methines at δ 5.67 (1H, d, 6.6Hz, H-19) and 3.57 (1H, d, 4.4, 11.7 Hz, H-12) were
also observed. The carbon signals in the low-field region at 98.6 (d, C-19), 127.3 (s, C-17),
136.3 (d, C-16), and 168.0 (s, C-20) were reminiscent of those of scalarin (Tsukamoto et al
2003). H-12 was assigned as axial on the basis of the coupling constant of 11.7 Hz between
H-11 and H-12. The Mol Wt of 20 is identical with that of 18 (both are 402 mass units),
showing the same formula (C25H38O4), the bresence of one olefenic proton resonating at 6.86
ppm indicated that the presence of double bond in the lactone ring should be different from
that of compound 18 and more likely to be between C-16 and C-17 (instead of C-17 and C-18
as in compound 18). This was confirmed by the coupling constant of H-16 (q, J= 3.1 Hz) due
to the adjacent methylene at δ 2.38 and 2.32. furthremore the assignment at the position 18 as
methine (δ 2.55, br s) was evident from the adjacent doublet (H-19, d, J = 6.6 Hz). The NMR
data of 20 were similar to those of 12-epi-scalarin (Tsukamoto et al 2003 and Cimino et al
1977) ) and suggested that 20 was a deacetyl derivative of 12-epi scalarin and identical with
the previously published 12-O-deacetyl-12-epi-scalarin which isolated from Spongia sp.
3.60
6.6
6.4
6.2
6.0
5.8
405.48
415.25
423.76
431.64
465.69
1157.38
1161.48
1166.52
1170.62
1177.56
1181.66
1186.70
1190.80
1261.73
1265.52
1271.19
1275.60
415.25
431.64
423.76
0.84
(ppm)
4.1103
3.2517
3.3616
3.9766
0.88
0.80
0.76
15-a & -b
18
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
Fig (3.4.37):1HNMR spectrum of compound 20.
188
2.8
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
4.1103
3.2517
3.3616
2.4
3.9766
2.6
3.1927
12
1.0772
5.6
405.48
1779.40
465.69
3.1927
0.92
1.6276
6.8
1783.81
1791.06
Integral
0.96
0.9991
1.0000
Integral
7.0
3.55
(ppm)
19
16
7.2
(ppm)
21
24
1.2105
(ppm)
1779.40
1.0772
0.9991
1.0000
6.88
23 22
25
12
16
7.4
1783.81
1795.16
19
1791.06
1795.16
2866.43
2873.05
2866.43
2873.05
3433.90
3437.06
3440.52
3443.99
3433.90
3437.06
3440.52
3443.99
(Tsukamoto et al 2003).
0.6
9.089
16.55
16.76
18.01
18.53
21.28
23.55
26.06
33.26
37.38
37.42
39.87
39.91
41.46
42.00
52.69
56.44
58.69
58.79
80.46
98.59
127.3
136.2
Results
HO
O
HO
19
25
20
18
12
11
23
17
24
9
16
14
15
1
2
10
H
5
3
4
22
O
13
8
H
7
H
6
21
18,
14
9 5
12
16
140
19
17
130
120
110
100
90
80
70
60
7
50
21,
4
1,
8 10,
3 13
40
24,
22 6, 23
2
11 15
30
20
25
10
Fig (3.4.38):13CNMR spectrum of compound 20.
Table ( 3.4.8): NMR data of compound 20 (CDCl3, 500 MHz)
No.
12-O-deacetyl-12-epi-scalarin
(Tsukamoto et al 2003)
(DMSO-d6)
13
1
C (mult.)
H [mult., J (Hz)]
1
40.2 (CH2)
2
18.2 (CH2)
3
41.3 (CH2)
4
5
6
33.5 (C)
56.5 (CH)
18.7 (CH2)
7
42.2 (CH2)
8
9
10
11
37.5 (C)
58.7 (CH)
37.4 (C)
27.4 (CH2)
12
13
14
15
78.6(CH)
37.4 (C)
52.9 (CH)
24.1 (CH2)
compound 21
(CDCl3)
13
1
C (mult.)
0.80 (1H, m),
1.62 (1H, m)
1.57 (1H, m),
1.63 (1H, m)
1.10 (1H, m),
1.33 (1H, m)
0.79 (1H, m)
1.38 (1H, m),
1.46 (1H, m)
0.88 (1H, m),
1.63 (1H, td, 3.3, 12.4)
39.9 (CH2)
*
18.0 (CH2)
*
41.5 (CH2)
*
33.3 (C)
56.5 (CH)
18.5 (CH2)
*
*
42.0 (CH2)
*
39.9 (C)
58.7 (CH)
37.4 (C)
26.1 (CH2)
0.80 (1H, m)
1.30 (1H, dd, 2.1, 13.2),
1.55(1H, m)
3.39 (1H, dd, 4.4, 12.0)
80.5(CH)
37.4 (C)
52.7 (CH)
23.6 (CH2)
1.20 (1H, m)
2.12 (1H,m),
2.25 (1H, m)
6.67 (1H, q, 2.9)
H [mult., J (Hz)]
*
*
3.57 (1H, dd, 4.4, 11.7)
*
2.38 (1H,m),
2.32 (1H, m)
6.86 (1H, q, 3.1)
135.7(CH)
136.3(CH)
16
128.9 (C)
127.3 (C)
17
58.1 (CH)
2.40 br s
58.8 (CH)
2.55 br s
18
100.5(CH)
5.72 (1H,t 5.9)
98.6(CH)
5.67 (1H,d, 6.6)
19
167.5 (C)
168.0 (C)
20
33.7 (CH3)
0.63 (3H, s)
33.3 (CH3)
0.81 (3H, s)
21
21.7 (CH3)
0.77 (3H, s)
21.3 (CH3)
0.83 (3H, s)
22
16.4 (CH3)
0.80 (3H, s)
16.6 (CH3)
0.85 (3H, s)
23
16.8 (CH3)
0.81 (3H, s)
16.8 (CH3)
0.86 (3H, s)
24
9.8 (CH3)
0.83 (3H, s)
9.1 (CH3)
0.93 (3H, s)
25
* chemical shifts can not be precisely characterized from 1HNMR spectrum.
189
0
Results
3.4.9- 7-dehydrocholesterol peroxide (21, Known compound):
0.87
18.55
35.92
0.77
12.6
35.2
56.40
23.39
27.97
28.22
51.55
51.04
22.79
0.84
20.59
34.67
36.89
30.09
3.94
66.47
82.14
HO
36.92
2.12,1.85
79.45
130.75
135.36
6.45
6.22
C27H44O3
Exact Mass: 416,33
Mol. Wt.: 416,64
O
O
100 ms051004 #12
mAU
0.83
22.52
23.78
39.41
44.71
0.85
18.14
39.41
ihbfck
UV_VIS_1
WVL:235 nm
70,0
8 - 38,395
Peak #8
%
38.32
80
50,0
204.7
60
40
25,0
243.0
4 - 1,123
20
566.7
6 - 1,251
1,284
5
1,075
-- -0,953
12
-370,149
1,440
min
-10
0,0
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
500
Fig ( 3.4.39 ):HPLC chromatogram and UV-spectrum of compound 21
Yield : 4 mg
[M-(O2+H2O)]+
[M-(O2)]+
[M-(O2+CH3+H2O)]+
[M+Na]+
Fig (3.4.40):FAB-MS spectrum of compound 21.
190
595
Results
Compound 21 [5α,8α-epidioxy-cholesta-6-en-3β-ol] was isolated as a white needle
crystals. Compound 21 has a UV absorption at λmax 204 and 243 nm. FAB-MS showed
pseudomolecular ion peak m/z 439 [M+Na]+ and characteristic fragment ions of typical 5α,8αepidioxy-cholesta-6-en-3β-ol (Gauvin et al, 2000) suggesting the molecular formula
C27H44O3. 1H NMR spectrum showed resonances for five methyl groups at δ 0.77 (3H, s, Me18), 0.85 (3H, s, Me-19), 0.87 (3H, d, J=6.5 Hz, Me-21), 0.83 (3H, d, J=6.6 Hz, Me-26), and
0.84 (3H, d, J=6.6 Hz, Me-27). The resonances at δ 3.94 (1H, m, H-3), 6.22 (1H, d, J=8.5
Hz, H-6) and 6.45 (1H, d, J=8.5 Hz, H-7) suggested ∆6, mono hydroxylated 5α,8αepidioxysteroidal compound (Gauvin et al , 2000). This was confirmed by the presence of a
fragment ion at m/z 384 (rel. int., 100 %) [M-O2] through the loss of O2 from the molecular
ion, presumably by a retro Diels-Alder fragmentation (Gunatilaka 1981), and by
13
C NMR
signals at 82.14 and 79.41 of C-5 and C-8 respectively (Yaoita et al 1998, and Yue et al
2001). The β-configuration of hydroxy group at position 3, δ 3.96 (1H, m, H-3) and 66.44
ppm (d, C-3) was suggested by comparison with the published data of [5α,8α-epidioxycholesta-6-en-3β-ol] (Gauvin et al, 2000). From the above discussion, compound 21 was
452.45
445.83
439.84
435.11
432.90
428.49
426.28
398.54
1064.69
1062.80
1059.65
1057.76
1050.82
1049.24
1045.78
1044.20
1842.13
1832.36
2002.29
1997.24
1990.94
1985.89
1980.54
1978.01
1974.55
1969.50
3124.63
3116.43
3257.67
3249.16
assigned as 5α,8α-epidioxy-cholesta-6-en-3β-ol.
21
22
18
12
13
17
18
26
25
16
14
1
2
10
8
5
7
6
4
27
15
9
3
HO
24
23
11
19
21,19,27,26
20
O
O
6.6
6
6.4
6.2
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
Fig(3.4.41): 1H NMR spectrum of compound 21
191
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
2.7143
3.3569
3.5351
3.5921
3.6926
9.4897
9.4172
2.9968
2.8445
2.5838
5.8412
1.5805
0.5055
1.1227
3
1.0273
1.0000
7
0.6
0.4
20.5957
18.5553
18.1472
12.6088
23.3941
22.7892
22.5269
23.7803
28.2256
27.9706
30.0912
35.2069
34.6749
36.9267
36.8976
35.9284
44.7169
39.4117
51.5598
51.0424
56.3986
66.4770
79.4486
82.1376
130.7589
135.3646
Results
21
22
18
23
12
11
19
13
1
5
4
26
25
27
15
9
22,
41
7
6
2, 16
12, 24
8
10
3
HO
24
17
16
14
2
20
O
23,11
15
O
14, 9
20
25
27, 26
21,1918
10
6
190
180
170
160
150
140
7
130
5
120
110
100
90
3
17
8
80
70
60
50
40
30
20
Fig(3.4.42): 13C NMR and DEPT spectra of compound 21
Table (3.4.9): 1H, 13C-NMR data of compound 21 in (CDCl3 , 500, MHz)
Compound 20
5α,8α-epidioxy-cholesta-6-en-3β-ol.
(Guavin et al , 2000)
No.
13
C (Multiplicity)
1
Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
34.74 t
30.16 t
66.52 d
37.00 t
82.24 s
135.46 d
130.83 d
79.54 s
51.10 d
37.00 s
23.46 t
39.49 t
44.80 s
51.63 d
20.68 t
28.32 t
56.46 d
12.69q
18.24q
35.29d
18.64q
36.00 t
23.86 t
39.49 t
28.06 d
22.62 q
22.89 q
13
H (Multiplicity,
C
1
H (Multiplicity, Hz)
(Multiplicity)
34.67 t
30.09 t
66.48 d
36.93 t
82.14 s
135.36 d
130.75 d
79.45 s
51.04 d
36.90 s
23.39 t
39.41 t
44.72 s
51.56 d
20.59 t
28.22 t
56.40 d
12.61q
18.15q
35.21d
18.55 q
35.93 t
23.78 t
39.41 t
27.97 d
22.53 q
22.79 q
3.94 (m)
6.22 (d, J=8.5 Hz)
6.45 (d, J=8.5 Hz)
0.77 ( s)
0.85 ( s)
0.87 (d, J=6.5 Hz)
0.83 (d, J=6.6 Hz)
0.84 (d, J=6.6 Hz)
192
3.94 (m)
6.22 (d, J=8.51 Hz)
6.45 (d, J=8.51 Hz)
0.77 ( s)
0.85 ( s)
0.87 (d, J=6.62 Hz)
0.83 (d, J=6.62 Hz)
0.84 (d, J=6.62Hz)
10
0
Results
Bioactivity:
Many scalarane-type sesterterpenoids have been isolated from marine sponges
belonging to the order Dictyoceratida. Scalarane-type sesterterpenes display a variety of
biological activities such as cytotoxic, antimicrobial, antifeedant, antimycobacterial,
ichthyotoxic, anti-inflammatory, and platelet-aggregation inhibitory effects, as well as nerve
growth factor synthesis-stimulating action (Youssef et al, 2005).
The isolated compounds from the Hyrtios erectus were tested for cytotoxic activity
against L5178Y cells and the results wer summarised in table (3.4.10). Compounds 14, 16,
18 showed mild antimicrobial activity against Bcillus subtilis and Saccharomyces cereviisae.
Table (3.4.10): cytotoxic activity of compounds 13-18 and 21 against L5178Y cells.
Compound
13
14
15
16
17
18
21
Cell growth %
Cell growth %
Cell growth %
(conc. of 3µg/ml)
(conc. of 10µg/ml)
(-ve control)
98.1
100
95.5
119
95.8
59.5
86.4
92.4
96.6
80.5
94.5
64
44.7
77
100
100
100
100
100
100
100
193
Results
3.5. Natural products from Petrosia nigricans
Marine sponges of the genus Petrosia are known as a source of diverse bioactive natural
products. Sponge species of genus Petrosia are rich in polyacetylenic compounds (Faulkner
1998, Faulkner 2002, Watanabe et al 2005), unusual bioactive steroids and steroidal sulphates
acting as anti-HIV, antivirals, and anti-inflammatory (Goud et al 2003, Giner et al 1999,
Reddy et al 1999, Shatz et al 2000, Sun et al 1992, Qureshi and Faulkner 1999).
Many alkaloidal natural products were isolated from genus Petrosia belonging to
various subclasses, examples are bioactive manzamines (Crews et al 1994, Yousaf et al
2002), mimosamycins (Kobayashi et al 1994, Kobayashi et al 1992), cardioactive pentacyclic
hydroquinones (Gorshkova et al 1999), dihydroisoquinolines (Ramesh
et al 1999),
pentacyclic pyridoacridines (Skyler et al 2002, Molinski et al 1988), isoquinoline quinones
(Venkateswarlu et al 1993), 3-alkylpyridinium polymers (Sepcic et al 1997), bis-quinolizidine
alkaloid (Braekman
et al 1982, Braekman
et al 1984), and dihydrotubastrines –
phenethylguanidine (Sperry and Crews 1998). Petrosia–derived fungal strain Penicillium
brevicompactum was reported to produce cyclodepsipeptides (Bringmann et al 2004).
However, this is the first report of Petrosia nigricans-derived natural products. An
Indonesian Petrosia sp. (Petrosia nigricans, family Petrosiidae) was chemically investigated
and several compounds were isolated. These compounds can be divided into the following
chemical classes:
1- steroidal compounds (24ξ-ethyl-cholesta-5-en-3β-ol, 24ξ-ethyl-cholesta-8(9)-en-3β-ol,
5α,8α-epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol)
2- phenylacetic acid derivatives (phenylacetic acid, p-hydroxyphenylacetic acid, phydroxyphenylacetic acid methylester, p-hydroxyphenylacetic acid ethylester, phydroxyphenylacetic acid butylester*)
3- primary metabolites ( adenosine, nicotinamide)
4- cerebrosides. (petrocerebroside 1*, petrocerebroside 2*).
5- purine derivatives (Nigricine 1*, Nigricine 2*, Nigricine 3*, Nigricine 4*)
6- indole alkaloid *
(* = new natural products).
194
Results
3.5.1- 24ξ-Ethyl-cholesta-5-en-3β-ol (22, known compound)
0.85
12.3
0.93
18.8
23.0
33.9
0.68
11.8
36.2
1.01
19.4
39.7
21.1
36.5
56.7
31.9
H
24.3
H
C29H50O
Exact Mass: 414,39
Mol. Wt.: 414,71
140.7
31.9
HO
19.6
0.83
28.2
31.6
3.52
71.8
19.0
0.81
28.9
46.0
56.0
42.3
H
50.1
37.2
26.3
121.7
5.35
42.2
-------------------------------------------------------Yield : 2g
[M-(CH2+H2O)]+
[M]+
[M-H2O]+
Fig (3.5.1):EI-MS spectrum of compound 22
19 18
29
28
22
21
26
24
20
25
23
18
12
11
27
17
13
16
14
15
19
8
1
9
3
7
10
6
HO
5
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
(
3.5
3.0
2.5
)
Fig (3.5.2): 1HNMR spectrum of compound 22 (in CDCl3)
195
2.0
1.5
1.0
2.7120
8.8947
4.0374
3.6896
7.5117
6.9580
9.6489
1.5152
3.0837
2.2593
2.0324
4
0.9684
3
1.0000
Integral
5
Results
Compound 22 [24ξ-ethyl-cholesta-5-en-3β-ol] was isolated as a white amorphous
powder, with [α]D of - 17° (c 0.1.2 CHCl3). EI-MS showed molecular ion peak m/z 414 [M]+
and fragment ions at 396 [M-(H2O)]+, 382 [M-(CH2 + H2O)]+, 368 [M-C2H5OH]+, 273 [Mside chain]+, 255 [M- (side chain+H2O)]+, suggesting the molecular formula C29H50O. 1H
NMR spectrum showed resonances for six methyl groups at δ 0.68 (3H, s, Me-18), 1.01 (3H,
s, Me-19), 0.93 (3H, d, J=6.94 Hz, Me-21), 0.81 (3H, d, J=6.93 Hz, Me-27), 0.83 (3H, d,
J=6.94 Hz, Me-26), 0.85 (3H, t, J=7.56 Hz, Me-29). The resonances at δ 5.35 (1H, m, H-6),
3.52 (1H, m, H-3) were indicative for ∆5-6 mono hydroxylated steroidal nucleus (Itoh et al ,
1983).13C NMR showed six methyl signals at δ 12.3, 11.8, 19.4, 18.8, 19.6 and 19.0 for the
methyl groups 29,18, 19, 21, 26, and 27, respectively.
13
C NMR spectrum showed one sp2
quaternary olefenic carbon resonance at δ 140.7 and one sp2 methine at 121.7, and an
oxygenated methine at 71.8. In addition,
13
C NMR spectrum shows 7 sp3 methines, and 11
aliphatic methylenes (see table 3.5.1). The above EI-MS, and NMR data were identical with
that of 24ξ-ethyl-cholesta-5-en-3β-ol (Wright et al 1978, Younus et al 1974). Compound 22
29
28
22
21
20
6
3
27
17
13
16
14
15
9
25
18
12
11
26
24
23
14, 17
7,8,2
1,10,20
13,4
28
12
15
22
19
9
1
3
5
140
HO
130
120
23
8
10
7
5
6
110
100
90
80
70
60
50
40
30
20
26, 19, 27, 21
24
20 8 25
22
7
16
11
1
Fig (3.5.3): 13CNMR and DEPT spectra of compound 22 (in CDCl3)
196
10
29
18
11.8452
12.3170
18.8171
18.9519
19.3853
19.5875
21.0705
22.9868
24.2964
26.3379
28.2254
28.9091
31.6535
31.9039
33.8876
36.2566
36.4973
37.2388
39.7522
42.2848
42.3040
46.0404
50.1138
56.0072
56.7390
71.8096
121.7205
140.7393
was also isolated from Petrosia (Strongylophora) durissima (Shen and Prakash 2000).
Results
3.5.2- 4α-Methyl-5α-cholesta-8-en-3β-ol (23, known compound)
0.92
18.72
36.14
0.6
11.22
39.5
54.88
28.0
27.45
135.0
51.84
22.81
0.86
23.73
35.08
36.25
31.2
3.1
76.5
HO
128.1
C28H48O
Exact Mass: 400,37
Mol. Wt.: 400,68
28.79
46.88
0.85
22.54
23.92
37.0
42.06
22.74
0.95
18.87
36.25
20.87
39..2
0.99
15.04
-----------------------------------------------------------
Yield : 15 mg
[M]
+
20
26
24
22
21
25
23
18
[M-(side chain+H2+C3H6O)]
27
+
12
17
11
19
9
13
16
14
15
1
10
[M-(side chain+H2O)]
3
+
8
7
5
6
HO
[M-(side chain)]
+
28
[M-(CH3)]
[M-(CH3+H2O)]
+
+
Fig (3.5.4): ES-MS spectrum of compound 23
19
27, 26
18
H2O
21
CHCl3
22
21
20
26
24
28
25
23
18
27
12
17
11
19
9
13
16
14
15
1
10
3
8
7
5
6
HO
28
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
(
3.5
3.0
2.5
)
Fig (3.5.5): 1HNMR spectrum of compound 23 (in CDCl3)
197
2.0
1.5
1.0
3.0000
5.0551
3.7806
3.5928
4.4096
1.2624
1.6507
4.1655
1.6963
10.283
1.4710
3.0901
2.7305
4.4408
1.7699
2.8405
1.5833
1.3219
Integral
3
0.5
12
22
24
17
3
20
26
24
22
21
14
4
5
7
1 2
25
20
16
11
11.2286
15.0420
18.7205
18.8746
20.8776
22.5436
22.7458
22.8132
23.7377
23.9206
27.4548
28.0037
28.7933
31.2007
35.0815
36.1408
36.2564
37.0075
39.2031
39.5016
42.0631
46.8877
51.8470
54.8804
128.1342
135.0098
Results
6
27, 26
25
23
18
27
12
17
11
13
16
14
15
19
9
10
8
9
3
8
5
HO
10
13
1
7
23,
15
6
19, 21 28
18
28
140
130
120
110
100
(
90
80
70
60
50
40
30
20
10
)
Fig (3.5.6): 13CNMR and DEPT spectra of compound 23 (in CDCl3)
Compound 23 [4α-methyl-5α-cholesta-8-en-3β-ol] was isolated as white amorphous
powder, with [α]D of + 44° (c 0.5 CHCl3). EI-MS showed molecular ion peak m/z 400 [M]+
and characteristic fragment ions at 385 [M-CH3], 367 [M- (CH3+H2O)], 287 [M-(side chain)],
269 [M-(side chain+H2O)], 227 [M-(side chain+C3H6O+H2)] suggesting the molecular
formula C28H48O. 1H NMR spectrum showed resonances for six methyl groups at δ 0.6 (3H, s,
Me-18), 0.95 (3H, s, Me-19), 0.92 (3H, d, J=6.31 Hz, Me-21), 0.86 (3H, d, J=6.6 Hz, Me-27),
0.85 (3H, d, J=6.61 Hz, Me-26), 0.99 (3H, t, J=6.31 Hz, Me-28). The presence of the typical
3β- hydroxyl group was indicated by the methine signal at δ 3.1. Furthermore, appearance of
the methine signal as a triple doublet with coupling constant of J=4.7, 9.9, 11.1 Hz indicated
that the methine proton is axial and is coupled to three adjacent hydrogens as in other 4αmethylsterols (König et al, 1998 & Gunasekera et al, 1989). The two axial–axial couplings of
9.9 and 11.1 Hz confirmed the α-equatorial nature of the 4-methyl group. The
13
C-NMR
spectrum revealed only two singlets in the olefenic region (δ128.1 and 135.0) indicating the
presence of tetrasubstituted double bond (Gunasekera et al, 1989). The mass spectrum
showed the characteristic peak at m/z 287 (M-side chain). The above NMR and MS data
confirmed compound 23 as 4α-methyl-5α-cholesta-8-en-3β-ol. The structure of 23 was
confirmed by comparing its spectral data with the literature data reported for the same
compound isolated from Agelas oroides and Agelas flabelliformis (König et al, 1998 &
Gunasekera et al, 1989 respectively).
198
Results
3.5.3. 5α,8α-Epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol (24, known compound)
0.85
12.29
0.92
18.67
22.96
33.66
0.79
12.60
45.99
56.27
28.9
28.24
51.04
51.55
19.56
0.80
20.61
34.66
36.90
30.09
3.96
66.45
82.14
HO
0.82
18.94
26.31
39.39
44.71
23.39
0.88
18.15
35.7
79.44
130.75
135.37
6.49
6.25
36.90
C29H48O3
Exact Mass: 444,36
Mol. Wt.: 444,69
O
O
---------------------------------------------------------------------------
Yield : 25 mg
[M-O2]
[M-(H2O+O2+CH3)]
[M-(O2+ side chain+H2O)]
+
+
+
[M-(O2+C3H7O)]
[M-(O2+ side chain)]
+
+
[M-(H2O)]
+
[M]
+
Me-18
Me-19
Me-29, 26, 27
Me-21
7.0
6.5
5.5
5.0
4.5
4.0
3.5
3.0
Fig (3.5.8): 1HNMR spectrum of compound 24 (in CDCl3)
199
2.5
2.0
1.5
1.0
4.4739
10.389
3.1150
4.0321
4.5610
1.1241
6.0
1.2376
H-3
H-6
0.9535
1.0000
Integral
H-7
398.86
405.48
409.26
416.19
424.39
439.84
451.50
458.12
601.57
605.35
611.34
615.76
1043.89
1045.78
1048.93
1050.51
1057.44
1059.33
1062.49
1064.38
1968.87
1973.92
1978.01
1980.22
1985.58
1990.62
1996.93
2001.97
3116.75
3125.26
3249.79
3258.30
Fig (3.5.7):EI-MS spectrum of compound 24
Results
Compound 24 [5α,8α-epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol] was isolated as white
needle crystals with [α]D of + 4° (c 0.70 CHCl3). EI-MS showed molecular ion peak m/z 444
[M]+ and characteristic fragment ion peaks at m/z 426 [M-H2O], 412 [M-O2], 379 [M-(H2O,+
O2+CH3)], 353 [M-(O2+ C3H7O)], 271 [M-(side chain +O2)] and 253 [M-(side chain + O2+
H2O)] suggesting the molecular formula C29H48O3. 1H NMR spectrum showed resonances for
six methyl groups at δ 0.79(3H, s, Me-18), 0.88 (3H, s, Me-19), 0.92 (3H, d, J=6.7 Hz, Me21), 0.80 (3H, d, J=6.6 Hz, Me-27), 0.82 (3H, d, J=6.90 Hz, Me-26), 0.85 (3H, t, J=7.5 Hz,
Me-29). The resonances at δ 3.96 (1H, m, H-3), 6.25 (1H, d, J=8.51 Hz, H-6) and 6.49 (1H,
d, J=8.51 Hz, H-7) suggested a ∆6 mono hydroxylated 5α,8α-epidioxysteroidal compound
(Gauvin et al , 2000). This was confirmed by the presence of an ion fragment at m/z 412 [MO2] through loss of O2, presumably by a retro Diels-Alder fragmentation (Gunatilaka 1981),
and by 13C NMR signals at 82.14 and 79.41 of C-5 and C-8, respectively (Yaoita et al 1998,
and Yue et al 2001). The β-configuration of hydroxyl group at position 3, δ 3.96 (1H, m, H-3)
and 66.44 ppm (d, C-3) was suggested by comparison with the published data of 3α- and 3βhydroxy steroids (Eggert et al 1976, Wright et al 1978, and Gauvin et al, 2000 ). Although the
NMR data of the position 24 were in comparison with those reported by Wright et al 1978
and Gauvin et al, 2000, the sterochemistry at this position is still undetected because the
chemical shifts of the side chain carbons which were not similar enough to those given for
1
12 4
7
22
20
6
2
23 11,28
16
12.3014
12.6075
18.1532
18.6779
18.9402
19.5742
20.6236
22.9629
23.3928
26.3224
28.2462
28.9021
30.0972
33.6680
34.6737
35.7085
36.9036
39.3959
45.9910
51.0338
51.5512
56.2734
66.4612
130.7577
135.3779
24R or 24S.
15
25
18,29
27,26,21,19
29
28
22
21
20
10,4
26
24
25
23
3
18
27
12
11
16
14
15
12
24
14
8
19
13
9
1
10
3
17
5
17
13
9
8
5
HO
7
O
6
O
135
130
125
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
45
Fig (3.5.9): 13CNMR and DEPT spectra of compound 24 (in CDCl3)
200
40
35
30
25
20
15
10
Results
Table (3.5.1):
1
H- and
13
C-NMR data of compounds 22, 23 and 24 in (CDCl3,
500, MHz)
Compound 22
No.
13
C
1
H (Multiplicity, Hz)
Compound 23
13
1
C
H (Multiplicity, Hz)
Compound 24
13
C
1
H (Multiplicity,
(Multi-
(Multi-
(Multi-
plicity)
plicity)
plicity)
1
37.2 t
35.08 t
34.66 t
2
31.6 t
31.2 t
30.09 t
3
71.8 d
4
42.2 t
39.50 d
36.90 t
5
140.7 s
46.88 d
82.14 s
6
121.7 d
20.87 t
135.37 d
6.25 (d, J=8.51)
7
31.9 t
28.79 t
130.75 d
6.49 (d, J=8.51)
8
31.9 d
128.1 s
79.44 s
9
50.1 d
135.0 s
51.03 d
10
36.5 s
36.25 s
36.90 s
11
21.1 t
22.74 t
23.39 t
12
39.7 t
37.0 t
39.39 t
13
42.3 s
42.06 s
44.71 s
14
56.7 d
51.84 d
51.55 d
15
24.3 t
23.73 t
20.61 t
16
28.2 t
27.45 t
28.24 t
17
56.0 d
54.88 d
56.27 d
18
11.8 q
0.68 ( s)
11.22 q
0.6 ( s)
12.60q
0.79 ( s)
19
20.4 q
1.01 ( s)
18.87 q
0.95 ( s)
18.15q
0.88 ( s)
20
36.2 d
21
18.8 q
22
33.9 t
36.14 t
33.66 t
23
26.3 t
23.92 t
26.31 t
24
46.0 d
39.5 t
45.99 d
25
28.9 d
28.0 d
28.89 d
26
19.6 q
0.83 (d, J=6.94 Hz)
22.54 q
0.85 (d, J=6.61)
18.94 q
0.82 (d, J=6.9 Hz)
27
19.0 q
0.81 (d, J= 6.93Hz)
22.81 q
0.86 (d, J=6.61 Hz)
19.56 q
0.80 (d, J=6.6 Hz)
28
23.0 t
15.04 q
0.99 (d, J=6.31)
22.96 t
29
12.3 q
3.52 (m)
5.35 (m)
76.5 d
3.1 (ddd, J=4.7, 9.9, 11.1)
36.25 d
0.93 (d, J=6.94 Hz)
66.45 d
Hz)
3.96 (m)
35.70 d
18.72 q
0.92 (d, J=6.31)
0.86 (t, J = 6.94 Hz)
18.67 q
12.29 q
201
0.92 (d, J=6.7 Hz)
0.85 (t, J=7.5Hz)
Results
3.5.4- Phenylacetic acid (25, known compound)
1
COOH
2
3
4'
4
5'
5
C8H8O2
Exact Mass: 136,05
Mol. Wt.: 136,15
δΗ−2 3.65 (2H, s)
δΗ(4,5, 6,4', 5') 7.3 (5H, m)
6
--------------------------------------------------------------------
Yield : 6.0 mg
250 ms050920 #8
mAU
tm7et6g
UV_VIS_1
WVL:235 nm
10 - 21,371
5 - 1,203
70,0
200
Peak #10
21.64
No spectra library hits found!
%
213.8
6 - 1,275
150
100
244.8
7 - 1,420
8 - 12,529
50
299.0
9 - 16,127
0
11 - 46,517
4 - 1,113
3 - 1,011
12 - 0,589
0,668
min
-50
0,0
10,0
20,0
30,0
40,0
50,0
-10,0
200
60,0
Fig (3.5.10): HPLC chromatogram and UV spectrum of compound 25
[M-(COOH)] +
[M] +
[M-(CH2COOH)]+
Fig (3.5.11):EI-MS spectrum of compound 25
202
nm
400
595
Results
Compound 25 [phenylacetic acid] was isolated as a white amorphous powder, UV
absorption at λmax 213, 244, 299 nm. EI-MS showed molecular ion peak at m/z 136 [M]+,
and fragment ion peaks at m/z 91 [M-COOH]+, 77 [M-CH2COOH]+, (see figure 3.5.22)
1
suggesting the molecular formula C8H8O2. A deceptively simple
H NMR spectrum
(meassured in CDCl3) showed two sets of protons resonating at δ 3.65 (2H, s) suggesting the
presence of lower field methylene group (i.e., attached to aromatic spin system), and at δ 7.3
(5H, m) suggesting monosubstituted phenyl group. The above NMR data together with the
EI-MS fragments confirmed compound 25 to be phenylacetic acid. 1H-NMR and 13C-NMR of
3641.66
3644.82
3649.23
3664.05
1
COOH
3641.66
3644.82
3649.23
3664.05
3670.98
3678.86
H-4, H-5,
H-6, H-4’
and H-5’
3670.98
3678.86
25 is identical with those reported for phenylacetic acid (Aldrich, 1993, 2/981).
2
CHCl3
3
4'
H-2
7.40
7.36
4
5'
5.3930
Integral
H-4, H-5,
H-6, H-5’
and H-4’
7.32
7.28
5
6
7.24
10.4
10.0
9.6
9.2
8.8
8.4
8.0
7.6
2.0000
5.3930
Integral
(ppm)
7.2
6.8
6.4
6.0
5.6
5.2
4.8
4.4
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
41.0004
127.3339
128.6310
129.3525
133.2294
177.6313
Fig (3.5.12):1HNMR spectrum of compound 25
CDCl3
C-4 & C-4’
C-5 & C-5’
C-2
C-6
C-1
205
195
185
C-3
175
165
155
145
135
125
115
105
95
85
Fig (3.5.13):13CNMR spectrum of compound 25
203
75
65
55
45
35
25
0.0
Results
3.5.5- p-Hydroxyphenylacetic acid (26, known compound)
173.4
COOH
3.4
39.0
C8H8O3
Exact Mass: 152,05
Mol. Wt.: 152,15
126.1
7.03
132.2
113.8
6.72
156.6
OH
------------------------------------------------------
Yield : 5.2 mg
1.400
ms041210 #13
mAU
et13a2
UV_VIS_1
WVL:235 nm
70,0
6 - 9,356
1.000
Peak #69.66
%
50,0
223.0
750
25,0
500
275.0
250
0
-0,574
1,156
12-345-0,229
- 1,083
1,266
562.1
10 - 30,569
7 - 810,274
- 12,513
-911,580
11 - 47,835
min
-200
0,0
10,0
20,0
30,0
40,0
50,0
60,0
10,0
200
nm
300
400
500
Fig (3.5.14): HPLC chromatogram and UV spectrum of compound 26
[M-(COOH)] +
phenyl
[M]+
Fig (3.5.15):EI-MS spectrum of compound 26
204
595
Results
Compound 26 [p-hydroxyphenylacetic acid] was isolated as a white amorphous powder, UV
absorption at λmax 201, 223, 275 nm. EI-MS showed molecular ion peak m/z 152 [M]+, and
fragment ion peaks at m/z 107 [M-COOH]+, 77 [M-(CH2COOH+OH)]+, (see figure 3.5.26)
suggesting the molecular formula C8H8O3. The difference between 25 and 26 is only 16 mass
units which indicates the presence of one oxygen atom in 26 more than 25. Comparable to
compound 25, 1H NMR spectrum of 26 in DMSO-d6 showed three proton resonances at δ
3.38 (2H, s) suggesting the presence of lower field methylene group, attached to an aromatic
spin system and at δ 7.03 (2H, d, J=8.52 Hz, H-4& H-4') and δ 6.72 (2H, d, J=8.2 Hz, H-5&
H-5'), suggesting p-disubstituted phenyl group instead of 5 proton resonances overlaping at
δ7.3 in 1HNMR of compound 25. The NMR data together with the ESI-MS fragments
suggested compound 26 to be p-hydroxyphenylacetic acid, which was confirmed by HMBC
correlations (see figure 3.5.17). The CH2 at 3.38 showed HMBC correlations to C-1, C-3, C-4
and C-4` at δ 173.4, 126.1, and 132.2, while H-4 and H-4` at δ 7.03 showed HMBC
correlations to C-2, C-5, and C-6 at δ 39.0, 113.8, and 156.6, respectively. The NMR data of
26 are identical to those of p-hydroxyphenylacetic acid (Aldrich, 1993, 2/1018). p-
Hydroxyphenylacetic has been previously isolated from many terresterial biological sources [
2
1665.57
1700.25
1787.89
3333.63
3341.83
H-2
OH
1
H-4, H-5,
H-4' H-5'
3
4
4'
3507.34
3515.86
O
4625.59
6085.89
e.g. Mellilotus officinalis and Taraxacum officinale (DNP 2005)].
5
5'
6
16
15
14
13
12
11
10
9
8
7
1.9969
2.0270
0.7733
6-OH
0.7281
Integral
Acidic H
2.0000
OH
6
5
4
Fig (3.5.16):1HNMR spectrum of compound 26
205
3
2
1
0
-1
-2
-3
-4
Results
H-4,
H-4'
H-2
H-5,
H-5'
(ppm)
40
H-4&H-4'
/C-2
O
2
OH
1
60
3
H-4& H-4'
/C-4' &C-4
H-4/C-5
4
5'
5
80
6
H-5&H-5'
C-5' /C-5
OH
100
H-2/C-3
H-5 & H-5'
/C-3
H-4&H-4'
/C-6
4'
120
H-2/C-4
H-2/C-4'
140
H-5 & H-5'
/C-6
160
H-2/C-1
(ppm)
7.2
6.4
5.6
4.8
4.0
3.2
2.4
Fig (3.5.17): HMBC spectrum of compound 26
206
Results
3.5.6- Methyl 2-(4-hydroxyphenyl)acetate (27, known compound)
O
3.65
O
3.51
7.06
6.72
C9H10O3
Exact Mass: 166,06
Mol. Wt.: 166,17
OH
------------------------------------------
Yield : 4.4 mg
350 ms041210 #7
mAU
et13b6
UV_VIS_1
WVL:235 nm
90
Peak #5
%
14.66
5 - 14,629
300
50
200
223.8
7 - 30,121
100
275.5
6 - 15,275
0
34 -- 1,085
1,152
0,911
12- -0,564
562.2
8 - 47,910
min
-50
0,0
10,0
20,0
30,0
40,0
50,0
60,0
-10
200
nm
300
400
500
595
Fig (3.5.18): HPLC chromatogram and UV spectrum of compound 27
[M-COOMe]+
[M-(CH2COOMe+OH)]+
[M]+
Fig (3.5.19):EI-MS spectrum of compound 27 (up) compared with standared p-hydroxyphenylacetic acid methyl ester (down).
207
Results
Compound 27 [methyl 2-(4-hydroxyphenyl)acetate] was isolated as a white amorphous
powder, UV absorption at λmax 201, 223, 275 nm. EI-MS showed molecular ion peak at m/z
166 [M]+, and fragment ion peaks at m/z 107 [M-COOMe]+, 77 [M-(CH2COOMe+O)]+, (see
figure 3.5.30) suggesting the molecular formula C9H10O3. It is clear that compound 27 is 14
mass units larger than compound 26 which indicates the presence of one CH2 group or
exactly, the replacement of the carboxylic proton with methyl group as evident from the 1H
NMR spectrum. The 1H NMR spectrum in methanol-d4 of 27 showed the same set of proton
resonances as compound 26, in addition to the presence of a methoxy proton at 3.65 ppm
which indicated the methyl ester of compound 26. Compared to the authentic sample of
methyl 2-(4-hydroxyphenyl)acetate, compound 27 showed the same molecular ion peak as
well as the same fragmentation pattern (as shown in figure 3.5.19). From NMR data and
EIMS compound 27 was assigned as methyl 2-(4-hydroxyphenyl)acetate which was
previously isolated from many biological sources [like Fusarium oxysporum (Kachlicki P., et
al . (1997)]
H-7
O
2
H-2
7
1
O
3
4
4'
H-4&H-4'
H-5 & H-5'
5
5'
6
7.6
7.2
6.8
6.4
6.0
5.6
5.2
4.8
(ppm)
Fig (3.5.20):1HNMR spectrum of compound 27
208
4.4
4.0
1.9595
3.0413
2.0959
2.0000
OH
3.6
3.2
2.8
Results
3.5.7 Ethyl 2-(4-hydroxyphenyl)acetate (28, known compound)
O
4.03
60.1
172.6
40.1
3.48
1.16
14.2
O
126.2
7.04
130.0
6.68
C10H12O3
Exact Mass: 180,08
Mol. Wt.: 180,2
115.2
156.8
OH
-----------------------------------------
Yield : 3.2 mg
900 ms050920 #7
mAU
tm7et6f
UV_VIS_1
WVL:235 nm
10 - 16,770
Peak #10
%
224.3
70,0
600
16.72
202.8
11 - 17,020
400
275.6
200
12 - 17,525
9 - 13,642
--0,946
1,322
1,461
678
1,197
1,257
1,093
1234
-5
-0,202
--0,597
-100
0,0
13 - 47,114
min
10,0
20,0
30,0
40,0
50,0
-10,0
200
nm
400
595
60,0
Fig (3.5.21): HPLC chromatogram and UV spectrum of compound 31
[M-(C2H5OCO)]+
[M-(CH2)]+
[M-(C2H4O)]
+
[M]+
Fig (3.5.22):EI-MS spectrum of compound 28
209
Results
Compound 28 [ethyl 2-(4-hydroxyphenyl)acetate] was isolated as white amorphous powder,
UV absorption at λmax 202, 224, 275 nm. EI-MS showed molecular ion peak m/z 180 [M]+,
and fragment ion peaks at 136 [M-C2H4O]+, 107 [M-COOC2H5]+, 91 [M-(COOEt+O)]+, (see
figure 3.5.33) suggesting the molecular formula C10H12O3. Compound 28 is 14 mass units
more than compound 27 which indicates the presence of one CH2 group or exactly elongation
of one carbon in the alkoxy group of 27 as evident from the NMR data. The 1H NMR
spectrum in DMSO-d6 of 28 showed proton resonances of ethoxy group at δ 4.03 (2H, q,
J=6.93 Hz, H-7) and 1.16 (3H, t, J=6.93 Hz, H-8) instead of a methoxy proton at 3.65 ppm as
in compound 27. The other proton resonances of 28 are the same as those of 27. The
methylene CH2-7 at δ 4.03 of 28 showed an HMBC correlations to C-8 and C-1 at δ 14.2 and
172.0, respectively. The other HMBC correlations were identical with those of compound 26,
see figure (3.5.24). p-Hydroxyphenylacetic acid ethylester is a synthetic product (Bhawal et al
1991, Meltzer et al 1957). However, according to our knowledge, it is the first report of phydroxyphenylacetic acid ethylester as natural product.
O
H-2
7
H-4&
H-4'
2
H-5 &
H-5'
O
1
8
H-8
H-7
3
4'
4
5'
5
6
2
7.0
6.8
6.6
6.4
6.2
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.0185
2.1965
2.1649
2.0000
1.9952
OH
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
Fig (3.5.23):1HNMR spectrum of compound 28
H-4&
H-4'
H-5 &
H-5'
H-2
H-7
H-8
(ppm)
H-7/C-8
H-4&H-4’
/C-2
40
H-8/C-7
80
O
H-5&H-5'
/C-3
H-4&H-4’
/C-6
7
H-2/C-3
2
H-2/
C-4&C-4'
H-5&H-5'
/C-6
O
1
8
120
3
4'
4
5'
5
H-2/C-1
H-7/C-1
6
160
OH
200
(ppm)
7.00
6.00
5.00
4.00
3.00
Fig (3.5.24): HMBC spectrum of compound 28
210
2.00
1.00
0.8
0.6
Results
3.5.8 - Butyl 2-(4-hydroxyphenyl)acetate (29, new compound)
O
3.99
18.4
1.27
63.8
172.6
40.1
3.49
O
124.8
7.03
130.5
0.85
13.1
1.51
30.2
C12H16O3
Exact Mass: 208,11
Mol. Wt.: 208,25
6.68
114.1
156.1
OH
---------------------------------------------------
Yield : 2.5 mg
600
ms051002 #15
mAU
tm7et6n
UV_VIS_1
WVL:235 nm
70,0
12 - 23,754
500
Peak #12
%
23.78
224.8
203.3
400
300
200
276.2
100
13 - 24,080
11 - 23,016
0
14 - 47,958
0,777
3456-789
-0,460
1,080
-0,401
0,961
- 1,362
--0,139
12
-10
1,249
-0,593
1,157
-100
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
595
Fig (3.5.25): HPLC chromatogram and UV spectrum of compound 29
[M-(C4H9OCO)]+
[M-(C2H4)]+
[M-(C3H6)]+
[M-(C4H8)]+
Fig (3.5.26):EI-MS spectrum of compound 29
211
[M]+
Results
Compound 29 [butyl 2-(4-hydroxyphenyl)acetate] was isolated as white amorphous powder,
UV absorption at λmax 203, 225, 276 nm. EI-MS showed molecular ion peak m/z 208 [M]+,
and fragment ion peaks at 180 [M-C2H4]+, 166 [M-C3H6]+, 152 [M-C4H8]+, 136 [M-C4H8O]+,
107 [M-COOC4H9]+, and 91 [M-(COOC4H9+O)]+, (see figure 3.5.37) suggesting the
molecular formula C12H16O3. The difference between compounds 28 and 29 is only 28 mass
units which suggested the elongation of the alkoxy group with C2H4 unit as evident from the
NMR spectra of 29. 1H NMR spectrum in DMSO-d6 showed the presence of four sequentially
correlated proton resonances (integrated as 2:2:2:3) at δ 3.99 (2H, triplet, J=6.63 Hz, H-7),
1.51 (2H, quintet, J=7.23 Hz, H-8), 1.27 (2H, sextet, J=7.56 Hz, H-9) and 0.85 (3H, t, J=6.93
Hz, H-8). This indicated the presence of a butoxy group instead of a methoxy group as in
compound 27 and ethoxy group as in compound 28. The other proton resonances of 29 are the
same as those of 27 and 28. The NMR data together with the ESI-MS fragments suggested
compound 29 to be butyl-2-(4-hydroxyphenyl)acetate, which was further confirmed by
HMBC correlations as shown in figure 3.5.28. To the best of our knowledge this is the first
O
7
O
1
2
417.12
424.38
431.94
619.21
626.46
634.03
641.59
649.16
656.41
742.16
748.78
756.35
763.60
770.22
1745.33
1990.92
1997.23
2003.85
3335.84
3344.35
3508.29
3516.80
report of a butyl-2-(4-hydroxyphenyl) acetate as a natural product.
9
8
10
3
4'
4
5'
5
2
10
6
7
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
2.0548
1.5481
1.8345
1.9370
2.0000
Integral
8
3.0
Fig (3.5.27):1HNMR spectrum of compound 29
212
2.5
2.0
1.5
9
2.9431
5 & 5'
2.0309
4 & 4'
OH
1.0
0.5
Results
H-4&
H-4'
H-5 &
H-5'
H-2
H-7
H-10
H-8 H-9
(ppm)
H-8 &H-9 /C-10
H-7/C-9
O
H-4&H-4’
/C-2
2
7
9
O
1
H-8 /C- 9
H-9 /C- 8
H-7/C-8
H-10 /C- 8
8
40
10
3
4'
4
5'
5
H-8 &H-9
/C-7
80
6
OH
H-5&H-5'
/C-3
H-2/C-3
120
H-2/C-3
H-4&H-4’
/C-6
H-5&H-5'
/C-6
160
H-7/C-1
(ppm)
7.00
6.00
5.00
H-2/C-1
4.00
3.00
2.00
1.00
Fig (3.5.28):HMBC spectrum of compound 29
Table (3.5.2): NMR data of compounds 25, 26, 27, 28 and 29
Compound 25
No.
1
2
3
4, 4'
13
C
(ppm)
177.6
41.0
133.2
129.3
5, 5' 128.6
6 127.3
7
8
9
10
1
H (ppm)
(Multiplicity,
Hz)
3.65 (2H, s)
7.3 (5H, m)
Compound 26
13
C 1H (ppm)
(ppm)
(Multiplicity, Hz)
173.4
39.0
126.1
132.2
3.38 (s)
7.03
(d,J=8.52)
113.85
6.72
(d,J=8.2)
156.6
-
Compound
27
1
H (ppm)
(Multiplicity,
Compound 28
13
C
(ppm)
Hz)
1
H (ppm)
(Multiplicity,
Hz)
Compound 29
13
C
1
H (ppm)
(ppm) (Multiplicity,
Hz)
3.51 (s)
7.06 (d, J=8.5)
172.0
40.1
124.3
130.0
3.48 (s)
7.04(d, J=8.5)
172.6
40.1
3.49 (s)
124.8
130.5 7.03 (d, J=8.51)
6.72 (d,J=8.5)
115.1
6.68 (d,J=8.5)
114.1 6.68 (d,J=8.51)
3.65 (s)
156.8
60.1
14.2
4.03 (q,J=6.93)
1.16 (t,J=6.93)
156.1
63.8 3.99 (t,J=6.93)
30.2 1.51 (t,J=6.93)
18.4 1.27 (t,J=6.93)
13.1 0.85 (t,J=6.93)
213
Results
3.5.9 - Adenosine (30, known compound)
7.33
NH2
N
N
8.34
5.41
HO
8.12
3.54,
3.66
N
N
O
3.95
C10H13N5O4
Exact Mass: 267,1
Mol. Wt.: 267,24
5.86
4.60
4.13
OH
5.17
OH
5.43
----------------------------------------------------
Yield : 23.0 mg
600 ms041017 #10
mAU
bu17-9
UV_VIS_1
WVL:235 nm
2 - 1,198
500
Peak #2
70,0
1.19
%
400
207.4
257.8
300
559.1
200
nm
-10,0
200
400
595
100
4 - 3,048
5 - 47,901
- 1,448
1 3- 0,950
0
min
-100
0,0
10,0
20,0
30,0
40,0
50,0
60,0
Fig (3.5.29): HPLC chromatogram and UV spectrum of compound 30
[M-(ribose)] +
[M-(C 4 H 7O 3 )] +
[M-(C 3 H 5 O 3 )] +
[M-(ribose+NH 2)] +
[M-(CH 2O+OH+H 2 O)] +
[M-(CH 2O+OH)]+
HN
HN
N
N
[M-(C 4 H 7O 4 )] +
Fig (3.5. 30):EI-MS spectrum of compound 30
214
[M-CH 2 O]+
[M]+
8
1660.53
1757.94
1761.41
1765.19
1769.61
1773.39
DMSO
H2O
2
1777.17
1780.96
1819.74
1823.83
1827.93
1831.72
1835.81
1839.91
1969.80
1973.27
1976.42
1979.89
2058.39
2063.12
2066.27
2071.00
2290.74
2297.05
2302.09
2308.08
2582.36
2586.77
2699.01
2703.74
2706.26
2711.30
2717.61
2928.52
2934.82
3667.50
4061.89
4168.14
Results
NH2
N
7
N
HO
5'
O
4'
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
(
4.5
4.0
1'
3.5
3.0
2.5
2.0
1.5
5’-OH
4'
3'
2'
5’a 5’b
H2O
(ppm)
NH2
H2O
N
5
7
6
8
5’b
N
HO
5'
O
5’a
4'
4'
3'
OH
9
4
3
3.2
N1
2
N
3.6
1'
2'
3’/4’
OH
4.0
5’a & 5’b
/4'
3'
4.4
2’/2'-OH
1’/2’
2'
2’/3’
4.8
3’/3'-OH
3’-OH
5’-OH
5’a & 5’b
/5'-OH
5.2
2'-OH
5.6
1'
6.0
(ppm)
5.6
5.2
4.8
4.4
4.0
3.6
Fig (3.5.32):COSY spectrum of compound 30
215
N
OH
)
3’-OH
4
2'
OH
5’a 5’b
9
1'
3'
Fig (3.5. 31):1HNMR spectrum of compound 30
2'-OH
N1
2
3
1.2373
4'
1.0212
3'
1.0159
1.0284
0.9751
2.0989
1.0053
1.8877
0.8886
1.0000
Integral
2'
1.0768
3’-OH
1.0777
1'
6
8
2'-OH 5’-OH
NH2
5
3.2
1.0
0.5
Results
Compound 30 was isolated as brownish-white amorphous powder, UV absorption at
λmax 207, 257 nm of a very polar peak in HPLC chromatogram suggested the nucleoside
nature of the compound. EI-MS showed molecular ion peak at m/z 267 [M]+, and fragment
ion peaks at 237 [M-CH2O]+, 220 [M-CH2O+OH]+, 202 [M-CH2O+OH+H2O]+, 178 [MC3H5O3]+, 164 [M-C4H7O3]+, 148 [M-C4H7O4]+, 135 [M-(ribose)]+, and 119 [M(ribose+NH2)]+, (see figure 3.5.41) suggesting the molecular formula C10H13N5O4. 1H NMR
spectrum which was meassured in DMSO-d6 showed resonances at δ 8.12 (s, H-2), 8.34 (s, H8), 7.33 (s, 6-NH2) suggesting the presence of 9H-purin-6-ylamine moiety. The existence of a
ribosyl moiety in this compound was evident by a number of protons resonating between δ
3.54 and 5.86 (table 3.5.4). Furthermore, the presence of hydroxyl functional groups at the
positions 2', 3', 5' were determined by resonances at δ 5.43(d, J=6.3) 2'-OH, 5.17 (d, J=4.41)
3'-OH, 5.41 (dd, J=7.25, 4.73) 5'-OH as shown in 1H-NMR and confirmed by its COSY
spectrum (figure3.5.31 & 3.5.32). The above NMR data together with the EI-MS fragments
confirmed compound 30 to be adenosine.
Table 3.5.3 1H-NMR data of Compound 30:
No.
2
8
6-NH2
1'
2'
2'-OH
3'
3'-OH
4'
5'a
5'b
5'-OH
1
H (ppm)
(Multiplicity, Hz)
8.12 (s)
8.34 (s)
7.33 (s)
5.86 (d, J =6.3)
4.60 (dd, J=11.35, 6.3)
5.43(d, J =6.3)
4.13 (dd, J =7.88, 4.73)
5.17 (d, J =4.41)
3.95 (dd, J =6.62, 3.47)
3.54 (ddd, J =3.47, 7.25, 11.67)
3.66 (ddd, J =4.09, 8.19, 11.98)
5.41 (dd, J =7.25, 4.73)
216
Results
3.5.10 Nicotinamide (31, known compound)
O
NH2
C6H6N2O
8.19
9.03
Mol. Wt.: 122,12
N
7.48
8.7
-------------------------------------------------
Yield : 2.9 mg
1.000 ms050503 #2
mAU
4 - 1,485
et17a
UV_VIS_1
WVL:235 nm
70,0
800
Peak #4 1.51
No spectra library hits found!
%
260.9
218.6
202.6
600
400
200
3 - 1,123
0,933
12--0,517
-100
0,0
5 - 30,581
min
10,0
20,0
30,0
40,0
50,0
nm
-10,0
200
6 - 47,553
400
595
60,0
Fig (3.5.33): HPLC chromatogram and UV spectrum of compound 31
[M-CONH2]+
+
[M-NH2]+
[M]+
[M-(CONH2 + HCN)]
O
NH2
1
2
3
4
7
N
6
5
Fig (3.5.34):EI-MS spectrum of compound 31 (up) compared to authentic sample of
nicotinamide (down)
217
Results
Compound 31 was isolated as white amorphous powder. It has UV absorption at λmax
202, 218, 260 nm. EI-MS showed molecular ion peak at m/z 122 [M]+, and showed the
same fragmention pattern of authentic sample of nicotinamide where it displays a fragment
ion peaks at m/z 106 [M-NH2]+, 78 [M-CONH2]+, and 51 [M- CONH2+HCN]+, (see figure
3.5.34) suggesting the molecular formula C6H6N2O. 1H NMR spectrum (meassured in
DMSO-d6) showed resonances at δ 9.03 (br s, H-3), 8.7 (br s, H-5), 7.48 (dd, J=7.88, 4.73),
8.19 (d, J=7.88 H-4). The 1H NMR spectrum of compound 31 is identical with those of
3737.80
3742.53
3745.68
3750.41
3796.44
4092.16
4093.74
4100.04
4347.21
4510.83
nicotinamide (Aldrich 1993, pp 3/338).
O
NH2
1
2
7
3
3
6
4
5
7
N
6
5
9.4
9.2
9.0
8.8
8.6
8.4
8.2
8.0
7.8
Fig (3.5.35):1HNMR spectrum of compound 31
218
7.6
7.4
7.2
7.0
6.8
6.6
6.4
Results
3.5.11 and 12 Petrocerebrosides 1 and 2 (32 & 33, new compounds)
O
C22H45
OH
HN
C12H25
HO
HO
H O
HO
HHO
OH
H
H
O
H O
O
HHO
OH
H
OH
H
C57H109NO14
Exact Mass: 1031,78481
H
H
O
C21H43
OH
HN
C11H23
HO
H O
HHO
H
HO
HO
O
OH
H
H
HHO
H O
H
H
O
OH
H
OH
C56H107NO14
Exact Mass: 1017,77
--------------------------------------------------------------------------------------------
Yield : 35.0 mg
M-Fatty
Petrocerebros
Petrocerebros
Fig (3.5.36):EI-MS spectrum of compounds 32 and 33
Cerebrosides are glycolipids composed of a long chain aminoalcohol, known as a
sphingoid base ( sphingosines), a fatty acid residue linked to its amino group (the resulting
amide is called ceramide), and a carbohydrate chain attached to the primary hydroxyl group of
the ceramide. The scientific interest in cerebrosides has recently increased on account of the
role they play as therapeutical immunomodulating agents (Costantino et al 2004, 2003, 1994).
Sphingolipids have come to be used as functional foodstuffs and sphingomyelin isolated from
219
Results
bovine milk has been found to have significant effects in colon cancer prevention. Sphingoid
bases isolated from plants and fungi have been shown to induce apoptosis in the caco-2 cellline. The ingestive cerebrocides from maize and Saccharomyces kluyveri prevent aberrant
crypt foci in mice adminstrated with N,N-dimethylhydrazine (Tanji 2004).
Compound 32 and 33 were obtained as mixture [in a ratio of 5:4, respectively as
evident from the fatty acid ratios (see figure 3.5.4)] and as an amorphous powder. The
molecular formulas were deduced as C57H109NO14 and C56H107NO14 based on a molecular ion
peak [M+H]+ 1032.1 and [M]+ 1031.2 for petrocerebroside 1 and [M+H]+ 1018.1 and [M]+
1017.2 for petrocerebroside 2 as obtained from the ESI-MS of the sample. The difference
between both cerebroside derivatives is only 14 mass unit which suggested an additional
methylene group in petrocerebroside 1 when compared to petrocerebroside 2. The position of
this CH2 group was deduced to be in the long chain fatty acid part of the compound as shown
below. The structure elucidation of petrocerebrosides 1 and 2 were obtained using extensive
1
H NMR,
13
C NMR, DEPT, and 2D NMR experiments, as well as by acid hydrolysis. GC
analysis determined the absolute configuration of the sugar moieties. GC-MS was done to
analyze the molecular weights both the free fatty acid and sphingosine base after acid
hydrolysis.
The cerebroside nature of the petrocerebrosides 1 and 2 were established from the 1H
NMR which showed a triplet-like signal at δ 0.82 for the terminal methyl units and broad
singlet at δ 1.20 for long chain (CH2)n group for both fatty acids and long chain bases. The
presence of several doublets in the region between 3.35-5.30 ppm indicated the occurrence of
the sugar moieties. Two coupling sp2 methines at δ 5.41 and 5.34 showed the presence of only
one double bond. Two doublets at 7.42 and 7.70 ppm exhibited the presence of two amide
NHs belonging to two different compounds.
13
C NMR showed two resonances at δ 172.3 and
170.15 indicating two amide carbonyls for petrocerebroside 1 and 2, respectively. Two
resonances at δ 131.5 and 128.1 were assigned for two sp2 methine carbons, while two sp3
methines resonating at 101.2 and 98.9 indicated the presence of anomeric carbons for two
monosaccharides.
220
1032.34
1028.82
1022.47
1014.77
985.99
979.80
965.58
959.39
952.70
425.67
418.97
411.95
1853.67
1849.82
1842.96
1838.61
1833.59
1794.94
1793.10
1789.09
1783.23
2148.80
2140.61
2334.02
2331.17
2722.35
2713.65
2707.12
2700.26
2684.70
2678.34
2671.65
2662.95
2656.26
3857.05
3848.52
3715.01
3706.31
Results
Terminal
methyls
(CH2)n
H-6, H-7
(E-coupling)
Amide NHs
7.5
7.0
6.5
6.0
5.5
Sugar protons
and other sp3-down
field
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Fig (3.5.37):1HNMR spectrum of compound 32 and 33
(comp-35) (comp-34)
NH NH
H-6,
H-7
(ppm)
H-7/H-8
2.0
(comp-35)
NH/H-2
H-5/H-6
comp-34)
NH/H-2
(
4.0
H-7/H-6
6.0
8.0
(ppm)
8.0
6.0
4.0
2.0
Fig (3.5.38):COSY spectrum of compound 32 and 33
221
1.0
Results
(ppm)
20
2 epimeric protons
40
60
2 epimeric protons/
2 epimeric carbons
80
100
120
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.5.39):HMQC spectrum of compound 32 and 33
CH2Cl2
(ppm)
OH
CH3
(Methanol peaks)
H-6 &H-7/C-5 and C-8
40
Sugar part
Hs/Cs
80
H-2" H-2```/C-1" &C-1```
H-8/C-6 &C-7
(comp-33)
NH/H-1'
(comp-32)
NH/C-1'
(ppm)
8.0
Hα/C-1'
6.0
4.0
2.0
Fig (3.5.40):HMBC spectrum of compound 32 and 33
222
120
160
Results
The sample was hydrolysed using 6N HCl and heated over a hot plate under reflux for
7 hours, then cooled with cold water stream. The hydrolysate were extracted with n-hexane to
obtain the fatty acid part which was then examined by GC-MS (figures 3.5.41, 42a &42b)
where mixture of two long chain fatty acids (pentacosanoic acid C25H50O2, m/z 382 and
tetracosanoic acid C24H48O2, m/z 368) were detected. The aqueous part were purified over a
Sephadex column chromatography and the Molish-positive fraction were separated and
utilised for determination of the absolute streochemistry of the monosaccharide units. The
absolute streochemistry of the monosuccharide units were established through butanolysis,
silylation, then GC analysis in comparison with authentic monosaccharides. This experiment
revealed that both sugar units are D-galactoses.
Fig (3.5.41):GC-chromatogram of the fatty acid-containig fraction of
compound 32 and 33 hydrolysate
Fig (3.5.42a):GC-MS of tetracosanoic acid Fig (3.5.42b):GC-MS of pentacosanoic acid
223
Results
Deduced NMR data of
both fatty acid and sphingosine moieties were confirmed after
methanolysis. 1H NMR of the fatty acid mixture showed a singlet resonating at δ 3.66
indicating a methoxy group of the derivatised fatty acid. A triplet at δ 0.88 indicated the
terminal methyl, a triplet at 2.3 ppm was assigned for the α-CH2, and the multiplet at 1.61
13
ppm represented the β-CH2. This data were confirmed by
C NMR spectrum where
characteristic signals of the long chain fatty acid methyl esters were obtained as shown below
431.96
438.89
445.51
625.53
792.30
799.87
807.12
814.06
821.62
1142.25
1149.81
1157.38
1832.36
(figures 3.5.43 and 44).
(CH2)n
O-CH3
Terminal
methyls
14.0
13.0
12.0
11.0
10.0
9.0
8.0
7.0
6.0
(
5.0
4.0
3.0
3.0000
41.688
1.6121
β-CH2
1.3118
1.6033
Integral
α-CH2
2.0
1.0
0.0
)
14.1178
22.6883
24.9512
29.1498
29.2461
29.3520
29.4483
29.5832
29.6506
29.6891
31.9136
34.1188
51.4427
174.3759
Fig (3.5.43):1HNMR spectrum of fatty acid mixture of compound 32 and 33 hydrolysates.
n`= 24 ---------------- tetracosanoic acid
n`= 25 ---------------- pentacosanoic acid
C-3` C-n`-2
C-1`
0
170
C-2`
Me-O
160
150
140
130
120
110
100
90
80
70
60
50
40
C-n`
(CH3)
C-n`-1
30
20
10
(ppm)
1764.60
1758.92
1521.53
1516.48
1514.28
1509.55
1502.30
1363.58
1356.33
1349.08
1342.14
1334.58
1025.93
1019.00
1012.06
1005.44
961.62
953.74
951.53
942.07
3032.90
3025.97
3017.77
3010.52
3003.58
2862.03
2855.09
2848.47
2839.96
2833.34
2470.47
2462.59
2147.01
2126.20
2125.57
2120.53
2110.13
2013.02
2005.77
1998.21
1994.42
1986.86
1979.61
Fig (3.5.44):1HNMR spectrum of fatty acid mixture of compound 32 and 33 hydrolysates.
(CH2)n
Solvent peaks
(pyridine)
sp3-down field
region
H-6,
8.5
8.0
7.5
7.0
6.5
6.0
Terminal
methyls
H2-8
H-5a, H-5b
H-7
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Fig (3.5.45):1HNMR spectrum of sphingosine part of compound 32 and 33
224
Results
H-1a, H-1b,
H-4
H-3
H-8
H-2
H-5a
(ppm)
1.00
H-8/H-9
H-7/H-8
2.00
H-4/H-5a & b
H-5a /H-5 b
H-6/H-5a & b
3.00
H-2/H-1a,H-1b,
H-3
H-4 /H-3
4.00
5.00
6.00
(ppm)
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.5.46):COSY spectrum of sphingosine part of compound 32 and 33
MeOH
H-6
H-7
H-1a, H-1b, H-3
H-4
H-2
H-8
H-5a H-5 b
(ppm)
0
40
80
120
6.0
4.0
2.0
0.0
Fig (3.5.47):HMQC spectrum of sphingosine part of compound 32 and 33
225
Results
Table 3.5.4 NMR data of compound 32 and 33, in addition to free fatty acids and
sphingosine
No.
1
Compounds 35 &36
Fatty acid
(DMSO-d6)
(CDCl3)
13
1
13
C
H (ppm)
C (ppm)
(ppm)
(Multiplicity, Hz)
63.5
4.20 and 4.35 (m & m)
55.2
54.5
**NHs
75.0
3
74.1
4
33.0
5
**2
6
128.1
7
131.5
8
9-19
34.3
33.029.0
14.29
172.2
170.0
35.8
31.7
20
**1´
2´
3´
*4´-(n1)´
n´
1´´
2´´
3´´
4´´
5´´
6´´
1´´´
2´´´
3´´´
4´´´
5´´´
6´´´
30.0 –
22.5
14.29
102.0
71.0
69.5
83.2
68.4
63.5
99.0
70.5
69.2
80.7
68.2
62.2
1
H (ppm)
(Multiplicity, Hz)
3.90 (m)
3.4 (m)
(d, 9.3 Hz)
7.72 (d, 8.2 Hz)
3.55 (m)
3.43 (m)
2.40 & 1.9 ( m &m)
Sphingosine
(Pyridine-d5)
13
1
C
H (ppm)
(ppm)
(Multiplicity, Hz)
65.0
4.25 & 4.35 (m &
m)
58.0
3.6 (m)
75.0
74.2
38.2
5.45 (ddd, 6.4, 8.2,
15.1 Hz)
5.71 (ddd, 6.6, 8.5,
15.1 Hz)
1.9 (m)
1.2 (br s)
128.1
132.5
34.3
33.029.0
14.2
0.85 (t, 6.9 Hz)
-
174.4
2.3 (m)
1.6 (m)
34.1
31.9
1.2 (br s)
30.0 – 22.7 1.3 (br s)
4.04 (m)
4.25 (m)
3.05 (m) &
2.72 (ddd, 14.8, 8.0,
6.8)
6.05(ddd, 15.13,
6.95, 8.19)
5.71 (ddd, 15.13,
6.5, 6.6)
1.99 (m)
1.26 (br s)
0.82 (t, 6.9)
2.3 (t, 7.6)
1.6 (m)
0.85 (t, 6.9 Hz)
14.1
4.3 (d, 8.5 Hz)
3.5 (m)
3.63 (m)
3.7 (m)
3.4(m)
3.32 &3.25 (m &m)
4.7 (d, 4.2 Hz)
3.2(m)
3.60 (m)
3.28 (m)
3.4(m)
3.30 &3.4 (m &m)
0.88 (t, 7.0)
* n =25 for petrocerebroside 1
n =24 for petrocerebroside 2
** these pairs of NMR values were assigned for both compounds and can be interchanged,
These differences attributed mainly to the differences in stereochemistry of the three chiral
centers in each cerebroside at C-2, C-3 and C-4.
226
Results
New purine derivatives isolated from Petrosia nigricans:
Marine organisms particularly sponges have proven to be an exceptionally rich source
of modified nucleosides. The isolation of spongouridine and spongothymidine from
Cryptotethia crypta (Bregman and Feeney 1950) served as models for the development of
adenine arabinoside (ARA-A) for treatment of Herpes simplex infection and cytosine
arabinoside (ARA-C) for the
treatment of leukemia (Lindsay et al 1999). Subsequent
development of antiviral analogues demonstrated the potential medicinal importance of these
compounds such as antifungal phidolopine which was isolated from the
bryozoan
Phidolopora pacifica (Ayer et al 1984), the hypotensive doridosine which was obtained from
the sponge Tedania digitata (Cook et al 1980) , and the cytotoxic mycalisines which was
found from the sponge Mycale sp. (Kato et al 1985). Many other purines and nucleosides
isolated from marine organisms particularly sponges, display potant bioactivities, such as the
marine derived 1,3-dimethylisoguanine from Amphimedon viridis which showed activity
against an ovarian cancer cell line (IC50, 2.1 µg/mL) (Mitchell et al 1997) and 3,7dimethylisoguanine from a Caribbean sponge Agelas lonigssima, which displayed mild
antibacterial activities (Cafieri et al 1995). Investigation of ethylacetate fraction of the
Indonesian sponge Petrosia nigricans, led to the isolation of four new purine derivatives
nigricines 1 to 4. Their structures were elucidated by extensive spectroscopic analysis, 2DNMR experiments, EI/MS, ESI/MS, and HRMS.
227
Results
3.5.13. Nigricine 1 (34 , new compounds)
3.62
31.0
3.91
34.2
N
O
141.5 N
154.9
7.57
139.1
115.0
N
N
153.6
7.67
36.3
3.59
NH
34.8
2.62
0.89
13.3
O
171.3
1.57
31.2
O
C14H21N5O3
Exact Mass: 307,165
Mol. Wt.: 307,348
4.08
65.3
1.31
20.1
-----------------------------------------------
Yield : 2.3 mg
350 ms040918 #12
mAU
tm7et16-7
300
UV_VIS_1
WVL:235 nm
70,0
9 - 16,453
Peak #9
%
50%
100%
999.94
999.95
-50%
210.3
50,0
200
290.2
25,0
100
0
-50
0,0
11 - 23,165
- 27,772
10 - 21,568 1213
- 28,905
5 - 1,214
1,301
1,335
1,425
1,053
1,133
1,566
1234678----0,954
560.9
14 - 47,406
min
10,0
20,0
30,0
40,0
50,0
60,0
nm
-10,0
200
400
Fig (3.5.48): HPLC chromatogram and UV spectrum of compound 34
Fig (3.5.49):ESI-MS spectrum of compound 34
228
595
Results
Compound 34, is the key structure for this group of purine derivatives. The
HRESIMS+ was in agreement with the molecular formula of C14H21N5O3 (measured, m/z
308.170, [M+H] ) with 7 degrees of unsaturation. The UV spectrum of 34 showed absorption
λmax (MeOH) at 210 and 290 nm. Simple 1H NMR spectrum (figure 3.5.51) showed an
exchangeable triplet signal at δ 7.73 ppm for an NH, in addition to signals at δ 7.61 (1H, s ,
H-8), 4.17 ( 2H, t , CH2), 1.67 ( 2H, m , CH2), 1.4 ( 2H, m , CH2), 0.95 ( 3H, t , CH3), 3.80 (
3H, t , N-3 CH3), 4.05 ( 3H, t , CH3). The basic structure of the purine skeleton was evident
through interpretation of 1H NMR and
13
C NMR as well as comparison with those of the
literatures of Lindsay et al 1999, Mitchell et al 1997, Lindsay et al 1999, Capon et al 2000,
Yagi et al 1994.
The
NMR measurement of compound 34 in deuterated methanol indicated the
presence of only one exchangeable triplet signal at 7.73 ppm for 6-NH suggesting a 6derivatized adenine structure, the methyl signals at δ 31.4 and 34.8 showed a characteristic
13
C chemical shifts for NCH3 resonances and excluding those of OCH3, often found between
50-60 ppm, the confirmation of the positions of both methyl groups were obtained from
HMBC correlation of both methyl groups to a quaternary carbon at δ 143.1 (C-4), one of
them, N(3)-CH3, showed further HMBC correlation to carbonyl at 159.0 ppm (C-2), while the
other, N(9)-CH3, showed an additional HMBC correlation to the methine carbon at δ 140.6
(C-8). Furthermore, ROESY experiment showed a correlation through space between both
methyl signals, thus, the existence of N(7)-CH3 was excluded. The methine proton signal, H8, showed HMBC correlations to both quaternary carbon at 143.1 (C-4) , and 115.0 (C-5).
The remaining carbon in the purine skeleton, C-6, was established through HMBC correlation
of the NH proton signal at δ 7.73 to a quaternary carbon signal at 157.7 ppm (C-6). The
position of β-propionyl side chain was confirmed by sequential COSY correlation between
the proton signals at δ 7.73, 3.80, and 2.72 of 6-NH, β-CH2, and α-CH2 of propionyl group,
and compared with chemical shifts of the same substructure of the previously described
marine derived purine compound, erinacean (5), which was obtained from the antarctic
sponge Isodictya erinacea (Moon et al 1997). In addition, HMBC experiment showed a
correlation of both α- and β- CH2 groups to the carboxyl at 173.0 ppm. The attachment of this
group to the purine skeleton was evident through HMBC correlation betwwen β-CH2 proton
signal and the quaternary carbon at 157.7 ppm (C-6). The last substructure (alkoxy group)
was confirmed through a sequential COSY correlations between 4 aliphatic proton signals at δ
4.17 ( 2H, t. CH2), 1.67 ( 2H, m. CH2), 1.4 ( 2H, m. CH2) and 0.95 ( 3H, t. CH3), the
229
Results
connection of this substructure to the
purine skeleton was established through HMBC
correlation between α-CH2 at δ 4.17 and the carboxyl at 173.0 ppm.
N
O
2
N
2
8
1
5
7
N
N
6
NH
2
3
11
12
O
N
4
3
5
3
N
N
4
11
12
O
6
N
H
NH
10
R = Et
R = Me
O
8
1
NH
R = Bu
R
7
H
N
N
9
8
1
6
1
10
O
N
O
N9
4
3
O
10
11
O 12 O H
5
New compounds, nigricines 1 to 4, as well as the known analogue erinacean (5)
LC/MS of compound 34 showed a positve pseudomolecular ion peak at m/z
308(M+1), and at m/z 615 (19 % , 2M+1) and a characteristic fragment ion at m/z 234 [Malkoxy group] and 192 [M-(alkoxy group+NCO)] indicating the loss of NCO fragment due to
retro Diels-Alder cleavage of N-1/C-6 and C-2/N-3 bonds which is a characteristic
fragmentation pattern for 2-oxopurines (Cafieri et al 1995). The presence of a fragment ion
peak at m/z 180 indicated 3,9-dimethyl isoguanine skeleton after loss of the side chain (m/z
138). Both MS fragmentaion pattern and NMR spectra corroborate with the structure proposal
and established the identity of 34 as butyl 3-(3,9- dihydro-3,9-dimethyl –2-oxo-2H-purin-6ylamino) propanoate. To the best of my knowledge this is the first report of 34 as a natural
product and also as far as I know this is the first report of an 2-oxo-3,9-dimethylpurin-6ylamino derivative, which we assign the trivial name nigricine 1.
h
i
d
Fig (3.5.50):13CNMR and DEPT spectra of compound 34
230
g
f e c
a
b
446.75
453.69
461.25
668.07
675.00
682.57
690.13
780.93
787.87
795.43
799.85
802.37
809.30
1334.53
1340.84
1347.78
2036.94
h
3. 8
3 1 .4
2043.88
3784.77
g
2050.18
Results
*
4. 05
3 4 .8
N
E
P
*
S
N
N
1 43. 1
1 1 5 .0
0.8050
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
1 .5 5
0 .8 9
c
1 .3
b
2.0
1.5
1.0
0.5
g
a
e
N H 7 .6 6
d
O
d
2.5
i
N
e
a
3.0
7 .6 1
2 .6 2
NH
h
N
N
f
3.5
3 .9
N
3 .6 0
c b
h
3 .6 3
i
4.0
a
e
(ppm)
g
O
4.5
f
3.0000
d
i
65. 6
g
h
2.3725
c d4 .1 7 O
Integral
b
O
173
2.0732
1 .4
2 0 .1
e
2.3389
1 .6 7
3 1 .8
NH
2 .7 2
3 4 .4
1.9562
a
0 .9 5
14. 0
3 .8
3 7 .2
I
N
1 5 7 .7
f
S
1.7362
N
*
i
1 4 0 .6
7 .6 1
2.7801
159. 0
3.5515
O
c
f
b
O
4 .0 9
Fig (3.5.51):1HNMR spectrum of compound 34 measured in methanol-d4 (up) and
DMSO-d6 (down).
b/a
b/c
NH/
i/h
long range
coupling
f/e
g
h
3.63
3.9
N
O
N
7.61
N
f
3.60
N
NH7.66
2.62
e
a
0.89
1.55
1.3
b
c
i
g/h
long range
coupling
O
d O
4.09
Fig (3.5.52):COSY spectrum of compound 34
231
Results
d
i
h
g
e
f
c
a
b
(ppm)
g
h
3.8
31.4
N
O
143.1
159.0
N
40
4.05
34.8
115.0
157.7
N
140.6
7.61
i
N
80
NH
3.8
f 37.2
2.72
e 34.4 O
14.0
173
1.67
31.8
a
0.95
O
c d
4.17
1.4
20.1
120
65.6
b
160
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.5.53):total HMBC spectrum of compound 34
(ppm)
i/115.0
120
g
h
3.8
31.4
i/143.1
4.05
34.8
N
O
143.1
159.0
N
115.0
157.7
N
140.6
7.61
i
h/i
N
g/143.1
NH
3.8
f 37.2
2.72
e 34.4
a0.95
14.0
1.67
31.8
1.4
20.1
b
140
h/143.1
O
173
O
c d
4.17
f/157.7
65.6
160
g/159.0
d/173
e/173
f/173
180
(ppm)
7.2
6.4
5.6
4.8
4.0
3.2
Fig (3.5.54):part of HMBC spectrum of compound 34
232
2.4
Results
Table 3.5.5. 1H and 13C NMR Data of compound 34 (Nigricine 1) (500 MHz)
13
1
HMBC
(DMSO-d6) (DMSO-d6)
(CD3OD)
(CD3OD)
(H--------- C)
2
154.9
-
158.9
-
N(3)-CH3
31.0
3.62
31.4
3.80 (s)
4
141.5
-
142.5
-
5
115.0
-
115.4
-
6
153.6
-
157.6
-
6-NH
-
7.67 (t,5.8)
-
-
C-6
8
139.1
7.57
140.6
7.61 (s)
C-4, C-5
N(9)-CH3
34.2
3.91
34.5
4.02 (s)
C-8, C-4
10
36.3
3.59 (dt,6.30&5.8)
37.2
3.81 (t,6.30)
C-6, C-12, C-11
11
34.8
2.62 (t,6.30)
34.8
2.72 (t,6.31)
C-12, C-10
12
171.3
-
173.5
-
13
65.3
4.08 (t,6.30)
65.4
4.17 (t,6.94)
C-12, C-15, C-14
14
31.2
1.57 (tt,6.93&6.94)
31.8
1.67, (tt,6.93&6.94)
C-13, C-15, C-16
15
20.1
1.31(tt,6.93&6.94)
20.6
1.40 (qt,6.93&6.94)
C-13, C-14, C-16
16
13.3
0.89 (t,6.94)
13.9
0.95 (t,6.94)
C-14, C-15
Position
13
C δ ,m
1
H δ,m, j(Hz)
C δ ,m
233
H δ,m, j(Hz)
C-2, C-4
Results
3.5.14 – Nigricine 2 (35, new compounds)
3 .6 5
2 9 .84
3 .9 2
3 3 .0 8
N
O
N
14 1 .1
1 5 5.81
1 3 8 .2 7
7.57
1 1 3 .1
N
N
1 55 .4 5
NH
3.5 5
3 5 .3 4
2 .5 8
3 3 .3
7 .73
C 12 H 1 7 N 5 O 3
E x act M ass: 27 9 ,1 3
M o l. W t.: 27 9 ,3
O
17 1 .3
1 .1 2
14.0 7
O
4 .0 8
5 9 .9 1
-----------------------------------------------------
Yield : 6.1 mg
400 ms050503 #7
mAU
et17f
UV_VIS_1
WVL:235 nm
70,0
Peak #7
%
8.54
7 - 8,892
300
209.9
50,0
289.7
200
25,0
100
0
-50
0,0
560.5
12 - 30,904
11 - 30,569
9 -10
24,177
- 25,281
8 - 15,756
1,205
56 -- 1,117
-0,584
0,957
1234
- --0,079
0,727
13 - 47,630
10,0
20,0
30,0
40,0
50,0
nm
-10,0
200
min
60,0
400
595
Fig (3.5.55): HPLC chromatogram and UV spectrum of compound 35
H+
H+
O
N
O
N
N
N
N
N
N
A
N
HN
HN
O
O
m/z 234
O
m/z 280
H+
N
-side chain
H+
B
O
N
N
N
C
A
1
2
N
NH2
m/z 180
Fig (3.5.56):ESI-MS spectrum of compound 35
234
C
N
-OEt
retro Diels-Alder
fragmentation
-[NCO]
N
HN
B
O
m/z 193
Results
Compound 35, showed molecular formula C12H17N5O3 through HRESIMS-TOF
analysis (measured, m/z 280.139, [M+H]+) with 7 degrees of unsaturation. The UV spectrum
of 35 showed absorption λmax (MeOH) at 210 and 290 nm indicating the same chromophoric
functionalities as 34. The difference in molecular weight between 34 and 35 was only 28
mass units indicating a loss of C2H4 group from the alkoxy group as evident from its 1HNMR
spectrum. ESI/MS spectrum showed pseudomolecular ion peak at m/z 280.1 (M+H) and at
m/z 559 (2M+H) and at m/z 838 (3M+H). Tandem MS fragmentation spectrum showed a retro
Diels-Alder fragment ion peak at m/z 192 [M-(ethoxy group +OCN)]+ which confirmed the
loss of an OCN group suggesting 2-oxopurine derivative (Cafieri et al 1995), and also
exhibited a molecular ion fragment at m/z 234 indicating the loss of an alkoxy group.The 1H
NMR and 13C NMR spectra showed signals at chemical shift the same as those of nigricine 1
with the exception of loss of 2 CH2 chemical shifts from the terminal alkoxy group.
The proton signals for 6-NH exhibited HMBC correlations to the adjacent quaternary
carbon signals at 155.45 and 113.1 ppm and also HMBC correlation to the β-methylene
carbon of propionc acid moiety at 35.34 ppm. The aromatic proton signal, H-8, at 7.57 ppm
showed HMBC correlation to N(9)-Me carbon signal at 33.08, quaternary aromatic carbon
signals at 113.1 and 141.1 ppm of C-5 and C-4 respectively. N(3)-Me proton signal exhibited
HMBC correlation to the carbonyl (C-2) at 155.81 ppm. The connection of the ethoxy group
was established through HMBC correaltion between CH2 proton signal to the carboxyl at δ
171.3. Both MS fragmentaion pattern and NMR spectra confirmed the proposed structure and
established the identity of 35 as ethyl 3-(3,9- dihydro-3,9-dimethyl –2-oxo-2H-purin-6ylamino) propanoate and was assigned the trivial name nigricine 2.
Table 3.5.6. NMR Data of compound 38 (Ashourine 2) [500 MHz]
Position
2
N(3)-CH3
4
5
6
6-NH
8
N(9)-CH3
10
11
12
13
14
13
C δ ,m
(DMSO-d6)
155.8
29.84
141.1
113.1
155.45
138.27
33.08
35.34
33.56
171.3
59.91
14.07
1
H δ,m, j(Hz)
(DMSO-d6)
3.65 (s)
7.73 (t,5.68)
7.57 (s)
3.91 (s)
3.56 (dt,6.63&5.8)
2.58 (t,7.25)
4.08 (q,7.25)
1.12 (t,7.25)
235
HMBC
(H-------- C)
C-2, C-4
C-6, C-5, C-10
C-4, C-5, N(9)-CH3
C-8, C-4
C-6, C-12, C-11
C-12, C-10
C-12, C-14
C-13
7.6
7.2
6.8
6.4
6.0
5.6
5.2
4.8
4.4
601.2
607.2
614.4
621.7
1329
1336
1343
1647
1648
3.6
3.2
3.0000
d
4.0
a
c
1.9663
4.9730
2.8805
2.1216
b
0.9230
8.0
1650
e
f
g
1652
1653
1871
1875
1877
1884
1985
1991
2054
2061
2068
2075
3782
Results
2.8
2.4
2.0
1.6
1.2
0.8
3.65
29.84
e
f
N
O
NH
2.58
33.3
c
NH g
a
576.02
582.95
590.20
1286.3
1293.5
1300.8
1770.8
1777.4
1784.1
1791.0
1945.2
2014.5
1798.3
e
7.73
a
O
b
171.3
1.12
14.07
2021.8
N
155.45
3.55
35.34
f
g 138.27
7.57
113.1
N
d
3.92
33.08
N
141.1
155.81
2028.7
2036.0
3793.6
3852.8
3858.5
3864.2
(ppm)
O
c
d
b
4.08
8.0
7.6
7.2
6.8
6.4
6.0
5.6
5.2
4.8
4.4
4.0
3.6
3.2
2.8
3.0000
2.1178
2.2291
2.5409
2.5627
2.1720
0.8013
0.8066
59.91
2.4
2.0
1.6
1.2
0.8
0
(ppm)
e
f
N
O
2.58
33.3
14.0744
29.8442
33.3349
33.5681
35.6085
59.9192
113.1024
138.2730
141.1078
O
dc
b
171.3
1.12
14.07
a
a
g
NH 7.73
3.55
35.34
c
138.27
7.57
N
155.45
d
f e
g
113.1
N
3.92
33.08
N
141.1
155.81
155.4566
3.65
29.84
155.8137
171.3066
Fig (3.5.57):1HNMR spectrum of compound 35
O
b4.08
59.91
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
Fig (3.5.58):13CNMR and DEPT spectra of compound 35
236
30
20
10
0
Results
f e
NH
g
b
c
d
a
a
(ppm)
2.00
c
3.00
d
e
f
3.65
29.84
e
b
f
N
O
g 138.27
7.57
113.1
N
NH
3.55
d 35.34
2.58
33.3
7.73
6.00
O
171.3
1.12
14.07
a
5.00
N
155.45
c
4.00
N
141.1
155.81
3.92
33.08
O
b4.08
7.00
59.91
g
NH
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.5.59):COSY spectrum of compound 35
NH
f e
b d
g
a
c
(ppm)
a
f& c
d
e
40
3.65
29.84
e
f3.92
33.08
b
N
O
141.1
155.81
113.1
N
N
155.45
3.55
35.34
2.58
33.3
a
1.12
14.07
g
N
g138.27
7.57
80
NH 7.73
d
c
O
171.3
b
120
O
4.08
59.91
160
(ppm)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fig (3.5.60):HMBC spectrum of compound 35
237
Results
3.5.15 – Nigricine 3, (36 , new compounds)
3 .6 0
2 9 .8 4
3 .9 2
3 3 .3 3
N
O
N
1 4 1 .1
1 5 5 .8 1
1 3 8 .2 7
7 .5 7
1 1 3 .1
N
N
1 5 5 .4 5
NH
3 .5 5
3 5 .6
2 .5 8
3 3 .4
7 .7 3
C 11H 15N 5O 3
E x a c t M a s s : 2 6 5 ,1 2
M o l. W t.: 2 6 5 ,2 7
O
1 7 1 .7 8
O
3 .6 1
5 1 .3 4
---------------------------------------------
Yield : 7.5 mg
200 ms050503 #13
mAU
9 - 4,528
et18f
UV_VIS_1
WVL:235 nm
150
70,0
50,0
Peak #9
%
4.68
210.4
289.7
100
25,0
50
560.3
15 - 30,910
12 - 15,506
7 - 1,214
8 - 1,367
6 0,598
- 1,125
123-45--0,188
- 0,964
1,057
-0,515
13 - 24,164
14 - 30,573
16 - 47,606
11 - 12,692
10 - 9,838
min
-20
0,0
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
Fig (3.5.61): HPLC chromatogram and UV spectrum of compound 36
[M+1]+
[M-side chain]+
[M-(OMe+NCO)]+H+
[2M+1]+
[M-OMe]+H+
Fig (3.5.62):ESI-MS spectrum of compound 36
238
595
Results
Compound 36, has the molecular formula C11H15N5O3 based on HRESIMS-TOF
(measured, m/z 266.123 [M+H]) with 7 degrees of unsaturation. The UV spectrum of 36
showed UV λmax(MeOH) absorption at 210 and 290 nm indicating the same chromophoric
functionalities as 34 and 35. The difference in molecular weight between 34 and 36 was only
32 mass units indicating a loss of C3H6 group from the alkoxy group as evident from its 1H
NMR spectrum. ESI/MS spectrum showed pseudomolecular ion peak at m/z 266.1 (M+H),
531 (2M+H) and 796 (3M+H). Tandem MS fragmentation spectrum showed a retro DielsAlder fragment ion peak at m/z 192 which confirmed a loss of OCN group indicating 2oxopurine derivative (Cook et al 1980, Mitchell et al 1997, Lin et al 1996), and also
exhibited a molecular ion fragment at m/z 234 indicating the loss of a methoxy group.
The 1H NMR spectrum of 36 ( see figure 3.5.63) showed four singlets at δ 7.57 (8CH), 3.60 (N(3)-CH3), 3.92 (N(9)-CH3), and 3.61 (O-CH3 ), in addition to a triplet signal at δ
2.58 for the α-CH2 of the propionic acid moiety, multiplet at 3.55 ppm for the β-CH2 of the
propionic acid and an exchangeable triplet signal at 7.73 ppm for 6-NH. DEPT experiment of
36 deduced a downfield methyl carbon signal at 51.34 ppm and absence of the upfieled
methyl carbon signals which were established for 34 and 35. The attachment of the methyl
group directly to the carboxyl of propionic acid part was confirmed through HMBC
correlation between the methyl proton signal and the carboxyl resonance at 171.78 ppm.
Additional HMBC correlations were assigned in figure 3.5.66 and presented in table 3.5.7.
Compound 36 was then elucidated as methyl-(3,9-dihydro-3,9-dimethyl-2-oxo-2H-purin-6-
N
N
8.0
7.6
6.8
6.4
6.0
5.6
5.2
4.8
4.4
4.0
3.6
129
130
2.0000
O
5.3497
c
51.34
7.2
130
b
O
2.7434
a3.61
177
NH 7.73
171.78
f
0.8451
0.8087
NH
c
b
d
e
2.2173
3.55
35.6
f138.27
7.57
113.1
155.45
2.58
33.4
a
N
141.1
155.81
177
3.92
e 33.33
N
O
178
d
179
3.60
29.84
179
194
379
385
386
387
ylamino) propanoate and was named nigricine 3.
3.2
2.8
2.4
(ppm)
Fig (3.5.63):1HNMR spectrum of compound 36
239
2.0
1.6
1.2
d
N
141.1
155.81
N
3.55
35.6
c
2.58 b
33.4
113.1
a
f138.27
7.57
113.1
c
N
155.45
f
141.1
171.78
29.8
33.3
3.92
e 33.33
N
O
33.3
3.60
29.84
35.6
51.3
113.
138.
141.
155.
155.
171.
Results
e d
b
NH 7.73
O
171.78
180
170
160
150
140
130
120
110
100
90
a3.61
(ppm)
O
80
70
60
51.34
f
50
40
30
e
a
Fig (3.5.64):13CNMR and DEPT spectra of compound 36c b
ad
c
e
f
b
(ppm)
2.4
b
d
a&c
e
3.2
3.60
29.84
4.0
d
e
N
O
N
141.1
155.81
c
2.58 b
33.4
138.27
7.57
N
155.45
3.55
35.6
4.8
f
113.1
N
3.92
33.33
5.6
NH 7.73
6.4
O
171.78
a3.61
f
O
7.2
51.34
8.0
(ppm)
8.00
7.00
6.00
5.00
Fig (3.5.65):COSY spectrum of compound 36
240
4.00
3.00
20
d
10
0
Results
d
e a
f
b
c
NH
d
e&bc
(ppm)
40
a
3.60
29.84
d
3.92
e 33.33
N
O
N
f
113.1
c
2.58 b
33.4
f
138.27
7.57
N
155.45
3.55
35.6
80
N
141.1
155.81
NH 7.73
120
O
171.78
a3.61
O
160
51.34
(ppm)
7.2
6.4
5.6
4.8
4.0
3.2
2.4
Fig (3.5.66): HMBC spectrum of compound 36
Table 3.5.7. NMR Data of compound 36 (Ashourine 3) [500 MHz]
13
1
(DMSO-d6)
(DMSO-d6)
2
155.81
-
N(3)-CH3
29.84
3.60 (s)
4
141.1
-
5
113.1
-
6
155.45
-
6-NH
-
7.73 (t,5.36)
C-6, C-5, C-10
8
138.27
7.57 (s)
C-4, C-5, N(9)-CH3, C-6
N(9)-CH3
33.33
3.93 (s)
C-8, C-4
10
35.6
3.57
C-6, C-12, C-11
Position
C δ ,m
H δ,m, j(Hz)
HMBC
C-2, C-4
(dt,6.94&6.0)
11
33.4
2.58 (t,6.94)
12
171.78
-
13
51.34
3.91 (s)
C-12, C-10
C-12
241
Results
3.5.16 – Nigricine 4 (37, new compounds)
3.62
3.45
N
O
N
3.58, 3.62
N
3.58
N
NH 6.88
O
2.6
3.58
2.55
C12H19N5O3
Exact Mass: 281,15
Mol. Wt.: 281,31
O
-------------------------------------------------
Yield : 1.1 mg
70,0
ms050503 #12
mAU
et18e
UV_VIS_1
WVL:235 nm
7 - 5,034
70,0
50,0
Peak #7
%
214.1
5.09
No spectra library hits found!
315.6
37,5
25,0
8 - 47,523
5 - 1,210
4 - 1,118
12,5
6 - 1,341
0,0
-0,375
0,592
123- -Peak
1 - 0,063
0,0
nm
-10,0
min
-10,0
10,0
20,0
30,0
40,0
50,0
60,0
200
400
595
Fig (3.5.67): HPLC chromatogram and UV spectrum of compound 37
[M+1]+
[M-OMe]+H+
[M-side chain]+ [M-(OMe+NCO)]+H+
[2M+1]+
Fig (3.5.68):ESI-MS spectrum of compound 37
Compound 37, was assigned molecular formula C12H19N5O3 based on ESIMS analysis
with 6 degrees of unsaturation. ESI/MS spectrum showed positive pseudomolecular ion peaks
at m/z 282.1 (M+H), 280 (M-H) and 563 (2M+H). Similar to the previous congeners, MS
fragmentation showed a fragment ion peak at m/z 250.2, [M-(OMe)]+ and a characteristic
242
Results
fragment ion peak at m/z 208, [M-(OMe+NCO)]+ which confirmed the loss of OCN group
indicating 2-oxopurine derivative (Cook et al 1980, Mitchell et al 1997, Lin et al 1996), and
also exhibited a fragment ion peak at m/z 196.2 (24 %) indicating the loss of the side chain.
This fragmentation pattern is typical as those of 36 (nigricine 3), [figures 3.5.62 & 3.5.68]
with the exception of a loss of one double bond and addition of one methyl group at position
7.
The UV spectrum of 37 showed bands at λmax (MeOH) 214 and 315 nm indicating the
same chromophoric functionalities as 34, 35, and 36. The difference in molecular weight
between 36 and 37 was 16 mass units. The additional substituent was deduced to be away
from the alkoxy group, because the methoxy group is still present at δ 3.58 as evident from
its 1H NMR spectra. The additional methyl singlet at δ 2.55 indicated an additional N-methyl
function [N(7)-CH3]. Disappearence of the sharp singlet at 7.57 ppm and presence of two
coupled protons at 3.58 and 3.62 ppm indicated the saturation at position 8. The presence of a
triplet signal at 6.88 ppm (J=5.7Hz) ensures the presence of NH which showed a COSY
correlation to adjacent ethylenes at δ 3.58 (2H, m) and δ 2.6 ( 2H, t, J= 6.6 Hz). The NH
signal was shifted upfield indicating the shielding effect of the new methyl group at position
7. From the above NMR data, MS fragmentation pattern and other spectral data, compound
37 was elucidated as methyl-(3,7,8,9-tetrahydro-3,7,9-trimethyl-2-oxo-2H-purin-6-ylamino)
3439.25
3.45
N
O
N
G1 G2
NH 6.88
3.58
B
6.0
5.5
5.0
4.5
4.0
3.5
2.6466
2.8198
3.6618
0.9131
6.5
5.7234
G2
NH
Integral
1292.30
B
G1
(ppm)
7.0
1298.92
C
A
6.90
7.5
1305.54
F
O
3.58
0.9131
2.55
O
2.6
8.0
1728.31
A
D
E
N
C
NH
1767.09
E
3.58, 3.62
N
1774.65
1779.38
1782.85
D
3.62
1785.69
1805.86
3439.25
propanoate and assigned the trivial name nigricine 4.
3.0
2.5
2.0
1.5
1.0
(ppm)
Fig (3.5.69) total 1HNMR spectrum of compound 37
243
0.5
Results
D
D
3.45
N
O
A
E
3.62
N
F
E
G1 G2
3.58, 3.62
N
N
NH 6.88
C
3.58
B
G1
2.55
F
B
G2
O
2.6
C
O
3.58
A
Fig (3.5.70): part of 1HNMR spectrum of compound 37
B/C
NH/C
G1/G2
D
3.62
N
O
E
3.45
N
G1 G2
3.58, 3.62
N
C
3.58
N
NH 6.88
O
2.6
B
3.58
2.55
F
O
A
Fig (3.5.71) COSY spectrum of compound 37
244
Results
3.5.17. Nigricinol [4-((1H-Indol-3-yl)methyl)-2-amino-5-(1H-indol-3-yl)-3H-pyrrol-3one] (38, new compounds)
NH2
O
137.0
176.5
3.72
7.53
111.0
119.5
7.0
1283
122.3
137.9
112.1
7.33
7.16
122.3
127.2
7.19
123.6
7.94
7.15
124.5
7.08
8.05
122.0
108.0
109.0
119.9
N
137.9
32.3
133.5
N
H
N
H
137.9
1125
7.43
C21H16N4O
Exact Mass: 340,13
--------------------------------------------------------------------
Yield : 3.2 mg
350 ms040918 #6
mAU
tm7et11f
UV_VIS_1
WVL:235 nm
70,0
300
8 - 18,275
Peak #8
%
18.37
214.8
200
25,0
280.0
100
558.9
9 - 19,081
0
10 - 23,016
4
57 --- 1,267
1,311
1,452
1,392
- 1,044
1,113
1 -236Peak
1 - 0,100
12 - 47,243
11 - 29,545
min
-50
0,0
10,0
20,0
30,0
40,0
50,0
-10,0
200
nm
400
595
60,0
Fig (3.5.72): HPLC chromatogram and UV spectrum of compound 38
Fig (3.5.73):GC-MS spectrum of compound 38
Compound 38 was isolated as brownish white amorphous powder. It has UV
absorbance of typical indole compound at λmax 214 and 280 nm. ESI-MS showed
245
Results
pseudomolecular ion peak m/z 339.1 [M-H] while GC-MS showed a molecular ion peak at
m/z 340 [M]+ and fragment ion peaks at m/z 284 [M-(COCNH2)]+, 207 [M-(indolyl group +
NH2)]+ and 177 [M-(3-indolylmethylene group + O + NH2)]+, suggesting the molecular
formula C14H18N2O2. 1H NMR spectrum showed characteristic spin systems for two indole
groups. The first indole group showed an ABCD spin system at δ 7.55 (d, J=7.56 Hz), 7.00
(dt, J=1.2,6.9 Hz), 7.08 (dt, J=1.2, 6.9 Hz), 7.33 (d, J=8.2 Hz) for H-4', H-5', H-6', and H-7',
respectively. Furthermore, the H-2' resonance at δ 7.15 (s) exhibited a COSY correlation to
an exchangeable NH signal at 10.9 ppm. Another indole group showed an ABCD spin system
as shown by 1H signals at 8.05 (dd, J=6.3, 1.2 Hz), 7.16 (dt, J=1.2, 6.9 Hz), 7.19 (dt, J=1.2,
6.9 Hz), 7.43 (dd, J=6.9, 1.2 Hz) for H-4", H-5", H-6", and H-7", respectively. The H-2''
signal at δ 7.94 (s) exhibited a COSY correlation to another exchangeable NH resonance at
11.8 ppm. The first 3-indolyl group is attached to a methylene group resonating at δ 3.72 (s)
which displays an HMBC correlations to carbons at δ124.5, 109.0, and 128.0 of C-2', C-3',
and C-3a', respectively. The methylene protons also exhibited an additional HMBC
correlations with two carbons resonating at 176.5 ppm (C-3) and 110.0 ppm (C-4). The H-2"
methine singlet showed HMBC correlations to carbons resonating at δ 108.0, 127.2, and
137.9 for C-3", C-3a", and C-7a", respectively. The NMR data and mass fragmentation
pattern confirmed 38 to be 4-((1H-indol-3-yl)methyl)-2-amino-5-(1H-indol-3-yl)-3H-pyrrol-
1856.61
3490.31
3491.57
3498.51
3505.44
3506.70
3532.56
3533.82
3540.75
3547.69
3548.95
3572.91
3576.69
3578.58
3579.85
3586.15
3588.04
3594.35
3595.61
3601.28
3602.54
3658.66
3666.86
3708.47
3715.41
3716.67
3759.55
3767.11
3967.62
4025.63
NH2
4030.67
4031.93
3-one and was assigned as nigricinol.
O
N 7a''
H
1''
10.8
10.4
10.0
9.6
9.2
8.8
8.4
8.0
7.6
7.2
2.4768
0.9776
1.2031
1.0000
Integral
11.2
8’
7''
3.4505
N
H
7a'
7'
6''
0.9923
6'
2''
2'
1.0870
3a'
5''
3a''
0.9888
5'
4''
3''
5
4
3'
N1
0.8266
4'
2
3
8'
6.8
6.4
6.0
5.6
5.2
4.8
4.4
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
3490.31
3491.57
3498.51
3505.44
3506.70
3532.56
3533.82
3540.75
3547.69
3548.95
3572.91
3576.69
3578.58
3579.85
3586.15
3588.04
3594.35
3595.61
3601.28
3602.54
3658.66
3666.86
3708.47
3715.41
3716.67
3759.55
3767.11
3967.62
4025.63
4030.67
4031.93
(ppm)
*
N
E
P
*
S
*
S
8.30
8.25
8.20
8.15
8.10
8.00
7.95
7.90
7.85
7.80
7.75
7.70
7.65
7.60
7.55
7.50
7.45
7.40
7.35
7.30
7.25
7.20
6’
5’
7.15
7.10
0.9776
3.4505
0.9923
1.0870
0.9888
0.8266
8.05
7’
7”
4’
1.0000
Integral
4”
1.2031
5”
2’
6”
2”
7.05
7.00
6.95
6.90
6.85
6.80
(ppm)
Fig (3.5.74):total 1HNMR spectrum of compound 38 (up) and part of 1HNMR showing
an aromatic region (down).
246
Results
4”
5”
6” 2’ 6’
4’ 7” 7’
2”
5’
(ppm)
6.8
7.2
7.6
NH2
O
4'
4
3'
5'
3a'
6'
N1
4''
3''
5
2''
2'
N
H
1''
N
H
7a'
7'
2
3
8'
5''
8.0
3a''
7a''
6''
7''
1'
8.4
(ppm)
8.0
7.6
7.2
6.8
Fig (3.5.75):Part of COSY spectrum of compound 38
2''
2'
6'',5''
7'
4' 7''
4''
8'
6',5'
NH2
(ppm)
O
4'
3a'
6'
7'
2''
2'
N
H
7a'
N1
5
4
3'
5'
2
3
8'
4''
3''
N
H
1''
3a''
40
5''
H-8'/C-3'
6'' & C-4
7a''
7''
H-8'/C-3a',C-2
80
1'
120
160
200
(ppm)
80
72
64
56
48
40
H-8'/C-3
32
Fig (3.5.76):Total HMBC spectrum of compound 38
247
Results
2''
4''
4'
2'
5''
6''
7'
7''
5'
6'
(ppm)
H-2"/C-3"
H-2'/C-3'
H-5'/C-7'
112
H-5"/C-7"
H-6'/C-4'
H-7'/C-5'
H-7"/C-5"
H-6"/C-4"
120
H-4'/C-6'
H-4"/C-6"
H-2'/C-3a' H-5'/C-3a'
128
H-2"/C-3a"
H-7'/C-3a'
H-5"/C-3a"
H-2'/C-7a'
H-2"/C-7a"
H-4"/C-7a"
(ppm)
H-4'/C-7a'
8.00
7.80
136
H-6'/C-7a'
7.60
7.40
7.20
7.00
1809.96
3475.82
3483.07
3490.64
3521.85
3529.73
3536.98
3560.94
3567.24
3574.81
3576.07
3583.95
3591.20
3605.39
3663.40
3671.28
3677.90
3719.20
3726.77
3989.07
3475.82
3736.23
3990.96
3483.07
3744.11
3997.90
3490.64
5444.01
5895.47
Fig (3.5.77):part of HMBC spectrum of compound 38
7'
3521.85
3529.73
3536.98
3560.94
3567.24
3574.81
3583.95
3591.20
3605.39
3663.40
3576.07
NH2
6''
7''
4'
3671.28
3677.90
3719.20
3726.77
3736.23
3744.11
NH2
O
5''
2'
4'
6'
5'
3a'
6'
7.40
7.30
7.20
7.10
5''
3a''
N
H
1''
N
H
7a''
6''
7''
8'
1'
2''
1.0000
1.2523
3.2953
1.0357
1.1610
1.2827
1.1969
Integral
7.50
4''
3''
2''
2'
7a'
7'
N1
5
4
3'
5'
2
3
8'
7.00
(ppm)
14.5
14.0
13.5
13.0
12.5
12.0
11.0
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
1.6345
1.0000
1.2523
3.2953
1.0357
1.1610
1.2827
2.2151
0.5724
11.5
1.1969
4''
1'
0.9706
Integral
1''
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
(ppm)
Fig (3.5.78):1HNMR spectrum of compound 38 (measured in DMSO-d6)
248
Results
2''
7''
4''
7'
4'
2'
6'' 5''
6'
5'
(ppm)
7.16
H-2'/C-3'
NH2
O
136.5
173.4
3.62
7.48
6.97
118.5
118.5
107.5
7.21
124.5
7.05
121.3
136.3
111.5
7.33
N
H
7.15
122.5
112
121.2
126.0
106.5
7.98
132.5
H-5'/C-7'
H-2"/C-3"
7.99
136.5
108.5
127.5
N
104
NH2/C-4
7.14
122.5
N
H
136.1
H-5"/C-7"
H-4"/C-4”
direct
H-7'/C-5'
1125
7.44
H-6'/C-4'
H-6"/C-4"
H-7"/C-5"
H-4"/C-6"
120
11.8
10.9
H-5"/C-3a"
C21H16N4O
Exact Mass: 340,13
H-2"/C-3a"
H-7"/C-3a"
H-2'/C-3a'
H-7'/C-3a'
H-2"/C-2”
direct
H-6"/C-7a"
H-2'/C-7a'
H-2"/C-7a"
(ppm)
8.0
H-6'/C-7a'
136
NH2/C-5
7.6
7.2
Fig (3.5.79):part of HMBC spectrum of compound 38
249
128
H-5'/C-3a'
6.8
Results
Table (3.5.9): NMR data of 38 (500 MHz, Methanol-d4),
Carbon
13
1
C NMR,
ppm
2
137.0
3
176.5
4
111.0
5
137.9
1
H NMR, ppm
(Multiplicity, J= Hz)
H NMR*, ppm
(Multiplicity, J= Hz)
(NH2), 7.15 br s
1'
10.9 (s)
2'
124.5 d
7.15 (s)
7.15 (s)
3'
109.0 s
3a'
128.3 s
4'
119.5 d
7.55 (d, J=7.56)
7.48 (d, J=7.9)
5'
119.9 d
7.00 (dt, J=1.2,6.9)
6.97 (t, J=8.2)
6'
122.3 d
7.08 (dt, J=1.2, 6.9)
7.05 (t, J=7.9)
7'
112.0 d
7.33 (d, J=8.2)
7.33 (d, J=7.8)
7a'
137.9 s
8'
32.3 t
3.72 (s)
3.62 (s)
1"
11.8 (s)
2"
133.5 d
7.94 (s)
7.98 (s)
3"
108.0 s
3a"
127.2 s
4"
122.0 d
8.05 (dd, J=6.3, 1.2)
7.99 (dd, J=7.00)
5"
122.3 d
7.16 (dt, J=1.2, 6.9)
7.15 (t, J=7.3)
6"
123.6 d
7.19 (dt, J=1.2, 6.9)
7.14 (t, J=7.00)
7"
112.5 d
7.43 (dd, J=6.9, 1.2)
7.44 (dd, J=7.5)
7a"
137.9 s
* meassured in DMSO-d6
Biological activity :
None of the isolated compounds from Petrosia nigricans showed significant
antimicrobial activity against B.subtilis, S. cerevisae, C. herbarum, and C. cucumerinum.
All purine derivatives (nigricines 1 to 4) showed no cytotoxic activity against L5178Y
cell line, while nigricinol (compound 38) showed 68.1 % cytotoxic activity at a concentration
of 10 µg/ml.
250
Results
3-6- Natural Products from Callyspongia biru
Sponges belonging to the genus Callyspongia are extensively investigated for
bioactive natural products. Many new bioactive natural products have been isolated from
different Callyspongia sp., these natural products includes unusual steroids (Theobald et al
1978, Agrawal and Garg 1997); acetylene derivatives ( Youssef et al 2003, Nakao et al 2002
and Youssef et al 2000); uncommon fatty acids (Carballeira and Pagan 2001, Toth and
Schmitz 1994); diterpenes (Garg and Agrawal 1995) and triterpenes (Fukami et al 1997).
Cyclic peptides have also been described from the genus Callyspongia which includes
phoriospongins A and B from C. bilamellata (Capon et al 2002), callynormine A from C.
abnormis (Berer et al 2004) and callyaerins from C. aerizusa (Min, et al., 2001). Many
natural product-producing fungi have been isolated from some Callyspongia sp., and
cultivated on artificial media for production of natural products [spiciferone derivatives,
macrolides, anthraquinones, and benzofuran have been isolated from C. aerizusa–derived
fungi (Edrada et al 2000, Jadulco et al 2001, Jadulco et al 2002)]. The most attractive
compound, that was isolated from Callyspongia sp., is callystatin A. Callystatin A is a potant
cytotoxic polyketide isolated from Nagasakian marine sponge, Callyspongia truncata
(Kobayashi et al 1997a), and due to the biological importance many studies have been carried
out to synthesize this natural product (Murakami et al 1997, Lautens and Stammers 2002,
Enders et al 2002, Vicario et al 2002).
Methanol extract of an Indonesian sponge Callyspongia biru was investigated in the
present study and five compounds were elucidated. These compounds includes indole-3carbaldehyde, p- hydroxyphenylacetic acid, p-hydroxyphenylacetic acid methylester, which
have been described earlier (see compounds 15, 26, and 27) in the present study, in addition
to indole-3-acetic acid, and 2`-deoxythymidine.
251
Results
3.6.1- Indole-3-acetic acid (39, known compound)
COOH
3.12
7.48
6.94
7.20
7.05
N
H
10.8
7.32
C10H9NO2
Mol. Wt.: 175,18
----------------------------------------------------------
Yield : 2.5 mg
900 ms050524 #5
mAU
58etj3
UV_VIS_1
WVL:235 nm
11 - 19,246
Peak #11 19.21
70,0
No%spectra library hits found!
224.6
600
400
279.6
286.6
200
- 20,077
-12
18,190
9 10
- 17,522
--0,961
1,236
1,309
1,390
1,114
--0,432
1,046
12345
-67-80,185
14 - 47,714
13 - 32,831
min
-100
0,0
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
400
Fig (3.6.1) HPLC chromatogram and UV spectrum of compound 39
M+1
M-COOH
2M+1
3M-1
M-1
2M-1
Fig (3.6.2) ESI-MS spectrum of compound 39
252
595
Results
Compound 39, [indole-3-acetic acid] was isolated as yellowish white amorphous
powder. Compound 39 shows pseudomolecular ion peak at m/z 176 [M+H]+ and in the
negative mode at m/z 174 [M-H], 349 [2M-H], and 524 [3M-H] suggesting the molecular
formula C10H9NO2. The UV λmax (MeOH) absorption of 39 was at 224, 279, 286 nm and
were indicative of an indole chromophore. The 1H NMR (measured in DMSO-d6) showed 7
proton resonances that included an aromatic ABCD spin system at δ 7.48 (1H, d, J = 7.5 Hz,
H-4), 6.94 (1H, t, J = 7.3 Hz, H-5), 7.05 (1H, t, J = 7.4 Hz, H-6), 7.32 (1H, d, J = 8.2 Hz, H7). In addition to three proton resonances at δ 7.20 (1H, s , H-2), a most downfield broad
singlet at δ 10.8 (1H, s , H-1) and singlet proton signal at δ 3.12 (2H, s , CH2-8) were also
observed. The NMR data and the UV absorption maxima were identical to those of indole-31577.93
3466.36
3473.61
3480.86
3514.28
3522.16
3529.41
3595.30
3656.15
3664.35
3740.32
3747.89
5418.79
acetic acid ( Aldrich, 1990 and Hiort, 2002).
**
N
E
P
S
3466.36
3473.61
3480.86
3514.28
3522.16
3529.41
3595.30
3656.15
3664.35
COOH
8
3740.32
3747.89
**
**
S
4
5
7.50
12.0
11.0
7.40
1.1937
7.30
5
7.20
7.10
7.00
10.0
9.0
8.0
2.2458
1.0452
1.2372
1.1937
1.1533
1.1962
(ppm)
1.0000
Integral
NH
1.1533
1.1962
7
2
1.0452
4
N
H
6
1.2372
1
6
8
7
2
7.0
6.0
(
5.0
4.0
3.0
)
Fig (3.6.3) 1HNMR spectrum of compound 39
253
2.0
1.0
Results
3.6.1- 2'-Deoxythymidine (40, Known compound)
O
1.77
11.28
NH
7.7
5.03
HO
N
3.55
O
O
3.75
H
4.22
6.18
H
H
H
OH
5.22
C10H14N2O5
Mol. Wt.: 242,23
H
2.08
--------------------------------------------------------
Yield : 10 mg
350 ms050727 #2
mAU
58etl8c
UV_VIS_1
WVL:235 nm
70,0
8 - 5,774
300
Peak #8
%
208.8
5.87
No spectra library hits found!
267.0
200
25,0
100
0
-50
0,0
557.0
10 - 47,215
9 - 32,699
5
1,226
1,395
67---0,957
1,279
1,042
1,100
1234- -0,329
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
nm
300
400
595
Fig (3.6.4) HPLC chromatogram and UV spectrum of compound 43
[2M+1]
[M+H-(deoxyribose)]
[M+1]
[M-(CHCH2OH+H)]
[M+ formic acid-1]
[M-1]
[M-(deoxyribose+H)]
[2M+ formic acid-1]
[2M-1]
[M-(CH2CHOHCHCH2OH+H)]
[M-(CHOHCHOCH2OH+H)]
[3M-1]
Fig (3.6.5) ESI-MS spectrum of compound 40
Compound 40, [2'-deoxythymidine] was isolated as white amorphous powder. It
showed pseudomolecular ion peaks at m/z 243 [M+H], 486 [2M+H], 241 [M-H], 287
[M+formic acid-H], 483 [2M-H], 529 [2M+formic acid -H], and 725 [3M-H] suggesting the
254
Results
molecular formula C10H14N2O5. The UV λmax (MeOH) of 40 at 208, and 267 nm was deduced
for a nucleoside nature of the compound. Molecular ion fragmentation pattern confirmed the
presence of thymine-2‘-deoxyribose (see figure 3.6.5 ). 1H NMR spectrum showed a broad
singlet at 11.28 (1H, s, 3-NH), 7.7 (1H, s, H-6), 6.18 (1H, t, J = 7.0 Hz, H-1'), 2.08 (2H, m, H2'), 4.22 (1H, br s, H-3'), 3.75 (1H, dd, J = 6.6& 3.1 Hz, H-4'), 3.55 (2H, m, H-5'), 1.77 (3H, s,
H3-7). The 1H NMR data were identical with those of thymine-2‘-deoxyribose (Aldrich 1992
). From the above NMR data, mass fragmentation patterns, and UV spectrum, compound 40
879.62
1008.56
1011.40
1014.87
1018.65
1022.12
1027.16
1033.15
1039.77
1046.39
1053.01
1758.26
1769.92
1773.71
1783.48
1787.89
1795.46
1867.02
1870.18
1873.65
1876.80
2110.72
2506.38
2510.79
2608.21
2611.99
3070.39
3077.32
3084.26
3843.10
5629.39
was concluded to be thymine 2´-deoxyribose.
O
7
5
7
4
6
3 NH
2
1
N
HO
5'
O
1'
H
1'
H
OH
H
4'
3’-
11 6
11 2
10 8
10 4
10 0
9 6
9 2
8 8
8 4
8 0
7 6
7 2
6 8
6 4
6 0
5 6
5 2
0.9703
0.8594
0.9741
0.9736
0.7653
Integral
12 0
0.8270
5’-
NH
4 8
4 4
2'
5'
3'
4 0
3 6
3 2
2 8
2 4
2.8184
2'
H
1.8595
3'
2.0458
H
1.0000
4'
6
O
2 0
1 6
1 2
0 8
0 4
Fig (3.6.6) 1HNMR spectrum of compound 40
Biological activity:
Total methanol extract, ethylacetate- and butanol-fractions along with n.butanol
subfractions were tested for cytotoxic activity using the brine shrimp assay. The butanol
subfraction (but-V) showed strong cytotoxic activity against the brine shrimp (Artemia salina
Leach). This fraction was further chromatographed and purified to gave (But-Ve3) which
seems to be alkylpyridinium compound. Howevere the structure elucidation of the compound
is still under revision.
Deoxythymidine (compound 40) showed cytotoxic activity 76.2 % and 41.7 % against
L5178Y mouse lymphoma cell line at concentrations 3 and 10 µg/ml respectively.
255
Discussion
IV- Discussion
The main target of the medicinal chemist is concerned about the exploration of new
effective and less toxic medicaments. However, the main objective of the pharmacognosist or
phytochemist is the presentation of this effective medicament from the natural sources. The
essential topics that the phytochemist will be dealing with through the achievement of this
task includes isolation, chracterisation, structure elucidation, and biological evaluation of the
targeted natural products (Exarchou, et al, 2005; Lambert, et al, 2005).
Concerning the above mentioned
Target identification
topics many problems will be faced.
- Cellular and m olecular biology
However, the employment of the new
- Genom ics
- Proteom ics
- Bioinform atics
technology in the field of natural product
chemistry solved many of these problems.
Target validation
All parts of the process leading to an
- Knock-in and knock-out anim al m odels
elucidated structure have experienced an
- A ntisense nucleic acid and antibodies
- Proteom ics
- Structural biology/structural genom ics
immense speed-up in the past fifty years.
Separation
technology,
analytical
Lead identification
and
- H igh throughput screening
- N atural products screening
- N M R-based screening
- Virtual screening
- Com binatorial chem istry
- Com pound library design
- Structure-based design
spectroscopic methods have improved
steadily and with good fortune, a chemist
might be able to go from a crude extract to
a full set of 2D NMR spectra in one day
Lead optim ization
(Steinbeck 2004). It should be borne in
- M edicinal chem istry
- Parallel synthesis
- Design of focus com pound libraries
- M olecular m odeling, Q SAR
- in vivo pharm acology
- Pharm acokinetic and toxicology
mind that the above mentioned tasks
(isolation,
characterisation,
structure
elucidation, and biological evaluation) are
Preclinical and clinical developm ents
only a part of the whole process of new
drug discovery. Scheme 1 (Giersiefen et
al., 2003),
summarizes the whole drug
discovery process.
Effective D rug
Scheme (1) phase of drug discovery process
(Giersiefen et al., 2003)
256
Discussion
4-1
Isolation, characterisation, structure elucidation, and biological evaluation of
natural products:
4.1.1 Isolation of natural products:
Unlike the medicinal chemist, who usually concentrates on a series of synthetic
compounds of known chemical and physical properties and hence is able to master the limited
number of separation techniques applicable to the specific chemotype, the natural product
chemist must be prepared to deal with molecules of the whole spectrum of bioactive
metabolites. These can vary in hydro- and lipophilicity, charge, solubility, and size.
(McAlpine and Hochlowski 1994). In general, the more hydrophilic metabolites may be
candidates for ion exchange chromatography, reversed phase silica gel chromtography, or size
exclusion chromatography on polysaccharide resins. The more lipophilic metabolites can be
further purified by chromatography on normal phase silica gel, florisil, alumina, or lipophilic
size exclusion resins such as sephadex LH-20. They may be also candidates for a variety of
high-speed countercurrent techniques or chromatography on polyresins (McAlpine and
Hochlowski1994). However, this branch of the main task of the natural product chemist was
advanced to large extent. The production of many new packing materials, new isolation
instruments with high degree of resolution and detection facilitated the chemist´s duty in this
branch of the task. Some problems are still unresolved, for example, separation of individual
pure compounds from mixture of cerebrosides, many trials were applied for achievement of
this goal but unfortunately, all failed. The sphingolipids (cerebrosides) are easy to be
separated as a mixture of closely similar chemical constituents from natural sources.
However, they are very difficult to be separated from each other or in another word they are
very difficult to be separated and purified to analytical purity. The difficulty arises probably
from their chromatographic properties being overshadowed by the polar nature of the
glycoside, thus making these metabolites difficult to separate to analytical purity (Jenkins
1999). Furthermore, the fact that, these metabolites are present oftenly as a series of very
similar chemical nature, where the difference in most cases is one or two methylene groups,
thus many of the published cerebrosides were reported as a groups of mixed compounds (e.g.,
Jenkins 1999& Inagaki 2003 ). However, HPLC in reversed-phase mode is now the standard
method for separation of molecular species of cerebroside mixture, often after benzoylation so
that the lipids can be detected by sensitive UV spectroscopy. Mass spectrometric methods are
now being used increasingly for charaterization purposes of such sphingolipids (Christie,
2003)
257
Discussion
4.1.2 Characterisation of natural products:
This branch of the task is the first step in structure elucidation. Therefore it is very
important for the natural product chemist, he has to spend his effort and time to drive the
structure proposal in the right direction. In contrast, the medicinal chemist, who is dealing
with the synthetic chemicals, has no need to spend his time and effort here because he knew
previously what type of chemicals he is dealing with. In the fact, there are many simple and
general chemical tests that are widely available and more applicable since a long time, for
example, Molish’s test for carbohydrates, Mayer`s and Dragendorf`s reagents for alkaloids,
alkali solution (e.g.NaOH, and KOH) for both anthraquinones and phenolic compounds.
However, these reagents were intended to dissolve a small part of the problem. The
phytochemist can not decide precisely and also confirm the structure using these simple
reagents. These reagents tells us about the general chemical class to which the isolated
compound may be related. But in most cases they give no idea about the subchemical class
rather than the smallest details of the structure. Metabolite profiling is not an easy task to
perform since natural products display a very important structural diversity. For each
compound, the order of the atoms and stereochemical orientations have to be elucidated de
novo in a complex manner and the compounds can not simply be sequenced as it is the case
for genes or proteins. Consequently a single analytical technique does not exist, which is
capable of profiling all secondary metabolites in the biological source (Wolfender, et al
2005). However, the employment of advanced analytical and spectroscopic methods like UVand IR- spectroscopy solved such problems to a large extent, where they can give a good idea
about the different substructures and/or functional groups of the structure. The hyphenated
techniques coupled to HPLC give not only ideas about the details of functional groups, but
also play an important role in what is called „Dereplication process“ in order to avoid the
tedious isolation of known compounds, and directed the chemist´s effort toward the targted
isolation of constituents presenting novel or unusual spectroscopic features (Wolfender, et al
2005).
In the present study, a lot of chemical compounds that share the same chromophoric functions
were examined by the hyphenated technique HPLC-UV-photodiode array detection (LC/UVDAD). This examination showed that, all chemicals having the same chromophoric functions
will show the same UV spectrum with the same absorption maxima even though they are
different in their additional non-chromophoric functions and their molecular weights.
258
Discussion
-
Kahalalide F, (compound 1, m/z 1478), R (8, m/z 1520) and S (9, m/z1536), all having the
same chromophoric function and therefore showing the same UV spectrum and having
approximately the same absorption maximum at ~ 203 nm.
-
Kahalalide E (2, m/z 836), Kahalalide D (2, m/z 596), N,N-dimethyl tryptophan
methylester. (6, m/z 246), indole-3-acetic acid (39, m/z 175), all having the same
chromophoric function (tryptophan amino acid) and therefore showing the same UV
spectrum and having approximately the same absorption maxima at ~ 220, 279 sh and 285
sh nm.
-
A group of 3-acyl indole derivatives, e.g., hyrtiosine A (13, m/z 191), 5-hydroxy-1Hindole-3-carbaldehyde (14, m/z 161), ), indole-3-carbaldehyde (15, m/z 145) and 5deoxyhyrtiosine A (16, m/z 175) all having the same chromophoric function (3-acyl
indole) and therefore showing the same UV spectrum and having approximately the same
absorption maxima at ~ 210, 250, 275 and 300 nm. The same UV spectrum was obtained
for the new indole derivative, isohyrtiosine A (17, m/z 191) with a little deviation in the
UV-absorption maxima. Isohyrtiosine A showed an absorption maxima at 214, 241 and
286 nm due to the presence of carboxyl group instead of a ketonic group.
-
New purine derivatives, nigricines 1-3 (34, m/z 307), (35, m/z 279), (36, m/z 265), all
having the same chromophoric function (2-oxo-purine) and therefore showing the same
UV spectrum and having approximately the same absorption maxima at ~ 210 and 290
nm. The same UV spectrum was obtained for both nigricine 4 (37, m/z 281), and
adenosine (30, m/z 267), with a little deviations in the UV-absorption maxima. Ashourine
4 showed an absorption maxima at 214 and 315 nm due to N(7)-methylation and loss of
one double bond at position 8, while adenosine showed an absorption maxima at 207 and
257 nm because it has no 2-oxo- group.
-
p-Hydroxyphenyl acetic acid, and its methyl-, ethyl- and butylesters, [(26, m/z 152) (27,
m/z 166) (28, m/z 180) and (29, m/z 208)] all having the same chromophoric function (4-
hydroxyphenyl) and therefore showing the same UV spectrum and having approximately
the same absorption maxima at ~ 202, 224 and 275 nm.
However, the compounds that have no chromophoric functions can not be characterised by
LC-UV hyphenated technique.
Also, NMR spectroscopy play a significant role in characterisation of natural products:
The peptide nature of isolated kahalalides (compounds 1-5, 8& 9) was suggested by:
259
Discussion
1) From 1HNMR, the presense of amide NHs resonating in the downfield region between
7.0-9.0 ppm, α-protons resonating arround 4.0 ppm and the CH3 groups resonating in the
higher field region arround 1.0 ppm.
2) From 13C NMR, The presence of amide carbonyls resonating arround 170 ppm, α- carbons
(sp3 methines) resonating between 50-60 ppm and methyl carbons in the higher region.
3) From a COSY, each amino acid could be distinguished by a sequential correlations
beginning from the amide NH, through α-, β-, γ- to the methyl protons which could be
confirmed by the total correlation spectroscopy (TOCSY).
-
The cerebroside nature of compounds 32 and 33 (petrocerebrosides 1 and 2) were
established from the 1HNMR which showed a triplet-like signal at δ 0.82 (terminal
methyls) and broad singlet at δ 1.2 ( long chain (CH2)n groups for both fatty acids and
long chain bases), the presence of several doublets in the region between 4.2-5.3 ppm
indicated the presence of many OHs (of the sugar parts), the presence of multiplet signals
between 3.35 and 5.0 ppm indicated the presence of sugar CHs. In addition to one amide
NH in the lower field region rather than the sp2 methines in aromatic region between 5.0 –
9.0 ppm region. This suggestion cauld be supported by the presence of amide carbonyl
signal in 13CNMR at approximately 170.0 ppm.
-
The steroidal nature of the isolated steroidal compounds and also the scalarane type
sesterterpenoids could be suggested from 1HNMR by the presence of a characteristic
methyl around 1.0 ppm and presence of overlaping methylenes and sp3 methines in the
higher field region between 1.0 and 2.0 ppm, which was supported by a characteristic 13CNMR spectrum.
-
p-Disubstituted phenyl group could be distinguished from 1HNMR by the presence of two
doublets inter-correlated at approximately 6.8 and 7.2 ppm (especially for 4-hydroxy-1substituted phenyl group), for example, tyrosine-containig natural products (kahalalide B,
compound 4), and p-hydroxyphenylacetic acid and its derivatives (compounds 26, 27, 28
and 29).
-
3-Substituted-indole could be easily distinguished from 1HNMR by the presence of
ABCD spin system in the aromatic reagion (approximately between 7.00 and 8.2 ppm) for
proton resonances of H-4, H-5, H-6 and H-7, in addition to a relatively sharp singlet or in
rare cases doublet with small coupling constant of H-2 between 7.0 and 8.0 ppm.
Furthermore the sharp singlet in the down field region between 10.0 and 12.5 ppm
indicating the indole NH. The present examples are compounds 2, 3, 6, 15, 16, 38, and
39. In contrast 5-hydroxy-3-substituted indole displays an ABM spin system
260
Discussion
(approximately between 6.80 and 8.0 ppm) for proton resonances of H-4, H-6 and H-7
instead of the above ABCD spin system (see 1H NMR of compounds 13, 14 and 17).
Mass fragmentation pattern, could play a significant role in characterisation of natural
products:
-
Comparison of the MS fragmentation of the isolated compound with those of the known
authentic samples may be enough not only for characterisation of substructures but also
for full structure elucidation of the compounds (e.g. compounds 27 and 31).
-
MS fragmentation pattern may be helpful in characterisation of some substructures which
seem difficult to be distinguished by other means, rather than they are also helpful in
establishing the relation between the closely related natural products, for example, the
characteristic loss of 42 mass unit through retro Diels-Alder fragmentation from the new
purines, nigricines 1-4, (compounds 34-37) indicated the presence of 2-oxo-purine
derivatives and consequently the loss of NCO fragment, thus, the closed relation between
them could be established.
-
MS fragmentation mechanisms (e.g. tandem ESI/MS and MALDI-TOF-PSD) are
described as the methods of choice in characterisation and confirmation of the amino acid
sequence of the isolated peptides, for example, kahalalides F, E, D, B, C, R and S
(compounds 1, 2, 3, 4, 5, 8 and 9 respectively).
4.1.3 Structure Elucidation of Natural Products:
After the characterisation of the isolated natural product and determination of the
subchemical class to which the compound is related, the phytochemist has to demonstrate
unambigiously the small details of the substructures and consequently the elucidation of the
complete structure. Structure elucidation of the isolated new natural product is still the
bottleneck of the objective achievement. Actually, the new technology in the field of NMR
spectroscopy (including 1D- and 2D- NMR experiments) and mass spectrometry facilitated
the chemist´s effort in this part of the whole task. Furthermore, the presence of commercial
and non commercial computer-assisted structure elucidation (CASE) programs even though
they are not widely available and less applicable nowadays but may be the method of choice
in the near future. It is important to say that the interaction between a spectroscopist and a
CASE system will remain important in order to generate the correct structure rapidly.
Therefore CASE will complement the skills of the spectroscopist, not replace them. The use
of CASE system is likely to increase in the near future, and this will enable the bottleneck so
often caused by structure elucidation to be removed from the natural product drug discovery
process (Jaspers 1999).
261
Discussion
4.1.4 Biological evaluation of natural products:
In general, drug discovery strategies can be trivially separated into three categories:
1. Chemically driven, finding biological activities for purified compounds.
2. Biologically driven, bioassay-guided approach begining with crude extracts.
3. Combination of chemically and biologically driven approaches (vide infra).
(McConnell et al. 1994)
Beginning in the 1970´s through today, the majority of the academic-based research
efforts has become essentially „biologically driven“ i.e., the object of the search has shifted to
discover natural products with biological activity. The biological activities include exploring
their potential as agrochemicals (Crawley 1988) and pharmaceuticals, as well as their possible
chemical ecological roles (Bakus et al. 1986; Hay and Fenical 1988).
Drug discovery in industry has evolved to the use of specific assays with target receptors
and enzymes involved in the pathogenesis of disease rather than cellular or tissue assays
(Johnson and Hertzberg 1989), and has benefitted immensely from breakthroughs in receptor
technology (Hall 1989; Reuben and Wittcoff 1989). These assays reflect new opportunities
due to the recent identification of previously unrecognized biomolecular targets for therapy
(Larson and Fischer 1989). More specifically, this approach for most disease areas is
characterized in industry by:
1. Essentially exclusive reliance on biological activity of crude extracts in numerous targetspecific assays, i.e., enzyme assays and receptor-binding assays, for selection of crude
extracts and bioassay-guided fractionation of the crude extracts (prioritization criteria
emphasize selectivity and potency).
2. High volume, automated screening, i.e., thousands of samples per year for smaller
companies and thousands per week for larger companies.
3. The use of „functional“ or whole-cell assays to confirm activity in a particular disease state
and to further prioritize samples for fractionation.
4. The use of genetically engineered microorganisms, enzymes, and receptors.
Because of low correlation between cytotoxity and antitumor activity, a number of
programs have utilized in vivo tumor models directly for drug discovery (Johnson and
Hertzberg 1989). From 1958 through 1985, National Cancer Institute (NCI) used in vivo
L1210 and P-388 murine leukemia assays as primary screens (Suffness et al. 1989; Boyd et
al. 1988) and was successful primarily in identifying compounds possessing clinical activity
against leukemias and lymphomas. Unfortunately, they were not very successful in finding
262
Discussion
compounds active against slow growing tumours in humans. Further, these in vivo assays
were expensive, time consuming, and relatively insensitive (Suffness et al. 1989). In other
disease areas it was shown that activity in in vitro antiviral assays does not translate well to in
vivo activity, e.g., using Herpes simplex. In contrast, reasonable correlations exist between in
vitro and in vivo antifungal activity, e. g., using Candida albicans.(McConnell et al., 1994).
General or non-specific cytotoxic assays could be insufficient to prove or disprove the
activity. For example, the known antitumour drug candidate (kahalalide F, compound 1)
shows no cytotoxic activity against Artemia salina using brine shrimp assay even though it
has potent antitumour activity.
Using bioactivity guided isolation, it should be borne in mind that the active substance
may be present at very low concentration in the total extract and/or subfractions otherwise
many active compounds will be rejected at an early stage because of lack of significant
activity. Firn and Jones 1996, mentioned some limitations that affect the biological
activity studies of the secondary metabolites and consequently minimize the chance of
detecting high biologically active compounds, specially for commercial use, for example:
-
The high selectivity required for a biologically active substance means that many
active compounds will be rejected at an early stage because of lack of specificity.
-
Some active compounds are unstable and are either lost during isolation or are
unsuitable for use .
-
Some compounds have already been isolated (and possibly patented before).
-
The type of biological activity found is not meaningful in terms of the particular
usage sought.
-
The screening process is inappropriate.
-
Some forms of biological activity are dependent on the presence of other compounds
(synergists) which are lost during the purification processes before the substance is
bioassayed.
4.1.5 Technology of HPLC:
High performance liquid chromatography (HPLC) including both normal and reversed
phases (RP) is now a well-developed and widely used technique for separation of complex
mixtures (Exarchou, et al, 2005). HPLC is now available in many forms of hyphenated
systems such as LC-UV, LC-mass spectroscopy (LC-MS, LC-MS-MS) and LC-NMR which
has been successfully and practically achieved in the last decade. (Wolfender, et al 2005, and
Exarchou, et al, 2005). With the aid of the modern HPLC-coupled spectroscopic techniques
263
Discussion
such as HPLC-UV, HPLC-MS as well as HPLC-NMR, the main constituents of plants that
are frequently used in traditional Chinese medicine have been characterized and identified. As
well as compared with reference compounds (Zschocke et al 2005; Albert 2002; Wilson, et
al 1999).
Analytical HPLC that is coupled to a UV detector is ideal for detection of most natural
products that possess chromophoric functions in their structures. However, in some cases,
HPLC-UV hyphenated systems fail to detect the important biological substances such as
purine derivatives, nucleotides and other similar small biologically active metabolites, the
presence of acidified water as a part of the mobile phase make the nitrogenous compounds
protonated (in other word, highly polar according to the affinity of that compounds to accept
protons), which in turn elute the compounds very rapidly and in some cases can not be
detected with the UV-detector). This problem prompted some analytical chemists (e.g.
Nordström et al 2004) to derivatise such basic compounds to be more hydrophobic,
consequently improve their retentions on reversed phase materials and enhance their ESI
response in the case of LC-MS analysis. The use of ortho-phosphoric acid in maintainence of
the mobile phase-pH around 2 give a good compound-separation, allowing the UV-detector to
impart sharp peaks with high resolution, but it is not compatible with all possible analytes. In
some cases of poly-nitrogenous compounds (new purine derivatives and known compound
adenosine in the present study), phosphorylated H2O (pH 2) rendered the compounds very
polar, providing very short retention time and consequently masking their detection by UV
detector. The following example will explain the effect of o-phosphoric acid in masking peaks
of purine derivatives during HPLC analysis.
From the above example, it was clear that, low concentrated analyte couldn`t be
detected by elution of the sample with eluant composed of pH 2 aqueous solution as in fig
4.1.A. In contrast, the same sample concentration could be easily detected (fig 4.1.B) through
elution with mobile phase composed of neutral H2O (free of phosphoric acid). Furthermore,
high concentrated sample (fig 4.1.C) provides a broad peak with relatively low resolution and
short retention time by elution with mobile phase that composed of pH 2 water. while sharp
peak with high resolution separation and relatively delayed retention time could be obtained
through elution with mobile phase composed of neutral water (fig 4.1.D).
264
Discussion
110
ms050503 #15
et18h
9.25
mAU
4.000
% No spectra library hits found!
UV_VIS_1
WVL:235 nm
300
250
19 - 36,233
22
23- -37,678
-38,213
38,651
21-24
-36,715
37,165
20
80
10 - 29,507
1.250
18 - 35,397
60
- 33,737
1516- 32,882
344.7
A
70,0
150
16.90
No spectra library hits found!
228.1
B
200
530.3
12- -30,426
30,899
551.711
Peak #10
%
209.0
281.3
nm
-500
200
40
UV_VIS_
WVL:235 nm
10 - 16,822
17 - 34,093
2.500
et18h
ms050506 #15
mAU
400
14 - 32,109
595
100
13 - 31,750
3 - 1,088
4 - 1,117
8 - 24,155
9 - 25,234
20
50
5 - 1,210
6
7 -- 1,258
1,318
0,950
12 -- 0,758
0
nm
-10,0
200
67 -0,778
1,237
1,360
-0,459
1,108
0,597
12345
----Peak
1 - 0,046
300
400
500
595
11 - 47,527
15,412
89- -14,673
min
-10
0,0
10,0
20,0
30,0
400 ms050503 #
mAU
40,0
50,0
60,0
et17f
UV_VIS_
WVL:235 nm
C
7 - 8,892
300
70,0
50,0
Peak #7 8.54
%
min
-50
0,0
10,0
30,0
2.000 ms050506 #
mAU
40,0
et17f
50,0
60,0
UV_VIS_
WVL:235 nm
8 - 16,660
D
1.500
209.9
70,0
Peak #8 16.58
No spectra library hits found!
%
228.3
289.7
200
20,0
210.1
1.000
281.3
25,0
560.5
100
0
8 - 15,756
6 -- 1,117
1,205
5
-0,584
0,957
0,727
1234- --0,079
-50
0,0
500
nm
-10,0
200
400
-10,0
200
595
12 - 30,904
11 - 30,569
9 10
- 24,177
- 25,281
min
20,0
30,0
595
13 - 47,630
7 - 15,490
910
- 17,849
- 18,311
1,236
1,345
0,795
1,117
123456----0,472
0,977
10,0
nm
400
40,0
50,0
60,0
-200
0,0
12 - 36,766
11 - 32,503
13 - 47,629
min
10,0
20,0
30,0
40,0
50,0
60,0
Fig(4.1) different HPLC chromatograms of a new purine derivative (compound 35,
ashourine 2); A: diluted solution, the chromatogram was obtained after running in HPLC
using acidic aqueous solvent (pH 2); B: the same sample of A, the chromatogram was
obtained after running in HPLC using neutral water (pH 7); C: concentrated solution, the
chromatogram was obtained after running in HPLC using acidic aqueous solvent (pH 2);
D: the same sample of C, the chromatogram was obtained after running in HPLC using
neutral water (pH 7).
This problem during analysis by HPLC hyphenated systems confirmed that some of
the important bioactive natural products may be undetected even though they have adequate
chromophoric functionalities especially when present at low concentrations (as the case
during an early stage of their isolation from biological sources).
The same problem would be faced during LC-MS analysis of such compounds. Two
major problems are associated with LC-MS analysis. First, no universal LC column packing
material can be used for all possible kinds of analytes. Second, no eluent system is compatible
with both all possible analytes and ESI. A compromise has to be made at some level
265
Discussion
regarding the packing material, eluent system, or analyte response. Positive ESI is an
excellent interface for reversed-phase chromatography. The solvents used, acidic
acetonitrile/MeOH-H2O mixtures, are suitable for the desolvation process. However, many
biologically important compounds do not separate readily on reversed-phase packing material
due to their high polarity. The need for acetate or formate buffers or ion pairing reagents often
hampers the ESI response (Nordström et al 2004). During the LC-MS analysis of the new
purine derivatives (compounds nigricines 1-4), adenosine (compound 30) and nicotinamide
(compound 31), it was noted that, only the more hydrophobic compound, nigricine 1, could
be measured by LC-MS while the sensitivity was decreased as the side chain (alkoxy group)
was decreased. Adenosine and nicotinamide couldn`t be analysed in the same condition.
Hyphenated HPLC systems play a significant role in comparison the components of
biological extracts or mixtures of substances and a previously known compounds. Thus, the
tedious isolation of known compounds can be avoided and a targeted isolation of constituents
presenting novel or unusual spectroscopic features can be undertaken. (Wolfender, et al
2005).
Furthermore, HPLC-UV-MS hyphenated system is the method of choice for detection
the absolute configuration of amino acids and thus helps in the unambiguous structure
elucidation of new peptides not only by indication of certain amino acids of which the peptide
composed but also by detection and confirmation of stereochemistry of the amino acid. HPLC
analysis of the kahalalide hydrolysates after derivatisation by Marfey`s reagent, N-(5-flouro2,4-dinitrophenyl)-L-alanine amide (FDAA), and comparison of their retention times and
molecular weights with those of authentic diastereoisomer amino acids which were treated in
the same manner confirmed the stereochemistry of the kahalalide amino acids (see figures
3.1.11-3.1.15). Due to some of an expected negative effects of factors such as concentration
of the sample, or minute changes in the solvent gradient or in the pH of the mobile phase
that could affect the retention times, peak enrichment technique was applied to give
unambiguous streochemistry determinations. This procedure was made by co-elution of the
kahalalide hydrolysates together with the expected standard amino acids at the same time.
Complete overlapping of both standard and test amino acid
peaks indicated complete
stereoidentity (see figures 3.1.11).
LC-ESI/MS/MS hyphenated systems supported online amino acid sequencing of a
mixture of peptides without separation and purification which in turn avoid the cost of
266
Discussion
expensive adsorbents and eluents, and save the time and effort that may be lost during
isolation of known peptides, and for cheking whether a compound that is intended to be
isolated is a genuine new peptide (Bringmann and Lang 2003). The same advantages could
be obtained by online LC-NMR analysis (Wolfender, et al 2005).
4.2. Isolated compounds from genus Elysia ( sacoglossan mollusc):
Genus Elysia is known as a rich source of bioactive natural products such as
diterpenoids (Paul and Van Alstyne 1988) polypropionates (Gavagnin, et al 1994, Cueto et
al 2005) and depsipeptides (Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997,
Horgen et al 2000, Dmitrenok et al 2006). Elysia-derived depsipeptides known as kahalalides
consist of 3 to 13 amino acid units, ranging from a C31 tripeptide to a C75 tridecapeptide, with
cyclic and linear components, the latter terminating in a saturated fatty acid moiety. Ten of
these derivatives are cyclic depsipeptides, kahalalides A to F, K, O, P,and Q while three
analogues, kahalalides G, H, and J are linear peptides. Kahalalide F and its linear analogue
kahalalide G as well as the two new kahalalides R and S are the only congeners that feature
the unusual amino acid, Z-dehydroaminobutyric acid (Z-Dhb). The methanol extract of Elysia
grandifolia showed a promising biological activity such as antimicrobial, cytotoxicity,
feeding deterrence and icthyotoxicity (Padmakumar 1998, Bhosale et al, 1999). These
bioactivities attracted our attention to investigate the methanol extract of the mollusc. The
investigation which was guided by online LC-MS analysis resulted in the detection of many
known kahalalides [kahalalides B, C, D, E, F, G, J, K, O (see figures 2-15 and 2-16)] with
regard to their molecular weights and amino acid sequences through interpretation of ESI-MS
fragmentation patterns. The presence of two unidentified peaks at m/z 1520.2 and 1536.0
aroused our interest to do further chemical work on the mollusc extract. A steady continuous
effort led to the isolation characterisation, and full structure elucidation of two new
depsipeptides, kahalalides R and S.
267
Discussion
4.2.1 Which of the following organism is actually responsible for kahalalides
production? The animal sacoglossan mollusc (Elysia sp.), the plant Bryopsis sp (algal diet
of the mollusc), or the procaryotic symbiont bacterium (Vibrio sp.) ?
The green algal diet, Bryopsis sp., has been found to yield kahalalides A, B, F, G, K,
P, and Q. Kahalalides A-K were isolated from the molluscan E. rufescens, while kahalalide O
was isolated from E. ornata. In the present study, the corresponding molecular ion peaks of
kahalalides B, C, D, E, F, G, J, K, O as well as the new congeners R and S have been
characterized from online LC-ESI-MS analysis of the methanol extract. Furthermore ,
kahalalide F was produced by a symbiont bacterium Vibrio sp. associated with both the
specialist herbivore mollusc Elysia sp and their algal diet Bryopsis sp (Ashour et al 2006).
The existence of the kahalalides in the extracts of these taxonomic unrelated groups
of organisms (animal, plant and procaryote) led the question: which of these organisms is
actually responsible for kahalalides production?
Vibrio sp.
?
Bryopsis pennata
?
H 2N
H 3C
O
H 3C
H 3C
N
H
O
O
H 3C
H 3C
O
HN
O
H 3C
O
H 3C
HN
O
H
HN
HO
NH
H 3C
H 3C
H3C
O
O
O
CH 3
HN
CH 3
CH 3
NH
O
H
N
CH 3 O
O
NH
O
?
CH 3
N
H
N
HN
Elysia sp.
NH CH3
O
CH 3
CH 3
CH3
Kahalalides
Fig (4.2) Sources for kahalalides production.
-
kahalalides, especially the major one (kahalalide F) play an important ecological role,
since it was reported that they show significant antipredatory effects against fish
predation (Becerro et al 2001). Thus, these compounds increase the fitness of the
mollusc even though they play no role in primary metabolism.
268
Discussion
-
Hamann and Scheuer 1993, stated that the herbivorous sacoglossan mollusc E.
rufescens has the ability to sequester from their algal diet „Bryopsis sp“ functioning
chloroblasts, which then perhaps may participate in the biosynthesis of secondary
metabolites (Hamann and Scheuer 1993).
-
It was hypothesized that E. rufescens acquires Kahalalide F–producing Vibrio from
the surface of the algal diet Bryopsis sp and maintain these microbes as symbionts
(Ashour et al 2006).
-
According to the natural product definitions (Bennett 1995 and Firn 2004) it is
considered that kahalalides are actually secondary metabolites or true natural products
since they are not active participants in primary metabolism and have no function in
growth. Consequently their production is restricted to certain closely related
taxonomic groups of living organisms. Therefore it is not accepted that the above
mentioned organisms of completely different taxonomic groups (animal, plant and
procaryotic members) are responsible for production of one chemical class of closely
related secondary metabolites.
This discussion led to the following conclusion:
1- Kahalalides may be procaryotic products, produced by Vibrio bacterium and
accomulated in both algae and mollusc. (the most likely scenario, but needs further
confirmation).
2- Kahalalides may be plant products, since their production by Vibrio sp is still
unconfirmed (there is no true publication that describe the natural production of the
kahalalides by procaryotic organisms)
3- Kahalalides may be procaryotic products, produced by Vibrio bacterium, and their
production by plant, Bryopsis sp, may be attributed to a possible spontaneous
transplantation of a responsible gene from the bacterium to the plant
4- It is not accepted that kahalalides are actually animal products because of their
production by another completely unrelated organisms, and the presence of
kahalalides in the animal extract may be attributed to accomulation of the secondary
metabolites by contineous intake of the algal diet Bryopsis sp.
5- It is possible that kahalalides are plant or bacterial products liable to modification
through the molluscan metabolism.
6- The isolation of β-sitosterol (compound 7) and a previously known plant secondary
metabolite (N,N-dimethyltryptophan methylester, compound 6) from the animal
extract may confirm the plant responsibility for the production of the kahalalides.
269
Discussion
However, this interesting point needs further study and explanation from the biosynthetically
oriented chemists.
4.2.2 The possible biosynthetic pathway of kahalalides:
In spite of the long debating discussion about the producing organisms, the
kahalalides are considered as non-ribosomal depsipeptides (Marahiel et al 1997). The possible
biosynthetic pathway of kahalalides (as depispeptide natural products) was obtained from the
references (Marahiel et al 1997 and Schwarzer et al 2003). The kahalalide biosynthesis may
be supported by large multifunctional enzymes called non-ribosomal peptide synthetases. The
depsipeptides are assembled from diverse groups of precursors including pseudo-, non
proteinogenic, hydroxy, N-methylated, and D-amino acids. These peptides then undergo
further modifications that lead to additional structural diversity. A typical enzyme module concerning the depsipeptide biosynthesis – consists mainly of three steps as shown in figure
4.2
a) Adenylation domain (A): responsible for amino acid activation .
b) Thiolation domain (T) or peptide carrier protein (PCP): responsible for thioestrification of
the activated amino acids by the attachment to the cofactor, phosphopantotheiene.
c) Condensation domain (C): responsible for the peptide bond formation to elongate the
growing peptide chain.
The modification (e.g. epimerization, cyclization, N-methylation, reduction and/or
oxidation) of the peptidyl substrate during this process takes place according to special
considerations regarding the essential properties of the individual species.
270
Discussion
SH
O
H 3C
n
H 3C
M g 2+
+ ATP
O
PP
PCP
O
H 3C
n
H 3C
O
H 3C
O -A M P
n
H 3C
S
PCP
A -D om ain
O
H 3C
n
H 3C
(T ) PC P -D om ain
O
S
S
PCP
H
N
H 3C
n
H 3C
SH
R
PCP
O
H 2O
H 2N
S
R
O
PCP
PCP
C -D om ain
- repeated condensation (C-D om ain)
- repeated Epim erisation (E-D om ain)
H 2N
H 3C
H 3C
CH3
H 2N
O
N
H
N
H
O
O
N
H 3C
HN
O
HN
O
H 3C
O
CH3
H 3C
H 3C
O
HN
O
CH3
CH3
NH
H 3C
CH3
HN
O
NH
H
HN
HO
O
O
H 3C
NH
H
N
H 3C
C yclization
O
H 3C
(C y-D om ain)
CH3
O
HO
H 3C
H
N
CH3
O
O
HN
CH3
O
O
O
HN
HN
CH3
O
HO
NH
H 3C
H 3C
H 3C
O
O
NH
CH3
O
CH3
SH
S
NH
H
CH3
H3C
PC P
on
in a ti
)
T e r mD o m a i n
e
T
(
CH3
K ah alalid e F
N
H
N
H
O
HN
CH3
O
O
NH
O
CH3
CH3
N
H
N
H 3C
CH3
O
O
N
H
H 3C
CH3
H 3C
H 3C
H 3C
O
PC P
SH
K ahalalid e G
PCP
Scheme (4.2) Possible biosynthetic pathway of
kahalalide F (modified from
Schwarzer et al 2003)
4.2.3 Structure-activity relationship:
In an agar diffusion assay, kahalalide R at a disc loading concentration of 5 µg,
showed strong antifungal activity against the plant pathogens Cladosporium herbarum and C.
cucumerinum with inhibition zones of 16 and 24 mm, respectively. These results were almost
identical to those of kahalalide F, which exhibited its fungicidal activity with inhibition zones
of 17 and 24 mm, respectively, at the same concentration as kahalalide R. Using the same
concentration, the fungicidal activity of kahalalides F and R were also comparable to that of
nystatin showing inhibition zones of 19 and 39 mm, respectively. other kahalalides exhibited
neither antibacterial nor antifungal activities.
The known derivatives, kahalalides B, D, E, F, together with the new congeners,
kahalalides R and S were assayed for their cytoxicity toward L1578Y, HELA, PC12, H4IIE,
271
Discussion
and MCF7 cancer cell lines. Kahalalides F and R were found to be comparably cytotoxic
toward MCF7 cells with IC50-values of 0.22 ± 0.05 µmol/L and 0.14 ± 0.04 µmol/L,
respectively. Kahalalide S and E were less cytotoxic in MCF7 cells with IC50-values of 3.55 ±
0.7 µmol/L and 4.5 ± 0.49 µmol/L, respectively. Kahalalide R was cytotoxic towards the
mouse lymphoma L1578Y cell line at an IC50 of 4.28 ± 0.03 nmol/mL, which is almost
identical to that of kahalalide F with an IC50 of 4.26 ± 0.04 nmol/mL. The kahalalides
including kahalalides F and R were found to be inactive toward HELA, H4IIE and PC12
cancer cell lines. This implied the cytotoxic selectivity and specificity of kahalalides F and R.
Furthermore the depsipeptide, kahalalide G, (linear analogue of kahalalide F) lack the
antitumour activity (Hamann et al 1996). This overview indicated that :
1) the presence of the sequential fragment [Val-1-Dhb-Phe-Val-2-Ile-1-Thr-1] with lactone
bond is essential for the antitumour activity.
2) The activity is not affected by substitution of Ile-1 with Valine (as in kahalalide R)
3) Opening of the lactone ring results in loss of the activity (as in kahalalide G)
4) The presence of terminal fatty acid (5-MeHex or 7-MeHex) is also responsible for the
activity
5) Hydroxylation at position 5 of the fatty acid (7-MeHex) is responsible for decreasing the
activity (as in kahalalide S)
6) The activity is not affected by substitution of The -2 and Val-5 of kahalalide F with Val
and Glu respectively (as in kahalalide R).
7) The streochemistry of individual valines is responsible for the biological activity.
4.3 Isolated compounds from unknown Pachychalina sp:
4.3.1 Epidioxy Sterols
Chemical investigation of unknown Indonesian sponge Pachychalina sp led to
isolation and structure elucidation of two 5α, 8α- epidioxy sterols (compounds 10 and 11), as
well as of the previously known Octopus derived natural product, 8-hydroxy-4-quinolone
(compound 12)
Many of the reported 5α, 8α- epidioxy sterols showed significant biological activity
including antimycobacterial activity (Cantrell et al 1999), inhibitory activity against the
human T-cell leukemia/lymphotropic virus type I (HTLV-I) and cytotoxic activity against the
human breast cancer cell line (MCF7 WT) (Gauvin et al 2000), and antitumor activity against
different tumour cell lines (Bok et al 1999). 8-Hydroxy-4-quinolone (compound 12) was
reported to be one of the ink components that is ejected by the giant octopus Octopus dofleini
272
Discussion
martini which was reported to have antipreditory activity (Siuda 1974). Some other quinolone
analogues (e.g. 3,8-dihydroxyquinoline) showed mild cytotoxic activity against the growth of
several human tumour cell lines( Moon et al 1996).
Cytotoxicity study of compounds 10 and 11 showed activity with growth inhibitory activity of
57.2 % and 38.6 % for compounds 10 and 11 respectively at concentration of 10µg/ml each
against L5178Y cancer cells. Compound 12 showed no significant antimicrobial activity.
Biosynthetic pathway of 5α, 8α- epidioxy sterols:
Steroids are formed biosynthetically from active isoprene, isopentenyl pyrophosphate, (see
figure 4.3) which involve the same pathway as those for terpenoid biocynthesis. The
triterpenoid squalene is an intermediate in the steroid biosynthesis (Dewick, 1997). Oxidation
of ∆5,7 steroid results in formation of 5-, 8- epidioxysterol, this oxidation can be produced
spontaneously or enzymatically (Gunatilaka et al 1981)
Until recently, reservation has been expressed that naturally isolated ergosterol
peroxides may be an artefact (Gunatilaka et al 1981). However White and co-workers (Bates
et al 1976) investigated the conversion of ergosterol into its epidioxide in two unrelated fungi
and demonstrated that both chemical (photooxidation) and enzymatic pathways are operative.
Gunatilaka et al 1981 suggested that the peroxidation of ergosterol and semilar ∆5,7 sterols is a
biological process and is aided by peroxidases. This suggestion was confirmed by the recent
isolation of polyketide peroxides from a number of marine sponges (Stierle and Faulkner
1980). Gauvin et al 2000 considered that these peroxides may be biosynthetic precursors for
∆4,7-3,6-diketones where both chemical groups were isolated together from the marine sponge
Raphidostila incisa. Furtheremore, Serebryakov et al 1970 isolated such epidioxy sterols from
fungi in the absence of oxygen. However, these epidioxysterols and their analogues were
previously isolated from many unrelated sponge species for example Axinella cannabina
(Fattorusso et al 1974), Tethya aurantia (Sheikh and Djerassi 1974), Raphidostila incisa
(Malorni et al 1978), Thalysias juniperina (Gunatilaka et al 1981), Haliclona rubens
(Calderon et al 1982), Hyrtios sp.(Koch et al 1983), Axinissa sp.(Iguchi et al 1993),and
Dysidea. fragilis. (Elenkov et al 1994).
273
Discussion
O PP
O
oxidosqualene
squalene
HO
R
L anosterol
HO
O2
R
HO
4α -m ethyl-cholesta-8en-3β-ol
(e.g. com pound 23)
O
HO
HO
O
24-ethyl-cholesta-5en-3β-ol
(e.g. com pound 22)
5α ,8α −epidioxysteroids
(e.g. com pounds 10, 11, 20 and 24)
Scheme (4.3) The biosynthetic pathway of steroids
The possible biosynthetic pathway of 8-hydroxyquinolin-4-one from Pachychalina
sp. may include acetylation and cyclization of o-aminobenzoyl CoA as explained in (scheme
4.4)
O
O
CoA
Ac-CoA
CoA
O
NH2
NH2
Cyclization
O
O
N
H
N
H
OH
Scheme (4.4) Biosynthesis of 8-hydroxy-quinolin4-one (modified from Torssell 1997)
274
Discussion
4.4 Isolated compounds from Hyrtios erectus :
Marine sponges of the genus Hyrtios (family Thorectidae, order Dictyoceratida) have
proven to be a rich source of secondary metabolites, including terpenoids, macrolides, and
tryptamine-derived alkaloids. The most important metabolites of the genus Hyrtios discovered
to date include the powerful anticancer agents spongiastatins 1-3 that were discovered by the
Pettit group in 1993 and 1994, (Youssef 2005).
In the present study, 210 g dry weight of Hyrtios erectus collected from the Red Sea –
Egypt were chemically investigated and nine compounds were structurally elucidated
including 8 known compounds and one new 5-hydroxy-1H-indole derivative.
4.4.1 Exessive harvesting of the wild-type living organisms:
The potent antitumour activity of spongiastatins 1-3 from H. erectus (Pettit et al 1993;
Pettit et al 1994) prompted the same authers to exhaustively recollect the wild-type of the
same sponge Hyrtios erecta (600 kg wet wt.) for further chemical investigations. The later
chemical investigations resulted in the isolation of antineoplastic sesterstatins 1, 2 and 3 in
very minute concentrations 3 x 10-7 %; 3 x 10-7 % and 5 x 10-7 % respectively (Pettit et al
1998b), sesterstatins 4 and 5 in concentrations of 1 x 10-6 % and 4 x 10-5 % respectively
(Pettit et al 1998c) and sesterstatin 6 in concentration of 8.3 x 10-7 % (Pettit et al 2005).
Concerning this point (excessive harvesting of a wild–type living organisms), I think
that, we should be aware of the ecological problems that might occur as a result of such
irresponsible collections. Otherwise a big environmental problem takes place, in this case, we
have to balance the advantages against the disadvantages of such exhaustive re-collections. In
order to overcome this problem, I predict the same as Prof. Dr. Faulkner (Faulkner 2000a) he
also predict a great impact of chemical and biological researches including genetic
engineering concerning to different forms of marine living forms ranging from marine
invertebrates to the marine-derived microorganisms, Faulkner also expect that, in the near
future we will be able to transfer biosynthetic genes from one marine organism to another and
imagine the marine natural product chemist of 2025 still involved in structural elucidation, but
considerable effort to the genetic engineering required to produce unique metabolites by
fermentation of genetically modified microbes. This will accomplish the goal of having the
marine organisms provide the inspiration for new compounds while avoiding their excessive
harvesting.
275
Discussion
4.4.2. Biosynthesis of scalaran-type sesterterpenoids:
Tetracyclic sesterterpenes with a scalarane skeleton occur frequently as metabolites of
sponges of the family Thorectidae (order dictyoceratida) which includes the genera Hyrtios,
Cacospongia and Ircinia (Faulkner, 1997). Sesterterpenoids are composed of a C25 backbone.
Sesterterpenoid is built-up of 5 isoprene units. Their biocynthetic pathway involves enzymatic
cyclization of all-trans pentaprenyl methyl ether (which is a detoxification product of
isoprenic alcohol) as illustrated in (scheme 4.5) to form scalarane derivative
(tetracyclopentaprenyl methyl ether). The conversion is aided by cyclase enzymes (Renoux
and Rohmer, 1986)
OCH3
OPP
isopentyl pyrophosphate
all-trans pentaprenyl methyl ether
O
OCH3
O
pentacyclic scalarane derivative
tetracyclopentaprenyl methyl ether
(scalarane derivative)
Scheme (4.5) Scalarane biosynthesis. (Renoux and Rohmer, 1986)
Although the origin of scalarane-type sesterterpenes is still highly obscure, and since
in most of sponges up to one third of the volume is occupied by symbiotic microbes and
various detritus filtered off by invertebrate Renoux and Rohmer 1986, suggested that the
scalarane-type sesterterpenes which are restricted to a few sponges, (dictyoceratidae family)
might be in fact microbial metabolites. The authors (Renoux and Rohmer 1986) exhibited the
conversion of all-trans pentaprenyl methyl ether to tetracyclopentaprenyl methyl ether
through incubation of the former with cell-free extract of the protozoan ciliate (Tetrahymena
pyriformis).
276
Discussion
4.4.3. The pharmacological activity of scalaran-type sesterterpenoids:
Many scalarane-type sesterterpenoids have been isolated from marine sponges
belonging to the order Dictyoceratida. Scalarane-type sesterterpenes display a variety of
biological activities such as cytotoxic, antimicrobial, antifeedant, antimycobacterial,
ichthyotoxic, anti-inflammatory, and platelet-aggregation inhibitory effects, as well as nerve
growth factor synthesis-stimulating action (Youssef
et al, 2005). The most important
scalarane derivatives are spongiastatins1-3 which displayed a potent antineoplastic effect.
(Youssef 2005). However compound 18 (16-hydroxyscalarolide) showed cytotoxic activity
59.5 and 44.7 % at concentrations 3.0 µg and 10.0 µg respectively against L5178Y cells.
4.4.4. Indole derivatives from Hyrtios erectus :
COOH
COOH
H2N CH
H2N CH2
HN
HN
Tryptophan
CH2
CH2
CH2
NH
CH2
Indol-3-acetic acid
CH2
OH
HN
Serotonin
COMe
OMe
N
H
Melatonin
During the 1930’s the essential aminoacid tryptophan was discovered as well as the
plant growth hormone indole acetic acid (Kogl 1933). Tryptophan is a constituent of most
proteins, and serves in man and animal as a biosynthetic precursor for a wide variety of
tryptamine and other indole derived metabolites, several of them of paramount physiological
importance. Tryptophan is ingested during the animal–diet or vegetarian-protein-diet where it
is converted to biologically active derivatives e.g. serotonin and melatonin. Melatonin is
thought to control the day and night rhythms, while serotonin is an important neurotransmitter
acting at the nerve cells to promote feeling of well being, calm, personal security, relaxation,
confidence and concentration. Serotonin neural circuits also help counterbalance the tendency
of brain dopamine and noradrenaline circuits to encourage over-arousal, fear, anger, tension,
aggression, violence, obsessive-compulsive actions, over-eating (especially carbohydrates),
anxiety and sleeping. Serotonin itself can not penetrate the blood brain barrier, but its
precursor tryptophan can penetrate it. The typical diet provide about 1-1.5 g. tryptophan daily,
this amount is sufficient to maintain the brain essential requirement of serotonin.
277
Discussion
The sponge Hyrtios erectus is known as rich source of tryptamine derived natural
products. Hyrtiosine A, hyrtiosine B, 5-hydroxy-3-formyl-1H-indole (Kobayashi et al 1990),
hyrtiosulawesine,
1,6-dihydroxy-1,2,3,4-tetrahydro-β-carboline,
serotonin,
6-hydroxy-1-
methyl-1,2,3,4-tetrahydro-β-carboline, 5-hydroxy-3-(2-hydroxyethyl)-indole, 6-hydroxy-3,4dihydro-1-oxo-β-carboline (Salmoun et al 2002), and hyrtioerectines A-C (Youssef 2005)
were previously isolated from Hyrtios sp.
In the present study, five indole derivatives were isolated (compounds 13-17).
Isohertiosine A, compound 17, is new, its molecular formula is the same as hyrtiosine A. The
difference has been assigned as a methoxy group attached to the carbonyl in 17 instead of
hydroxymethylene group in hyrtiosine A ( compound 13). The known compounds 15 and 16
were not previously reported for H. erectus. Some of 5-hydroxy indole derivatives have been
reported to exert cytotoxic activity against tumour cell models (Youssef 2005). However, the
indole derivative of such basic structure are known to exert their biological effects on CNS.
Compound 17 (isohyrtiosine A) showed growth inhibition activity 64 % at concentrations
10.0 µg/ml against L5178Y cells while other compound has no effect at concentrations up to
10 µg/ml.
4.4.5 Biosynthetic pathway of indole derivatives from Hyrtios erectus
The possible biosynthetic pathway of Hyrtios-derived indole includes hydroxylation
at position 5, then decarboxylation leading to the formation of the intermediate 5hydroxtryptamin (serotonin) which has been already isolated before from the same sponge
(Salmoun et al 2002).
278
Discussion
O
H2N
CH C
OH
CH2
Tetrahydrobiopterin
(BH4)
HN
Hydroxylase
NAD
Dihydropteridine reductase
Dihydrobiopterin
(BH2)
NADH
O
H2N
CH C
OH
O
CH2
CH2
OH
HO
HO
OH
HN
NH2
N
H
CO2
N
H
Hyrtiosine A
5-Hydroxytryptamine
(Serotonin)
O
O
CH3
HO
O
H
HO
O
O
CH2
H
OH
N
H
Isohyrtiosine A
N
H
5-Hydroxy-indol-3carboxaldehyde
N
H
N
H
Indol-3-carboxaldehyde
5-Deoxyhyrtiosine A
Scheme (4.6) Possible biosynthetic pathway of 3-acyl-indole derivatives
4.5 Isolated compounds from Petrosia nigricans :
The total methanol extract of Petrosia nigricans yielded 17 compounds, (compounds
22-38) belonging to different chemical classes which included steroids, aromatic acid
derivatives, new cerebrosides, new purine derivatives and new indole derivative, in additon to
adenosine and nicotinamide. Although there are a lot of new active metabolites isolated from
genus Petrosia, this is the first report of isolated compounds from Petrosia nigricans. None
of the isolated compounds from Petrosia nigricans showed significant antimicrobial activity
against B. subtilis, S. cerevisae, C. herbarum and C. cucumerinum. All purine derivatives
(nigricines 1 to 4) showed no cytotoxic activity against L5178Y cell line, while nigricinol
(compound 38) showed 68.1 % cytotoxic activity at conc. 10 µg/ml.
4.5.1 Biosynthetic pathway of aromatic acids:
Although the isolated aromatic acids are described as catabolic products (waste) of
aromatic amines and amino acids metabolism (O’connor et al 2001), they exhibit a significant
ecological role. For example, p-hydroxybenzaldehyde, which is common in marine sponges,
exhibited a significant antipredetory activity against the major predator Perknaster fuscus of
antarctic sponges (Moon et al 1998). However, the following scheme demonstrates the
possible biosynthetic pathway of aromatic acids.
279
Discussion
O
H 2N
CH
C
OH
CO2
CHO
NH2
CH2
Dehydrogenase
COOH
Dehydrogenase
tetrahydrobiopterin
4-Monooxygenase
O
H 2N
CH
CH2
C
dihydrobiopterin
OH
O2
NH2
CHO
COOH
CO2
Dehydrogenase
OH
Dehydrogenase
OH
OH
COOH
OH
3-Hydroxylase
CHO
O
R2
COOR1
OH
OH
OH
OH
OH
R2 =H, OH
OH
R1= H, Me, Et or Bu
Scheme (4.7) Biosynthetic pathway of aromatic acids and aldehydes. (chosen
from O’connor et al 2001, Cuskey and Olsen 1988 )
4.5.2 Cerebrosides from Petrosia nigricans:
Cerebroside is the principal glycosphingolipid in the brain tissue, hence the trivial
name „cerebroside“, which was first conferred on it in 1874, although it was much later
before it was properly characterized. In fact, glycosphingolipids are found in all nervous
tissues, but they can amount to 2% of the dry weight of grey matter and 12% of white matter.
Cerebroside are found at low levels in animal tissues, such as spleen and erythrocytes, skin
lipids as well as in nervous tissues. Cerebrosides containing α-D-galactose are not found in
humans, but are known to occur in a marine sponge, they are potent stimulators of
mammalian immune systems (Christie, 2003). In the present study cerebroside mixture were
separated from total methanol extract of the sponge Petrosia nigricans, after many trials using
different methods of separation, a mixture of two cerebrosides were separated and elucidated
as petrocerebrosides1 and 2.
4.5.3 Biosynthesis of cerebrosides
The basic mechanism for the biosynthesis of sphinganine involves condensation of
acyl-coenzyme A with serine, catalysed by the enzyme serine acyltransferase as illustrated, to
280
Discussion
form 3-keto-sphinganine. The keto group is then reduced to hydroxyl by a specific reductase.
The free sphinganine is rapidly acylated to form a dihydroceramide, by a dihydroceramide
synthase, which can utilize a range of acyl-CoAs. Insertion of the trans-double bond in
position 4 to produce sphingosine occurs only after the sphinganine has been esterified in this
way to form a ceramide. The biosynthesis of cerebrosides takes place through direct transfer
of the carbohydrate moiety from a sugar-nucleotide, eg uridine-5-diphosphate (UDP)galactose, UDP-glucose, etc, to the ceramide unit. During the transfer, which is catalysed by a
glycosyl-transferase, inversion of the glycosidic bond occurs (from α to β).
O
COOH
R
SCoA H
CH2OH
Long chain acyl-CoA
NH2
Serine
OH
CH2O
R
OH
R1
O
O
CH2OH
R
Gal-Gal
NH
Cerebroside
NH2
3-keto-sphing. reductase
NADPH+H
NADP
OH
R
Sphinganine
NH2
OH
Fatty acid
R1
Dihydroceramide
O
Dihydroceramide
synthase
OH
OH
R
NH
R1
O
CH2OH
R
OH
R1
OH
Sphingosine
CH2OH
CH2OH
Desaturase
NH
CH2OH
R
NH
R1
O
Ceramide
O
Scheme (4.8) Biosynthesis of cerebrosides (Christie, 2003)
4.5.4 New purine derivatives from Petrosia nigricans:
Marine organisms, particularely sponges, have proven to be an exceptionally rich
source of modified nucleosides. The isolation of spongouridine and spongothymidine from
Cryptotethia crypta (Bregmann, and Feeney 1950) which served as models for the
development of adenine arabinoside (ARA-A) for treatment of Herpes simplex infection and
cytosine arabinoside (ARA-C) for the treatment of leukemia (Lindsay et al 1999), and
subsequent development of antiviral analogues demonstrated the potential medicinal
importance of these compounds such as antifungal phidolopine which was isolated from the
bryozoan Phidolopora pacifica (Ayer et al 1984), the hypotensive doridosine which was
isolated from the sponge Tedania digitata (Cook et al 1980), and the cytotoxic mycalisines
which were obtained from the sponge Mycale sp. (Kato et al 1985). Many other purines and
281
Discussion
nucleosides isolated from marine organisms particularly sponges display potant bioactivities,
such as the marine derived 1,3-dimethylisoguanine from Amphimedon viridis which showed
activity against an ovarian cancer cell line (IC50, 2.1 µg/mL) (Mitchell et al 1997); 3,7dimethylisoguanine from a Caribbean sponge Agelas longssima, which displayed mild
antibacterial activities (Cafieri et al 1995). Figure 4.3 shows the structure of some purine and
pyrimidine derivatives that used as antiviral agents (Baba 2005).
NH2
NH2
N
N
N
N
N
HO
NH2
N
N
N
N
N
HO
N
O
Br
CH3
N
O
O
HO
O
N
O
HO
HN
O
N
O
HO
Sorivudine (BVaraU)
NH2
CF3
N
N
O
N
O
O
HO
OH
Ganciclovir (GCV)
O
I
HN
O
HO
Acyclovir (ACV)
O
O
HN
HO
O
OH
Adefovir (PMEA)
Tenofovir (PMPA)
HN
O
P
N
N
O
OH
O
H 2N
N
N
HO
O
OH
OH
Vidarabine (Ara-A)
H2 N
N
HN
N
HN
OH
O
P
HO
N
O
O
O
HO
N3
Zidovudine (AZT)
OH
Idoxuridine (IDU)
OH
Trifluridine (TFT)
OH
O
P
OH
Cidofovir (HPMPC)
Fig (4.3) Purine and pyrimidine derivatives used as antiviral agents (Baba 2005).
In the present study, investigation of ethylacetate fraction of the Indonesian sponge
Petrosia nigricans, which was collected from Pulau Baranglompo - Indonesia led to the
isolation of four new purine derivatives nigricines 1-4. Their structures were elucidated by
extensive spectroscopic analysis, 2D-NMR experiments, EI/MS, ESI/MS and HRMS.
Erinacean is a related purine derivatives with unusual 6-β-alanine amino acid was isolated
before from the antarctic sponge Isodictya erinacea (Moon et al 1997). To the best of our
knowledge, Purine derivatives with alkyl 3-(3,9-dihydro-3,9-dimethyl–2-oxo-2H-purin-6ylamino) propanoate were not reported neither as a synthetic nor as a natural product.
282
Discussion
O
N
N
NH
O
nigricines:
1
2
3
R
5 7
N
NH
R = Bu
R = Et
R = Me
O
N
4 N9
2
1
N
N
N
O
N
O
O
nigricine-4
N
H
N
8
O
N
H
NH
10
11
O 12 OH
Erinacean
Fig ( 4.4 ) Nigricines 1-4 as well as the related compound erinacean
4.5.5 The possible biosynthetic pathway of nigricines 1-4:
Synthesis of the purine ring is a central metabolic function of all cells. The products,
AMP and GMP, provide purine bases for DNA and RNA, as well as for a number of essential
coenzymes (NAD, NADP, FAD and Coenzyme A) and signaling molecules (e.g. cAMP). The
purine pathway also functions in pathways that are different and distinct from these
„housekeeping roles.“ It is employed in specialized tissues to assimilate and detoxify NH3
(Smith and Atkins 2002). The proposed biosynthetic pathway of nigricines 1-4 seems to
follow the same biosynthetic pathway of the normal adenylated nucleotides until the
formation of adenylsuccinate then the later substance (adenylsuccinate) is more likely to be
used in de novo nigricines biosynthesis via a lateral pathway as explained in scheme (4.9).
283
Discussion
N-3
2
3
C-8
N
9
4
1
5
6
N
N -formylTHF
CO 2
(O)
Glycine
H
N
O
-Ribose
CH
N
COOH
Adenylsuccinate
NH 2
H
N
-P
N
CH
N
N
HN
nucleotidase
nucleosidase
N
Ribose P
HN
COOH
N
O
COOH
N
CH
N
N
NH
N
O
O
N
N
O
NH
OH
CH
N
OH
N
N
NH
N
CH 2
N
O
N
O
N
CH 2
N
Ribose P
HOOC
COOH
H
N
CH
N
HN
Aspartate
Ribose P
O
N
N
7
N
C-6
O
AMP
Fumarate
10
8 CH
N-1
CO 2
CH
N
NH 2 Ribose P
N
HN
N
N
N-9
C-2
Aspartate
N
Glutamine (amide N)
N 10 -formyl-THF
N
O
O
R
NH
Nigricines 1-3
O
O
Nigricine 4
Scheme (4.9) Possible biosynthetic pathway of new purine derivatives
from Petrosia nigricans (modified from Smith and Atkins 2002)
4.6
Isolated compounds from Callyspongia biru:
Sponges belonging to the genus Callyspongia are extensively investigated for
bioactive natural products. Many new bioactive natural products have been isolated from
different Callyspongia sp., these natural products include unusual steroids (Agrawal and Garg
1997); acetylene derivatives ( Youssef et al 2003); uncommon fatty acids (Carballeira and
Pagan 2001); diterpenes (Garg and Agrawal 1995); triterpenes (Fukami et al 1997); many
cyclic peptides have been isolated from the genus Callyspongia [ phoriospongins A and B
from C. bilamellata (Capon et al 2002), Callynormine A from C. abnormis (Berer et al 2004)
and callyaerins from C. aerizusa (Min, et al., 2001)]. Many natural product-producing fungi
284
Discussion
have been isolated from some Callyspongia sp., and cultivated on artificial media for
production of natural products [spiciferone derivatives, macrolides, anthraquinones and
benzofuran have been isolated from C. aerizusa –derived fungi (Edrada et al 2000, Jadulco et
al 2001, Jadulco et al 2002)]. The most attractive compound, that was isolated from
Callyspongia sp., is callystatin A. Callystatin A is a potant cytotoxic polyketide isolated from
Nagasakian marine sponge, Callyspongia truncata (Kobayashi et al 1997). Methanol extract
of Indonesian sponge Callyspongia biru was investigated and p-hydroxyphenylacetic acid, phydroxyphenylacetic acid methylester, indole-3-carbaldehyde, indole-3-acetic acid, and 2‘deoxythymidine were isolated. Deoxythymidine (compound 40) showed cytotoxic activity
76.2 % and 41.7 % against L5178Y mouse lymphoma cell line at concentrations 10 and 3
µg/ml respectively.
285
V- Summary
The modern techniques as well as the advanced instruments that employed in the
discovery programms of bioactive natural product facilitated the task of the natural product
chemists and permitted them to detect, target, isolate and elucidate the structure of the
pharmacologically active natural product in significantly short times compared with that done
in the not too distant past. The targeted natural product may show novel or unusual
spectroscopic features and/or promising biological activities. These methodologies are now
applicable to achieve the task without the need of tedious isolation procedures of known
compounds. The application of these new methodolgies resulted in saving the time, efforts
and economy during the processing of the bioactive natural product drug discovery programs.
The present study deals with the application of the modern techniques including NMR, MS,
and hyphenated HPLC systems as very efficient and applicable tools in the achievement of
the above mentioned aim. The biological materials that were employed in this study included
six different soft bodied marine animals which were collected from four different
geographical zones. The study resulted in isolation, purification, and structure elucidation of
40 compounds. Some of them showed a very promising biological activity and recommended
as drug candidates in the near future.
1- The mollusc Elysia rufescens
The sacoglossan molluscs Elysia rufescens were collected from the black point „Kahala“ in
Oahu Island in Hawaii. The animal extract provided 5 known kahalalides (kahalalides B, C,
D, E and F) in addition to β-sitosterol and a previously known fabaceous secondary
metabolite, N,N-dimethyl-tryptophan methylester.
2- The mollusc Elysia grandifolia
The sacoglossan molluscs Elysia grandifolia were collected from the Gulf of Mannar and
Palk Bay, Rameswaram, India. The hyphenated system (LCMS) detected the molecular ion
peaks that corresponding to kahalalides B, C, D, E, F, G, J, K, O as well as two new
molecular ion peaks (corresponding to kahalalides R and S) in the total methanol extract. The
isolation process was directed to target the new ones, and resulted in structure elucidation of
them in addition to another two known kahalalides (kahlalaides D and F).
286
3- The Indonesian sponge Pachychalina sp.
The hithertho new sponge species, Pachychalina sp, was collected from Pulau
Baranglompo, Indonesia. Chemical investigation of the methanol extract of the sponge led to
isolation of two 5α, 8α-epidioxysteroids, as well as the previously known compound, 8hydroxy-4-quinolone, which was reported as an ink component that is ejected by the giant
octopus „Octopus dofleini martini“.
4- The Indonesian sponge Petrosia nigricans
The Indonesian sponge Petrosia nigricans was collected from Pulau Baranglompo,
Indonesia. Seventeen compounds were isolated from the total methanol extract. These
compounds included 10 known natural products, 2 new cerebrosides, one bis-indole
derivative and four new 2-oxo-purine derivatives.
5- The Indonesian sponge Callyspongia biru
The Indonesian sponge Callyspongia biru was collected from Taka Bako, Indonesia. The total
methanol extract of the sponge provided five known compounds (indol-3-carbaldehyde,
indol-3-acetic acid, p- hydroxyphenylacetic acid, p-hydroxyphenylacetic acid methylester,
and 2`-deoxythymidine).
6- The Red Sea sponge Hyrtios erectus
The Red Sea sponge Hyrtios erectus was collected from El-Quseir, in the Red sea , Egypt.
The total methanol extract of the sponge provided nine compounds including one
epidioxycholesterol derivative, three scalaran-type sesterterpenes, four known indole
derivatives and one new 5-hydroxy-indole derivative.
287
References
Agrawal S., Garg H.S., (1997) Indian J. Chem. Sect. B 36 343-346
Albert K., (2002), In : On-line LC-NMR and related techniques (edited by Klaus Albert),
John Wiley.
Aldrich library of 13C and 1H Ft NMR spectra (1992) Sigma-Aldrich.
Aldrich Library of
13
C and 1H FT NMR spectra (1993) 1st edition, (ed., Pouchert C J and
Behnke J)
Ashcroft, A. E., (2005) Nat Prod Rep., 22, 452-464.
Ashcroft, A.E. (1997) : In (“Ionization Methods in Organic Mass Spectrometry”, The Royal
Society of Chemistry, UK, and references cited therein, and availableat websites,
http://WWW.astbury.leedsac.uk/Facil/Mstut/mstutorial.htm,
and
http://www.jeol.com/ms/docs/ionize.html .
Ashour M., Edrada R, Ebel R., Wray V., Wätjen W., Padmakumar K., Müller W. E. G., Lin
W. H. and Proksch P. (2006) J. Nat. Prod., 69, 1547.
Ayer S.W., Andersen R.J., Cun-Heng H., Clardy J. (1984) J. Org. Chem. 49, 3869-3870.
Baba M. (2005) Uiusu, Jun;55(1):69-75.
Bakus G. J., Targett N.M., Schulte B., (1986) J. Chem Ecol. 12, 951-987.
Barnes R.D. (1986) In : Invertebrate Zoology 5th edition, Saunders College publishing New
York.
Bates M.L., Reid W.W., White J.D., (1976) J. Chem. Soc., Chem. Commun. 44-45.
Becerro M.A., Goetz G., Paul V.J., and Scheuer P.J. (2001), J. Chem. Ecol., 27, 2287-2299.
Bennett J.W. (1995) Can. J. Bot. 73 : S917-S924.
Bennett J.W., and Bentley R. (1989) Adv. Appl. Microbiol., 34, 1-34.
Berer N., Rudi A., Goldberg I., Benayahu Y., and Kashman Yoel (2004) Org. Lett., 6, 25432545.
Bernart M., Gerwick WM. (1990) Phytochemistry, 29, 3697
Bhawal B.M., et al (1991) synthesis 112-114.
Bletsos I.V.;Hercules D.M.; vanLeyen D.; Hagenhoff B., Niehuis E.; Benninghoven A.
(1991) Anal. Chem., 63, 1951.
Bohsale S. H. Jagtap T.G., and Naik C.G. (1999): Mycopathologia 147, 133-138.
Bok J.W., Lermer L., Chilton J., Klingeman H. G. and Towers G. Neil (1999)
Phytochemistry, 51, 891-898.
Bonnard, I.; Manzanares, I.; Rinehart, K. L. (2003) J. Nat. Prod. 66, 1466-1470.
288
Bourel-Bonnet L., Rao K.V., Hamann M.T., and Ganesan A., (2005), J. Med. Chem., 48,
1330-1335.
Bourguet-kondracki M.-L. and Kornprobst,J. –M. (2005) In: Marine Pharmacology:
potentialities in the treatment of infectious diseases, Osteoporosis and Alzheimer’s
Disease. [book series : Advances in Biochemical Engineering/Biotechnology, 97,
2005, SpringerLink Berlin / Heidelberg, Book : marine biotechnology II].
Boyd M.R., Schoemacker R.H., Cragg G.M., and Sffness M., (1988) in pharmaceuticals and
the sea (Jefford C.W., Reinhart K.L., and Shield L.S., eds.), pp. 27-44, Technomic
Publishing Company, Lancaster, PA.
Boyd R.M. (1989) Principles Practices Oncol. 3, 1-12.
Braekman J.C., Daloze D. Macedo D., Abreu P. (1982) Tetrahedron Lett. 23, 4277-4280
Braekman J.C., Daloze D., Defay N., Zimmermann D. (1984) Bull. Soc. Chim. Belg 93, 941944.
Bratt K. (2000) , In :Secondary Plant metabolites as Defense against Herbivores and
Oxidative Stress, (a Thesis presented by Katharina Bratt 2000) ,
Department of
organic chemistry, Institute of chemistry, university of Uppsala, sweden.
Bregman W., Feeney R.J. (1950) J. Am. Chem. Soc. 72, 2809- 2810.
Bregmann W., and Feeney R., (1951), J. Org. Chem., 16, 981-987.
Bringmann G., Lang G., Steffens S., Schaumann K. (2004) J. Nat. Prod. 67, 311-315 .
Brown A.P.; Morrissy R.L.; Faircloth G.T.; Levine B.S. (2002) Cancer Chemother.
Pharmacol., 50, 333-340
Bu´Lock J.D., (1980). Mycotoxins as secondary metabolites, In: the
Biosynthesis of
mycotoxins (Steyn, P.S. ed.) Academic Press. New york. p 1-16.
Burlingame A.L., Baillie T.A., Russell D.H. (1992) Anal. Chem., 64, 467R.
Cafieri F., Ernesto F., Mangoni A., Tagliatela-Scafeti O. (1995) Tetrahedron Lett. 36, 78937896.
Calderon J.S., Quijano L., Gomez F., Acosta F. and Rios T. (1982) Rev. Latinoam. Quim. 13,
49.
Cantrell C.L., Rajab M. S., Franzblau S.G., Fronczek F.R. and Fischer N.H.(1999) Planta
Medica, 65 732-734.
Capon R.,J., Ford J., Lacey E., Gill J.H., Heiland K., Friedel T., (2002) J. Nat. Prod. 65, 358363
Capon R.J., Rooney F., Murray L.M. (2000) J. Nat Prod. 63, 261-262.
Carballeira N.M., Pagan M. (2001) J. Nat. Prod. 64, 620-623
289
Carlson C. & Hoff P.J., 1999 (Aug 15) photos of Elysia rufescens. [Message in] Sea Slug
Forum. Australian Museum, Sydney. Available from
http://www.seaslugforum.net/find.cfm?id=1189
Chaurand P., Luetzenkirchen F., Spengler B. J. Am. Soc. Mass Spectrom. (1999), 10, 91-103.
Cimino G., De Stefano S., Minale L., Trivellone E. (1977) J. Chem. Soc., Perkin Trans. 1
1587-1593.
Ciruelos C.; Trigo T.; Pardo J.; Paz-Ares L.; Estaun N.; Cuadra C.; Domínguez M.; Marín A.;
Jimeno J.; Izquierdo M (2002) . Eur. J. Cancer, 38, Suppl. 7, S33.
Clardy J. and Walsh C., (2004) Nature,432, 829-837 and litratures therein.
Constantino V., Fattorusso E., Imperatore C., and Mangoni A.(2003) J. Org. Chem. 68, 14331437 .
Constantino V., Fattorusso E., Imperatore C., and Mangoni A.(2004) J. Org. Chem. 69, 11741179 .
Constantino V., Fattorusso E., Mangoni A., Aknin M. and Gaydou E.M.(1994) Liebigs Ann.
Chem. 1181-1185 .
Cook A.F., Bartlett R.T., Gregson R.P., Quinn R.J. (1980) J. Org. Chem. 45, 4020-4025.
Copp B. R. (2003), Nat. Prod. Rep. 6, 535-557.
Corely D.G., and Durley R.C. (1994) J.Nat. Prod. 57, 1484.
Cotter R.J. (1980) Anal.Chem., 52, 1589A.
Cragg GM., Newmann DJ.; and Snader K.M. (1997) J. Nat. Prod., 60 , 52.
Crawley L.S. (1988) : In pharmaceuticals and the sea (Jefford, C.W., Reinhart, K.L., and
Shield, L.S., eds.), pp. 101-107, technomic publishing company, Lancaster, PA.
Crews P., Cheng X.C., Adamczeski M., Rodriguez J., Jaspars M., Schmitz F.J., Traeger S.C.,
Pordesimo E.O., (1994) Tetrahedron 50, 13567-13574
Cristie W.W. (2003) In: Lipid Analysis (3rd edition). Oily Press, Bridgwater. Available at
http://www.lipidlibrary.co.uk/Lipids/cmh/index.htm
Cueto M.; D'Croz L.; Mate J. L.; San-Martin A.; Darias J. (2005) Org. Lett., 7, 415-418.
Cuskey S M and Olsen R H (1988) J. Bacteriology, 170, 393-399
Davies J. (1985) Recombinent DNA and the
production of small molecules. In :
Microbiology. (Leive L., Washington A.S.M. eds.) D.C. pp. 364-366.
Dewick P.M.(1997) In: Medicinal natural products, a biosynthetic approach, third avenue,
New York, USA, 153-173.
Dillman R.L., Cardellina J.H.(1991) J. Nat. Prod. 54, 1056-1061.
Dmitrenok A., Iwashita T, Nakajima T., Sakamoto B., Namikoshi M., Nagai H (2006)
Tetrahedron, 62, 1301-1308.
290
DNP (2005), from DNP „Dictionary of natural products“ on CD-Rom version 13:2, Copyright
©1982-2005 Chapman & Hall/CRC.
Edrada R.A., Wray V., Berg A., Grafe U., Sudarsono Brauers G., Proksch P. (2000) Z.
Naturforsch. C Biosci. 55, 218-221
Eggenberger U., and Bodenhausen, G. (1989), 61, 2298.
Eggert H., Vanantwerp C., Bhacca N., Djerassi C. (1976) J. Org. Chem. 41, 71-78.
Elenkov I., Milkova T., Andreev S. and Popov S. (1994) Comp. Biochem Phsiol. 107B, 547.
El-Sayed K.A., Bartyzel P., Shen X., Perry T.L., Zjawiony J.K., Hamann M.T., (2000),
Tetrahedron 56, 949-953.
Enders D., Vicario J.L., Job A., Wolberg M .M.,Ÿller M., (2002) Chem. Eur. J. 8, 4272-4284
Evans J.N.S. (1995) In : Biomolecular NMR spectroscopy, Oxford University Press.
Exarchou V., Krucker M., Beek T.A., Vervoort J.,Gerothanassis I. P., and Alert K. (2005)
Magn. Reson. Chem. 43, 681-687.and references therein.
Fattorusso E., Magno S., Santacroce C. and Sica D.(1974) Gazz. Chim. Ital. 104, 409
Faulkner D. J. (1997). Nat. Prod. Rep. 14. 259-302, and previous reviews of this series.
Faulkner D. J. (1998) Nat. Prod. Rep., 15, 113-158, and earlier reviews cited therein.
Faulkner D. J. (2002) Nat. Prod. Rep., 19, 1-48, and earlier reviews cited therein.
Faulkner D. J., (2000a) Nat. Prod. Rep., 17, 1-6.
Faulkner D.J., (2000b) Antonie van Leeuwenhoek 77: 135-145.
Fenn J. B., (2003) Angew. Chem. Int. Ed., 42, 3871-3894.
Fenn J., (1984) J. Phys. Chem., 88, 4451.
Firn R.D. (2004) natural products- module 867. Lectures 1-9, available at world web:
www.users.york.ac.uk/~drf1/867_1.htm
Firn R.D. and Jones C.G , (2003) Nat. Prod. Rep., 20, 382-391.
Firn R.D., (2003) Biodiversity and Conservation, 12, 207.
Firn R.D., and Jones C.G. (1998). Nature, 393, 617.
Firn R.D., and Jones C.G. (1999) Nature, 400: 13-14.
Firn R.D., and Jones C.G. (2000) Mol. Microbiol., 37, 989.
Firn R.D., and Jones, C.G. (1996) : An explanation of secondary product "redundancy". In
Recent Advances in Phytochemistry. Romeo J.T., Saunders J.A., and Barbosa P.
(eds). New York: Plenum Press, pp. 295-312.
Fitzgerald J.S. (1963), Aust. J. Chem., 16, 246.
Fleming I.D., Nisbet L.J. Brewer S.J. (1982). In Bioactive Microbial products : Search and
discovery. (J.D. Bu`lock, N.J.Nisbet, D. J. winstanley , eds.,) Academic press,
london, pp107-130.
291
Fukami A., Ikeda Y., Kondo S., Naganawa H., Takeuchi T., Furuya S., Hirabayashi Y.,
Shimoike K., Hosaka S., (1997) Tetrahedron Lett. 38, 1201-1202
García-Rocha, M.; Bonay, P.; Avila, J. (1996) Cancer Lett. 43-50.
Garg H.S., Agrawal S., (1995) Tetrahedron Lett. 36, 9035-9038
Gates P., (2005a), available at web page , http://www.chm.bris.ac.uk/ms/theory/maldiionization.html .
Gates P., (2005b), available at web page , http://www.chm.bris.ac.uk/ms/theory/cidfragmentation.html .
Gauvin A., Smadja J., Aknin M., Faure R., and Gaydou E-M. (2000) Can J. Chem. 78, 986992.
Gavagnin M., Spinella A., Castelluccio F and Cimino G. (1994), J.Nat. Prod., 57, 298-304.
George J.D., and George J.J. (1979) In : Marine Life an Illustrated Encyclopedia of
Invertebrates in the Sea , John Wiley Sons, New York U.S.A.
Gerrit J., Johannis P. and Johannis V. (1977) Carbohydrate Research. 62, 349-357.
Gerwick W.H. and Bernart M.W. (1992) Pharmaceutical and bioactive natural products In :
Marine biotechnology 1. (Attaway D.H. and Zaborsky O.R. eds) Plenum Press, New
York.
Giersiefen H., Hilgenfeld R., Hillish A.(2003): Natural products for lead identification: nature
is a valuable resource for providing tools, In: modern methods of drug discovery (ed,
Hillish A., Hilgenfeld R) .
Giner J.L., Gunasekera S.P., Pomponi S.A., (1999) Steroids 64, 820-824
Giorgiani F.; Cappiello A.; Beranova- Giorgiani S.; Palma P.; Trufelli H.; Desiderio D. M.
(2004) Anal. Chem., 76, 7028-7038.
Goetz G.; Yoshida W. Y.; Scheuer P. J. (1999) Tetrahedron, 55, 7739-7746.
GoetzG.; NakaoY.; Scheuer P. J. (1997) J. Nat. Prod., 60, 562-567. ,
Gorshkova I.A., Gorshkov B.A., Fedoreev S.A., Shestak O.P., Novikov V.L., Stonik V.A.,
(1999) Comp. Biochem. Physiol. C Comp. Pharmaco 122, 93-99 .
Goud T.V., Reddy N.S., Swamy N.R., Ram T.S., Venkateswarlu Y. (2003) Biol. Pharm.
Bull. 26, 1498-1501
Gullo, V.P. (1994) : In The Discovery of natural products with therapeutic potential (Gullo,
V. P. ed.), Butterworth-Heinemann press.
Gunasekera S.P., Cranick S., Longly R.E., (1989) J. Nat. Prod., 52, 757-761
Gunatilaka A., Gopichand Y., Schmitz F. and Djerassi C. (1981) J. Org. Chem. 46, 38603866.
292
Gunatilaka A.A.L., Kingston D.G.I. and Johnson R.K Pure & Appl. Chem., 1994, 66, Nos
10/11, pp. 2219-2222.
Hall M.J. (1989) biotechnology 7, 427-430.]
Hamann M. T.; Scheuer P. J. (1993) J. Am. Chem. Soc. 115, 5825-5826.
Hamann M.T.; Otto C.S.; & Scheuer P. J., Dunbar D.C. (1996) J.Org.Chem..61,6594-6600.
Harborne J.B. : In Introduction to Ecological Biochemistry. Academic Press, 1988.
Harborne J.B., (1986) Nat. Prod. Rep. 3, 323-344.
Haslam E.(1986) Secondary Metabolism , fact or fiction. Nat. Prod. Rep., 3, 217 – 249.
Hay M.E., and Fenical W. (1988) Annu. Rev. Ecol. Syst. 19, 111-145.
Hensens O. D, (1994) : In The Discovery of natural products with therapeutic potential,
(Gullo, V. P., ed.) Butterworth-Heinemann press. pp390.
Hill R.T., Hamann M.T., Enticknap J., and Rao K.V. (2005) Kahalalide-producing bacteria
and methods of identifying kahalalide-producing bacteria and preparing kahalalides..
PCT Int. Appl., 41 pp. CODEN: PIXXD2 WO 2005042720.
Hiort J. (2002) Neue Naturstoffe aus Schwamm-assoziierten Pilzen des mittelmeeresIsolierung
strukturaufklärung
und
biologischen
aktivität.,
(Heinrich-Heine
Universität. 2002).
Ho C.S., Lam C.W.K., Chan M.H.M., Cheung R.C.K., Law L.K., Lit L.C.W., Ng K.F., Suen
M.W.M., and Tai H.L.; (2003), Clin. Biochem. Rev., 24, 4.
Hooper J.N.A., and van Soest R.W.M. (2002) In : Systema Porifera : A Guide to the
Classification of Sponges, Kluwer Academic/Plenum publishers, New York.
Horgen F. D.; de los Santos D.B.; Goetz G.; Sakamoto B.; Kan Y.;Nagai H.; Scheuer P. J
(2000) J. Nat.Prod., 63, 152-154
Hunter I.S. (1992) Tibtech, 10, 144-146.
Hussar D.A. (2006): Nursing 2006, 36, 54-55
Iguchi K., Shimura H., Yang Z., and Yamada Y. (1993) Steroid, 58, 410
Inagaki M., Nakamura K., Kawatake S., Higuchi R. (2003) Eur. J. Chem. 325-331
Ireland C. M., Copp B.R., Foster M.P., McDonald L.A., Radisky D.C., Swersey J.C., In :
Marine Biotechnology,(Attaway,D.H., Zaborsky, O.R., eds) Plenum Press, New
York, 1993; Vol. 1, Chapter 1 pp 1-43.
Itoh T., Sica D., and Djerassi C. (1983) J.Chem. Soc. Perkin Trans 1, 147-153.
Jadulco R., Brauers G., Edrada R.A., Ebel R., Wray V., Sudarsono., Proksch P., (2002) J.
Nat. Prod. 65, 730-733
293
Jadulco R., Proksch P., Wray V., Sudarsono Berg A., Graefe U., (2001) J. Nat. Prod. 64, 527530
Janmaat M. L; Rodriguez J. A; Jimeno J.; Kruty F. A.; Giaccone G. (2005) Mol Pharmacol.,
68, 502-510.
Janmaat ML,Rodriguez JA, Jimeno J, Kruty FA, Giaccone G., (2005) Mol Pharmacol.
68(2):502-10.
Jaspars M. (1999) Nat. Prod. Rep. 16, 241-248.
Jenkins K.M., Jinsen P.R., Fenical W. (1999) Tetrahedron Lett., 40, 7637-40.
Jensen K.R. (1992) Anatomy of some Indo-Pacific Elysiidae (Opisthobranchia: Sacoglossa
(=Ascoglossa), with a discussion of the generic division and phylogeny. Journal of
Molluscan Studies, 58(3): 257-296.
Jimeno J. M.; Lopez-Martin J. A.; Casado A. R.; Izquierdo M. A.; Scheuer P. J.; Rinehart K.
(2004) Anti-Cancer Drugs, 15, 321-329.
Johnson R.K., and Hertzberg R.P.(1989) in Annual Reorts in Medicinal Chemistry, vol. 25
(Bristol, J.A., ed.), pp.129-140, Academic Press, san diego, CA.
Jones C.G., and Firn R.D. (1991) On the evolution of plant secondary metabolite chemical
diversity. Phil Trans Roy Soc B, 333: 273-280.
Kachlicki P., et al . (1997) Proc. Eur. Fusarium semin., 5th , 853-855.
Kan Y.; Fujita T.; Sakamoto B.; Hokama Y.; Nagai H.; (1999) J. Nat.Prod., 62, 1169-1172.
Karas M.; Hillencamp F. (1988) Anal. Chem., 6, 2301.
Karas M.; Hillencamp F., Beavis R.C., Chait B.T. (1991) Anal. Chem., 63, 1193.
Kato Y., Fusetani N., Matsunaga S., Hashimoto K. (1985) Tetrahedron Lett. 26, 3483-3486.
Kay, E.A. (1979)- Hawaiian Marine Shells,reef and shore fauna of Hawaii, section 4 :
Mollusca. Bernice P. Bishop Museum Special Publication 64(4),xviii +653 pp.
Kelaart E.F. (1858). Description of new and little known species of Ceylon nudibranchiate
molluscs and zoophytes. Journal of the Ceylon Branch of the Royal Asiatic Society,
columbo, 3(1): 84-139.
Knox J.P.; Dodge A.D. (1985), Phytochemistry 24, 889.
Kobayashi J., Murayama T., Ishibashi M., Kosuge s., Takamatsu M., Ohizumi Y., Kobayashi
H., Ohta T., Nozoe S., Sasaki T. (1990) Tetrahedron Lett. 46, 7699-7702.
Kobayashi M., Higuchi K., Murakami N., Tajima H., Aoki S., (1997) Tetrahedron Lett. 38,
2859-2862
Kobayashi M., Okamoto T., Hayashi K., Yokoyama N., Sasaki T., Kitagawa I. (1994a) Chem.
Pharm. Bull. 42, 265-270.
294
Kobayashi M., Rao S.R., Chavakula R., Sarma N.S., (1992) J. Chem. Res. Synop. 282-283 .
Kobayashi M., Rao S.R., Chavakula R., Sarma N.S., (1994) J. Chem. Res. Synop. 282-283
Koch P., Djerassi C., Lakshmi V., Schmitz F. J. (1983) Helv. Chim Acta, 66 2431.
Kogl F., Haagen-Smit A. J., Erxleben . (1933) Z. Physiol, 214, 241.
König G.M., Wright A.D., Linden A (1998) Planta medica 64, 443-447.
Kreuter M. H., Robitzki A., Chang S., Steffen R., Michaelis M., Klijajic Z., Bachmann M.,
Schröder H. C. and Müller W.E.G (1992) Comp. Biochem. Physiol., 101C, 183-187.
Lambert M.; Staerk D; Hansen S H.; Jaroszewski J W. Magn.Reson. Chem. (2005), 43, 771775 and references therein.
Larson E.R., and Fischer P.H. (1989) in Annual Reorts in Medicinal Chemistry, vol. 24
(Allen. R., ed.), pp.121-128, Academic Press, san diego, CA.
Lautens M., Stammers T.A., (2002) Synthesis 1993-2012
Leontein K., Lindberg B. and Lönngren J. (1977): Carbohydrate Research. 62, 359-362.
Lin Y.L., Lee H.P., OU J.C., Kuo Y.H. (1996) Heterocycles 43, 781-786.
Lindsay B.S., Almeida A.M.P., Smith C. J., Berlink R.G.S., Da Rocha R.M., Irland C.M.
(1999a) J. Nat Prod. 62, 1573-1575.
Lindsay B.S., Battershill C.N., Copp B.R. (1999b) J. Nat Prod. 62, 638-639.
Loo J. A.; Loo R. R. O. (1997) In Electrospray Ionization Mass Spectrometry of Peptides and
Proteins, edited by Cole, R. B., John Wiley & Sons, Inc. New York.
López-Maciá A.; Jiménez J. C.; Royo M.; Giralt E.; Albericio F (2001a). J. Am. Chem. Soc.,
123, 11398-11401.
López-Maciá A.; Jiménez J. C.; Royo M.; Giralt E.; Albericio F (2001b). Tetrahedron Lett.,
41, 9765-9769.
Luckner M.,(1990). Secondary metabolism in plants and animals. Third edition, Springer
Verlag, Berlin.
Malorni L., Minale L. and Riccio R. (1978) Nouv. J. Chim. 2, 351
Mann J. (1987) : In Secondary Metabolism. Clarendon Press, Oxford.
Mann J. (1994) : In Chemical Aspects of Biosynthesis, Oxford University Press.
Marahiel M.A., stachelhaus T. and Mootz H.D.(1997) Chem. Rev., 97, 2651-2673
Marfey P. (1984) Carlsberg Res. Commun. 49, 591-596.
Martin J.F., and Demain A.L. (1978) Fungal development and metabolite formation. In : The
Filamentous Fungi. Vol. III. Developmental mycology. ( Smith J.E. and Berry D.R.
eds.) Halstead Press, New York.
Mayer A.M.S., and Hamann M.T., (2004) Mar. Biotechnol. 6, 37-52.
295
McAlpine J.B., and Hochlowski J.E., (1994) : In The Discovery of natural products with
therapeutic potential (Gullo, V. P., edit.) 1994, Butterworth-Heinemann press.
pp349.
McConnell O. J., Longley R.E., and Koehn F. E., (1994) : In The Discovery of natural
products with therapeutic potential (Gullo, V. P. ed.), Butterworth-Heinemann
press, pp109-172.
Meltzer R.I. et al (1957) J.Org. Chem. 22, 1577-1581.
Meyer B.N, Ferrigni N.R.,Putnam J.E., Jacobsen L.B., Nichols D.E., and McLaughlin J.L.
(1982) : Planta Medica, 45, 31-34.
Min C., Lin W.H., Edrada R.A., Ebel R., TeusherF., Wray V., Nimtz M., and Proksch P.
(2001): Poster.
Mitchell S.S., Whitehill A.B., Trapido-Rosenthal H.G., Ireland C.M. (1997) J. Nat Prod. 60,
727-728.
Miyaoka H., Nishijima S., Mitome H., Yamada Y. (2000) J. Nat. Prod.63, 1369-1372.
Molinski T.F., Fahy E., Faulkner D.J., Van Duyne G.D., Clardy J. (1988) J. Org. Chem. 53,
1340-1341 .
Moon B., Baker B.J., McClintock J.B. (1997) J. Nat Prod. 61, 116-118.
Moon S.S., et al (1996) J.Nat Prod. 59, 777-779.
Moore R.E, Bornemann V., Niemczura W.P., Gregson J.M., Chen J., Norton T.R., Patterson
G.M.L., Helms G.L.J., (1989) J. Am. Chem. Soc., 111, 6128-6132.
Mosmann, T. (1983) J. Immunol. Methods. 65, 55-63.
Müller H., Brackhagen O., Brunne N., Henkel T., and Reichel F. (2000) : Natural products in
drug discovery, ernst Schering research foundation workshop 32: The role of natural
product in drug discovery, Springer-Verlag, Germany, 205-216.
Murakami N., Wang W.Q., Aoki M., Tsutsui Y., Higuchi K., Aoki S., Kobayashi M., (1997)
Tetrahedron Lett. 38, 5533-5536
Nakao Y., Uehara T., Matsunaga S., Fusetani N., Van Soest R.W.M. (2002) J. Nat. Prod. 65,
922-924
Nelson T.C. (1961) Proceeding of the National Academy of Research Council Supplement.
(1961) .p. 891.
Neudrt R. and Penk M. (1996) J. Chem. Inf. Comput. Sci. 36, 244-248.
Newmann D.J. and Cragg GM., (2004) J. Nat. Prod. 67, 1216-1238.
Newmann DJ., Cragg GM., and Snader K.M. (2000) Nat. Prod. Rep. 17, 215-234]
296
Nisbet L.J. (1992) In: Secondary Metabolites: Their Function and Evolution. ( D.J.
Chadwick, J.Whelan, eds,.) John Wiley, Chichester, P299.
Nordström A., Tarkowski P., Tarkowska D., Dolezal K., Astot C., Sandberg G., Moritz T.
(2004), Analytical Chem. 76, 2869-2877.
O’connor K. E., Witholt B., Duetz W. (2001) J. Bacteriol. 183, 928-933
Ojima N., Masuda K., Tanaka K., Nishimura O. (2005) J. Mass Spectrom., 40, 380-388.
Padmakumar K. (1998) Phuket Marine Biological Center Special Publication., 18, 187.
Paul v.J., and Van Alstyne K.L (1988), J.Exp.Mar.Biol.Ecol., 119, 15-29.
Pearse V., Pearse J., Buchsbaum M. and Buchsbaum R. (1987) In : Living Invertebrates,
Blackwell Scientific Publications, California.
Pease W.H. (1871). Descriptions of Nudibranchiate Mollusca inhabiting Polynesia. American
Journal of Conchology,6(4): 299-305.
Pechenik J.A. (2000) In : Biology of the Invertebrates, 4th McGraw-Hill companies.
Pedpradab S., Ederada R., Ebel R., Wray V., Proksch P. (2004) J. Nat. Prod.67, 2113-2116.
Pettit G.R., Cichacz Z.A., Gao F., Herald C.L., Boyd M.R., Schmidt j.M., Hooper J.N.A.
(1993) J. Org. Chem. 58. 1302-1304.
Pettit G.R., Cichacz Z.A., Herald C.L., Gao F., Boyd M.R., Schmidt j.M., Hamel J., Bai R.
(1994) J. Chem. Soc. Chem. Commun. 1605-1606.
Pettit G.R., Cichacz Z.A., Tan R., Herald D.L., Melody N., Hoard M.S., Doubek D.L.,
Hooper J.N.A. (1998a) Collect. Czech. Chem. Commun. 63, 1671-1677
Pettit G.R., Cichacz Z.A., Tan R., Hoard M.S., Melody N., Pettit R.K. (1998b) J. Nat.
Prod.61, 13-16.
Pettit G.R., Kamano Y., Herald C.L., Dufresne C., Cerny R.L., Herald D.L. Schmidt T.M.,
Kizu H.J.(1989), J. Am. Chem. Soc., 111, 5015-5017.
Pettit G.R., Tan R., Cichacz Z.A. (2005) J. Nat. Prod.68, 1253-1255.
Pettit G.R., Tan R., Melody N., Cichacz Z.A., Herald D.L., Hoard M.S., Pettit R.K., Chapuis
J.C. (1998c) Bioorg. Chem. Lett. 8, 2093-2098.
Proksch P., Ebel R., Edrada R.A., Wray V., Steube K., (2003) : In Bioactive Natural Products
from Marine Invertebrates and Associated Fungi.,
Prog. Mol. Subcell. Biol., 37,
117-142.
Proksch P., Edrada R.A. and Ebel R. (2002). App. Microbiol.and Biotech., 59, 125-134.
Puapaiboon U.; Tylor R.T.; Jainhuknan J. (1999) Rapid Commun. Mass Spectrom. 13, 516520.
Qureshi A., Faulkner D.J., (1999) Tetrahedron 55, 8323-8330 .
297
Rademaker-Lakhai J M., Horenblas S., Meinhardt W., Stokvis E., de Reijke T M., Jimeno J
M., Lopez-Lazaro L., Lopez-Martin J A., Beijnen J H. and Schellens JH. (2005)
Clinical Cancer Research, 11, 1854-1862
Ramesh P., Reddy N.S., Venkateswarlu Y. (1999) J. Nat. Prod. 62, 780-781 .
Reddy N.S., Ramesh P., Venkateswarlu Y. (1999)
Nat. Prod. Lett. 14, 131-134
CA133:28562 A .
Reddy N.S., Ramesh P., Venkateswarlu Y., (2000) Nat. Prod. Lett. 14, 131-134 .
Renoux J-M., Rohmer M., (1986) Eur. J. Biochem. 155, 125-132
Reuben B.G., and Wittcoff, H.A. (1989) Pharmaceutical Chemicals in Perspective,John
Wiley & Sons, New York.
Rodricks J.V. (1992) : In Calculated Risks. Cambridge university press, cambridge, p. 256.
Rosenthal G.A.and Janzen D.H. (1979) : In Herbivores. Academic press. New York.
Rudman W.B., 1999 (Aug 10). Comment on Elysia rufescens from Hawaii by Juan Lucas
Cervera. [Message in] Sea Slug Forum. Australian Museum, Sydney. Available from
http://www.seaslugforum.net/find.cfm?id=1165
Rudman W.B., 1999 (November 4) Elysia ornata (Swainson, 1840). [In] Sea Slug Forum.
Australian
Museum,
Sydney.
Available
from
http://www.seaslugforum.net/factsheet.cfm?base=elysorna
Rudman W.B., 2001 (November 19) Elysia grandifolia Kelaart, 1858. [In] Sea Slug Forum.
Australian Museum, Sydney. Available from
http://www.seaslugforum.net/factsheet.cfm?base=elysgran
Rycroft D.S., (1988) In : Natural Product Chemistry III ; Atta-ur-Rahman A., Le Quesne P.
W., Springer –Verlag Berlin Heidelberg, pp. 43.
Salmoun M, Devijver C, Daloze D., Braekman J-C, van Soest R.W.M (2002) J. Nat. Prod.
65, 1173-1176.
Schwarzer D., Finking R. and Marahiel M.A. (2003) Nat. Prod. Rep., 20, 275-287.
Seal A.N., Partley J.E., Haig T. and An M. (2004) J.Chem., Ecol., 30, 1647-1662.
Seigler D.S (1998) : In Plant Secondary Metabolism. Kluwer Academic Publisher. London.
759 P.
Sepcic K., Batista U., Vacelet J., Macek P., Turk T. (1997) Comp. Biochem. Physiol. C Comp.
Pharmaco 117, 47-53 .
Serebryakov E., Simolon A., Kucherov B. (1970) Tetrahedron, 26, 5215
Sewell J.M., Mayer I, Langdon SP, Smyth JF, Jodrell DI, Guichard SM. (2005) Eur J Cancer.
41(11):1637-44.
298
Shamma M.,1989 : In Studies in Natural products chemistry , Volume 5, Structural
elucidation (Part B) , (Atta-ur-Rahman, A., edit.), Elsevier Science Publishers B.V.,
1989., pp V.
Shatz M., Yosief T., Kashman Y., (2000) J. Nat. Prod. 63, 1554-1556 .
Sheikh Y.M., and Djerassi C. (1974) Tetrahedron, 30, 4095.
Shen Y-C ., Prakash C.V.S. (2000) J. Nat. Prod. 63, 1686-1688 .
Silverstein R.M.; Bassler G.C.; Morrill T.C. (1991) : In Spectrometric identification of
organic compounds, John Wiley & Sons pub., Chpt 2.
Siuda J.F. (1974) J. Nat. Prod. (Lloydia) 37, 501- 503.
Skyler D., Heathcock C.H. (2002) J. Nat. Prod. 65, 1573-1581 .
Smith P.M.C., and Atkins C.A. (2002) Plant Physiology, 128, 793-802.
Spengler B. (1997) J. Mass Spectrom. 32, 1019-1036.
Sperry S., Crews P. (1998) J. Nat. Prod. 61, 859-861 .
Steinbeck C. (2004) Nat. Prod. Rep., 21, 512-518
Stierle A A.(1988) Investigation of Biologically Active Metabolites from Symbiotic
Microorganisms, Ph.D. Dissertation, Montana state University, Bozeman (1988).
Suarez Y, Gonzalez L, Cuadrado A, Berciano M, Lafarga M, Munoz A. (2003) Mol Cancer
Ther. , 2(9):863-72.
Suckau D., and Cornett D.S., Analusis Magazine, 1998,26, M18-M21.
Suffness M., Newman D.J., and Snader K.(1989) : In Bioorganic Marine Chemistry, vol.3
(Scheuer, P.J., ed.), pp. 131-168, Springer-Verlag, New York.
Sun H.H., Cross S.S., Gunasekra M., Koehn F.E., (1991) Tetrahedron 47, 1185-1190 .
Sun H.H., Cross S.S., Koehn F., Gunasekera M. (1992) US Pat. Appl. 0 0,
CA116(21):207800v
Swain T. (1977) Ann. Rev. Plant. Physiol., 28, 479-501.
Tanaka K., (2003) Angew. Chem. Int. Ed., 42, 3861-3870.
Tanji M., Namimatsu K., Kinoshita M., Motoshima H., Oda Y., and Ohnishi M. (2004)
Biosci. Biotechnol. Biochem., 68 (10), 2205-2208.
Theobald N., Shoolery J.N., Djerassi C., Erdman T.R., Scheuer P.J., (1978) J. Am. Chem.
Soc. 100 5574-5575
Torssell K.B.G. (1997) : In Natural Product Chemistry. A mechanistic ,biosynthetic and
ecological approach. Apotekarsocieteten-Swedish Pharmaceutical Press.
Toth S.I., Schmitz FJ. (1994) J. Nat. Prod. 57, 123-127
Tsukamoto S., Miura S., van Soest R.W.M., Ohta T.(2003) J. Nat Prod. , 66, 438-440.
299
Turner W.B. (1971) : In Fungal Metabolites . Academic Press. London.446 p.
Venkateswarlu Y., Reddy M.V.R., Srinivas K.V.N.S., Rao J.V. (1993) Indian J. Chem. Sect.
B 32, 704.
Vicario J.L., Job A., Wolberg M. M.,Ÿller M., Enders D., (2002) Org. Lett. 4, 1023-1026
Vining L.C. (1992) Gene, 115, 135-140.
Walker R.P., Thompson J.E., Faulkner, D.J. (1980), J. Org. Chem. 45, 4976-4979.
Watanabe K., Tsuda Y., Hamada M., Omori M., Mori G., Iguchi K., Naoki H., Fujita T., Van
Soest R.W.M. (2005) J. Nat. Prod. 68, 1001-1005
Webb AG. (2005) Magn.Reson. Chem. 43, 688-696 and references therein.
Whitehead R. (1999) Annu. Rep. Prog. Chem., Sect. B.95, 183-205.
Will M., Fachinger W., and Richert J.R. (1996), J.Chem.Inf. Comput. Sci., 36, 221.
Wilm M., Mann M. (1996) Anal. Chem.; 68, 1.
Wilson, I.D., Morgan, E.D., Lafont, R., Shockcor, J.P., Lindon, J.C., Nicholson, J.K. and
Wright B., (1999) Chromatographia, 49 374.
Wolfender JL., Queiroz EF.; Hostettmann K. (2005) Magn.Reson. Chem. 43, 697-709 and
references therein.
Woodruff H.B., Hernandez S., Stapley E.D. (1979) Hindustan Antibiotic Bulletin 21:71-84.
Woodruff H.B., MacDaniel A.E. (1958) : In Antibiotic approach in strategy of chemotherapy.
Society of general microbiology symposium 8: 29-48.
Wright J., Mcinnes A., Shimizu S., Smith D. and Walter J. (1978) Can J. Chem. 56, 18981903.
Xiao D., Deng S. and Wu H. (1997) Tianran Chanwu Yanjiu Yu Kaifa, 9, 1-4; C.A. 130:
107540f.
Xu F.and Alexander AJ. (2005) Magn.Reson. Chem. 43, 776-782 and references therein.
Yagi H., Matsunaga S., Fusetani N. (1994) J. Nat Prod. 57, 837-838.
Yang l., Andersen R.J., (2002) J. Nat. Prod. 65, 1924-1926 .
Yaoita Y, Amemiya K., Ohnuma H., Furumura K., Masaki A., Matsuki T., Kikuchi M.,
(1998) Chem. Parm. Bull. 44 , 944-950.
Younus M.S. and Djerassi C. (1974) Tetrahedron 30, 4095-4103 .
Yousaf M., El Sayed K.A., Rao K.V., Lim C.W., Hu J.F., Kelly M., Franzblau S.G., Zhang
F.Q., Peraud O., Hill R.T., Hamann M.T. (2002) Tetrahedron 58, 7397-7402 .
Youssef D.T. (2005) J.Nat. Prod.68, 1416-1419.
Youssef D.T. A., Shaala L. A., Emara S. (2005) J. Nat. Prod., 68 (12), 1782 –1784
Youssef D.T., Yamaki R.K., Kelly M., Scheuer P. J.(2002) J. Nat. Prod.65, 2-6.
300
Youssef D.T.A. Yoshida W.Y., Kelly M., Scheuer P.J., (2000) J. Nat. Prod. 63, 1406-1410
Youssef D.T.A., Van Soest R.W.M., Fusetani N. (2003) J. Nat. Prod. 66, 861-862
Yue J-M., Chen S-N., Lin S-W., Sun H-D., (2001). Phytochemistry 56, 801-806.
Zeng Z. (2000) Redai Haiyang, 19, 86-89; C.A. 133: 278967s.
Zeng Z., Zeng L. and Su J. (1996) Zhongshan Daxue Xuebao, Ziran Kexueban, 35, 52-57;
C.A. 125: 190994x.
Zschocke S., Klaiber I., Bauer R., Vogler B. (2005) Molecular Diversity 9: 33–39
301
List of Abbreviations
[α]D :
br :
CI :
COSY :
d:
dd :
ddd :
DEPT :
ED :
EI :
ESI :
eV :
FAB :
HMBC :
HMQC :
HPLC :
Hz :
LC :
LC-MS:
m:
MALDITOFPSDMS:
MeOD :
MeOH :
mg :
mL :
MS :
m/z :
µg :
µL :
NMR :
ppm :
Prep. HPLC :
q:
ROESY :
RP-18 :
s:
t:
TFA :
TLC :
UV :
VLC :
specific rotation at the sodium D-line
broad signal
chemical ionization
correlation spectroscopy
doublet
double of doublets
double double of doublets
distortionless enhancement by polarization transfer
effective dose
electron impact
electro spray ionization
electronvolt
fast atom bombardment
heteronuclear multiple bond connectivity
heteronuclear multiple quantum coherence
high performance liquid chromatography
herz
lethal concentration
Liquid chromatography-mass spectrometer
multiplett
Matrix-assisted laser dessorption ionization-time of flight-post source decay
mass spectrometer
deuterated methanol
methanol
milligram
millilitre
mass spectroscopy
mass per charge
microgram
microliter
nuclear magnetic resonance
part per million
preparative HPLC
quartet
rotating frame overhauser enhancement spectroscopy
reversed phase C-18
singlett
triplett
trifluoroacetic acid
thin layer chromatography
ultra-violet
vacuum liquid chromatography
302
Publication
Ashour M., Edrada R, Ebel R., Wray V., Wätjen W., Padmakumar K., Müller W. E. G., Lin
W. H. and Proksch P. (2006): „Kahalalide Derivatives from the Indian Sacoglossan Mollusc
Elysia grandifolia“ , J. Nat. Prod., 69, 1547.
303
Biographic data
Name:
Mohamed Abdelghaffar Ali Ashour
Date of birth:
18. December 1969
Place of birth:
El-Sharkiya, Egypt
Nationality:
Egyptian
Civil status:
Married, two children
Address:
Universitätsstrasse 1, 40225 Düsseldorf
Home Address:
Al-azhar University, Cairo, Egypt
Educational Background:
1975 – 1987:
Grade School - Higher School, Belbies Schools for Al-Azhar
Sciences, Belbies, Egypt.
1987 – 1992:
Bachelor of Science degree in Pharmacy, Al-Azhar University,
Cairo, Egypt.
1994 – 1999:
Master of Science degree in Pharmacy, Al-Azhar University,
Egypt
Thesis:
Pharmacognostical Studies of some Sophora sp., belonging to
family Fabaceae
2002-2002:
ZMP certificate of German language, Goethe Institut, Cairo,
Egypt.
2003 – present:
Ph.D. candidate, Institute of Pharmaceutical Biology, HHU,
Düsseldorf, Germany.
Employment Record:
1994 – 1999:
Demonstrator, Faculty of Pharmacy, Al-Azhar Univeristy,
Cairo, Egypt.
1999 – present:
Assistant Lecturer, Pharmacognosy Department, Faculty of
Pharmacy, Al-Azhar University, Cairo, Egypt.
304