report 2004

Transcrição

report 2004

Laboratório Associado
para a Química Verde
Tecnologias e Processos Limpos
REPORT
2004
ii
Executive Summary
REQUIM TE (Rede de Química e Tecnologia)results from a long-standing collaboration between
two university based research Centers - “CQFB: Centro de Química Fina e Biotecnologia da
Universidade Nova de Lisboa” and - “CEQUP: Centro de Química da Universidade do Porto“.
The association of the two centers was recognized as the Laboratório Associado para a Química
Verde by the Portuguese Ministério de Ciência e do Ensino Superior in November of 2001, and
was formerly chartered as a nonprofit scientific organization in January of 2003.
The scientific expertise and complementary knowledge available, put together by CQFB and
C E Q U P, has been focused on the topic GREEN CHEMISTRY - CLEAN TECHNOLOGIES A N D
PROCESSES with a wide range of tools and from different perspectives.
In general terms, the existing competencies will be drawn from the areas identified as expertise
fields within REQUIM TE: chemistry, (micro)biology, biochemistry and molecular biology,
molecular modeling, bio(catalysis) and reaction mechanisms, (bio)conversion and
bioremediation, transport phenomena, separation processes, sensor development, monitoring
and control.
The available expertise will be used to implement the use of cleaner products and cleaner
technologies, preventing pollution at its source. By working with manufacturers, governmental
agencies, consumers and general public new ideas and new attitudes can be implemented.
REQUIM TE can act as a promoter and is available to answer questions and problems placed
from outside. REQUIM TE will look forward to assist manufacturers in order to develop
cooperative efforts to design and redesign products and processes that will reduce life cycles
environmental impacts. Activities wil be implemented to provide reliable information on the
environmental benefits, performances and economic feasibility of green chemistry and clean
processes.
The year 2004 was the third year in which REQUIMTE was fully operational: frequent meetings
of the board of directors (monthly), creation of formal incentives to strengthen the cooperation
between researchers of the two Centers (seed money for joint projects) and the recruitment of
two more researchers(Investigadores Auxiliares) to carry out activities under the contract of
Laboratório Associado.
The activities of REQUIMTE were presented on a joined meeting between CQFB and CEQUP in
Fátima, 9/10 January 2004. The book of abstracts of the meeting is delivered together with this
report.
Laboratório Associado para a
Química Verde
iii
Mission Statement
a.
REQUIMTE, which is recognized as the Laboratório Associado para a Química Verde by the
Portuguese Ministério de Ciência e do Ensino Superior, is a voluntary association of two research
Centers which have freely opted to collaborate in research and postgraduate activities, whilst
still being fully committed to their respective Universities.
b.
REQUIMTE considers itself to have made a very important contribution to the way in which
Chemistry, Biology and Chemical Engineering being carried out in Portugal is viewed
internationally – as judged by the quality and quantity of its scientific publications and also by
the number and quality of ongoing research projects.
c.
REQUIMTE will focus the activities of its reaserchers to implement the principles of Green
Chemistry.
d.
REQUIMTE also aims to optimize internal collaboration and to identify the best international
strategic partners.
e.
REQUIMTE, whilst wishing to preserve its well-founded and successful roots in traditional
chemical, biological and engineering sciences, intends fully to strengthen its international
research in innovative and novel topics and to become a pool of attraction for young and well
established national and international scientists.
f
.
REQUIMTE views its involvement in the postgraduate training of young people as a very
important function. REQUIMTE policy is to select projects in such a way that students working
within the organization will gain, in the course of their researches, the skills required by the
employment market.
g.
REQUIMTE is presently the largest chemical network in Portugal, with approximately 340
researchers, of which more than 180 hold a Ph.D. degree.
h.
REQUIMTE provides an optimal environment to explore the synergies of the their expertise in
answering the needs of the productive sector and providing specialized services and consulting.
.
i
REQUIMTE is currently pursuing an environmentally driven reasearch in association with the
chemical industry to develop closed loop industrial processes.
.
j
REQUIMTE is working to foster public awareness of key chemical and biochemical concepts,
to help understand the costs and benefits of technology in the modern world and to develop a
balanced global appreciation of environmental issues.
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History
REQUIM TE stands for Rede de Química e Tecnologia and names the Chemistry and
Technolology Network that has been formed by two Research Units, the Centro de Química da
Universidade do Porto - CEQUP and the Centro de Química Fina e Biotecnologia da Universidade
Nova de Lisboa – CQFB.
Since November 2001 REQUIM TE became the “Laboratório Associado para a Química Verde
– Tecnologias e Processos Limpos” of Fundação para a Ciência e a Tecnologia of MCTES, in
which 341 researchers(184 holding a Ph.D. Degree) exploit the basic principles of Green Chemistry and aim to contribute to the practice of Sustainable Chemistry.
The need of practicing a sustainable development in order to reach the social, economic and
environmental objectives of the modern society is well accepted by governments, industrial
sector and general public. Within this scope, Chemistry which is commonly associated with
harmful products and not with materials absolutely essential for everyday life, must have a
decisive role in the maintenance and improvement of living and environmental conditions.
The awareness of contemporary society for the inevitability of the use of chemicals and chemical
processes and the understanding of the concept of sustainability lead to a new way of thinking
Chemistry. Having in mind the implementation of clean practices and clean industrial processes
in which the amount of raw materials, energy, costs and risks are reduced a scientific movement,
known as Green Chemistry, has emerged in the last quarter of the 20th century.
Green Chemistry aims to redevelop laboratory and industrial processes in order to make them
cleaner and economical viable. For that purpose scientists have to design new reactions and
processes in which principles like an economy of atoms, use of renewable materials, use of nontoxic solvents and use of clean energy sources must be followed.
The objectives of Laboratório Associado para a Química Verde – Tecnologias e Processos Limpos
are: a) to encourage the use of clean products and technologies; b) to assist industry in the
design and implementation of non-aggressive chemical processes; c) to train young researchers
in interdisciplinary areas related with the practice of sustainable chemistry; d) to make public
the principles of green chemistry and to alert society for the necessity of a sustainable practice
in everyday life.
Research is presently focused in the following thematic areas of: i) natural products, (ii) food
quality and safety, (iii) clean production technologies and processes, (iv) environmental control
and remediation and (v) catalysts, solvents and non-toxic compounds.
The sharing of multidisciplinary scientific knowledge, technology and equipment between
researchers of the two centers that formed the network has significantly contributed to the
development of new projects in green chemistry and to the enrichment of training of graduate
students by making easier the mobility of human resources.
At present the network REQUIM TE can be described as a big Laboratory that has two operating
sites, one at Universidade Nova de Lisboa and other at Universidade do Porto.
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REQUIM TE in 2004
2004 was the third year in which REQUIMTE operated as a — Laboratório Associado“, although
the cooperation existed since 1996. The strategic plan was to focus expertise in Analytical,
Biological, Inorganic, Organic, Physical and Theoretical Chemistry and in Chemical Engeeniring
in a contemporary unifying concept œ that of Green Chemistry.
2004 was the third year when the funding contract with FCT-MCTES was operational and
REQUIM TE hired new researchers and technicians in order to carry out activities included in the
contract with the Ministry.The profiles of the two new researchers are provided in Annex.
The scientific production was also considered a key aspect of the activity of REQUIMTE, and the
total number of articles in scientific journals, chapters in books and articles in proceedings has
grown to 274 (242+18+14), much to the dedication of its graduate students, 24 of which have
completed their doctoral thesis and 10 their master thesis in 2004.
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People at REQUIM TE
Board of Directors
Baltazar de Castro (UP)
Isabel Moura (UNL)
Manuel Nunes da Ponte (UNL)
Co-ordinating Comittee of the Scientific Council
Ana Lobo (UNL)
Baltazar de Castro (UP)
Isabel Moura (UNL)
José Costa Lima (UP)
José Moura (UNL)
Manuel Nunes da Ponte (UNL)
Maria de Lourdes Bastos (UP)
Maria Rangel (UP)
Advisory Comittee
Prof. William B. Motherwell
University College, Christopher Ingold Laboratories, 20 Gordon St., London WC 1 H OAJ, UK
Organic synthesis; Reaction mechanisms in organic chemistry
Prof. Luigi Marzilli
Department of Chemistry, Louisiana State University, Baton Rouge, LA70803-1804, USA
Spectroscopy; Inorganic synthesis; B12 chemistry; Bioinorganic chemistry; Metal based drugs
Prof. Jean L. Rivail
Université Henri Poincaré, Laboratoire de Chimie Théorique, B.P. 239, 54506 Vandoeuvre-les-Nancy,
France
Theoretical chemistry
Prof. Marek Trojanowicz
University of Warsaw, Faculty of Chemistry, Pasteura 1, PL-02-093,WA R S AW , Poland
Analytical Chemistry
Professor James Clark
Clean Technology Centre, Department of Chemistry, University of York, Heslington,York, YO10 5DD,
UK
Green Chemistry
Prof.Harry Kuiper, Programme Leader and International Account Manager at the State Institute for
Quality Control of Agricultural Products (RIKILT, UR Wageningen NL)
Food Safety
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http://www.requimte.pt
L A B O R ATÓRIO ASSOCIADO
QUÍMICA VERDE – TECNOLOGIAS E PROCESSOS LIMPOS
REQUIMTE, is the biggest network in Chemistry and Chemical Engineering established in Portugal and is recognized as the
Laboratório Associado para a Química Verde by the Portuguese Ministério de Ciência e do Ensino Superior since November 2001.
The scientific expertise and complementary knowledge available, put together by the two research centres that form the network
(Centro de Química Fina e Biotecnologia-UNL and Centro de Química-UP), allowed us to deal with the topic GREEN CHEMISTRY
- C L E A N T E C H N O L O G I E S A N D P R O C E S S E S with a wide range of tools and from different perspectives.
REQUIMTE has the mission of corporate in a continuous form, in a competent and efficient way in the prosecution of the specific
aims of the national scientific and technologic politics in the areas of:
1
N AT U R A L PRODUCTS: SCREENING AND SYNTHESIS
Screening of biologically active compounds of traditional plant-based Medicine and support to certification on natural
phamaceuticals.
Synthesis of pharmacologically active compounds.
Chromatographic purification of new compounds and spectroscopic studies of structure-function.
2
FOOD QUALITY AND SAFETY
Food Quality and Safety regulations and inter-laboratory validation of analytical and certification methodologies.
Screening of pharmaceutical residues and secondary metabolites
Survey of critical points for microbiological control in food processing and development of microbiological methodologies
for fast pathogen detection.
3
CLEAN PRODUCTION TECHNOLOGIES AND PROCESSES
Implementation of clean separation processes – supercritical fluids, membranes, adsorption.
Reaction/separation process integration.
Monitoring, automation and control of bio/chemical processes.
Scale-up.
4
ENVIRONMENTA L CONTROL AND (BIO) REMEDIATION
Advanced analytical tools (AAS-EA, ICP-MS, GC-MS, HPLC-MS, A A and DNA sequencing, NMR, EPR, X-Ray Crystallography
and MS) and implementation of good practices in chemical analysis.
Development of control systems, probes, sensors and transducers (mainly oriented to environmental problems).
Implementation of novel processes (including physical and biological) for treatment of water and industrial wastes, as well
as soils.
Energy recovery from waste and to recycljng of materials.
5
C ATA LYSTS, SOLVENTS AND NON-TOXIC COMPOUNDS
Green synthetic routes of chemicals and pharmaceuticals.
Spectroscopic / computational techniques and molecular structure.
Alternative solvents and catalysis.
Enzymes in non-aqueous solvents.
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Events
The Day of Chemistry is organized every year bringing more than 600 students to the Campus
of Caparica. In 2004 it occurred on the 8th of February. In the morning there is a session in the
auditorium with live experiments and in the afternoon the students visit several laboratories in the
Chemistry Deparpment.
Every year the Faculty of Sciences and Technoly (Campus of Caparica) organizes an Open Day
with visits to the laboratories of the Chemistry Department). In 2004 a Forum of Chemistry was
organized by students and teachers of Chemistry.
The VII Jornadas Tecnológicas de Engenharia Química took place on the 19th and 20 th of
Abril 2004
Program Ciência Viva
In the week of the FCT Scientific Culture visits to several laboratories were organized.
In the summer period two weeks courses, for students presently attending the secondary school,
were organised at CEQUP and on the themes of Treatment of Laboratory Wastes and Toxicology.
Monographic Courses (5 days) are ministrated in the themes:
Determination of Molecular Structures by X-Ray Diffraction Methods
Nuclear Magnetic Resonance: From Theory to Practice
Liquid-liquid and gas-liquid chromatography
Organization of Conferences, Symposiums and Workshops
XIX Reunião Ibérica de Adsorção, July 2004
QUITEL 2004 – 30th Congress of Theoretical Chemists of Latin Expression, Porto,Sep 2004
3rd Portuguese-Spanish Biophysics Congress, Lisboa, Outubro 2004.
Workshop on MALDI-TOF-MS
Curso de cianótipos, Instituto Superior de Engenharia do Porto, 25 e 26 de Fevereiro, 2004.
SMEs Go LifeSciences and SMes for Food, FFUP, July 2004
1as Jornadas de Alimentação e Saúde, FFUP, Dez de 2004
The III Meeting of REQUIMTE was organized in January 9-10, 2004 in Fátima. This event
brought together all researchers in REQUIMTE and sessions included: i) a main session in
which the achievements of the Laboratório Associado were reported, highlights on the green
chemistry field were presented and discussed and in which the Investigadores Auxiliares employed
by REQUIMTE presented their work and projects; ii) a poster session which made possible the
presentation of the work developed by all the reaserchers.
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Report Organization
The present report, after this introduction, is organized in Parts A and B and Annexes.
Part A contains the achievements of the Associated Laboratory under its main themes.
Part B contains the individual contributions of all the research groups from CQFB and CEQUP
in a more detailed form.
Annexes contain listings of the researchers, publications and on-going projects in 2004.
Química Verde
RESEARCH REPORT
2004
Part A
2
AREA 1
NATURAL PRODUCTS: SCREENING AND SYNTHESIS
Within the scope of this theme, the Associate Laboratory has been working in the following
topics:
- Screening of new compounds isolated from natural products and identification of molecules
with established or potential biological activity. Plants studied originated from Africa, Central and
South Americas, as well as Portugal, and the type of compounds ranged from isoprenoids, to
shikimic acid derivatives, and include alkaloids and complex glycosides. Salient observed activity
include antileishmanial, antioxidant and anti-inflammatory.
- Synthesis (hemisynthesis) of new structures for the pharmaceutical, agrochemical and energy
industries employing routes that avoid production of toxic residues. Having previously established
a simple route to the aglycone of one the most active compounds used in cell cycle control
(staurosporin) methods were devised for effective linkage of the sugar moiety. Asymmetric induction
induced by Barton esters holding chiral substituents was noticed while producing gammaaminoesters. A new enantioselective method for the hydroxylation of terminal alkenes was achieved
by an oxymercuration-demercuration phase transfer reaction using cyclodextrin.
- Characterization of the biological activity (toxic or beneficial, such as anti-inflammatory and
anti-oxidant) of natural and synthetic compounds. These were isolated from natural extracts or
from synthetic compounds (hemisynthesis) employing in vitro methods with cells isolated from
animal or human models. The metabolites of ecstasy were found to be particularly toxic towards
human neural cells. The mechanism of radical oxidation of DNA bases by OH radical was studied
using EPR and reaction products profiling.
- Characterization of complex organic mixtures in natural matrices. These were characterised for
application in the fields of agriculture and forestry, geology and water management (ocean and
freshwater). In particular the hopanoid composition of sediments from the lower Tagus basin was
used in the study of the fate of spilled oil. The profile of toxic compounds in railway sleepers was
done as part of a collaborative program. To study the process of host-tree selection by insects,
a detailed isoprenoid profile of pine varieties cultivated in Portugal was correlated with the
susceptibility of the trees towards attack by the Mediterranean defoliator winter pine processionary
moth (collaboration with environmental scientists at Monte da Caparica).
- Patrimony Conservation - Safeguard of our cultural heritage.
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AREA 2
FOOD QUALITY AND SAFETY
- Participation in International Collaborative study ISO/CD 6885 “Animal and Vegetable fats and
oils. Determination of Anisidine value” (2004).
- Membership of Technical Committees, and participation in the elaboration of the industry code
of Hygienic Practices and NP EN ISO documents.
- Implementation of collaborative projects with producer associations, in order to chemically
characterize traditional food products and evaluate their safety, taking into account agricultural
practices.
- Quality Control of food products answering to industry questions and consumers concerns,
including the presence of GMOs and acrylamide.
- Correlation of compositional analysis and beneficial health effects.
- Physical characterization of food products and food structure studies.
- Implementation of in vitro assays to evaluate the safety/toxicity/protection of food components
and beverages for human consumption.
- Elaboration of toxicity/safety reports of food additives requested by Industry in order to obtain
Market Authorization.
- Patented voltammetric method for the determination of diacetyl directly in beer fermentation
vessels.
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AREA 3
CLEAN PRODUCTION TECHNOLOGIES AND PROCESSES
- Membranes were used to extract amino acids with supported ionic liquids, to recover compounds
from aqueous streams by pervaporation and nanofiltration, and with bioreactors for Bioremediation.
- DEGDMA was polymerised in supercritical CO2 in the presence of a drug, in order to produce
molecularly imprinted polymers. A dynamic model of heat exchanger for supercritical CO2 was
developed. A pilot-scale demonstration of a large-scale ANG storage system was performed and
a chromatographic process with recycle, mimicking a Simulated-Moving-Bed unit, was built.
- Production of polyhydroxyalkanoates was experimentally evaluated and mathematical modelled.
- Biocatalysis in ionic liquids/supercritical CO2 systems was established. Model edible films
were prepared using calcium alginate and carrageenan by casting, and microspheres containing
non steroid anti-inflammatory drugs were produced.
- The catalytic depolymerisation of polymethylmethacrylate was studied.
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AREA 4
ENVIRONMENTAL CONTROL AND (BIO) REMEDIATION
- Development of control systems, probes, sensors (Chemo and Photo based on supramolecular
compounds) and transducers (mainly oriented to Development of advanced Analytical Tools/
Automation and Instrumentation) - Prototypes for laboratorial control and analytical detection.
Electroanalysis. Development of continuous flow systems and dedicated equipment in on-line
control. External services.
- Quality Control and Food - Quantification of additives.The determination of antioxidant and
acidity regulators in beverages.
- Development of electrochemical devices and amperometric wall-jet cells to flow injection analysis
set-ups.Development of Transducers. Photo and chemosensors. Synthesis of cavitands and
hemicarcerands with metal complexing units. Collaboration with a private the company Y dreams.
- Characterization of Biocatalysts and redox partners- (Peroxidases, Pseudoazurin, Cobalt, Zinc,
Molyddenum and Tungsten containing enzymes). Oxidative (superoxide reductases, transferin
and desaturases) and denitrification pathways (nitrate and nitrite reductases).
- Towards Biosensors - Direct electrochemistry of aldehyde oxidoreductase and nitrite reductase.
- Environmental Control and Bioremediation –Membrane bioreactors for water decontamination.
- Remediation of vaccine production wastewaters containing Hg compound. Pesticides analysis.
Laboratory management of wastes. Soils remediation
- Drug Design – Biomimetic systems and Simulation. Proteomics and computational genomics.
Non steroidal anti-inflamatory drugs (NSAIDs), Disease Related Research (AIDS, Hypertension,
Malaria, Alzheimer’s disease).
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AREA 5
CATALYSTS, SOLVENTS AND NON-TOXIC COMPOUNDS
- Lactams were synthesised in water by intramolecular C-H insertion of diazo substrates. Sugars
were used as sources for new chiral materials and microwave irradiation was used in accelerated
synthesis and/or in solvent free reactions;Several novel C1- and C2-symmetric chiral ligands
were developed.
- Equilibrium properties of ionic liquids were measured. Osmium catalysed dihydroxilations in
ionic liquid + high pressure CO2 without leaching of catalyst were carried out and phase behaviour
of polymers+ carbon dioxide system was measured.Pt and Pd nanoparticles/nanoassemblies
and metallosurfactants based SAMs for catalytic applications were prepared.Heterogenised
transition metal complexes in several supports were tested in the epoxidation (limonene and
other alkenes) and aziridination of alkenes; Pd and Pt on anthracites and synthetic carbons
were used for liquid phase hydrogenation.
- Calculations on the adsorption of radical methoxide on clean and oxygen modified Ru(001)
surfaces has given a reliable assignment of the experimental C-H stretching region of CH3O on
the Ru.
- Determination of inhibition mechanisms of RNR enzyme by anti cancer drugs and design/
optimisation of new leads for the treatment of cancer, malaria, AIDS and hypertension have been
done by molecular modelling and computer simulation.
- Structure determination of several components of the Cellulosome assembly in particular of the
noncatalytic carbohydrate-binding modules (CBMs) and structural analysis by Cytochrome c
peroxidases in active and inactive forms.
Química Verde
RESEARCH REPORT
2004
Part B
2
ANALYTICAL AND BIOORGANIC CHEMISTRY
Head of Laboratory: Elvira Maria M. Gaspar, Assistant Professor
Staff Members:
Angela M.S. Relva
Assistant Professor
Marco Diogo R.G. da Silva
Assistant Professor
João Paulo Noronha
Assistant Professor
Ph.D. Students:
Laila H. Ribeiro
Nº articles in scientific journals: 2 (1,2)
Scope and description of on-going research
During 2004 the group was focused in some environmental problems studying the hopanoid composition of
sediments from lower Tagus basin and the profile of toxic compounds in railway sleepers. Hopanoids are a
sub-class of steroids, important molecular fossils used as marker compounds in the study of the fate of
spilled petroleum. To the study GC/TOF-SiM MS technique has been employed. The creosote volatiles have
been analyzed by 1D GC-TOF MS and also by GCxGC-TOF MS in a collaborative program. To decode the
process of host tree selection by herbivore insects (a collaborative program with environmental scientists),
namely the Mediterranean defoliator winter pine processionary moth, a detailed isoprenoid profile of pine
varieties cultivated in Portugal was achieved as an indicator for insect attack in the environment. New natural
oligossacharides evolved in allelopathic interference and also with pharmacological properties were isolated
from an European Convolvulaceae plant. The compounds will be structurally characterized by high resolution
methodologies as NMR and MS. Some chiral HPLC separation of active compounds or fragment groups of
complex molecules have been developed as new separation techniques.
Future Activities
During 2005 the group will be involved in environmental / human health problems. Human health will be
focused by food analysis, monitoring the volatile profile to access the quality of important food matrixes and
also by the analysis of lipidic compounds, potential tumoral bio-markers in colo-rectal cancer. Extraction of
added value compounds from orange peel wastes and the potential of fruit stones to be used as adsorbents
for the selective removal of organic compounds will be topics of interests. A new member of the team, will
introduce a new research area related with clean analytical methodologies for organometallic compounds
speciation in food and environmental samples.
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PHOTOCHEMISTRY AND SUPRAMOLECULAR CHEMISTRY
Head of Laboratory: Fernando Pina, Full Professor
Staff Members:
A. Jorge Parola
Assistant Professor
J. Carlos Lima
Assistant Professor
M. João Melo
Assistant Professor
Carlos Lodeiro
Assistant Researcher
Alexandra Bernardo
Associate Professor
Post-Doct Fellows:
Berta Covelo, Damián Fernández
Ph.D. Students:
Margarida Moncada, Sérgio Alves, Letícia Giestas
Number of articles in scientific journals: 25 (3-27)
Photochromic systems
In the framework of supramolecular chemistry, the bottom up approach can be associated to the building of
more or less complex molecular entities, starting from simple molecules. The objective of this strategy is to
obtain new properties and functions not present on the building blocks. However the search of new properties
and functions through increasing of complexity, can also be achieved by using a simple molecule capable of
chemical transformations upon the input of different external stimuli; for example light (photochromism), pH
variations (acidichromism), electric current (electrochromism), or heat (thermochromism) the so-called multistate
molecules. The cis-trans photochromic molecules are an example of a dual state molecule responding to
light, while a di-acid can be viewed as a three fold state molecule responding to a pH input. On this basis, the
stimuli can be considered as an input of energy, leading to a chemical response of the multistate molecule,
for example through a change of colour. The different responses of a multistate molecule can be employed to
accomplish functions, such as write upon a light input, or erase after a pH jump, and in that case a multistate/
multifunctional system can be envisaged.
The need for complex multistate/multifunctional chemical systems that can be actuated to process multiple
information, through colour changes, is in pace with the predictions of a third wave of computing, usually
referred as ubiquitous computing. The ubiquitous computing will pass through computers imbedded in walls,
chairs, clothing - in everything, and is fundamentally characterized by the connection of things in the world
with computation, that will take place at many scales, including the microscopic.
A step further on this strategy was achieved by incorporating photochromic compounds in ionic liquids and
polymeric matrices which are permeable to water. These to vectors will be developed in the future, in particular
the use of multistate systems applied to the ubiquitous computing concept. A pH student will start to work in
2005 in collaboration with a private the company YDreams.
Chemosensors
Our group has reached a relatively high theoretical knowledge concerning chemosensors, and we feel able to
go a step further, maintaining our scope of doing fundamental research, but thinking on practical applications.
On this line the chemosensors project will be developed in collaboration with the private the company YDreams.
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The ideas that we are carrying out are based on the concept of chemosensor in particular fluorescent
chemosensors. A chemosensor should posses a receptor unit, which can bind target molecules, a spacer
and a fluorophoric unit, which should quench or enhance the fluorescence emission. A step further is the use
of chemosensors supported in solid or gel matrices, or even paper, in order to allow applications.
Synthesis
Several synthetic projects are currently underway in the Group of Photochemistry and Supramolecular
Chemistry that fuel the other areas with appropriate compounds.
i) synthesis of new flavylium salts to be used as photochromic materials, photoresponsive switches and
fluorescent probes.
The open forms of flavylium salts - cis- and trans-chalcones - are well known photochromic molecules. They
can be incorporated in supramolecular systems upon appendage of molecular moities at both ends of the
chalcones and exploited as photoresponsive switches.
Flavylium salts lacking hydroxy groups as substituents are strongly fluorescent and can be explored as
fluorescent probes upon inclusion of electron donor substituents in position 4 that prevent opening of the
pyrylium ring.
ii) synthesis of cavitands and hemicarcerands with metal complexing units and corresponding metal complexes.
Potential materials for metal induced self-assembled hemicarcerands.
ii) synthesis of fluorescent and/or colorimetric chemosensors involving bis-cromophoric podands provided
with 8OH-quinoline and pyrene units or β-naphtol-pyrene units, bis-cathecol devices, polyoxa-aza macrocyclic
systems with imines or amines. These compounds were studied with Al(III), Ga(III), In(III), Zn(II), Ru(II), etc and
their adducts formed by interaction with halide ions. Some macrocyclic ligands were synthesised for their
interaction with lanthanide(III) cations and Ca(II) in collaboration with the group of Professor Rufina Bastida
(Universidade de Santiago de Compostela, Spain (4 papers published). With the group of Professors Lluis
Escriche and Jaume Casabó from the Universidade Autónoma de Barcelona UAB (Spain) we have been
involved in the study of macrocycles with nitrogen and sulphur donor atoms, more appropriated for heavy
pollutant metals such us Hg(II), Cd(II), Pb(II), Ag(I) as well with Ni(II), Zn(II) and Pd(II); several X-ray structures
were determined. (3 submitted manuscripts and 4 manuscripts in preparation). In collaboration with Professor
Teresa Avilés (DQ-Requimte) have been synthesised a new group of rigid imine biscromophoric systems for
their interaction with Co(II), Cu(I) and Zn(II) with potential applications as catalysts and fluorescent systems.
In the field of crystal engineering we have been working in the develop of new salt metal complexes with
phenanthroline, α-hydroxicarboxylates and imidazol units with Cu(II), Ni(II) and Zn(II), in collaboration with
Professors Rosa Carballo and Ezequiel Vazquez (Universidade de Vigo, Spain) (2 papers published).
Biomolecules and Macromolecules
We are interested in the use of time resolved and steady state fluorescence techniques, in the study of the
structural changes in proteins, using intrinsic fluorescent probes, such as tyrosine and tryptophan. This can
be achieved in a pure phenomological approach, since the fluorescence decay times are signatures of the
protein state, and the pre-exponential coefficients can be used to evaluate the molar fractions of the folded
and unfolded states, but in the case of proteins bearing a single tryptophan or a single tyrosine, information
about the local flexibility changes can be obtained from the kinetic data associated to electron transfer
quenching from nearby residues or backbone carbonyls. The same strategy is well established for the study
of the molecular dynamics of polymers using the kinetic data of excimer formation. The solution of the kinetic
scheme allows to retrieve information concerning electron transfer rate constants (strongly dependent on the
dielectric environment) and rotational freedom of the residues.
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Cultural Heritage
The safeguard of our cultural heritage will contribute to a more sustainable environment and a better access
to the art and history of the past.
A better conservation will demand a better knowledge of the materials used in the past for the production of
Works of Art. The complexity of these materials and evolution in time demand an interdisciplinary team for
their study. Therefore, in the projects described bellow, Art Historians, Conservators and Chemists use the
best of their knowledge to construct a better understanding of Medieval Illuminations and Modern Art; namely,
in which concerns the pictorial materials used, such as historic dyes, pigments and binding media, and their
life time.
Currently, we are involved in the following projects:
i) “The colour of medieval Portuguese illumination: an interdisciplinary approach”, (POCTI/EAT/33782/2000)
in collaboration with the Department of Conservation and Restoration and the Department of Art History of the
New University Lisbon.
This project will bring together expertise from Chemistry, Art History and Conservation, aiming to establish an
interdisciplinary team as well as an interdisciplinary approach to the study of Portuguese medieval illumination.
Research on the origin, genealogy and circulation of the medieval manuscripts, subjects more related to Art
History, will be examined in the light of the nature and source of the materials used in the medieval scriptorium.
The study of the pictorial materials and the technology of colour production in the medieval workshop will be
carried out using techniques and methodologies from the chemistry and material science domain. The final
aim of this project is to obtain a better understanding of medieval Portuguese miniature, that in turn will permit
a better Conservation and Preservation of these documents.
ii) “The Molecules of Colour in Art: a Photochemical Study”, (POCTI/QUI/55672/2004) in collaboration with the
Department of Chemistry of Coimbra University and the Department of Conservation and Restoration and the
Department of Art History of the New University Lisbon.
In this project, a full photophysical and photochemical characterization of historical dyes, such as dragon’s
blood, brazil wood, alizarin, crocetin, indigo and derivatives such as purple, will be carried out.
This study will contribute to a better understanding of outstanding historical organic molecules, that are still
of economic significance nowadays. A better understanding will permit a better conservation and access to
our cultural heritage, namely manuscript illuminations and ancient textiles.
Furthermore, UV-Vis fluorescence emission, one of the most sensitive spectroscopic techniques, will be
tested, in order to ascertain its viability as a powerful, non destructive method of analysis for organic colorants
in objects of historical and cultural interest.
iii) “Liaisons dangereuses: a study of acrylics and vinyl polymers used in Portuguese Modern Art”, in collaboration
with the Department of Conservation and Restoration of the New University Lisbon and the National Modern
Art Museum- Museu do Chiado
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ORGANIC SYNTHESIS AND CHEMISTRY OF NATURAL PRODUCTS
Head of Laboratory: S. Prabhakar, Full Professor / Ana M. Lobo, Full Professor
Staff Members:
Pedro Abreu
Assistant Professor
Paulina Mata
Assistant Professor
Manuela Pereira
Assistant Professor
Ana M. Lourenço
Assistant Professor
Paula S. Branco
Assistant Professor
Luisa M. Ferreira
Assistant Professor
João Aires de Sousa
Assistant Professor
Maria Manuel Marques
Post-Doct Fellows:
Yuri Binev, Sunil Gupta, Qingyou Zhang, Yong Hong Liu, Susan Matthew
Ph.D. Students:
Maria M. Santos, Mariana Duarte, Mário Gomes, Marta Corvo, Paulo Glória, Luís Pinto, Vasco Bonifácio,
Rita Noronha, Susana Gaudêncio, Goncalo Carrera, Diogo Latino
M.Sc. Students:
Ana Margarida Fonseca
Project Trainee:
Marta Vilela, Artur Abreu, Valdemar Figueira, Carla Macedo, Catarina Soares
Nº articles in scientific journals: 18 (28-45)
Number of Ph.D Thesis: 2
Four major lines of interest have evolved in the group, namely the discovery of new natural products, the
organic synthesis of biomolecules by novel routes, the mechanism of important reactions and the use of
computational methods for molecular properties prediction and crude oil spills geographical origin.
1- The area of natural products, which continues to attract the interest of research workers of this group,
has been focused on the search for bioactive natural products of plant origin. In collaboration with the Faculty
of Pharmacy of Lisbon, new cytotoxic diterpenes with reversal effect on multidrug resistance, have been
isolated from Euphorbia species of Portuguese origin. A scientific collaboration with the University of Monastir
(Tunisia) has been developed, leading to the isolation of new antioxidant phenolic glycosides from Tunisian
plants. Following the signature of a scientific protocol with the Facultad de Ciencias Naturales, Universidad
de Oriente (Santiago de Cuba), a new project, aiming at the isolation of anti-inflammatory and antioxidant
compounds from Cuban medicinal plants, is now being developed. In the area of Food Chemistry, a study on
the glycoalkaloid content in varieties of marketed Portuguese potatoes from conventional, integrated, and
organic crop systems, was concluded.
An on going interdisciplinary study of Brazilian medicinal plants (through a collaboration with the Metodista
University of Piracicaba – São Paulo), to assign the principles responsible for gastric ulcer protecting activity
of Solanum and Cissus species, led to the isolation of active peptides, terpenoids and xanthin compounds.
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2- The aim of the efforts in organic synthesis is to devise novel, simple, economic and non-polluting ways
of producing biomolecules or derivatives thereof of established pharmacological interest.
Methods were tested for the specific N-glycosidation of a staurosporinone precursor, but the obtention of
reaction in the lactam oxygen forced development of an alternative procedure.
The enantiospecific synthesis of optically active gamma-amino acids can be based on the stereoselective
addition of the radical generated by homolysis of thiohydroxamate esters, to electrodeficient olefins. Chiral
induction was studied in derivatives of natural occurring amino acids holding chiral units bounding to the alfacarbon atom. The chiral induction was analysed on the corresponding gamma-aminoester. We studied
aspartic and threonine derivatives. The best stereoinduction was oobtained with the (2S,3R)-(1’-terc-butyl-1’dimethylsilyloxi)-2-terc-butoxicarbonylamino)-butanoic with 30% de on the chiral C(4) carbon atom on the
resulting gamma-aminoacid derivative, as resulting from 1,2 chiral induction. To studied the asymmetric
induction by Barton ester holding chiral substituents on the N moiety, we sintesised chiral pyrrolidine derivatives
of alfa-amino acids. These compounds will be tested and the asymmetric induction studied towards the
syntheses of chiral gamma-aminoacids.
The compounds sharing a 2,2-dimethylbenzopyran structural motif were tested for their ability to inhibit the
hypoxic activation of an alkaline phosphatase reporter gene under the control of hypoxia responsive elements
in human glioma cells. This effort led to the discovery of the 1-[1-(5-methoxy-2,2-dimethyl-2H-chromen-6-yl)ethyl]-1H-benzoimidazole (103D5R), a novel small-molecule inhibitor of Hypoxia-Inducible Factor 1 (HIF-1).
Methodologies for the semisynthesis of homopumiliotoxin alkaloids from quinolizidine analogues were
developed using piperidine model substacts. A new enantioselective method for the hydroxylation of terminal
alkenes was achieved by oximercuration-demercuration phase transfer catalysis using cyclodextrins. With
this methodology it was possible to obtain conversion of 65% and ee of 35%.
Preliminary works for the REQUIMTE project “Synthesis and Neurotoxic Evaluation of
Methylenedioxymethamphetamine (MDMA; Ecstasy) and its Metabolites in Rat Cortical Neurons” in
collaboration with the Faculty of Pharmacy (REQUIMTE-PORTO) allow the synthesis of putative metabolites
of MDMA: N-methyl-alpha-methyldopamine, alpha-methyldopamine and its conjugates with sulphur
compounds biologically relevant (glutathione, cysteine, and N-acetylcysteine). The synthesis was performed
involving enzymes (tyrosinase) or environmentally friendly oxidants, such as ferric chloride, which may
emulate biotransformations. The compounds were subjected to biological assays at the Faculty of Pharmacy
(Porto).
3- As a paradigm of atom economy, rearrangements continued to be studied, and the influence of substituents
assessed. Thus, in the mechanistic area, heteroatom containing sigmatropic rearrangements were studied,
and two Ph.D. theses were completed. One dealt with the search for aza-Cope sigmatropic rearrangements
involving the N—O—silyloxy group and the other suitable systems derived from N-hydroxyindoles.
Polycyclic nitro-aromatic hydrocarbons (Nitro-PAHS), which are environmental pollutants, result from
incomplete combustion processes and are reported to be carcinogens. Our study was focused on the
reaction of model compounds like 3,4-dimethyl-(2-hydroxyethyl)thiazolium triflate and the immediate biological
metabolites of nitroaromatics, the aromatic nitroso compounds. The main products of the reaction were Oacyl N-aryl hydroxylamines and the corresponding thiazolium salt. Azoxyaromatics and hydroxamic acids
were also isolated in variable amounts. In order to elucidate the reaction mechanism, EPR studies were
undertaken and phenylnitroxide and species derived from the thiazolium salt were detected. Some experiments
were carried in the presence of TEMPO. By careful reaction control TEMPOH, that had formed in the
reaction by reduction, could be trapped with Ac2O and its O-acetyl derivative isolated (43% yield).
The term photoreproduction, when applied to architectural drawings, indicates a copy from the original
drawing that was produced using a process similar to the photographic process. Usually, collections of
architectural drawings from the last century contain original drawings and photoreproductions. In the
architectural archives these prints are the conservation staff main preservation concern, especially the diazotype
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prints. As result of collaboration with Direcção Geral dos Edifícios e Monumentos Nacionais a central
administration body of the Ministry of Public Works, Transports and Housing, for the study of photoreproduction
conservation it was concluded that the reddish pink discoloration in a diazotype print is caused by the
alkaline conditions due to ammonia presence and the residual coupling agent, which may give rise to
polymerization reactions. The products from the polymerization reaction are coloured chemical species,
with high molecular weight. There is no evidence that these polymers or azo dyes migrate in the media used.
Our study demonstrates that discoloration results from any coupling component, used in diazo process,
with capacity to migrate through the document. When exposed to oxidative conditions it will induce discoloration
by polymerization reactions. At the same time these results show that the alkaline reserve in the interleaving
paper is totally inadequate, since it will induce polymerization reactions of the remaining coupling agents.
4- Computational methods were developed in response to practical chemical problems related to quantitative
structure-property relationships and pollution related issues and involve: application of chirality codes to the
automatic assignment of absolute configuration from 1H-NMR data using neural networks; application of the
previously developed SPINUS program to the prediction of 1H NMR chemical shifts of sesquiterpene lactones;
estimation of the melting point of potential ionic liquids using regression trees; development of empirical
descriptors of chemical reactivity and its application to QSAR, reaction classification and reaction prediction;
automatic classification of the geographical origin of crude oils from GC-MS parameters, in collaboration
with the Minister of Defense (Navy).
The group was also involved in the specification of stereogenic units in organic chemistry nomenclature,
which resulted in the preparation of a paper already submitted, and a critical evaluation of the new IUPAC
proposals at the request of such organisation.
5- Pedagogic tools
Given the academic activities of the majority of the elements of this group, teaching materials were produced.
The work in science teaching and divulgation continued. It hopes to contribute to improve science-teaching
practices, to motivate students to continue their studies in science and to develop the general scientific
culture. Science teaching activities involve working with other educational levels (production of teaching aids
and teacher training) and collaboration in research activities with other institutions, particularly Instituto
Superior de Psicologia Aplicada. Concerning science divulgation main activities refer to a) our collaboration
with Ciência Viva / Pavilhão do Conhecimento by organizing events (debates and children activities) and
participating in demonstrations (Energy Day, Pavilhão Anniversary and Science Week); b) our participation
in activities in Açores by invitation of Direcção Regional da Ciência e Tecnologia dos Açores; c) the publication
of science divulgation articles in a Portuguese national newspaper.
Our interests in teaching and research of Molecular Gastronomy were pursued by establishing international
connections with researchers in the area through the participation in the 6th International Workshop on
Molecular Gastronomy and in the “Course de Gastronomie Moléculaire 2004/2005”.
Plans for 2005
The syntheses of cancer relevant antimitotic compounds will pursue, with the development of methods to
achieve the glycosidation of the aglycones in good yields, and the syntheses of novel aglycones systems
recently discovered.
Plans are in place to improve the potency of the 103D5R through structure-activity relationship studies. This
will include building and screening a follow-up library in which the different regions of 103D5R will be refined:
(1) the substitution pattern in the left-hand benzopyran ring system will be varied; (2) the right-hand heterocyclic
aromatic system will be substituted by other heterocyclic rings; and (3) the alkyl group in the linker region
will be replaced with other aliphatic and aromatic substituents. As a result of such systematic structure
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activity relationship studies on 103D5R structure, we expect to design a new molecule, which will have a
more potent inhibitory activity against the HIF-1 pathway.
Systems will be chosen to test the anionic acceleration of hetero-Cope rearrangements in view of their wide
synthetic application.
In the computational front the following projects will be developed: application of descriptors of chemical
reactivity based on self-organizing maps to the estimation of mutagenicity from the molecular structure;
1
automatic classification of organic reactions from H NMR spectra of reactants and products; further
development of the SPINUS program for the prediction of 1H NMR spectra and its application to structure
validation.
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SELECTIVE SYNTHESIS AND STRUCTURAL CHEMISTRY
Head of Laboratory: Maria Teresa Barros, Associate Professor
Staff members:
Maria Teresa Avilés Perea
Assistant Professor
António Gil de Oliveira Santos
Assistant Professor
Eurico José da Silva Cabrita
Assistant Professor
Ana Maria Madeira M. Faísca Phillips Principal Researcher
Post-Doct Fellows:
Krasimira Petrova, Snezhana Bakalova
Ph.D. Students:
Marta Morais Saraiva de Andrade, Vitor João Salgueiro Rosa, Paula Correia da Silva, Maria João Morgado,
Filipe José dos Santos Duarte
M.Sc. Students:
Paula Alexandra Carvalho Rodrigues
The principles of Green Chemistry as applied to organic synthesis (economy and efficiency) needs advanced
principles of organic synthesis applied to the efficient production of complex organic compounds from renewable
natural (chiral) starting materials. We have continued to investigate the reactivity of saccharose and to
apply our results to the study and application of sugar as a chiral auxiliary for asymmetric synthesis. Our final
goal is to apply this chemistry to diverse problems under unusual but ecologically favourable reaction
conditions.
Saccharose is an abundant highly functionalised chiral molecule. Several strategies have been adopted for
the connection of the prochiral p system and other primary or secondary hydroxyl groups selected for linkage
to the unsaturated moiety. Studies on varying the reactive unsaturated moiety at the selected hydroxyl group
were carried out for a limited range of dienes and dienophiles.
The chemistry of azides includes a useful synthetic applications in the preparation of 1,2,3-triazolines via 1,3
dipolar cycloaddition reactions. We have reported the preparation of several azides of low molecular weight,
which have been useful for this work.
We have developed an efficient strategy to attach vinylic groups and dienes to the sugar nucleus. The
selectively deprotected hydroxyl groups were converted to the formate and we have found in our group a very
clean process for doing this. Wittig reactions at formates are known. In this field we have some preliminary
results under microwave activation. Water soluble dienes will also be prepared by exposure of the hydroxyl
groups of the sugar auxiliary.
We have been successful in the selective connection of acrylate to the sugar molecules (in some positions)
and the copolymerisation with simple acrylates to form more or less dense sugar polymers. By attaching
the sugar to a polymer it is possible to alter some of its properties.
Our group is mainly interested to use alternative energy source (microwaves) and alternative reaction conditions
(use of ecologically friendly solvents such as water or no solvent).
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Asymmetric synthesis and homogeneous catalysis Several novel C1- and C2-symmetric chiral compounds
containing N,N or N,S or S,S groups were synthesized from (R,R)-(+)-tartaric acid. Many chiral compounds
containing these functional groups have found applications in stereoselective synthesis and catalysis, i.e. in
hydrosilylation reactions, H-transfer reactions, hydrogenations, dihydroxylations, aldol reactions, Michael
additions, etc. At present, research is being conducted towards developing applications for these compounds
in stereoselective synthesis and metal catalyzed processes, and also for some novel related diamines which
are being synthesized.
The work undertaken during 2003 on the enantioselective alkylation of benzaldehyde by diethylzinc, catalyzed
by mono-oxazoline carbinols, was concluded and published.
The synthesis of chiral bis-oxazolines derived from tartaric acid and their application as ligands in the
enantioselective Michael addition of diethylzinc to enones was continued.
Stereoselective behavior of chiral a-haloacyl compounds in nucleophilic substitution reactions,
under dynamic kinetic resolution (DKR) conditions. We studied, theoretically, the stereoselective behavior
of nucleophilic substitution reactions of a-haloacyl compounds, using a chiral auxiliary approach, based on
ephedrine derived imidazolidinones, under DKR conditions. New chiral auxiliaries were proposed and some
derivatives were synthesised and tested, confirming the theoretical previsions of expected high final
diastereoisomeric excesses (results published in 2005).
Catalysts in asymmetric synthesis: A complexation study. The use of Lewis acids (LA) to enhance the
reactivity and stereoselectivity of a number of reactions involving N-acyloxazolidinones and Nacylimidazolidinones is well known. For some transformations the stereoselectivity induced by the LA
complexation can’t be entirely explained by the accepted models. We have undertaken a NMR and molecular
modeling study of this type of complexation with different LA. Since the molecules in question have two key
Lewis base sites, several solution complexes are possible, including chelated ones, which render the study
very complex. In an attempt to simplify it, we have prepared several model compounds and studied their
complexation with non-chelating (BF3.OEt2) and chelating (AlEtCl2, TiCl4 and Mg(ClO4)2) LAs. Only for Mg(ClO4)2
evidence for chelated forms in solution was found. This information can be of major importance in the revision
of the accepted mechanisms for all reactions where N-acyloxazolidinones and N-acylimidazolidinones are
used, prompting us to the proposal of new mechanistic pathways.
Asymmetric additions to alkenes. Our interest in this subject has been mainly related with Diels-Alder
reactions and the addition of electrophilic species (e.g amines) to unsaturated N-acyloxazolididinones and Nacylimidazolidinones. The accepted mechanism, proposed by Evans, more than 20 years ago is unable to
explain many unexpected experimental observations made during these years. Based on our previous work
on DKR reactions, we started the development of a full theoretical and experimental study (based on NMR
techniques), with the aim of clarifying possible mechanisms and to propose structural variations, able of
increasing the final diastereoisomeric excesses. Our studies brought us to a new proposal, which can explain
the observed stereochemistry, both in Diels-Alder and in electrophilic additions. We also studied similar
additions to alkenes connected to different chiral auxiliaries (as, for instance, Ethyl-S-lactyl acrylate) with
more flexible structures, showing that even in these cases no chelated transition states are theoretically
expected.
Intramolecular asymmetric aldol reactions. Based on our previous experimental results, we are currently
studying the mechanistic aspects of asymmetric aldol reactions, using theoretical and experimental tools.
Our aim is the understanding of the large amount of literature information on chiral and achiral systems. Until
the moment, in spite of many reports describing the use of chiral catalysts and auxiliaries, there is no real
understanding of the factors governing the mechanistic pathways of even the achiral processes. Our first
studies indicate a strong dependence on electronic effects which, if confirmed, can reduce substantially the
variety of structures able to work as starting materials.
Application of NMR diffusion and NOE techniques to the study of intermolecular interactions. The
complementary between the information obtained from NOE with that from diffusion NMR is being explored in
3 different projects: In the study of 1-buthyl-3-methylimidazolium (BMIM) ionic liquids, where the
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determination of internuclear distances and relative strength of ion pair association were interpreted in terms
of rotational motion, molecular structure and molecular properties; in the study of the molecular determinants
of ligand specifity in family 11 Carbohydrate Binding Module (CBM11), where NMR Saturation transfer
experiments are being performed in order to study the enzyme complexed with its carbohydrate ligands; and
in High-Pressure NMR spectroscopy of polymers and biopolymers in scCO2. where the interaction of
fluorinated surfactants and supercritical CO2 and the interactions of small peptides dissolved in stabilized
emulsions in supercritical CO2 are being studied.
In Organometallic compounds, we have synthesized a series of cobalt(II) complexes of the general formula
CoX2(á-diimine) where X=Cl, or I and the á-diimines are represented below (1 and 2), in order to study their
structural and magnetic properties, as well as their redox behavior. The X-ray structure of the Co(II) compounds
show a tetrahedral geometry, two of these structures are shown below. All the synthesized compounds are
paramagnetic and their magnetic properties are under investigation.
2005 activities to be developed
In a related project we pretend to study the use of concentrated saccharose solutions in accelerating
stereoselective reactions. It is hoped that the structurally ordered sugar solution will both direct the reagents
and accelerate the reaction in such a way that the product is obtained with high stereoselectivity and affords
optically active products. Pericyclic reactions which can be carried out under aqueous conditions should be
influenced by a chiral environment and organization of the reagents should occur.
In another part of this project we hope to study the glycosylation of steroids. A number of important groups
of drugs are glycosides. The sugars seem to play a key role in the interaction of the drugs with their receptors
All the synthetic methodologies will be designed in order to develop cleaner technologies as a major
emphasis in green chemistry. Among the several aspects, when is possible, the synthetic methodologies will
be performed in solvent free conditions, or water-based medium, and/or under microwave radiation as
alternative energy source.
In another project, we intend to produce environment-friendly, ready-biodegradable complexones at a
low cost, which may compete at industrial scale production with the well established and cheap synthetic
NTA and EDTA compounds. For this purpose, the synthesis pathway will be based mainly in the life cycle;
block units of stereo specific amino acids will be used for asymmetric synthesis of natural
aminopolycarboxilates ligands (or derivates) potentially better biodegradability (i.e. they will be
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decomposed into non-toxic compounds, environmentally benign substances). In addition, the synthesis will
be designed in order to be performed in a water-based medium, for reducing the use of organic solvents.
Collaboration in a European project related to the synthesis and physical properties of azides will continue.
In the field of DKR, since we reached the point where a good knowledge of the ionic mechanisms was
obtained, we expect now to invest our efforts in the understanding of similar mechanistic pathways in radical
systems.
The Lewis acids - carbonyl compounds complexation studies are in a final stage, and shall be finished during
the next few months. The results will be directly used on the clarification of the mechanisms involved in
asymmetric Diels-Alder reactions and other additions to double bonds.
The project dealing with intramolecular aldol reactions shall have a strong evolution during this year. We
expect to clarify several chemical aspects, based on molecular modelling, NMR and synthetic work. As soon
as the achiral systems are understood, efforts will be made in order to understand part of the literature
information and, in special, the experimental results obtained in our research group during the last couple of
years.
In the area of NMR we will continue to apply NOE techniques and diffusion methods to the study of intermolecular
interactions. In the area of ionic-liquids the studies will be mainly focused on solute-solvent interactions. The
study of the molecular determinants of ligand specifity in CBM11 will continue in strong collaboration with
groups integrated in other research themes: Protein Crystallography and Biomimetic Systems and Simulation.
HP-NMR studies in scCO2 will be mainly directed towards the determination of site-specific information about
the interaction of scCO2 with different surfactants.
In the next year we intend to prepare compounds containing both the a-diimine ligands and the 5ciclopentadienyl ligand. For that purpose we will try two different ways: 1- Reaction of CpCo(L)I2 (L=CO,
triarylphosphine), with a-diimines to see if compounds of the type [CpCoL(?-diimine)]+, could be obtained 2Reaction of the synthesized compounds CoX2 (?a-diimine) with Tl Cp or NaCp.
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PHYSICAL ORGANIC CHEMISTRY / RADICAL CHEMISTRY
Head of Laboratory: Abel J. Vieira, Associate Professor
Ph.D. Students:
Pedro Manuel C. C. P. dos Santos, João Adalberto A. Lourenço
·Study of radical induced oxidation of xanthine derivatives (Pedro Santos, Chemistry) Photolytic generation of
hydroxyl and sulphate radicals. In situ reaction of radicals with methylated xanthines.Identification of products
by HPLC. Characterization of transient radicals by ESR spectroscopy. Evaluation of relative redox properties.
Study of the antioxidant activity of non-steroidal anti-inflammatory drugs (NSAIDs). Radical oxidation (OH,
NO) of antipyrine and 4-aminoderivatives. Identification of products by HPLC. Characterization of transient
radicals by ESR spectroscopy. Synthesis of thiohydroxamic acid esters as precursors of alkyl radicals.
Reaction calorimetry in the scale-up of a chemical process (João Lourenço, Chemical Engineering) Multi step
synthesis of pirlindole chlorhydrate. Optimization of each synthetical step. Characterization of intermediates.
Calorimetric study of the reactions. Development of analytical methods and quality control assays.
Research activity for 2005
·Continuation of the study of radical degradation of caffeic and cynnamic acids. Evaluation of the potential
antioxidant activity of non-psychotropic cannabinoids. Continuation of the study of the antioxidant activity of
NSAIDs. Study of oxygen effect on radical oxidation of xanthines. Evaluation of superoxide elimination from
hydroxyl adducts. Study of the reaction of xanthines with alkyl radicals. Identification of products by HPLC
and Mass Spectrometry. Characterization of transient radicals by ESR spectroscopy. Natural products research:
Bivalves of the Portuguese Coast - Organic Constituent and its Biological Potential. Analysis/identification of
Flavonoids derivatives from Genista tenera by HPLC-DAD-MS. Screening the HPLC retention chiral compounds
for computational prediction of enanteoselectivity from the molecular structure. Conclusion of the PhD work
on reaction calorimetry in a scale-up process.
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B6
Head of Laboratory: Joao Carlos Sotomayor, Assistant Professor
CHEMICAL ENGINEERING SCIENCE / PHOTOCHEMISTRY AND SUPRAMOLECULAR CHEMISTRY
A irradiation set up was mounted using a Photomax light source from Oriel equipped with a 100 W mercury
arc lamp. The set up was vertically mounted in order to allow the irradiation of films of liquid phase samples.
A stainless steel filter filled with water was used as a heat remover and a 366 nm interference filter from Linos
was incorporated to obtain a monochromatic light. The focus of the monochromatic light can be altered in
order to tune the out put intensity from 3 to 7 mW cm-2. The light intensity was measured by iron actinometry.
In the photopolymerisation of TrEGDMA, 0.1% or 1% of 2,2’-dimethoxy-2-phenylacetophenone (DMPA) was
used as an ultraviolet sensitive initiator. The photopolymerisation of TrEGDMA was tested on several solid
supports and it was followed by using different spectrometric techniques: glass and HDPE using UV-Vis
spectrophotometry, NaCl and KBr disks using FTIR, and between two ITO coated glass plates using DRS.
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CHEMICAL ENGINEERING SCIENCE
Head of Laboratory: Manuel Nunes da Ponte, Full Professor
Staff Members:
Luís Sousa Lobo
Full Professor
Pedro Brito Correia
Invited Full Professor
João Crespo
Associate Professor
Susana Barreiros
Associate Professor
Joaquim Vital
Assistant Professor
Maria Ascensão Reis
Assistant Professor
Isabel Ligeiro da Fonseca
Assistant Professor
Ana Maria Ramos
Assistant Professor
Pedro Simões
Assistant Professor
Henrique Guedes
Assistant Professor
Isabel Coelhoso
Assistant Professor
Ana Aguiar Ricardo
Assistant Professor
Madalena Dionísio
Assistant Professor
José Paulo Mota
Assistant Professor
Maria Margarida Cardoso
Assistant Professor
Rui Oliveira
Assistant Professor
Svetlozar Velizarov
Post-Doct Fellows:
Svetlana Lyubchik, Paulo Lemos, Pavel Izak, Thomas Schäfer, Teresa Casimiro, Luisa Serafim, Gilda Carvalho,
Vesna Najdanovic-VisaK, Ewa Bogel Lukasik.
Ph.D. Students:
Mário Eusébio, António Rodrigo, Carla Portugal, Cristina Matos, Filomena Freitas, Isabel Esteves, José Luís
Santos, Luísa Serafim, Raquel Fortunato, Víctor Alves, Sílvia Garcia, Célia Peres, Pedro Vidinha, Joao Dias,
Ana Teixeira, Carla Brazinha, João Araújo, Maria Teresa Plaza, Liliana Guerreiro, Sofia Barata, Patrícia
Oliveira, José Castanheiro.
Project Grantee:
Márcio Tentem, Carlota Macedo, Ana Bráz, Ângela Machado
Number of articles in scientific journals: 36 (61, 63-97)
Number of articles in books: 9 (1-9)
Number of patents: 3
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Clean Solvents and Clean Processes
Clean Solvents –Ionic Liquids
Measurement of thermodynamic properties of Ionic Liquids, such as the density and the speed of sound and
determination of several derived thermodynamic and thermophysical properties in broad pressure and
temperature ranges. Also, excess volumes of mixtures of ionic liquids have been measured.
Clean Solvents –Ionic Liquids and Supercritical Carbon Dioxide
Studies on enzyme activity and selectivity in supercritical carbon dioxide and in ionic liquids.
Membrane Processing and Monitoring
1- Extraction of amino acids and derivatives using ionic liquids. The transport mechanism of water and small
ions, through supported liquid membranes (SLM), using ionic liquids was studied. To elucidate whether this
also regulates the transport of species with higher molecular weight, additional experiments using amino
acids and amino acids esters, were carried out. It was observed that the ionic liquid [C8MIMPF6] exhibits a
high selectivity towards amino acid esters, while almost completely excluding amino acids.
2- Enantioselective separation of propranolol by fixed carrier membranes. Polysulfone (PS) based membranes
have been prepared using N-hexadecyl-L-hydroxyproline (HHP) and L-di-n-dodecyltartrate (L-DDT) as chiral
carriers, respectively. Kinetic experiments have been carried out and the influence of both the solute/carrier
ratio and of the flow rates of the feed and stripping phases on the extraction and on the selectivity of the
process was evaluated for both chemical systems. Modelling of kinetic experiments has been performed and
mass transfer coefficients obtained for both systems were compared.
3 - Recovery of aroma compounds from diluted aqueous streams by pervaporation. The research was focused
on the understanding of the molecular interactions between target solutes and the membrane material. Realtime monitoring of the permeating stream, by on-line mass spectrometry allowed the description of the
process of solute permeation in steady-state and in transient operating conditions.
4 - Development of integrated pre-enrichment techniques for sample discrimination by electronic sensorial
systems (electronic nose). Pervaporation was integrated with electronic sensorial systems, in order to enhance
discrimination of wine samples, in an automated mode. This approach allowed the improvement of sample
discrimination using electronic nose, even when they present a high ethanol content.
5 - Study of membrane processes for clean and selective recovery of biological products from dilute streams.
This study involved different membrane processes, namely liquid membrane extraction using selective carriers
(chiral selectors), pervaporation for integrated reaction and recovery of solutes from dilute aqueous streams
and from ionic liquids, ultrafiltration for fractionation of proteins with pharmaceutical interest, and nanofiltration
for recovery and fractionation of antioxidant compounds from agroindustrial waste streams.
6 - Development of non-invasive, on-line monitoring techniques. On-line mass spectrometry, 2D-fluorescence,
Confocal Laser Scanning Microscopy and Raman Confocal Microscopy. Confocal Laser Scanning Microscopy
in combination with molecular probes was employed for investigating pH-gradients above dense membranes
used in the dialytic mode. Concentration gradients of solutes, in particular ionic liquids, within the membrane
polymer was investigated by means of Raman Confocal Microscopy due to the lack of suitable molecular
probes for this purpose (collaboration with the Group of Raman Spectroscopy, ITQB, Universidade Nova de
Lisboa).
Catalysis
Polymeric Catalytic Membranes & Heterogeneous Catalysts
The studies on the epoxidation of limonene over catalysts such as vanadium oxide supported on titania, and
vanadyl, cobalt or manganese acetylacetonates encaged in Y type faujasite, were continued. Hydrogen
peroxide and t-butylhydroperoxide were used oxygen suppliers.
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When the oxidation of limonene is carried out with concentrated t-butylhydroperoxide, over the vanadium
oxide catalysts, the main reaction product is also polymer. However, when the oxidant is diluted hydrogen
peroxide, limonene glycol is obtained, probably as a consequence of the acid catalysed opening of the
epoxide ring.
The zeolite Y encaged acetylacetonates were prepared in two steps: 1) adsorption of the Me(acac)2 in the
supercages of the zeolite Y; 2) Schiff condensation reaction between an aliphatic triamine and the metal
complex. Reaction studies were carried out with diluted tbutylhydroperoxide and diluted hydrogen peroxide
but the catalysts did not show any significant catalytic activity with this last reactant. The central metal
seems to play a key role, since for the vanadyl complex the oxidation reaction yields polymer, while for the
manganese and cobalt complexes beyond the polymer, which is still the main product, limonene glycol and
limonene oxide, are also obtained in significant amounts.
When the catalyst was a composite catalytic membrane consisting of Y zeolite encaged cobalt acetylacetonate
embedded in a PDMS matrix, the main reaction product was limonene oxide, in contrast with the results
reported above, where the main product was polymer.
A novel solid acid catalyst consisting of activated carbon bearing sulfonic acid functions was developed and
tested on the transesterification reaction of ethyl benzoate with methanol. Its performance was compared to
that of sulphonated MCM-41 and a polymeric catalytic membrane consisting of PVA cross-linked with
sulfonicsuccinic acid.
Environmental catalysis and adsorption
Studies on the removal of dioxin model compounds from water were continued using activated carbon as
adsorbent. Several model compounds have been selected, namely 2’,7’-dichlorofluorescein, chlorophenols
and phenols. The adsorption isotherms and the kinetics studies were carried out at pH=9. The surface of the
activated carbon was chemically modified by treatment with HNO3, Hydrogen and NH3, in order to obtain acid
or basic carbons. The data obtained showed that the basic carbons are better adsorbents.
Studies on adsorption of platinum group metals on activated carbon were performed in order to take in account
their properties, i.e., texture and surface functionality. The activated carbons were obtained from of apricot
stones, from mixture of the co-mingled natural organic liquid and solid wastes and anthracites. The adsorption
was investigated in static and dynamic modes at different sorption time, metal ions concentrations and
acidity of solutions. The anthracites chemically modified exhibited the higher adsorption capacity and selectivity
towards Pt and Pd.
Pd and Pt supported on anthracites and synthetic carbons (SCN) were also used as catalyst for liquid phase
hydrogenation of chlorobenzenes and cyclohexene. The data obtained showed that Pd supported on synthetic
carbon is very active for hydrodechlorination of chlorobenzene at room temperature.
Process Simulation and Modelling
Process Simulation And Modelling / Clean Solvents
A dynamic model of a heat exchanger for heating supercritical carbon dioxide under turbulent conditions was
accomplished. The model takes into account the resistances to heat transfer in the gas as well as in the
heating fluid and across the stainless steel wall of the inner tube. Experimental data on convective heat
transfer to supercritical carbon dioxide was measured in the pressure range 10–21 MPa, temperatures ranging
from 313 K to 343 K, and carbon dioxide mass flow rates from 3 to 12 kg/hr. The corresponding Reynolds and
Prandtl numbers ranged from 5´103 to 3104 and from 1.5 to 3, respectively. This model was later incorporated
in a previously validated model of a supercritical extraction packed column.
Modelling of biological processes
1- Mathematical modeling of a mixed culture cultivation process for the production of Polyhydroxybutyrate. A
two compartments cell model was developed. Experiments were performed with and without ammonia limitation
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enabling the analysis of PHB formation independently of the cell growth process. The experimental yields
were found to deviate considerably from the theoretical values proposed in the literature. The final model
exhibited high accuracy in describing the process state of most experiments performed, thus opening good
perspectives for future model-based optimisation studies.
2- Modelling and optimisation of a recombinant BHK-21 cultivation process. In this work a model-based
optimisation study of fed-batch BHK-21 cultures expressing the human fusion glycoprotein IgG1-IL2 was
performed. It was concluded that due to the complexity of the BHK metabolism it is rather difficult to develop
a kinetic model with sufficient accuracy for optimisation studies. An alternative more cost-effective methodology
based on hybrid grey-box models was adopted.
3- Modelling and Control of Rotavirus Virus-Like Particle Production. Rotavirus-like particles (VLP) seem to
be an excellent vaccine candidate to prevent rotavirus infection. For real scale production, experimental data
shows that less than 20% of the proteins produced find their way into a correctly assembled particle. In this
work, a mathematical model was developed attempting to describe the intracellular dynamics for DNA,
corresponding to the three structural genes of rotavirus, the respective mRNA concentration, single protein
concentration (VP2, VP6 and VP7), and assembled VLPs concentration.
Ab initio Process Modelling and Design
1 – Characterization of carbon nanotubes by molecular simulation. A novel procedure, combining molecular
simulation, Raman spectroscopy, and standard nitrogen adsorption data, has developed for structural
characterization of single-walled carbon nanotube (SWNT) samples. The fraction of open-ended nanotubes in
a sample can be estimated by scaling the simulated internal adsorption inside nanotubes to obtain a near
perfect fit between simulated and experimental isotherms.
2 - Viscous fluid mixing by chaotic advection. Viscous fluid mixing by chaotic advection in both time-periodic
and spatially-periodic 3-D prototype flows has been studied experimentally and theoretically. We have
demonstrated that chaotic advection can be regarded as a frequency-selective amplifier where external
perturbations are amplified for a certain frequency range. For values above or below this range, perturbations
are damped and the system is stable.
Polymeric Materials
Formulation of edible films for foods
Model edible films were prepared using 80%(w/w) calcium alginate (Sigma-Aldrich) and carrageenan (SKWBiosystems, Ltd) by casting. The water vapour permeance of the films was determined gravimetrically, based
on the ASTM E-96-95 method in triplicate, and the values obtained were 3.3±0.3 mol m-2 s-1 Pa-1. In order to
increase the hydrophobicity, films were also prepared including 0.2% - 0.4% (w/w) of two different fatty acids,
caprylic and palmitic acids (Fluka, Germany) but the values of the water vapour permeance measured were
very similar to the previous ones.
Drug controlled-release systems
Studies on polymeric formulations for drug controlled release have been performed. Microspheres containing
non steroid anti-inflammatory drugs (NSAID) have been prepared by conventional methods, namely solvent
evaporation method, as well as by technology using supercritical fluid in different conditions and drug release
studies were performed
Production of Biopolymers
Production of polyhydroxyalkanoates (phas) from different volatile fatty acids was experimentally evaluated.
Results showed that it is possible to control the polymer composition, and consequently their mechanical
properties, by varying the fatty acids proportion in the culture medium. The ongoing research in this field is
focused on the development of a reactor operating strategy for the obtaining of high polymer volumetric
productivities.
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The use of cane sugar molasses for pha production was initiated. The system consists of a first phase
fermentation followed by a polymer accumulation phase. In the first phase it was observed production of
hydrogen and this will explored as a second valuable product from the process of molasses fermentation.
Depolymerisation studies and characterisation of biopolymers
Under a cooperation project with Petrobrás, Brasil, it was studied the catalytic depolymerisation of
polymethylmethacrylate (PMMA), in order to obtain the pure monomer for further polymerisation. Catalytical
experiments with pure polymer and industrial waste were performed over six industrial FCC catalysts, with
very different acidity and accessibility properties, at previous optimized conditions, and the results were
compared. All the catalysts were not deactivated by the additives of the used PMMA, and the values of
activity and conversion obtained were similar to the correspondent obtained with the pure polymer.
Several highly dealuminated Y catalysts (USY) exchanged with La, Ce and Cs were prepared and tested in
the depolymerisation with pure PMMA, with very good results. Mesoporous carbon xerogels prepared with
the incorporation of La, Ce, Cs and Cu during the polymerisation of the precursor resorcinol/formaldehyde
polymer (further pyrolysed to obtain the mesoporous carbon support) were tested in the same conditions, as
well. Most of the results obtained with USY and mesoporous carbon catalysts were very promising and
similar to those obtained with the industrial FCC catalysts.
Under a project in cooperation with other group of the same CQFB scientific area, it was performed the
characterisation of polyhydroxyalkanoates (PHAs) obtained by biosynthesis, using mixed cultures, and different
process strategies. The thermal characterisation, involving the determination of glass transition and melting
temperatures, melting enthalpy and cristallinity degree, was achieved by differential scanning calorimetry
(DSC). The determination of average molecular weights and polydispersity was also carried out, by size
exclusion chromatography (SEC).
Also in the ambit of co-operation projects, the characterisation by SEC of natural polysaccharides, carrageenans
obtained from seaweeds of the Portuguese coast was performed. The characterisation of average molecular
weights and polydispersity of biopolymers used in the preparation of microspheres for controlled drug release
was carried out, as well.
Acrylates polymerization: kinetic and molecular dynamics studies
Temperature modulated differential scanning calorimetry, TMDSC,(Universidad Politecnica de Valencia) was
used to study the kinetics of the free radical isothermal polymerization of n-ethylene glycol dimethacrylates
using AIBN as initiator. The different stages of the polymerization process were identified with special attention
to the autoacceleration and vitrification steps. The influence of the temperature of polymerization in the final
glass transition temperature of the formed polymer was evaluated.
Parallel studies using dielectric relaxation spectroscopy were performed in our laboratories to monitor the
molecular changes that occur during polymerization of the tri-ethylene glycol dimethacrylate monomer. Both
glass transition and secondary relaxation processes were characterized prior to, during and after polymerization.
Polymer characterization / Thermodynamics
The phase behaviour of L-lactide + carbon dioxide system was measured at temperatures between 40 and
60ºC and pressures up to 26 MPa for compositions up to 10w/w% of L-lactide with respect to carbon dioxide
in order to obtain the p,x,T conditions where the system is homogeneous. The measurements were carried
out using a high pressure variable volume cell. Typically L-lactide was synthesized at 40ºC and 24 MPa (ca 7
w/w% of monomer with respect to CO2), conditions at which the system exhibits one single phase.
The acoustic technique developed by the host laboratory was tested for the determination of cloud points. The
cloud-point curve of the system CO2+MMA was obtained for temperatures between 40 and 65ºC and MMA
molar fractions up to 0.6 The technique was successfully validated for cloud-point measurements comparing
the results with literature data.
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Polymer processing in clean solvents
Polymerisation of polydiethylene glycol dimethacrylate a highly cross-linked biocompatible polymer, has
been developed in scCO2. The polymer was synthesized in the presence of different amounts of drug (template
molecule) in order to test the possibility of producing molecularly imprinted polymers, with specific molecular
arrangement especially focused for the template drug. Two templates were tested: salicylic acid and
acetylsalicilyc acid (initial weight/weight % of the template with respect to the monomer in the polymerization
up to 0.5%). The produced polymers were then extracted to empty the specific sites, followed by their
impregnation with the drug. The release profiles of the controlled release systems in a phosphate buffer
solution (pH=7.4) show that there is a correlation between the amount of template present during the
polymerization step and the percentage of impregnation.
Supercritical technology is of increasing interest in this area since the final product is completely free of
solvent and remaining residues of monomer and other additives are easily extracted by supercritical carbon
dioxide at the end of the process.
Development of polymer dispersed liquid crystals (PDLC) devices
The performance in terms of electro-optic response of differently prepared polymer dispersed liquid crystals
(PDLC) has been evaluated. This work is part of an extensive study of the relationship between the phase
separation processes and the electro optic properties upon changing the polymer matrix nature through the
use of different monomers, the polymerization process: thermal vs photo-induced phase separation, and the
mixture composition by studying different ratios monomer/liquid crystal. These studies are being performed in
conventional conditions and by using supercritical technology. The solubility of liquid crystal E7 in scCO2 was
determined at 40 ºC, 50 ºC and 60 ºC and pressures from 5 up to 19 MPa.
The mobility changes during the thermal induced polymerization process (TIPS) are being evaluated by
dielectric relaxation spectroscopy as the characterization of the final composites systems prepared differently.
The dielectric study of TrEGDMA60%/E740% is complete and the electro-optic properties of different ratios
TrEGDMA/E7 (70/30, 60/40, 50/50 and 40/60) are already characterized (in collaboration with Prof. João Luís
Maia Figueirinhas, CFMC-UL).
In the near future, we intend to compare the electro-optic properties of the PDLC devices prepared conventionally
with devices produced in supercritical conditions. Another aim of our research is to build-up the phase diagram
of the composites systems so prepared by using DSC (CSIC, Madrid) and to characterize the resulting
morphologies by SEM (FCT/UNL).
Integration of Processes
Development of reaction processes in clean solvent media
Biocatalysis in ionic liquids/supercritical CO2 systems using continuous stirred-tank reactors.
2005 activities to be developed
Process Simulation And Modelling / Clean Solvents
The use of static mixers as heat exchangers in supercritical fluid extraction processes will be assessed and
their performance evaluated against conventional double-pipe heat exchangers. A dynamic model of the given
heat exchanger will be developed. CFD modelling of the internal mixer flow field will be inserted in the model.
A previously developed model of a supercritical extraction packed column will be upgraded by incorporating
the momentum and energy balances in the packed bed. CFD numerical characterization of the multiphase
flow dynamics in the structured packing will be used to accomplish this objective.
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Thermodynamic Properties of Ionic Liquids
It is also planned the measurement of excess volumes of mixtures of (ionic liquids + alcohols) in broad
temperature and pressure ranges, as well as phase equilibria.
Polymeric Materials
Development of new biomaterials and polymers.
In particular, we plan to produce a new polyurethane biomaterial, with non-thrombogenic properties, using a
clean supercritical fluid technology.
Further work concerning molecularly imprinted polymers synthesized using supercritical technology will focus
in the preparation of less cross-linked polymers. A compromise between the cross-linking agent (DEGDMA,
EGDMA) and the functional monomers (L-lactide, caprolactone, methacrylic acid) has to be established so
that the structures of the imprinted cavities show enough stability to maintain the conformation in the absence
of template, but flexible into some extend to ease the impregnation and release of the drug. Different operation
conditions including different ratios of monomer-crosslinker and several commercial available polymerization
stabilizers (Fluorolink D, krytox FSL, ethylene glycol-copolymers) will be tested in the polymerization of
poly(lactide) and poly(caprolactone) in order to optimize the reactions and enhance the release profile of the
impregnated systems.
Development of polymer dispersed liquid crystals (PDLC) devices
In the near future, we intend to compare the electro-optic properties of the PDLC devices prepared conventionally
with devices produced in supercritical conditions. Another aim of our research is to build-up the phase diagram
of the composites systems so prepared by using DSC (CSIC, Madrid) and to characterize the resulting
morphologies by SEM (FCT/UNL). The differences in kinetics of neat acrylate monomers vs acrylate/E7
polymerization will be evaluated.
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BIOCHEMISTRY AND BIOPHYSICAL OF PROTEINS
and
BIOINORGANIC CHEMISTRY AND PROTEIN ENGINEERING
Head of Laboratories: Isabel Moura, Full Professor / José J. G. Moura, Full Professor
Staff Members:
Jorge Lampreia
Assistant Professor
Cristina Costa
Assistant Professor
Anjos Macedo
Assistant Professor
Jorge Caldeira
Associate Professor
Pedro Tavares
Assistant Professor
Alice Pereira
Assistant Professor
Carlos Brondino
Sergey Bursakov
Stephane Besson
Assistant Professor
Sofia Pauleta
Assistant Professor
Gabriela Almeida
Assistant Professor
Post-Doct Fellows:
Rui Duarte, Anders Thapper, Patricia Sousa, Cristina Timóteo, Francoise Auchere, Ludwig Krippahl, Celina
Santos
Ph.D. Students:
Teresa Alves, Patricia Raleiras, Cristina Cordas, Gabriela Rivas, Pablo Gonzales, Jorge Dias, Ana Martins,
Filipe Folgosa, Carlos Martins, Joana Raimundo, Márcia Guilherme, António Nunes, Teresa Tiago
Project Grantee:
Américo Duarte, Andreia Barateiro, Miriam Sousa, Vanessa Cabral, Célia Silveira, Ana Teresa Lopes
Number of articles in scientific journals: 20 (10,12, 98-115)
CYTOCHROME C PEROXIDASE – CCP
A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase.
Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d)
of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change.
Sedimentation velocity experiments confirmed the presence of a single site, although results at higher
pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry,
ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and
cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular
docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift
perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were
situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with
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the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electrontransferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome
c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons
required to restore the enzyme to its active state after turnover are delivered one-by-one to the electrontransferring heme
Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase.
The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a
heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were
shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was reevaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in
a weak monomer/dimer equilibrium that is ionic-strength-dependent. The pseudoazurin was shown to be a
highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence
consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of
which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a
wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of
NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These
lysines are associated with acidic residues just back from the rim, the resonances of which are also affected
by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of
the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.
Structural basis for the mechanism of Ca(2+) activation of the di-heme cytochrome c peroxidase
from Pseudomonas nautica 617
Cytochrome c peroxidase (CCP) catalyses the reduction of H(2)O(2) to H(2)O, an important step in the
cellular detoxification process. The crystal structure of the di-heme CCP from P. nautica 617 was obtained in
two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH
4.0, does not contain Ca(2+) and was refined at 2.2 A resolution. This inactive form presents a closed
conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction
from taking place. Form OUT is Ca(2+) dependent and was crystallized at pH 5.3 and refined at 2.4 A
resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand,
providing peroxide access to the active site. This is the first time that the active and inactive states are
reported for a di-heme peroxidase.
COBALT AND ZINC ENZYMES
Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas.
A novel adenylate kinase (AK) has recently been purified from D. gigas and characterized as a Co(2+)/Zn(2+)containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these
enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as
a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic
fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently
reversible in the 9-11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic
model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well
as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the
highly sensitive but integral DSC-method.
STRUCTURE AND MECHANISMS IN MOLYBDENUM AND TUNGSTEN ENZYMES
The groups add relevant contributions in the past to the characterization of mononuclear Mo and W containing
enzymes. Molybdenum and Tungsten (Group 6) are the only members of 4d and 5d metals series with known
biological functions. Their importance for biological systems has been recognized since 75 and 25 years,
respectively. Molybdenum is used by archae, bacteria, fungi, plants and animals including humans (more
than 50 enzymes are known) and Tungsten by only a few organisms mainly, but not exclusively, thermophyles.
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Mononuclear Molybdenum and Tungsten pterin containing enzymes cover different functions such as Aldehyde
oxidoreductase, Formate dehydrogenase and Nitrate reductase. An interplay of spectroscopic and
crystallography data to functional aspects has been the main stream for discussion. Novel arrangements of
molybdenum sites in biology have been discovered, opening the participation of this metal to novel performances.
Antagonists Mo and Cu in a heterometallic cluster present on a novel protein (orange protein)
isolated from Desulfovibrio gigas
An orange-coloured protein (ORP) isolated from D. gigas, a sulphate reducer, has been previously shown by
extended X-ray absorption fine structure (EXAFS) to contain a novel mixed-metal sulphide cluster of the type
[S(2)MoS(2)CuS(2)MoS(2)] [J.Am.Chem.Soc. 122 (2000) 8321]. We report here the purification and the
biochemical/spectroscopic characterization of this novel protein. ORP is a soluble monomeric protein (11.8
kDa). The cluster is non-covalently bound to the polypeptide chain. The presence of a MoS(4)(2-) moiety in
the structure of the cofactor contributes with a quite characteristic UV-Vis spectra, exhibiting an orange color,
with intense absorption peaks at 480 and 338 nm. Pure ORP reveals an Abs(480)/Abs(338) ratio of 0.535. The
gene sequence coding for ORP as well as the amino acid sequence was determined. The putative biological
function of ORP is under investigation.
Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis
NCIMB 13491
EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases
from sulfate-reducing bacteria.
We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase
(FDH) from the sulfate-reducing organism D. alaskensis NCIMB 13491. FDHs are enzymes that catalyze the
two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis
FDH is a heterodimeric protein with a molecular weight of 126+/-2 kDa composed of two subunits, alpha=93+/
-3 kDa and beta=32+/-2 kDa, which contains 6+/-1 Fe/molecule, 0.4+/-0.1 Mo/molecule, 0.3+/-0.1 W/molecule,
and 1.3+/-0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of FDH is typical of an
iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced
samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic
centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of
[4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less
than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively,
were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value,
as expected for tungsten in a d(1) configuration, albeit with an unusual relaxation behavior. The EPR parameters
of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several
Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at
temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center
magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the
crystal structure of a homologous enzyme.
EPR spectra of “arsenite-inhibited” Desulfovibrio gigas aldehyde dehydrogenase: a member of the
xanthine oxidase family.
Aldehyde dehydrogenase (ADH, or aldehyde oxidoreductase, also known as MOP) from D. gigas belongs to
the xanthine oxidase (XO) family of mononuclear molybdenum enzymes.The crystal structure contains a Mo
coordinated by one pyranopterin, two oxo ligands, and one hydroxo/water molecule,consistent with X-ray
structures of other XO family members. Arsenite is a kown inhibitor of this family of enzymes. The arsenite ion
reacts at the molybdenum site, and the enzymatic activity is lost. The Mo(V) ion EPR signal obtained upon
reduction of the as isolated XO incubated with arsenite was shown to have both hyperfine and quadrupole
couplings of a single arsenic nucleus (I ) 3/2) to Mo(V) (S=1/2). We present here the first molecular structure
of an arsenite-inhibited complex of a member of the XO family and the EPR properties of a paramagnetic
species detected in the reduced form of the active enzyme in the presence of arsenite. In this work, We show
that (1) arsenite coordinates to the molybdenum via an oxygen bridge in the desulfo form of the enzyme; (2)
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the proposed chemical pathway for electron transfer is conserved; and (3) although the presented structure
represents the inactive form of the enzyme, the site of binding of the arsenite is at the position of substrate
binding, indicating that the inhibition mechanism occurs through a competition with the substrate.
Direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase.
This work reports on the direct electrochemistry of the D. gigas aldehyde oxidoreductase (DgAOR), a
molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S]
centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was
analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were
used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of
the cases studied, electron transfer took place, although different redox reactions were responsible for the
voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of
the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the
reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor
occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.
DENITRIFICATION AND THE DINITROGEN BIOCYCLE
Denitrification is a stepwise sequencial pathway that transforms nitrate in dinitrogen, having nitrite, NO and
N(2)O has intermediates. Further insights in nitrite and N(2)O reduction were obtain. We have also interest in
the past on nitrate reductase.
Copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135.
The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type
1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct
determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper
(T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be
inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to
the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum
of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu
is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of
T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO(2) (-). Electrochemical experiments show that azurin from
the same organism can donate electrons to this enzyme.
Development of an electrochemical nitrite biosensor
Nitrate and nitrite are widespread contaminants, often appearing simultaneously in natural milieus. As a
consequence of nitrite toxicity and the easy conversion of nitrate to nitrite by bacterial action, there is a
growing demand to quantify nitrite ions in food, physiological and environmental samples. A large number of
methods have been proposed but most of them are lengthy and matrix sensitive. One attractive way out is the
development of analytical probes based on the recognition properties of highly selective nitrite reducing enzymes,
such as the multiheme nitrite reductase (ccNiR) from the sulfate reducing bacterium Desulfovibrio desulfuricans
ATCC 27774. The redox nature of the substrate recognition event turns out the electrochemical tools as the
natural choice for the transducing element. Therefore, we have been developing different ccNiR-based electrodes
envisaging the reliable determination of nitrite in complex samples. The enzyme was entrapped in different
matrices, such as the commercial ionomer Nafion, an electrogenerated polypyrrole film, N-substituted by
viologen groups, and sol-gel glasses. The direct electron transfer between ccNiR and a pyrolitic graphite
electrode was also explored. The electrochemical behavior of these bioelectrodes was investigated by cyclic
voltammetry. All of them displayed catalytic currents in presence of nitrite, with comparable sensitivities.
However, the remaining analytical parameters (detection limit, linear range, selectivity and operational stability)
were strongly dependent on the electrode design.
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Two azurins with unusual redox and spectroscopic properties isolated from the Pseudomonas
chlororaphis strains DSM 50083T and DSM 50135
Two azurins (Az624 and Az626) were isolated from the soluble extract of two strains of Pseudomonas
chlororaphis, DSM 50083(T) and DSM 50135, respectively, grown under microaerobic conditions with nitrate
as final electron acceptor. The azurins, purified to electrophoretic homogeneity in three chromatographic
steps, exhibit several peculiar properties. They have high reduction potentials and lower pI than most azurins
described in the literature. As previously observed for Pseudomonas aeruginosa azurin, their reduction potentials
are pH-dependent, but the pK values of their oxidized forms are lower, which suggests that deeper structural
changes are associated with the oxidation process of these novel azurins. A hitherto undescribed pHdependence of the diffusion coefficient was observed in Az624, that could be caused either by conformational
changes, or by the formation of supramolecular aggregates associated with a protonation process. Both
azurins exhibit axial X-band electron paramagnetic resonance spectra in frozen solution showing a typical
hyperfine with the copper nucleus (I=3/2) and a well-resolved superhyperfine structure with two equivalent 14N
nucleus (I=1), which is not usually observed for azurins from other species.
SIMPLE METAL SITES
Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxidemediated electron transfer with the superoxide reductase neelaredoxin.
Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of
the superoxide anion [Formula: see text] into hydrogen peroxide and water. Treponema pallidum (Tp), a
syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron
protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we
present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic
characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm
feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin,
determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized
upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the
two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per
one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion,
the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic
amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the
concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio
vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the
existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed
the physiological character of this electron transfer reaction.
Ligand K-edge X-ray absorption spectroscopy and DFT calculations on [Fe3S4]0,+ clusters:
delocalization, redox, and effect of the protein environment
Ligand K-edge XAS of an [Fe3S4]0 model complex is reported. The pre-edge can be resolved into contributions from the mu(2)S(sulfide), mu(3)S(sulfide), and S(thiolate) ligands. The average ligand-metal bond covalencies obtained from these pre-edges are further distributed between Fe(3+) and Fe(2.5+) components using
DFT calculations. The bridging ligand covalency in the [Fe2S2]+ subsite of the [Fe3S4]0 cluster is found to be
significantly lower than its value in a reduced [Fe2S2] cluster (38% vs 61%, respectively). This lowered
bridging ligand covalency reduces the superexchange coupling parameter J relative to its value in a reduced
[Fe2S2]+ site (-146 cm(-1) vs -360 cm(-1), respectively). This decrease in J, along with estimates of the
double exchange parameter B and vibronic coupling parameter lambda2/k(-), leads to an S = 2 delocalized
ground state in the [Fe3S4]0 cluster. The S K-edge XAS of the protein ferredoxin II (Fd II) from the D. gigas
active site shows a decrease in covalency compared to the model complex, in the same oxidation state,
which correlates with the number of H-bonding interactions to specific sulfur ligands present in the active site.
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The changes in ligand-metal bond covalencies upon redox compared with DFT calculations indicate that the
redox reaction involves a two-electron change (one-electron ionization plus a spin change of a second electron)
with significant electronic relaxation. The presence of the redox inactive Fe(3+) center is found to decrease
the barrier of the redox process in the [Fe3S4] cluster due to its strong antiferromagnetic coupling with the
redox active Fe2S2 subsite.
DECAVANADATE AS A BIOCHEMICAL TOOL IN THE ELUCIDATION OF MUSCLE CONTRACTION
REGULATION.
Recently reported decameric vanadate (V(10)) high affinity binding site in myosin S1, suggests that it can be
used as a tool in the muscle contraction regulation. In the present article, it is shown that V(10) species
induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin
and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and
decavanadate solutions, indicating that V(10) and tetrameric vanadate (V(4)) have the same binding sites in
myosin S1. Concentrations as low as 50 muM decavanadate (5 muM V(10) species) induces 30% of protein
cleavage, whereas 500 muM metavanadate is needed to attain the same extent of cleavage. After irradiation,
V(10) species is rapidly decomposed, upon protein addition, forming vanadyl (V(4+)) species during the
process. It was also observed by NMR line broadening experiments that, V(10) competes with V(4) for the
myosin S1 binding sites, having a higher affinity. In addition, V(4) interaction with myosin S1 is highly affected
by the products release during ATP hydrolysis in the presence or absence of actin, whereas V(10) appears to
be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers
to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin
ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher
vanadate anions, such as V(10). e spectra due to alignment of the “spin-packets”.
EPR SPECTROSCOPY OF PROTEIN MICROCRYSTALS ORIENTED IN A LIQUID CRYSTALLINE POLYMER
MEDIUM.
The EPR study of samples with partially aliened paramagnetic crystals was improved in order to determine a
mathematical model based on the shape of the crystal aligned in liquid crystal polymer (HPC) thin films.
Study of insulin derivatives (chain A and B) chemically modified with nitric oxide to yield nitrosothiol adducts
with potential physiological role in type 2 diabetes.
Quantification of GSH and GSSG compounds in plasma and liver under physiologically RIST tests of insulin
sensitivity
OXYGEN ACTIVATION
Mechanistic and Structural Studies of Iron Oxidation and Storage by Fast Ferritins.
Iron is an important nutrient, required in almost every aspect of cellular function. However, at physiological pH
and under oxidizing conditions, the predominant form is Fe3+, which is highly insoluble. Its functional importance,
coupled with its generally poor bioavailability, makes it essential for living organisms to husband this element.
How living organisms sequester iron for cellular utilization is therefore a fundamental question of vital importance.
Also, in the presence of O2, free Fe2+ ions are extremely toxic, capable of generating hydrogen peroxide,
superoxide, and other reactive oxygen species that can attack and destroy important cellular molecules. How
living organisms sequester and mobilize iron for cellular utilization is therefore a fundamental question of vital
importance.
Ferritin is unique in the sense that it performs dual functions of iron detoxification, by oxidizing the Fe2+ ions
in solution, and iron sequestration, by storing the oxidized Fe3+ ions in its inner protein cavity in the form of
ferrihydrate mineral. However, despite the importance of ferritin functions and decades of research efforts, the
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mechanism by which ferritin catalyzes the Fe2+ oxidation (ferroxidation) and directs the oxidized products to
form the mineral core (mineralization) is still poorly understood. The ferritin ferroxidase activity is associated
only with the H (or M subunits). This project seeks to further understand the kinetic and spectroscopic
observations on the iron uptake mechanism of the recombinant fast ferritins (H and M polymers). Our goal is
to understand how ferritin binds Fe2+ ions in solution, catalyzes their oxidation, and directs the oxidized Fe3+
ions into the inner protein cavity to form the ferrihydrite mineral core. Experiments to correlate spectroscopic,
kinetic, and functional effects of site-specific mutations were designed to better define the ferroxidase site
and to elucidate the ferroxidase mechanism.
Detailed kinetic investigations on the iron uptake and nucleation of the ferrihydrite mineral core were carried
out with recombinant frog H and M ferritins. In particular, the
experiments described in task 1 and 2 referring to the 36 Fe/
ferritin loading ratio. As a result a new intermediate was characterized (see bellow - Indicadores de realização física).
Results of experiments proposed in task 4 are also under analysis. A partial data collection was already done (see figure) and
results seem to be the direct proof that the ferroxidation site
plays a catalytic role In ferroxidation.
Left: High energy peak of ferrous in different samples and Right:
Fe chase experiment, lines over experimental spectra are
the sum of theoretical simulation of oxydimers species.
57
Structural and mechanistic studies of fatty acid
desaturases.
Looking for reactivity of diiron clusters.
The project contains two major goals:
(i) elucidate the catalytic steps of oxidative desaturation;
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(ii) explain the role of diiron clusters in this catalytic mechanism.
A multidisciplinary approach was proposed to complete the goals. Two proteins from Arabidopsis thaliana (a
known plant model) and one from castor seeds should be studied. In each case the gene sequence is known
which facilitates the cloning steps. Subsequently several overexpression systems will be tested. It is of
major importance to have a good overexpression system since milligram quantities of pure protein are needed.
After accomplish successful overexpression the proteins under study need to be purified from cellular extracts.
Then, a biochemical and spectroscopic (visible, EPR and Mössbauer spectroscopies) characterization will
be done. This will serve as a starting point for the mechanistic studies, where the use of visible stopped-flow
and rapid-freeze quench techniques will be applied to identify and characterize the intermediates of each
catalytic cycle. A model will be constructed and relevant comparisons with other biological significant diiron
cluster catalysis made. Lastly mutant proteins will be done to modulate enzyme activity.
As proposed, we are working in the cloning and overexpression of several desaturases from different sources.
Since this is a time consuming, trial and error, task we chosen an A. taliana clone to clone and express in a
variety of expression systems. Due to the lack of manpower this approach seemed the most reasonable. The
expression trials were done in the followings vectors: i) pET vectors; ii) CASS3 vector; and iii) pT7-7 vector. In
each case no results were obtained that would be acceptable for the purposes in mind. In fact, for the best
cases we were only able to produced inactive apo-protein. These results were obtained independently of
growth or induction conditions. A new approach was designed to overcome this problem. We are currently
trying a fusion protein expression system that gives more encouraging results. In effect the Glutathione STransferase (GST) Gene Fusion System (Amersham Biosciences) yields good amounts of fusion protein with
high purity and in a reproducible way. Currently we are testing different E. coli strains to optimize overexpression.
Also, using the known crystalographic structure of stearoyl-acyl carrier protein desaturase from castor seeds
a structural model of the protein coded by the At2g4710.1 clone was generated. The modeling project was
done using the DeepView/Swiss-PdbViewer (v. 3.7 SP5) in conjunction with the Swiss-Model protein modeling
server. We found this approach very useful since both sequences have more than 70% identity. The obtained
model has a RMSD smaller than 1 Å and, although probably not without faults, can serve as a working
hypothesis during tasks 3, 4 and 5.
THE MURINE 5-AMINOLEVULINATE SYNTHASE, THE FIRST ENZYME OF THE HEME BIOSYNTHETIC
PATHWAY
5-aminolevulinic acid synthase, ALAS, catalysis the condensation of glycine and succinyl-CoA to yield ALA.
NMR spectroscopy can be used as a tool to probe structural changes in the active center and to study the
enzymatic mechanism of an active truncated form of ALAS (from Q109 to V465, 43kDa MM). The optimal
experimental conditions to obtain 15N/2H labelled protein were achieved. The protein is sensitive to aggregation
and precipitates at typical concentrations for NMR. Doubly labelled samples, at concentrations in the micromolar
range were used to acquire TROSY and CRINEPT-TROSY spectra, at 600MHz and 800MHz.
A 22kDa Heme binding protein (HBP) from mouse liver cell extracts, was expressed in E.coli. The protein is
a monomer that can bind tetrapyrroles in general. NMR is used to determine structural features of the isotopically
labeled 15N, 13C and 2H HBP.
HIGH-PRESSURE NMR SPECTROSCOPY OF POLYMERS AND BIOPOLYMERS IN CO2 EMULSIONS
NMR experiments were performed in a specially designed High-Pressure cell, of several surfactants/CO2 and
surfactants/CO2/polymers mixtures at supercritical conditions The variations of the 1H and 19F resonances
chemical shifts with temperature were measured at variable pressures, for the same mixture. We were able to
record NMR spectra of a model peptide in supercritical solvent with the addition of a fluorinated surfactant,
opening the perspective of using it for the solubilisation of larger biopolymers
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PROTEIN CRYSTALLOGRAPHY
Head of Laboratory: Maria João Romão, Associate Professor
Staff Members:
Ana Luisa Carvalho
Cecília Bonifácio
Laboratory Technician
Luis Gomes
Computer Systems Administrator
Post-Doct Fellows:
José Trincão, Shabir Najmudin, Roeland Boer
Ph.D. Students:
Teresa Santos Silva
Number of articles in scientific journals: 7 (101-103, 113,114, 116,117)
We have been particularly interested in the study of molybdopterin-containing enzymes and their structure/
functional implications including those belonging to the xanthine oxidase family and mammalian xanthine
oxidase, isoquinoline oxidoreductase, nitrate reductase and formate dehydrogenases.
Other metalloproteins include heme proteins such as cytochrome c peroxidases, a multi-heme nitrite reductase
and a sixteen-heme cytochrome. The structural data have contributed to the study and understanding of the
Ca 2+ influence on the enzymatic activity.
Other systems under study are non-heme iron-sulfur proteins, a superoxide reductase, and Co-containing
proteins involved in sulphate metabolism (ATP sulfurylase, Adenylate kinase).
Since 2002, in collaboration with the Faculty of Veterinary Medicine of Lisbon, we have been actively involved
in the structure determination of several components of the Cellulosome assembly, a complex machinery
responsible for plant cell wall degradation.
METALLOPROTEINS
1-
Metalloproteins - Molybdopterin-containing enzymes
MOP- Aldehyde oxido reductase from Desulfovibrio gigas:
Correlation of EPR and X-Ray Structural Analysis: Crystal Structure and EPR Properties Of Arsenite
Inhibited Forms Of Aldehyde Oxidoreductase
Arsenite is a known inhibitor of the enzymatic activity of mononuclear molybdenum enzymes of the xanthine
oxidase family. The arsenite ion reacts at the molybdenum site and the
enzymatic activity is lost. The Mo(V) ion EPR-signal obtained upon reduction
of the as isolated XO incubated with arsenite was shown to have both hyperfine
and quadrupole couplings of a single arsenic nucleus (I=3/2) to Mo(V) (S=1/
2).2a On the basis of these studies, a structure was proposed in which the
arsenic and the molybdenum atoms are connected by a sulfido bridge, while
other authors proposed a structure with a double bridge (oxo and sulfido).
We have solved and described the first molecular structure of an arseniteinhibited complex of a member of the XO-family and the EPR-properties of
the paramagnetic species were detected in the reduced form of this complex.
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Periplasmic nitrate reductase (NAPA) of Ralstonia eutropha –mutagenesis
studies on the aminoacdis around the molybdenum center of NAP A
Molybdenum enzymes containing the pterin cofactor are a diverse group of
enzymes that catalyse in general oxygen atom transfer reactions. Aiming at
studying the amino acid residues, which are important for the enzymatic
specificity, we used nitrate reductase from R. eutropha (R.e. NAP) as a model
system for mutational studies at the active site. We mutated amino acids at
the Mo active site (Cys 181 and Arg 421) as well as amino acids in the funnel
leading to it (Met 182, Asp 196, Glu 197, and the double mutant Glu 197-Asp
196). The mutations were made on the basis of the structural comparison of
nitrate reductases with formate dehydrogenases (FDH), which show very similar
three-dimensional structures, but clear differences in amino acids surrounding
the active site. For mutations Arg421Lys and Glu197Ala we found a reduced
nitrate activity while the other mutations resulted in complete loss of activity.
In spite of the partially or totally loss of nitrate reductase activity, these mutants
do not however display FDH activity.
2-Metalloproteins - Heme-containing enzymes
Cytochrome c peroxidases: Structural basis for the mechanism of Ca2+ activation of the di-heme
CCP from Pseudomonas nautica 617
Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular
detoxification process.
The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two redox states.
The inactive form was refined at 2.2 Å. It presents a closed conformation where the peroxidatic heme adopts
a six ligand coordination, hindering the peroxidatic reaction to take place. The active form was refined at 2.4
Å. It shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide
access to the active site. This activated form contains a bound Ca2+ ion essential for enzymatic activation
and it shows several conformational changes.
Ca
Inactive form (IN)
Active form (OUT)
16-heme cytochrome from Desulfovibrio gigas
High molecular weight cytochromes (Hmc) belong to a large family of multiheme cytochromes in sulfate
reducing bacteria and HmcA is the first cytochrome reported to have sixteen type c hemes arranged in its
polypeptide chain. The function of this cytochrome is still unknown although it is clear that it belongs to a
membrane bound complex, involved in electron transfer from the periplasm to the membrane. HmcA from D.
gigas has been purified and successfully crystallized. The crystals diffracted X-rays to beyond 2.07 Å. The
structure was solved by MAD methods and the good quality of the electron density map allowed tracing most
of the polypeptide chain. The structure was fully refined.
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The CELLULOSOME
3- Plant cell wall degrading enzymes
A recent project, in collaboration with the Faculty of Veterinary Medicine of Lisbon, involves the structure
determination of several components of the “Cellulosome” assembly, a complex machinery responsible for
plant cell wall degradation.
Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydratebinding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended
catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize
single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed
by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate
specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile
of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM
(CtCBM11) in Clostridium thermocellum, an enzyme that contains two catalytic domains that display beta1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. We have shown that CtCBM11 binds
to both beta-1,4- and beta-1,3-1,4-mixed linked glucans with a preference for mixed linked glucans. To determine
whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of
the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that
forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located
in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein.
We proposed, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and
beta-1,3-1,4-mixed linked glucans.
Planned research activities for 2005
Metalloproteins
Formate dehydrogenase from D. vulgaris - Crystallization conditions will be optimized.
Periplasmic nitrate reductase (NAPA) of Ralstonia eutropha – Mutagenesis studies and purification of the
wild-type and mutants. Crystallographic studies of the purified proteins.
Co/Zn containing Adenylate kinase (AK) – suitable crystals are available and MR will be attempted.
Plant cell wall degrading enzymes
The molecular determinants of protein-protein interactions in cohesin-dockerin complexes will be studied by
mutagenesis studies and crystallography. Novel insights into the role of carbohydrate-binding modules (CBM)
in enzyme function will be obtained by the analysis of several CBM structures.
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BIOLOGICAL TRANSPORT
Head of Laboratory: Teresa Maria Fonseca de Moura, Associate Professor
Staff Members:
Hugo Gil Ferreira
Full Professor
Karin Tonnies Gil Ferreira
Associate Professor
Isabel Borges Coutinho de Medeiro
Assistant Professor
Maria da Graça Soveral Rodrigues
Assistant Professor
Ana Isabel Dias Bicho dos Santos
Project Grantee:
João Filipe da Custódia Dias
Membrane Transport
Role of aquaporins in different strains of S. cerevisiae
(in collaboration with Prof. Conceição Dias from Instituto superior de Agronomia)
The presence of active aquaporins is being investigated in S. cerevisiae. Genes coding for aquaporins have
been detected in the genome of this yeast. The functionality of proteins coded by these genes is being
investigated by measuring energy of activation of water fluxes in protoplasts. Strains of S. cerevisiae lacking
genes AQY1 and AQY2 and strains over expressing these genes are being used.
Inhibition of the mithocondrial pyruvate carrier by valproate and cetovalproate
Pyruvate driven oxygen consumption and ATP synthesis is severely inhibited by the antiepileptic Valproic
acid (VPA). To test whether this effect results from an inhibition of the mitochondrial pyruvate carrier by VPA
or by its â-oxidation metabolite 3-keto-VPA we used inverted submitochondrial vesicles (ISMV) prepared from
rat liver cells. We found a 30 to 40% inhibition on pyruvate transport for vesicles treated with both these
reagents.
Patch Clamp studies: Cells Obtained by Nasal Brushing From Non-Cystic Fibrosis (CF) Individuals:
(in collaboration with Drs. Colete Maurício from ICBAS and Débora Penque and Margarida Amaral from Instituto
Ricardo Jorge)
Tall Columnar Epithelial Cells obtained by a noninvasive procedure were studied functionally using the Patch
Clamp technique. Using the sub-apical region three different Cl- channels were identified: One with large
conductance, rectifying, the most frequent one; a second type with small conductance, activated by cAMP
and IBMX, in excised inside-out configuration, and voltage independent; and finally a third type with conductance
around 25ps, voltage independent, also in inside-out configuration. This experimental work using freshly
obtained TCL demonstrates that these cells may be a valid tool to study Cl- channels, as a model for the
lower respiratory epithelium.
Functional characterization of a P-ATPase from Nicotiana tabacum: Patch Clamp studies
(in collaboration with the Plant Development group of Prof. José Feijó from Institute Gulbenkian for Science)
The proton P-ATPase from Nicotiana tabacum is responsible for proton fluxes observed during pollen tube
growth (4µm/sec, one of the fastest polarized cellular growth in nature). The electrophysiological patch-clamp
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technique can be applied to access the proton currents through this ATPase and to access the mechanisms
by which pump activity is regulated.
To start this project the cell-culture room and the patch-clamp set ups were adapted to this project, namely for
the possibility of expression of proteins in heterologous cell systems suitable for patch-clamp studies. A fast
perfusion system that allows accurate temporal control of application of solutions and drugs to the cells was
built. A laser light (with 473 nm wavelength) was adapted to the microscope to access GFP, a positive marker
for transfected cDNA. The cell- culture room was adapted for the use of DNA.
Mathematical models of biological systems
Mathematical models for cells and epithelial systems are being developed. Numerical simulations are
implemented in the Berkeley Madonna package (http://www.kagi.com/authors/madonna).
Regulation of the Cellular Proton Balances in an Epithelial System
(in collaboration with Prof. Augusta Rebelo da Costa, from ICBAS, Porto)
The mathematical model of the outer mantle epithelium (OME) of the mollusk Anodonta sygnea is being built
in order to simulate the results obtained in vitro. It consists of three compartments (two external and semiinfinite bathing the epithelium faces and the intracellular compartment) separated by two barriers and seven
intracellular pools (Na, K, Cl, CO2, bicarbonate, non-volatile acids, protons). Movements across the two
barriers occur through the transport systems identified in vitro with the application of specific inhibitors.
Tubular organ - thick ascending limb of the Henle´s loop - TAL
Numerical simulations were obtained using the mathematical model developed for the behaviour of the thick
ascending limb of the Henle´s loop of mammalian kidney. Perturbations in the parameters were introduced
and the calculated results compared with experimental data from the literature. The purpose of this work is to
gain a better insight on the function of TAL by analyzing the results of the numerical simulations as a function
of tubule length and time, when testing different experimental conditions reported in the literature.
Calcium homeostasis - A minimal model (col. with J. Raposo and L. Sobrinho from Instituto Português de
Oncologia Lisboa)
In mammals the calcium concentration in the extracellular compartment (ECC) is finely regulated (Calcium
Homeostasis) and controls a large number of physiological processes: blood coagulation, the excitability of
nerve and muscle cells, the epithelial secretion, the secretion of parathormone (PTH), the functional state of
many ionic channels, etc. As a result of the development of reliable and easy techniques of measurement of
the main variables of this control system (Calcium, PTH and Calcitriol) it is now possible to characterize with
some detail many of the system’s disturbances which are frequently seen by the endocrinologists. Although
there is now published information on the behaviour of a number of the components of the calcium control
system we lack tools that may help us to analyze the behaviour of the system as a whole. This is an attempt
to develop such a tool. A first version of the model covering acute situations was published (J. Clin. End. Met.,
2002, 87, 4930-40). The model is now expanded to cover chronic situations.
Studies on Food Components
Analysis of metals and trace elements (in collaboration with Faculdade de Farmácia de Lisboa)
As stated in the report of 2003 an extensive screening of the total amount of metals and trace elements in the
most consumed beverages of the Portuguese population was concluded.
The same elements studied in the beverages are being screened in human milk samples from a group of
breast feeding mothers subjected to a nutritional assessment. All these studies are being performed using
the ICP (Induced Coupled Plasma) technique.
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DEVELOPMENT OF TRANSDUCERS
Head of Laboratory: José Luís Costa Lima, Full Professor
Staff Members:
Alberto Nova Araújo
Associate Professor
Rui Alexandre Santos Lapa
Associate Professor
Mª Conceição B. S. M. Montenegro
Full Professor
Maria Beatriz V. N. Q. G. Junqueiro
Associate Professor
Agostinho Almiro Almeida
Assistant Professor
João Luís Machado Santos
Assistant Professor
Maria Lúcia M. F. Sousa Saraiva
Assistant Professor
Marcela Alves Segundo
Assistant Reseracher
Ivone Valente Oliveira
Assistant Professor
Cristina Maria C. Morais Couto
Assistant Professor
Adriana Martins Pimenta
Assistant Professor
João Rodrigo da Silva Santos
Ph.D. Students:
Maria Luísa Soares Silva, Sofia Alexandra Lopes da Piedade Gomes
M.Sc. Students:
Branca Sofia Teixeira
New trends were investigated based on exploitation of different transducing schemes such us optical and
electrochemical. Studies on immobilization techniques were implemented for the monitoring low levels of
drugs, food and pesticides. Additionally studies were developed concerning the construction of flow-through
voltametric electrodes dedicated to the implementation of manifolds with multi-side detection or
multicommutated flow systems.
In more detail it can be referred the following results:
- Development of a voltametric detector with tubular connected to a automatic FIA system dedicated to the
determination of uric acid in urine. Samples were diluted in supporting electrolyte before analysis and no other
pre-treatmentwas employed. Such dilution enabled the matrix effects to be reduced and therefore improving
the sensitivity provided by the activation step of the electrodes that allows the uric acid to be detected a lower
concentration levels after dilution. The uric acid determination was developed in a single channel FIA manifold,
which provided reproducibility of the sample transport to the detector and enabled a sampling rate of 120
samples per hour.
- The stat of the art of voltametric and amperometric methods used in the study and determination of pesticides
incrops, food, phytopharmaceutical products, and environmental samples was been reviewed. The main structural
groups of pesticides were considered with some degradation products. The advantages, drawbacks and
trends in the development of voltametric and amperometric methods for study and determination of pesticides
in the referred samples were discussed.
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- The construction, evaluation and analytical application of ion selective electrodes sensitive to penicillin-G
antibiotics dedicated to pharmaceutical products analysis was implemented. Different types of polymeric
membranes based on PVC and EVA, without internal reference solution, were prepared using 5,10,16,20tetraphenylporphyrinate manganese (III) as electro active material. Differente additives such as tetra-noctylammonium bromide and sodium tetraphenylborate were incorporated into membranes to evaluate their
influence on the electrodes performance.
- The determination of giggerellic acid in growth formulations used in agriculture by sequential injection analysis
and potentiometric detection. The potentiometric detectors with improved characteristics used a PVC membrane
prepared with Mn(III)tetraphenylporphyrin-Cl as electroactive specie. Different membranes with several ionic
additives were compared in order to select the most suitable in what concern slope, response time, reproducibility
and selectivity.
- Two quasi-independent methods for potentiometric and optical determination of chloride were simultaneously
implemented in a flow system, providing real time assessment of the quality results. A potentiometric and an
optical polymeric membrane doped with the same indium (III) octaethyl porphyrin were used as sensor
ionophore. The working mechanism and the analytical characteristics of these porphyrin-based sensors with
respect to dynamic range, selectivity, repeatability and life time are discussed. These sensors utilised as
detectors in a flow system, were applied for the analysis of chloride in pharmaceutical solutions. The quality
of the results obtained was evaluated by comparation with those provided by the reference method and no
significant statistical differences were observed. The simultaneous attainment of the two measurements
permitted the standardisation of the results in real time and the detection of failures in the procedure.
- A sol-gel optical sensor coupled to multicommutated flow sytem was been proposed for direct
spectrophotometric determination of Cu(II) in urine. The optical sensor was developed by physical entrapment
of 4-(2-pyridilazo)resorcinol in sol-gel thin films by means of base-catalysed process. The immobilised PAR
formed a red 2:1 complex with Cu (II) with a maximum absorbance at 500 nm. Optical transduction was based
on a dual-colour light-emitting diode (LED) (green-red) light source and a photodiode detector. The sensor had
a optimum response and good selectivity towards Cu(II) and its generation was accomplished with picolinic
acid.
- A Zn (II) optical sensor was developed by incorporating 4-(2-pyridylazo)resorcinol (PAR) in a sol-gel film.
Acid- and base-catalyzed methods to prepared the sol-gel layers have been study, as well as different tupes
of precursors and different PAR concentrations. Sensors based on co-polymerizations of tetraethoxysilane
(TEOS) with 3-amynopropyltriethoxysilane (3-APTES), basic catalysis, water:alkoxyde ratio of 4 and PAR
concentration of 1.0 gl-1 showed optimum performance in the proposed working conditions. Optical transduction
was based on the use of a dual-color LED and a photodiode.
- A new sol-gel Bi (III) sensor was developed by incorporating xylenol orange (XO) into sol-gel thin films coated
on glass slides. Several sols were produced in order to evaluated the effect of different processing parameters
on the final characteristics of the sensor. Sensor films based on tetramethoxysilane (TMOS) as precursor,
nitric acid catalysis water:alkoxide ratio of 2 and XO concentration of 1.5 gl-1 were found to be the most
suitable to be used as Bi (III) sensors. They presented good sensitivity, reversibility and stability, low leaching
and fast response time in the proposed working conditions.
In 2005, in this topic, research efforts of the group will be made to developed new potentiometric, electrochemical
and optical transducers dedicated to the quantitative evaluation of inorganic and organic species in biological,
environmental, food and pharmaceutical matrices: (i) New potentiometric sensors dedicated to inorganic and
organic species will be developed and evaluated in batch and flow conditions. As membrane sensors calixarenes
and metalloporphyrins derivates will be used and tested several solvent mediators dedicated to a specific
application; (ii) Microelectrodes and flow-through voltametric electrodes will be developed for direct determination
without reagent consumption applied to determinations in human tissues, food and pharmaceutical products;
(iii) Optical sensing devices based on sol-gel techniques to produce membranes to support the sensing
elements controlling their morphology, porosity and silanol groups contents. Additionally the immobilization
of the developing colour reagent and the leaching characteristics, sensitivity and response time will be evaluated.
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AUTOMATION AND INSTRUMENTATION
Head of Laboratory: José Luís Costa Lima, Full Professor
Staff Members:
Alberto Nova Araújo
Associate Professor
Associate Professor
Mª Conceição B. S. M. Montenegro
Full Professor
Maria Beatriz V. N. Q. G. Junqueiro
Associate Professor
Assistant Professor
João Luís Machado Santos
Assistant Professor
Maria Lúcia M. F. Sousa Saraiva
Assistant Professor
Assistant Professor
Assistant Professor
Assistant Professor
Ph.D. Students:
João Alexandre Velho Prior, Paula Cristina de Azevedo Gomes Pinto, Cristina Manuela Pinto Vieira, Pedro
Rodrigues Marques Maia, Marta Filipa Teixeira Ribeiro, Karine Lopes Marques, Luís Miguel Andrade de
Magalhães
Number of M.Sc Thesis: 5
Continuous flow systems and dedicated equipment were developed and applied in automatic laboratorial
determinations or in on-line control of industrial processes. Special attention was been dedicated to procedures
based on the multicommutated flow methodologies and to the new concept of multi-pumping flow analysis.
In this theme the following activities can be stressed:
- We work on the concept related to the generation of ultrasound-assisted reagents in flow systems. To
evaluated the efficiency of the ultrasound-assisted reagent generation, the conversion of Fe (II) to Fe (III)
associated with 1,10-o-phenantroline specrophotometric method was tested to compare the oxidizing power
of the produced species from pure water and aqueous solutions saturated with CCl4 and CHCl3 irradiated
ultrasonic waves. A borosilicate reactor was used in batch studies, while PTFE tube reactor was used for
setting up the flow system, with controlled temperature during irradiation. The sonochemical production of
oxidizing agents was demonstrated to be efficient for chemical analysis in batch and flow systems.
- Between-method carry over due to reagent replacement in flow systems devoted to multiparametric
determinations was been critically evaluated. For this task, a novel system involving the spectrophotometric
procedure for iron speciation in natural waters based on the known reaction of Fe (II) with 1,10-phenantroline
was initially designed. Furthermore, other systems for different bi-parametric assays were highlighted.
Potentialities and limitations of the different strategies for compensation and/or reducing between-methods
carryover are discussed, and guidelines for laboratory management are withdrawn.
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- A continous flow methodology for chemiluminometric determination of clomipramine in pharmaceutical
preparations is described. Cloripramine acts as s sensitizer on the chemiluminescent oxidation of sulphite by
Ce (IV). The developed procedure is based on the multicommutated flow concept whose versatility was fully
exploited by using solution propelling device for the insertion of multiple solutions in propulsion mode. The use
of a binary sampling approach enable sample/reagent mixing within the flow cell with a significant reagent
saving. A time-based sample insertion assured an effective control of sample dispersion, enabling the attainment
of the distinct working concentration ranges by means of the selection of the appropriate sampling times.
- A multicommutated flow system was developed for the determination of aluminium in crystallized fruit samples.
Spectrophotometric determinations were based on the reaction with chromeazurol S. The binary sampling
techenique was implemented to improve mixing conditions and to minimize reagent consumption. Three
different concentrations working zones were established using the zone sampling approach, allowing us to
adapt the extent of the in-line dilution. The influence of the chemical and physical parameters on the performance
of the system was been studied. Detection limits of 0.1,0.6 and 0.8 ppm were obtained for the lowest, the
medium, and the highest dispersion system, respectively. The procedure was applied to the determination of
aluminium in crystallized fruit extracts. The results were in agreement with those obtained by the reference
flame atomic absortion procedure. Repeatability was better than 2.4 % in all three concentration ranges.
- It was been developed an automatic flow procedure for the determination of glycerol in wines by employing
a flow system based on multicommutation and enzymatic reaction. Glycerol dehydrogenase was immobilized
on aminopropyl glass beads and packed and packed into a column that was coupled to the flow system. The
NADH produced by enzymatic reaction was monitored by spectrophotometry. The system manifold comprised
a set of three-way solenoid valves controlled by a computer, which was provide with electronic interfaces and
runs a software designed to carry out on-linesample dilution, reagent addition and data acquisition. The
procedure allows the determination of glycerol in wine samples without any prior pre-treatment. The results
were compared with those obtained using a reference method.
- A multi-syringe system for spectrophotometric determination of total phosphorus involving in-line disgestion
was been developed. Sample and digestion solution were dispensed and directed towards a digestion vassel
located inside a domestic oven where sample digestion take place. Afterwards, the digested sample was
merged with the necessary reagents for the colorimetric determination based on the molybdenum blue method.
Several digestion conditions were studied regarding composition of the digestion solution, digestion time and
power set on the microwave oven. The system was applied to waste water samples and results shown a good
agreement with the reference method.
- A new separation method for simultaneous determination of paracetamol, caffeine, acetylsalicylic was
developed based on a novel reversed-phase sequential injection chromatography technique with UV detection.
The mobile phase used was acetronitrile/phosphate buffer at a flow rate of 0.6 ml per minute and the detection
was performed at 210 and 230 nm. The analysis time was less than 6 min and the method was found to be
applicable for routine analysis of paracetamol, caffeine and acetylsalicylic acid in pharmaceutical tablets.
- Accurate simultaneous analysis of different anionic species using ion-selective electrodes can be achieved
even for non-specific sensors by resorting to an ordinary least squares multiple regression in the vicinity of the
predicted concentration. In this work, the potentialities of this approach, are evidenced by the determination
of nitrate and chloride in synthetic and real water samples in which chloride concentration was significantly
higher than nitrate. An AgCl/Ag2S electrode based on homogeneous crystalline membrane together with a
PVC electrode based on tert-octylammonium bromide dissolved in dibutylphthalate were used as potentiometric
detectors for chloride and nitrate, respectively. For the implementation of the procedure, an automatic system
based on sequential injection analysis was used. The results obtained by the standard addition method were
biased for low concentrations of nitrate and were dependent on the relative proportion of nitrate/chloride. The
results obtained by the proposed methodology for chloride determination were slightly better when compared
to those obtained by the standard addition method.
- A sequential injection analysis system was developed to quantify pH, chloride and nickel in electrolytic
baths. To enable pH and chloride determination, potentiometric detection with two ion-selective electrodes in
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a tubular configuration was used. Nickel concentrations were assessed using colorimetric detection at 660nm.
pH was determined prior to nickel determination and just after sample injection into a phosphate buffer carrier
stream. For chloride determination, on-line dialysis through a cellulose membrane was used to enable sample
dilution and matrix separation. A fractional factorial design based on the carrier solution composition and the
levels of the hydrodynamic parameters was used for system optimization. At the optimized conditions a
sampling rate of 40 samples per hour was attained, with a precision and accuracy statistically indistinguishable
from those achieved with conventional procedures.
- The versatility of sequential injection analysis systems as a sampling handling approach combined with the
flexibility of the individual solenoid valves was conjugated for the automation of the proposed new enzymatic
method for the determination of aspartame in commercial sweetner tablets. The method involves the enzymatic
conversion of aspartame in hydrogen peroxide by the chymotrypsin-alcohol oxidase system, followed by the
use ABTS as electron donor for peroxidase. Chymotrypsin and alcohol oxidase enzymes were immobilised
on activated porous silica beads. No activity loss in the reactors was detected throughout 60 days. The
method was applied to the determination of aspartame in commercial sweetener tablets; the results obtained
were compared to those provided by a HPLC method and revealed no statistical differences.
- Sequential injection systems for wine analysis was been reviewed. Special focus is given to implementation
of in-line sample treatment and adaptation of system operation through software control to enable determination
in different types of wine. The strategies used to enhance selectivity and capacity for multi-parameter
determination were also addressed.
- An automated set-up based on sequential injection analysis concept with potentiometric detection for the
determination of chloride and iodide at low concentrations. The assessment of both ion concentrations was
accomplished by titration with silver ions using Gran’s plot approach. The proposed procedure enables chloride
and iodide to be determined simultaneously.
- An automatic monosegmented flow titration procedure based on Gran linearization approach has been
developed. The controlling program can estimate the end point of the titration after the addition of three or four
aliquats of titrant. Alternatively, the end point can be determined by the second derivative procedure. In this
case, additional volumes of titrant added until the vicinity of the end point and three points before and after the
stoichiometric point are used for for end point calculation. The performance of the system was assessed by
the determination of chloride in isotonic beverages and parenteral solutions.
- The development of a sequential injection analysis manifold for colorimetric determination of lead in water
samples was been described. The concentration of lead was assessed from the catalytic effect on the reaction
of resazurine reduction caused by sulphide in an alkali medium. To that effect, the reaction zone was stopped
at the detector, and the time interval required for the attainement of an absorbance decrease was estimated.
Interference of other transition metals of the samples was minimized by adding potassium iodide to the
sample and retaining the iodocomplexes formed in an on-line anionic resin. A good agreement was been
obtained comparing the results of the analysis of ten water samples given by the proposed system and by
electrothermal atomization atomic spectrometry.
- Multi-pumping flow systems was been reviewed considering the most recent developments in terms of
design and implementation of continuous flow methodologies, for sample and reagent handling and for the
automation of analytical procedures. Based on the utilisation of multiple solenoid micro-pumps they enable
the configuration of fully automated and easily controlled and operated analytical systems since all the
fundamental operations involved in carrying out a sample analysis, including sample insertion, reagent addition
and signal measurement could be carried out by the same manifold component, reducing the number of
system parts and minimising its control or the occurrence of mal-functions. On the other hand, micro-pumps
actuation produce a pulsed flow characteristics by a chaotic movement of the solutions, which contributes to
a fast sample/reagent homogenisation with low axial dispersion yielding improved analytical signals. The
combination of such advantageous features result in simple, compact, versatile, fast, low-cost analytical
procedures, exhibiting low reagent and low sample consumption, reducing the production of undesirable
wastes and minimising operator intervention.
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As activities during 2005 we plan the development of automated flow systems and dedicated equipment will
resort several flow concepts like flow injection analysis, sequential injection analyses, multi syringe approach
and mainly the techniques developed by our research group, namely, multicomutated flow analysis and the
very recent multi-pump flow analysis. Some studies will be made on the possibility of coupling in the same
manifold some of the previously referred techniques in order to improve the performance of the systems in
what concern with the saving of chemical and rate of the determinations.
Special interest will be devoted to the coupling of the multicomutated and multipump flow techniques to
several detection devices for fluorimetric, chemiluminescence, potentiometric and voltametric measurements
for the control of pharmaceutical products and pollutants.
Instrumentation dedicated to the on-line chemical generation in flow systems based on ultrasonic formation of
species will be constructed and evaluated. The instrumentation will be, in a first step, applied to the production
of oxidant species originated by the ultrasonic waves.
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QUALITY CONTROL AND AUTHENTICITY OF FOOD PRODUCTS
Head of laboratory: Rosa Seabra, Associate Professor / Beatriz Oliveira, Assistant Professor / Isabel Ferreira,
Assistant Professor
Staff Members:
Paula Andrade
Assistant Professor
José Fernandes
Assistant Professor
Patrícia Valentão
Assistant
Susana Casal
Assistant
Rui Alves
Professor Coordenador
Olívia Pinho
Assistant Professor
Isabel Mafra
Cristina Maria Delerue-Matos
Maria do Carmo Veiga Fernandes Vaz Professor Coordenador
Maria Goreti Ferreira Sales
Assistant
Luísa Maria Sobreira Vieira Peixe
Assistant Professor
Helena Maria Neto Ferreira
Assistant Professor
Miguel Freire de Albuquerque Cabral
Assistant Professor
Ph.D. Students:
Sara Cunha, Miguel Faria, Joana Amaral, Sandra Maria Basílio Quinteira, Carla Alexandra Novais Oliveira e
Silva, Patrícia Sofia Carneiro Antunes, Elisabete Maria Pereira Machado
Number of articles in books: 2 (10, 11)
Number of patents: 1
Our group has large experience in the field of food quality control and, more recently in the authenticity
concerns. Some PhD thesis and papers were presented with emphasis on macronutrients (fatty acids, proteins,
triglycerides), micronutrients (vitamin E, phytosterols, phenols, organic acids) and contaminants (biogenic
amines, PAH’s) of conventional and traditional food products, with “Protected Origin Denomination”, POD.
1. Development of molecular biology techniques for the evaluation of food safety and food
authenticity
The research includes the use of DNA based techniques (PCR Polymerase Chain Reaction) applied to food
products. The identification of species of origin for milk in dairy products, namely cheeses PDO (Protected
Denomination of Origin), has been carried out for the detection of fraudulent procedures. By means of a
duplex PCR technique discriminating simultaneously bovine and ovine species, it was possible to detect
(0.1%) and to quantify (1-50%) the addition of bovine milk to ovine cheeses. This method was applied to
developed is the detection of bovine milk in caprine cheeses by means of simple PCR and by means of duplex
PCR.
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Vitis vinifera clonal identification is of great importance in the process of clonal selection. The precise
identification and use of the best clones have obvious effects in wine quality and authenticity. The
methodology developed by our group, in collaboration with CIBIO and ISA, could be used for clonal selection
of grapevine cultivars. An optimized and highly reproducible procedure for the detection of differences among
Vitis vinifera L clones using the StSy–CHS gene family primer combination was achieved. With this methodology,
21 clones and a commercial cultivar, ‘Chardonnay’, were evaluated with three StSy–CHSmarkers. It was
possible to obtain 14% discrimination within Touriga Nacional (TN) clones, identifying two different groups.
The polyclonal origin of TN clones was previously verified with eight microsatellite markers. In this work a
novel pure DNA extraction methodology, which occludes the extract in an agarose matrix, thus allowing the
removal of the remaining hydrophilic impurities, was successfully used.
Development of analytical methods for the authenticity evaluation of Portuguese products, DOP and IGP,
throughout the analysis of constituent nucleic acids (e.g. wines and olive oils) with emphasis on the achievement
of an efficient wine residual DNA extraction methodology. We intent to apply to wine a few concepts of DNA
extraction and repair usually applied to the extraction of much degraded mummified DNA. These concepts
are based in the use of silica magnetic particles, membranes for the selective sorption of DNA and repair
methodologies based on DOP-PCR - Degenerate Oligonucleotide Primed Preamplification – and Whole Genome
Amplification techniques.
The detection of genetically modified (GM) soybean RR (Roundup Ready) with herbicide tolerance (glifosato)
and derived products is also being developed by means of PCR technique. Firstly, it was developed a method
of DNA extraction able to extract DNA from raw materials (soybeans and corn powders). The method was
improved to be applied successfully to real foods obtained in the local market, such as drinks, protein isolates,
hamburgers, sausages, biscuits, etc containing small amounts and/or degraded DNA. The evaluation of DNA
amplification of extracted DNA was done by means of PCR of soy lectin gene (Le1) with specific primers to
produce the fragments of 414 and 118 bp. The detection of GM soybean was performed by amplification of
sequences of NOS terminator and 35S promoter in a first stage. The amplification of RR insert was performed
with specific primers to produce the fragments of 447 and 169 bp. The use of certified reference materials
allowed verifying the detection of at least 0.1% of RR soybean. The results indicate that the contamination of
foods with RR soybeans is real, even in products labelled with no-GMO. The development of real time PCR
techniques for quantification of GM foods derived from soybeans is the research planned to do in the near
future. The application to maize derived food products will be also performed in a near future.
2. Cheese authenticity by means of Chemical Markers
Characterization of Terrincho and Transmontano PDO cheeses: evaluation of physico-chemical parameters,
lipolysis, proteolysis and authenticity. Cheese lipolysis: Study of cheese volatile fraction by solid phase
microextraction coupled with GC/MS. Quantification of the major volatile free fatty acids in ewe cheese.
Correlation between volatile components and sensory characteristics.
Determination of free amino acids and biogenic amines in PDO cheeses for hygienic and food safety reasons.
3. Human milk composition
The fatty acid composition of human milk was evaluated form 7 days to 16 weeks of lactation in order to
access the composition stability of the fatty acids during this period. The results are now being treated and
published.
4. Coffee
Some important coffee constituents were determined in both green and roasted coffee and their behaviour
with roasting evaluated and discussed. In particular the biogenic amines, free and conjugated, were useful in
the green coffee varieties discrimination. These compounds also seem to constitute important geographical
marker for Angolan coffees.
5. Olive and vegetable oils
Olive oil has a social and economic relevance. This fact has been taken in account by growers in order to
implement new groves and increase the number of phytossanitary treatments to obtain enhanced yields. The
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application of organophosphorus pesticides (dimethoate, fenthion, phosmet) to control olive pests and its
detection, in trace amounts, in olives and olive oils is common.The pesticides are known to accumulate in the
tissue of the plant, metabolized or not into more toxic compounds.
A GC/NPD methodology is implemented for the multirresidual determination and quantification of
organophosphorus pesticides being applied to olives and virgin olive oils from organic and conventional agriculture.
A GC/MS methodology is also implemented and applied to similar samples in order to determine the contents
of phosmet and its metabolites. Samples harvested in different regions of Portugal and submitted to
phytossanitary treatments with the referred product are in evaluation.
Several quality parameters were determined in varietal olive oils in order to characterize some cultivars of Olea
europaea (fatty acids, sterols, tocopherols, triglycerides) and were applied to POD olive oils “Azeite de Trásos-Montes”. Some obtained results are in publication. An HPLC methodology was in development aiming to
characterize the carotenoid profile of olive oils. The chlorophyll pigments of this food are also in study to
posterior implementation.
The quality of frying oils, along the frying process of several foods, will be determined with the implementation
of a HPSEC/ELSD methodology. The polymerised triacylglycerides, oxidized triacylglycerides, and
diacylglycerides will be some of the compounds evaluated with this technique. Another chemical parameters
will be included in the referred study.
Nutritional studies
Food, and more directly fat, have a central role in the consumer’s sustenance and pleasure, as well as a
predominant part in the world’s economy, politics and culture. Due to nutrients interactions and alterations
that can occur during cooking and industrial food processing, our studies also included the analysis of
processed diets as well as the raw materials and the used ingredients.
In order to obtain data on the nutritional value of some dishes largely consumed in Portugal, traditional
Portuguese dishes and largely consumed fast food meals (including pizzas, chinese food and “francesinhas”)
were evaluated, indicating that our feeding style is already far from the original and health claimed “Mediterranean
diet”. The nutritional composition of the traditional Portuguese sausage “alheira” was determined and evaluated
the modifications that occur with different cooking processes. The results obtained were in treatment for
publication. In this field are also in evaluation the nutritional compositions of confectionery cakes.
Nuts are also relevant components of the Mediterranean diet, correlated with health benefits mainly by coronary
heart disease prevention. A study on the chemical composition (including fatty acid and sterols) of different
cultivars of hazelnuts (Corylus avellana L.) and walnuts (Juglans regia L.), grown in different geographical
areas is concluded and some results publiched. This research line continues and the tocopherol and tocotrienol
contents and the triacylglycerol profiles were evaluated as well as the importance of these parameters in the
discrimination of the cultivars in study.
Sheep milks of two autochthonous Portuguese breeds were evaluated for their conjugated linoleic acid (CLA)
levels. CLA has recently been recognised as a nutrient with important physiological effects, including anticarcinogenic, anti-atherogenic, lean body mass promoting, and anti-diabetic. The results suggest that this
milk can be considered an interesting source of CLA. In prosecution of this line a similar study with Spanish
breeds will be performed in collaboration with a Spanish faculty. Our objective will be the detection of interesting
differences for genetic improvement of the breeds. The study will include the study of fat composition of the
baby animal.
In order to evaluate the influence of the diet in the composition of serum, plasma and red cells lipids, their fatty
acid profiles will be evaluated in some restricted populations, including weight loss or pathologies like diabetes.
Quince
To finish a series of works involving the chemical characterization of quince fruit and its jam, that constituted
the work plan of one of our PhD students, a group of chemically related terpenoid compounds were identified
by spectroscopic means. These compounds were described for the first time in nature and can be used as a
tool for the characterization of quince and its jam.
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Quince fruit (pulp, peel and seed) and jam antioxidant activity was also evaluated. The phenolic fraction
always exhibited a stronger antioxidant activity than the whole extract, while organic acid extracts always
displayed the weakest potential. The results showed that peel extract was the one with the highest antioxidant
capacity. Among the phenolic fraction, the seed extract exhibited the stronger activity, while for organic acids
fractions the peel extract presented the highest antioxidant potential. The antioxidant activity was correlated
with the caffeoylquinic acids and ascorbic and citric acids contents.
Edible mushroom species
The organic acids composition of 6 wild edible mushroom species (Amanita caesarea, Boletus edulis, Gyroporus
castaneus, Lactarius deliciosus, Suillus collinitus and Xerocomus chrysenteron) was determined. The results
showed that all of the samples presented a profile composed by at least 5 organic acids: citric, ketoglutaric,
malic, succinic and fumaric acids. Several samples also contained oxalic, ascorbic, quinic and shikimic
acids. The relative amounts and the presence/absence of each identified compound may be useful for the
differentiation of the species.
In order to check the influence of the conservation procedure in the chemical composition of another wild
edible mushroom species, the chanterelle (Cantharellus cibarius), phenolic compounds and organic acids of
samples preserved under four different conditions (drying, freezing, conservation in olive oil and in vinegar)
were determined. The results showed that chanterelle is characterized by the presence of 6 phenolic compounds
(3-, 4- and 5-O-caffeoylquinic acid, caffeic acid, p-coumaric acid and rutin) and 5 organic acids (citric, ascorbic,
malic, shikimic and fumaric acids). Samples preserved in olive oil also exhibited hydroxytyrosol, tyrosol,
luteolin and apigenin, while conservation in vinegar lead to the detection of hydroxytyrosol, tyrosol and tartaric
acid in the analysed samples.
Tronchuda cabbage
Within the scope of a project regarding the chemical characterization and the evaluation of the antioxidant
potential of tronchuda cabbage (Brassica oleracea L. var. costata DC) the phenolic compounds from its
external leaves were determined and 14 glycosylated kaempferol derivatives were identified, some of them
acylated with cinnamic acids These acylated derivatives are reported for the first time in nature, with the
exception of kaempferol 3-O-(sinapoyl)-sophoroside. Quantification of the identified compounds carried out in
samples cultivated under conventional or organic practices and collected at different times showed that, in
general, samples from organic production exhibited higher total phenolics content than those from conventional
practices, collected in the same period.
The evaluation of the antioxidant potential, against superoxide and hydroxyl radicals and hypochlorous acid,
is in course.
Plants of possible biological interest
The influence of 3 different radiation intensities in the phenolic composition of Lavandula angustifolia in vitro
material (calli and shoots), grown in 4 culture media, was determined and qualitative differences were noticed:
calli samples revealed the presence of caffeic, 2-O-glucosylcoumaric, o-coumaric and rosmarinic acids, coumarin
and herniarin, while in shoot neither caffeic or rosmarinic acids were noticed. In general, phenolics production
was higher in shoots than in calli. In addition, shoot samples subjected to the highest radiation intensity
produced more phenolic compounds. The phenolic composition in the growth curve of calli samples was also
determined; the results suggested that its content increases during the first 10 days and stabilises from then
on.
In the course of a project regarding the valorization of Salvia officinalis, Melissa officinalis, Mentha piperita
and Lavandula angustifolia the phenolic profiles of in vivo material collected at 4 different vegetative stages
were established. L. angustifolia was characterized by the presence of 2-O-glucosylcoumaric acid, luteolin 7O-glucoside, apigenin 7-O-glucoside, coumarin, herniarin, luteolin and apigenin. M. piperita presented eryodictiol
7-O-glucoside, hesperidin, luteolin 7-O-glucoside, apigenin 7-O-rutinoside adn rosmarinic acid. In S. officinalis
only rosmarinic acid was detected. M. officinalis exhibited 3-O-caffeoylquinic and rosmarinic acids. In addition,
the phenolic composition of M. officinalis shoots (reproduced in 2 culture media) and plantlets (grown with 4
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distinct hormonal conditions) was also determined and 5-O-caffeoylquinic and rosmarinic acids and luteolin
were detected.
Contaminants
In the field of food contaminants we started a project (approved and financed by FCT) that aims the assessment
of the dietary exposure of Portuguese consumers to acrylamide, a toxic compound that results from the
reaction of asparagine and sugars in heat-treated carbohydrate-rich foods. With this goal we developed an
isotope labelled GC-MS analytical methodology that allowed the accurate quantification of the compound in
distinct kind of food matrixes. The referred method was applied to determine the levels of the compound in
some starchy food products that include chips, potato crisps, breakfast cereals, biscuits and crisp bread.
Furthermore, we started to study the role of some raw material properties in acrylamide formation during food
processing, namely in cheap frying.
Quantification of food additives
Global use of food additives has promoted an extensive concern in human population. Each commercialized
product must be safe and in agreement with legal requirements. Industries must therefore perform routine
analyses to their products. Simple, quick and selective methods with emission of low amounts of effluents of
small toxicity should therefore be implemented.
Electrochemical techniques were explored for this purpose, and applied to the determination of antioxidant
and acidity regulators in beverages. Vitreous, carbon paste and chemically modified electrodes were evaluated
in flow set-ups of amperometric detection. Optimization of injection volumes, flow-rates and reactor lengths
were performed to ensure optimum response of the detection device. At least one of these electrodes provided
suitable operating characteristics for the analysis of commercial food samples. This line of research is presently
under development, though it was already applied to the analysis of commercial samples. Accurate and
precise results were obtained for each analyte under study: ascorbic and citric acids.
Microbiology
The rapid emergence and spread of several antibiotic resistant bacteria in both clinical and community settings
during the last years have been facilitated by the use of antimicrobial agents in hospitals and in human
husbandry. In an attempt to control the problem of antibiotic resistance the European Union ban the use of
antibiotics that are analogues to those used in human medicine for purposes of growth enhancement in foodproducing animals. Our main objective is to characterize the contribution of food products to the spread of
resistant bacteria or resistance genes as well as the impact of antibiotics restrictions politics in their containment.
Following we express the achievements obtained in this field:
The evaluation of antibiotics residue incidence in edible tissue of healthy pigs and the occurrence of antibiotic
resistance in their enteric E. coli indicates a large use of sulphonamides in pig production in our country as
well as a high prevalence of E. coli resistant to b-lactams, sulphonamides and tetracyclines in their enteric
flora.
A high incidence of sulphonamide resistant Salmonella was observed in food-animal products. The presence
in these isolates of class 1 and class 2 integrons suggest that the persistence of several sulfonamide resistance
genes may be the result of the successive pressure exerted by sulfonamides and other antimicrobial agents
that are also commonly used and may be mitigated by the fact that not all sulfonamide-resistant determinants
exert a fitness cost.
he establishment and dissemination through the food chain of a Salmonella typhimurium clone that is different
from the widespread multi-drug resistant clone of S. typhimurium DT104, with decreased susceptibility to
therapeutically important broad-spectrum b-lactams, is a cause of concern.
Future work will establish the diversity of both resistant bacterial strains and their genetic elements involved
in antimicrobial resistance in Portugal, one of the EU countries with the highest antibiotic use in veterinary
medicine and with the highest rates of vancomycin resistance in enterococci.
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PHYSICOCHEMICAL CHARACTERIZATION OF FOOD PRODUCTS
1. Peptides from whey proteins
Separation and characterization of bioactive peptides resulted from pepsin, trypsin and Alcalase® hydrolysis
of whey protein concentrates were separated according to their polarity and weight, using chromatographic
methods
2. Beer composition and beer foam stability
Comparison between composition of two types of beer: with alcohol and without alcohol. Study of beer foam
stability, by characterization of protein profiles using reversed phase and size exclusion –HPLC. Contribution
of iso-alpha acids, organic acids, sugars, amino acids and others to foam stability.
Green chemistry methodologies were developed for characterization of volatile fraction of food matrices (beer,
honey, chocolate), these methodologies included headspace-SPME extraction and chromatographic separation
of more than 100 volatile compounds (alcohols, fatty acids, hidrocarbons, esters, ketones, aromatic compounds,
sulphur compounds, halogen compounds and aldehydes). Identification of these constituents was performed
by comparing the experimental spectra with those of the Nist 98 data bank.
Development of an HPLC method using a “light scattering” detector was developed for separation and
quantification of sugars in food matrices (beer, honey, dairy products).
Monitorization of aflatoxins in spices by HPLC/Fluorescence after suitable sample extraction steps. (1 artigo
A GC-MS method was developed for the quantitative analysis of heterocyclic aromatic amines in coffee.
Based on the results achieved the major carcinogenic amines seem to be absent in roasted coffee. On the
other hand, two co-mutagenic beta-carbolines, harman and norharman, were detected in high amounts. Studies
are now being conducted by an M Sc. to develop a faster and accurate methodology for their quantification in
both green and roasted coffees. This student has received a scholarship from IBESA and the studies are
being conducted in collaboration with a local coffee industry.
Na expedite methodology was developed for the accurate determination of ethylcarbamate in alcoholic
beverages.
Planned research activities for 2005
Volatile compounds fraction from honey, beer and chocolate: Correlation with its sensory characteristics.
Quality and nutritional value of ice creams from market, integration of this food in a healthy diet.
Characterization of two varieties of barley Hordeum vulgare L. cv. Scarlett e cv. Prestige: protein profile at
different germination time, evaluated by SDS-PAGE and RP-HPLC/UV.
Separation and characterization of bioactive peptides from whey hydrolysis to be applied in food and
pharmaceutical industries: peptides resulted from pepsin, trypsin and Alcalase® hydrolysis of whey protein
concentrates will be separated and characterized according to their polarity and weight, using chromatographic
methods. Sensory analysis will also be performed to assess whether these peptides present or not bitter
characteristics. The study of amino acid composition and sequence will be performed by automated Edman
degradation using a Applied Biosystems LC 491 Protein Sequencer. Sequences can be obtained from small
amounts of material with high yield and, in optimum conditions, for ca. 40 N-terminal amino acid residues.
Thus, it is possible a complete, or quasi complete, primary structure determination. The sequences obtained
will be compared to known whey protein sequences. These results will unambiguously identify peptides
potential bioactive peptides obtained from b-LG and a-LA as well as from other less abundant whey proteins.
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ENVIRONMENTAL CONTROL AND REMEDIATION
Head of laboratory: Cristina Delerue Matos, Professor Coordenador / Helena Soares, Assistant Professor
Staff Members:
Maria Leonor Madureira Pinto
Maria do Carmo Veiga Fernandes Vaz
Simone Barreira Morais
Assistant
Assistant
Sónia Adriana Ribeiro da Cunha Figueiredo
Assistant
Maria Isabel Branco Alves Martins
Professor Adjunto
Maria Teresa Pereira de Oliva Teles Moreira Professor Adjunto
Jorge Manuel Pinto Jesus Garrido
Professor Adjunto
Abel José Assunção Duarte
Assistant
Florinda Figueiredo Martins
Assistant
Hendrikus Petrus Antonius Nouws
Assistant
Maria de Fátima de Sá Barroso
Assistant
Maria João Dantas Ramalhosa Ferreira
Assistant
Maria Manuela Barbosa Correia
Assistant
Olga Manuela Matos de Freitas
Assistant
Valentina Maria Fernandes Domingues
Assistant
Maria Isabel Viana de Brito Limpo de Serra
Assistant
José Tomás Veiga Soares de Albergaria
Technician
Maria Aurora Soares da Silva
Technician
Sérgio Alberto Cruz Monteiro de Morais
Technician
Paula Celeste Baptista Paíga
Technician
Ph.D. Students:
Carina Machado, Cristina Maria Rodrigues Ferreira Alves, Oriza Paula Guedes Tavares
Pesticides analysis
Determination of pesticide residues in food and environmental samples has received much attention in the
last few decades. Following the need to develop faster, cleaner and more reliable analytical methodologies
new chromatographic and electroanalytical-based methods have been developed.
Concerning chromatographic procedures such as HPLC-DAD, GC-ECD or MS, they have been applied for the
analysis of wine, must, grape and water. Solid-phase extraction (SPE) and microextraction (SPME) have
been used to extract the analytes studied for chromatographic analysis.
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Voltammetric procedures for the determination of atrazine, ametryn, dialifos, chlorfenvinphos and tribenuronmethyl in soil samples using gold or mercury film ultramicroelectrodes were developed. Ultramicroelectrodes
exhibited several attractive features. Before analysis, microwave-assisted solvent extraction was performed.
This strategy is effective when compared to traditional extraction techniques, providing reduced extraction
times, reduced solvent consumption and increased sample throughput.
Other electroanalytical techniques were used to analyse natural waters and commercial phytopharmaceuticals.
Voltammetric, amperometric and potentiometric based procedures were used for this purpose. All experiments
were adapted to flow conditions, with the main goal of reducing time and costs, as well as operator intervention.
These electroanalytical approaches generated also effluents of small toxicity.
Laboratory Waste Management
“Greening” of chemistry has clearly brought many advantages. Still, wastes from chemical analytical laboratories
are typically hazardous and present a high diversity. The fact that only small quantities are generated has
justified negligence of their existence or has induced laboratories to hire specific companies to perform this
task.
Thus, a waste management program (WMP) was created and is being implemented for supporting all laboratory
activities of students in a Chemical Engineering degree. The WMP is responsible for waste separation/
disposal strategy, collection, and characterisation of all inorganic liquid toxic wastes produced. Once entering
the research laboratory, a proper chemical treatment is investigated and implemented. Resulting solids are
isolated, purified, and evaluated in terms of purity and contaminants. When possible, solids are redirected as
reagents towards another laboratory experiment. This feature provides a sustainable perspective to the WMP.
Before discard in the public sewage system, all liquid effluents emanating from suitable treatment are chemically
controlled.
Still, fulfilling chemical regulations does not ensure lack of adverse effects in living organisms when aquatic
environment is chosen as final destiny for liquid treated wastes. Thus, Chlorella vulgaris and Daphnia magna
have been exposed to these wastes in order estimate inherent chronic toxicity effects. As comparison,
untreated wastes have also been subject of ecotoxicity trials.
Soils remediation
Existence of contaminated soils is an undeniable environmental distrustful fact. This has led scientific and
technical communities to search solutions for their remediation. Here, the possibility of using in-pulp solvent
extraction technique for remediation of soils contaminated with petroleum hydrocarbons was explored. Ternary
systems of ethyl acetate–ketone–water were studied for this purpose. These solvents are of small environmental
concern and present the ability of establishing a single phase mixture thus providing a more efficient contact
with soils. Contaminants used in this study were 2,4-trimethylpenthene, xylene, naphthalene and hexadecane.
Analytical control was done by gas chromatography. Parameters under evaluation are: kinetics of extraction;
extractant/soil phase ratio; and efficiency of single or multiple contacts on the extraction process. The effect
of some soil parameters such as organic matter content and grain size distribution were also assessed. The
solvent regeneration by distillation was also appraised. Global results are promising, demonstrating that inpulp solvent extraction is technically a feasible option for remediation of aromatic, polyaromatic and linear
hydrocarbons.
Removal of toxic compounds by means of adsorption strategies
Adsorption plays a key role in modern industries, especially in the field of environmental protection engineering,
with the increasing environmental awareness of people all over the world. Many companies are looking at
tertiary treatment processes to remove contaminants from their effluents.
Several studies are in development with (inexpensive) natural adsorbents: cork, algae and wastes from corn
production.
One of them explores the adsorption capacity of cork (Quercus suber L.) to remove pyrethroids from waters,
in order to attenuate the impact of this compound in aquatic life. An ecotoxicological evaluation showed that
synthetic pyrethroids are amongst the most toxic pesticides for aquatic organisms.
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Other study aims to explore the ability of Portuguese macro algae species (Laminaria hiperborea, Bifurcaria
bifurcate, Sargassum muticum and Fucus spiralis) for the removal of toxic metals (Cd(II), Zn(II) and Pb(II))
from aqueous solutions.
Finally the wastes (Zea mais) from corn production have been tested successfully as adsorbents for textile
dyestuffs and heavy metals Cu (II) and Pb (II).
Kinetic studies were conducted using batch adsorption experiments.
Environmental studies of “Good” Buffer
The influence of TAPS on solutions containing Cd, Cu, Pb and Zn and of AMPSO on solutions containing Cd,
Pb and Zn was studied by GEP and DCP. Several complexes were identified for each ligand-metal ion pair and
their global stability constants were determined (three papers in preparation).
In 2005, the work will pursue with the complexation study of additional ligand-metal ion pairs (POCTI/QUI/
39950/2001).
Recycling of aluminium salt from sludges
Total metals concentrations (Al, Ca and several heavy metals) in two types of sludges were determined. Then,
acid and alkaline dissolution for leaching aluminium from sludges was performed. The results obtained have
shown that acid leaching is more efficient and less expensive.
In 2005, conditions for recovering alum will be optimized. (Project: U. do Porto/Fundação Ilídio Pinho, 2003).
Biorremediation of wastewaters using microorganisms
The main aim of this project, which starts in 2005, is to develop a clean technique, using yeast brewer’s cells,
for the treatment of wastewaters containing heavy metals (POCTI/CTA/47875/2002).
Control of heavy metal pollution
Monitorization of heavy metals in natural waters and sediments collected nearby abandoned mines was
performed. Results evidenced contamination for several metals, particularly for arsenic.
New environment-friendly chelating agents for industrial and domestic applications
This is a multidisciplinary project, recently financed by REQUIMTE, which involves researchers from the two
centers of REQUIMTE.
The project starts in 2005 with the synthesis of the ligands. Then, biodegradation and metal complexation
studies will be performed to check the environmental impact of these ligands and their strength as metal
chelators, respectively. (Project: REQCHI, 2004).
Environmental Microbiology
The surveillance of the evolution of antibiotic resistance is an actual priority that includes, as fundamental
points, the investigation of the risk factors for the propagation and understanding the spreading of resistant
strains in different ecological niches, as in the environment. Wastewater, especially from hospitals, can work
as reservoirs of resistant bacteria and of transferable resistance genes in the environment. In that way,
sewage may function as an ecological niche suitable for antimicrobial resistance dissemination and wastewater
systems may contribute for environmental spreading of resistance. The detection of extended-spectrum betalactamases producers carried on sewage system downstream hospital discharges, confirmed a method in
that extended-spectrum beta-lactamases producers may function as biomarkers for detection of antibiotic
multi-resistant bacteria in the environmental compartment. This biomarker may be used to track the origin
and fate of these multi-resistant pathogens or genes, in different ecological niches, providing a tool for public
health and environmental protection.
Future work in environmental dissemination of antibiotic resistant bacteria and resistance genes, will be
assessed by screening of resistant bacteria, characterization of antimicrobial resistance mechanisms and
detection of antibiotics in different ecological niches, namely urban sewage plants, river water, seawater and
in three types of swine productions (intensive, domestic and sustainable).
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ANALYTICAL METHODOLOGIES
Head of laboratory: Aquiles de Barros, Associate Professor
Staff Members:
José António Maia Rodrigues
Assistant Professor
Paulo Joaquim Ferreira de Almeida
Assistant Professor
Luís Guilherme de Lima Ferreira Guido
Assistant
Maria Isabel Afonso Rocha
Assessor
Maria Fernanda Rocha M. L. O. Cabral
Associate Researcher
Cristina Maria F. Delerue Alvim de Matos
Assistant
Maria do Carmo Vaz
Assistant
Assistant
Maria de Fátima Sá Barroso
Assistant
Hendricus Petrus Antonius Nouws
Assistant
Paula Celeste Baptista Paíga
Assistant
Post-Doct Fellows:
Pedro Miguel Gonçalves Rodrigues
Ph.D. Students:
Andreia Filipa da Silva Curto
M.Sc. Students:
Marta Sofia Roma Pires, Maria Fernanda Andrade Resende, Sérgio José Pinto Teixeira
Number of Ph.D thesis: 1
Number of M.Sc thesis: 1
The strong lines of the project announced for the triennium 2003-2005 are the consolidation of the growing
investigation related with beer and the diversification of the analytical techniques used. In this context a
special reference is made to the research that is being devoted to the studies related with the problem of beer
ageing, no doubt the main particular topic considered in the development of the project. Anyway, it is important
to note that voltammetric analysis still represents an important field of investigation in the context of the
project, as a continuation of the research produced before 2003.
The main activities under development during 2004 have been:
The cooperation with Unicer, Bebidas de Portugal SGPS, S A - Strengthening of the good relationship that
already exists with the main Portuguese brewery. As result of this cooperation, several papers and
communications in congresses were produced and a three years joint research approved by “Agência de
Inovação” was signed. This research, initiated last April, is based on a patent meanwhile submitted (already
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approved in Portugal and in phase of EC approval). It consists in the development of an equipment for the
voltammetric determination of diacetyl directly in the fermentation vessels and involves two other companies:
Carlsberg S/A and CAI.
The cooperation with Carlsberg S/A, Denmark - The joint work previously referred involving this important beer
company will allow the intensification of a collaboration that exists since the year 2000.
The cooperation with Institut Français de la Brasserie et de la Malterie, I. F. B. M., Nancy, France In the
context of a collaboration involving Unicer and I. F. B. M., the Ph. D. student of this group Andreia Curto went
to UCL (Université Catholique de Louvain, Belgique) to attend a 3 months course in beer and is now going to
I. F. B. M. to do post-graduate training of 6 months, in the context of a Ph. D. “mix grant” that she obtained
from FCT. The main objective of this joint research is the study of the impact of several technological factors
of the malting and brewing processes on the organoleptic stability of beer.
Another branch of the research worthy of mention is:
The cooperation with the Department of Civil Engineering of the Faculty of Engineering of Porto – It is a
collaboration essentially devoted to studies on the accelerated degradation of geosynthetics; the work involves
the investigation of the behaviour of these materials after being submitted to the effect of some chemical
agents, under especially intense degradation conditions.
As a result of the investigation activity of the group in 2004, some papers were published in international
journals and several communications were presented in congresses; it was also possible to finish 1 Ph. D.
thesis and 1 M. Sc. thesis. For 2005 a similar production is expected. In 2004 it is worthy to note the
beginning of the work of 2 new Ph. D. students, Andreia Curto (FCT grant, initiated in March 2004) and Henri
Nouws (PRODEP grant, in collaboration with ISEP, where he is Assistant).
Green chemistry methodologies were developed for characterization of volatile fraction of food matrices (beer,
honey, chocolate), these methodologies included headspace-SPME extraction and chromatographic separation
of more than 100 volatile compounds (alcohols, fatty acids, hidrocarbons, esters, ketones, aromatic compounds,
sulphur compounds, halogen compounds and aldehydes). Identification of these constituents was performed
by comparing the experimental spectra with those of the Nist 98 data bank.
Development of an HPLC method using a “light scattering” detector was developed for separation and
quantification of sugars in food matrices (beer, honey, dairy products).
Monitorization of aflatoxins in spices by HPLC/Fluorescence after suitable sample extraction steps.
A GC-MS method was developed for the quantitative analysis of heterocyclic aromatic amines in coffee.
Based on the results achieved the major carcinogenic amines seem to be absent in roasted coffee. On the
other hand, two co-mutagenic beta-carbolines, harman and norharman, were detected in high amounts. Studies
are now being conducted by an M Sc. to develop a faster and accurate methodology for their quantification in
both green and roasted coffees. This student has received a scholarship from IBESA and the studies are
being conducted in collaboration with a local coffee industry.
An expedite methodology was developped for the accurate dtermination of ethylcarbamate in alcoholic
beverages.
Electroanalysis
Electrochemistry is often required in the analytical chemistry field for its selectivity, sensitivity, and generation
of effluents of small toxicity. It may also provide information concerning oxidative/reductive mechanisms.
Different voltammetric-based methods were developed, exploiting oxidation/reduction of the analyte as well
as its adsorption, occurring at glassy carbon or hanging mercury conventional electrodes or ultramicroelectrodes.
Development of flow methods was done by coupling amperometric wall-jet cells to flow injection analysis setups. Different working electrodes were used, including glassy carbon, carbon paste and chemically modified.
This strategy was applied to the analysis of different compounds within pharmaceutical and environmental
fields.
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Ion-selective electrodes (ISEs) are selective and low cost detection devices responding to wide concentration
ranges. However, their analytical features depend on the configuration and on the selective membrane
composition. ISEs were constructed with a cylindrical configuration to provide a laminar flow inside a FIA
system, decreasing dead volumes and the response time of the detector. For preparing selective membranes
nature of ionophore and of plasticizer employed were considered. Detectors with the best performance were
applied to the analysis of pharmaceuticals.
Plan for 2005
For 2005, the objective of the group is to continue the activity of 2004, with special focus on:
— the consolidation of the work conducting to the construction of an equipment for the determination of
diacetyl on line (in collaboration with the companies Unicer, Carlsberg and CAI); also, it is expected that there
are some news about the European Patent that is pending on this subject;
— the continuation of the study of the impact of several technological factors of the malting and brewing
processes on the organoleptic stability of beer (also involving IFBM and Unicer);
— the continuation of the studies on the accelerated degradation of geosynthetics, in collaboration with the
Department of Civil Engineering of the Faculty of Engineering of Porto, involving a Ph. D. student.
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PHYSICOCHEMICAL CHARACTERIZATION OF FOOD PRODUCTS
Head of laboratory: Maria do Pilar Gonçalves, Associate Professor / Alberto Sereno, Associate Professor
Staff Members:
Loïc Hilliou
Post-Doct Fellows:
Marta Isabel de Glória V. M. Silva
Ph.D. Students:
Luis Mayor Lopez, Wancheng Sittikijyothin, Fabio Donato Soares Larrotonda
Number of articles in books: 6 (12-17)
Development and chemical, structural and rheological characterisation of protein/ polysaccharide
mixed aqueous systems
Studies were conducted on the heat-set gelation of whey proteins triptic hydrolysates, at pH 7.0, and on the
rheological properties of the cured gels. Protein/ peptides concentration and degree of hydrolysis were varied
in the experiments. Increasing the degree of hydrolysis resulted in a decrease of the gelation capacity of the
proteins, probably due to a variation of the covalent bonds/ hydrogen bonds contribution to the network
formation.
The effect of the addition of tara gum (a neutral polysaccharide) to the whey proteins or their hydrolysates on
the rheological properties and microstructure of heat-set gels, at pH 7.0, were also studied. Different
microstructures were observed by confocal laser scanning microscopy, depending on the protein/polysaccharide
ratio; in all cases, a segregative phase separation was observed with a protein enriched continuous phase.
The rheological properties of the final gels
reflected these differences. (Project POCTI/QUIM/36452/2000).
Rheological behaviour and morphology of protein-polysaccharide mixed systems
a) Studies were conducted on the viscoelasticity of b-lactoglobulin / galactomannan (locust bean or tara
gums) mixed solutions and gels, at two different pHs (7.0 and near the isoelectric point of the protein). The
microstructures of these systems were analysed using confocal laser scanning microscopy. All the studied
systems were two-phase even before gelation. Their microstructures depended on the protein/ galactomannan
ratio and on the pH, as well as their viscoelastic properties.
In 2005, the effect of shear on b-lactoglobulin gels, pure or mixed with galactomannans, will be investigated
using dynamic oscillatory measurements. Different shear rates, time of shear and temperature conditions will
be tested.
b) The rheological behaviour of dispersions of partially hydrolysed waxy maize starch (HWS), whey protein
concentrate (WPC) and urea was followed by small-amplitude oscillatory measurements, under heating to
80°C and cooling to 25°C, at pH 7.5. At the lowest HWS/WPC ratio, a thixotropic structure was characterised
under steady-shear flow, and visualised by light microscopy as a continuous network. With increasing HWS/
WPC ratio within a certain range, gelling and non-gelling mixtures resulted in phase-separated structures,
which hindered viscous flow. At the highest HWS/WPC ratio, a shear-thinning dispersion was formed, consistent
with the liquid-like small deformation properties and the homogeneous image obtained by light microscopy. In
2005, this work will be concluded with the analysis of the influence of urea and/or WPC over HWS dispersions
(project GRICES / CAPES).
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Cold gelation of globular proteins
a) The textural properties of several WPC cold-gels with and without tara gum were studied. The samples
were subjected to penetration, compression and TPA tests in a TA-XT2 texture analyser. Each sample was
characterized through values of rigidity, stress at rupture, toughness, cohesiveness, etc. It was verified that
the gel properties were mainly affected by the protein and tara gum concentrations.
b) The Mg2+-induced gelation of a whey protein isolate (WPI) was studied. A phase diagram was determined
by varying heat-denatured protein and salt concentrations. The phases were characterized through rheological
and textural measurements. In 2005, the influence of Mg2+ and tara gum concentrations will be studied, at
25ºC, through rheological dynamic measurements at fixed frequency, and the gels obtained will be characterized
through frequency and strain sweep tests.
Technology for edible biodegradable films and coatings for foods
In the frame of a project aiming at engineering new green edible films for food coatings and packaging (POCTI/
EQU/45595/2002), the potential use of seaweeds collected on the Portuguese coast as a source of natural
thickening and gelling agents has been screened. Infrared spectroscopy and proton NMR spectroscopy
performed at CQFB revealed that Mastocarpus stellatus is essentially a k-i hybrid carrageenan, whereas
Gigartina Pistillata is from the l type. For both phycocolloids, minor seasonal variation of the chemical structure
was noticed. For Mastocarpus Stellatus, extraction parameters strongly influenced the ultimate gel strength,
gel elasticity and gel melting temperature, which were determined by DSC, linear and non linear rheology, and
texture analysis. Preliminary results have been presented at IBEREO 2004, and 3 papers are to be prepared.
In 2005, the formulation of edible films which will incorporate the carrageenans extracted and characterized
above will be developed. Strategies for film production and on-line characterization will be experimented,
possibly in the frame of an industrial cooperation.
Controlling the microstructure of food products by rheo-optical methods.
A project aiming at transferring rheo-optical techniques to model food product characterization has been
submitted and granted by the FCT (POCTI/EQU/58064/2004). The associated work plan will be initiated
whenever funding will become available. This project is connected with two international cooperation projects
which have been submitted (GRICES/DAAD and GRICES/GREECE projects). They propose a new model
food characterization by NMR imaging and Fourier Transform Rheology, and an investigation of the gel-setting
mechanism in model carrageenan biopolymers.
Physical characterization of PHA biopolymers
A project aiming at synthesizing new PHA biopolymers by mixed microbial cultures has been proposed by
and granted by the FCT (POCTI/BIO/55789/2004). The group participation to this project involves the mechanical
and thermal characterization of PHA which are to be first produced.
In 2005, it is intended to implement new equipment on existing machines in the laboratory, in order to allow
the planed characterization of forthcoming PHA samples.
Seaweed integrated aquaculture for the production of new Portuguese algal biopolymers.
A project aiming at extracting biopolymers from seaweeds utilized in aquaculture for cleaning processes has
been proposed to the FCT (POCTI/MAR/59635/2004). Unfortunately, this proposal (where 4 institutions will
merge their research efforts, namely REQUIMTE-CEQUP, REQUIMTE-CQFB, University of Aveiro, and CIIMARPORTO) has not yet been reviewed and consequently no research activity is to be reported.
Studies in Food Structure
Several studies were performed at different structural levels. The food materials used in these tests were
apples and pumpkin crops.
Macrostructural level: changes in mechanical properties (compression tests), density and porosity of vegetable
tissue during different dehydration processes (osmotic dehydration and convective drying).
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Microstructural level: changes in cellular vegetable tissue during the aforementioned dehydration processes.
These changes were studied by means of image analysis with computer tools.
In 2005, it is planned to establish relations quality-macrostructure-microstructure of processed food materials,
using mathematical models.
The studies will be focused on the osmotic dehydration processing of vegetable tissue, namely pumpkin
fruits.
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TOXICITY AND PROTECTION STUDIES
Head of Laboratory: Maria de Lourdes Bastos, Full Professor
Staff Members:
Felix Carvalho
Assistant Professor
Fernando Remião
Assistant Professor
Helena Carmo
Assistant
Maria Elisa Soares
Assessor Principal
Eulália Mendes
Assessor Principal
Carla Rodrigues
Technician
Maria João Marques
Researcher
Ph.D. Students:
Márcia Cláudia Dias Carvalho, João Paulo Soares Capela, Ricardo Jorge Dinis Oliveira , Joana Andrea Soares
Amaral
M.Sc. Students:
Luis Correia, Renata Silva
Number of articles in scientific journals 17 (144,147, 186-200)
Number of articles in books: 1 (18)
Studies of mechanisms of toxicity using in vitro and in vivo models
Contributing for the universal aim of reducing the number of animals used in the toxicological evaluation of
compounds, the in vitro models proportionate the establishment of the mechanisms of expression of toxicity
at the cellular and molecular levels.
The studies performed in vitro were carried out in suspensions of isolated cells, namely freshly isolated rat
hepatocytes and cardiomyocytes. By using these cell suspensions, the toxicity of 3,4methyledioxymethamphetamine (MDMA; ecstasy) and its catecholic metabolites, á-methyldopamine, and
N-methyl-á-methyldopamine as well as the formation of glutathione adducts of these toxic compounds, was
evaluated.
Other validated in vitro models, namely the microcrustaceous Daphnia magna and Artemia parthenogenica,
as well as the microalgae Tetraselmis chuii and the mosquitofish Gambusia holbrooki were used for the
evaluation of environmental contamination by acethylcholinesterase inhibitors and pharmaceutical drugs.
Interference with xanthine oxidase activity can result in an imbalance in antioxidant/oxidant production, either
by increasing the production of reactive oxygen species (ROS) or by decreasing antioxidant defences.
The toxic effects elicited by d-amphetamine and ecstasy at the skeletal muscle level, in rested and exercised
mice, was evaluated and the respective mechanisms involved were elucidated.
A comparative study also using isolated rat hepatocytes was performed for the evaluation of the toxicity/
safety of metal complexes (Cu, Va and Zn) which are potential antidiabetic drugs. This study will be pursued
in order to clarify the mechanism of cellular lesion of some of these complexes.
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Studies in rat cardiomyocyte suspensions are being performed and will be continued in order to clarify the
mechanism of toxicity reported for catecholamines oxidation as well as the mechanism of toxicity of ecstasy
and metabolites in this in vitro model. It was evaluated, using an in vitro model, the interaction between 1,3dipropyl-8-sulfophenylxanthine (DPSPX) and xanthine oxidase in order to ascertain its putative contribution to
the hypertensive state and cardiovascular injury observed in DPSPX-treated rats.
An HPLC methodology was implemented for the quantification of MDA in cell suspensions and liver, after
complexing this compound with thiobarbituric acid (TBA). The implemented methodology was validated and
allowed a reliable separation and quantification of MDA, with high precision and recovery, and a low detection
limit.
Biotransformation of compounds: in vivo and in vitro studies
It is very well established that for many compounds, their biological effects are mainly due to their
biotransformation products. Also, the knowledge of the biotransformation profile of new compounds constitutes
one of the first requirements in order to find the animal model most similar to human for further toxicological
evaluation and reliable extrapolation for the human situation.
These biotransformation studies have been performed by using in vitro models, namely, the cryopreserved
hepatocytes from several animal species (monkey, dog, rabbit, rat and mouse) including human. These cells
were used to evaluate the comparative metabolism of two designer drugs of abuse, 4-MTA and 4-bromo-2,5dimethoxyphenethylamine (2C-B), alongside with the evaluation of their toxic effects as evaluated by the loss
of ATP hepatocyte content.
For the study of the biotransformation of 2C-B it was also used an in vivo model, namely the mouse, and the
main metabolic pathways were constructed. It was found a great similarity between the mouse and human
metabolism which enable further toxicokinetic in vivo or in vitro studies with this species.
An in vivo biotransformation study was also performed to characterize the metabolism of adrenochrome
(reactive metabolite of adrenaline).
In line with these biotransformation studies, we are planning to evaluate the possible influence of genetic
polimorphism of the main metabolizing enzymes of the designer drugs of abuse 4-MTA and 2C-B in their toxic
effects. By using cell lines that express human metabolizing enzymes we will be able to identify which
enzymes are mostly involved in the detoxification of these drugs. Once the relationship between metabolism
and toxicity is well established, we will use human microsomal fractions profiled for the enzymes of interest
and incubate them with susceptible cell lines. We will then be able to determine if genetic polimorfism is in
fact related with the overexpression of the toxic effects of these designer drugs of abuse. Furthermore, an in
vivo model with transgenic mice, which is knock-in for the main human metabolizing enzymes will also be
used.
Biological activity of plant extracts and compounds
In recent years, there has been a worldwide trend towards the use of the natural phytochemicals present in
medicinal plants to which are attributed antioxidant properties.
In vitro models, both chemical and biological, were used for the evaluation of the antioxidant and radical
scavenging activities of plant infusions and isolated compounds.
The toxic versus antioxidant protective activities of the Essential Oil from Salvia officinalis was evaluated on
freshly isolated rat hepatocytes. The results showed that the EO is not toxic when present at concentrations
below 200 nl/ml; it was only at 2000 nl EO/ml that a significant LDH leakage and GSH decrease were
observed indicating cell damage. In the range of concentrations tested, the EO did not show protective effects
against t-BHP-induced toxicity.
The Hypericum androsaemum infusion was evaluated for its hepatoprotective effect against t-BHP-induced
toxicity in isolated rat hepatocytes, and in vivo studies, in mice. In vitro assays showed that the pre-incubation
of the cells with the infusion prevented t-BHP induced loss in cell viability and lipid peroxidation. The
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experiments will pursue in order to compare the effects in both models, to clarify the mechanisms of toxicity
and to verify if results obtained in vitro are extrapolable for the in vivo situation.
It was also shown that hesperidin and lipoic acid provided protective effects in vivo against As(III)-induced
acute toxicity in the liver and kidneys of mice. These compounds may potentially play an important role in the
protection of populations chronically exposed to arsenic.
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BIOMIMETIC SYSTEMS AND SIMULATION
Head of Laboratory: Maria João Ramos, Associate Professor
Staff Members:
André Melo
Assistant Professor
José Augusto Pereira
Assistant Professor
Pedro Fernandes
Invited Assistant Professor
Agostinho Antunes Pereira
Nelson Brito
Technician
Post-Doct Fellows:
Elsa Henriques
Ph.D. Students:
Susana Pereira, Rute Fonseca, Nuno Cerqueira, Fátima Lucas, Akapong Suwattanamala, Vineet Pande,
Sérgio Sousa, Ricardo Branco, Zenaida Mourão, Irina Moreira, Alexandre Carvalho
Project Grantee:
Alexandra Teresa Carvalho
Molecular Modelling and Simulation
Just as in the past few years, we have been generally interested in the molecular modelling of biological and
chemical systems. To further these studies, we have been using both molecular simulations and quantum
mechanics techniques, as well as quantum mechanics/molecular mechanics (QM/MM) or quantum mechanics/
quantum mechanics (QM/QM) hybrid methods. This year however, we have added to our present, general
interest in proteomics, a new line of computational genomics. Our idea is to eventually establish a close link
between both areas, which we have already started doing. A more detailed description concerning our main
present interests, which have derived from year 2003 and will continue in 2005, follows:
Disease Related Research
Cancer
(i)
The study of the catalytic and inhibition mechanisms of enzymes P450 1A2 (an enzyme involved
in the metabolic pathway of carcinogenesis) and RNR 1,2,3,4 (ribonuclease reductase, an enzyme thought to be
involved in cancer), resorting to both quantum mechanics, QM/QM and QM/MM methods, has advanced very
much. These studies have been financed by the National Foundation for Cancer Research, U.S.A. (P450
1A2) and by the Fundação para a Ciência e Tecnologia, Portugal (RNR) via project POCTI/35376/QUI/2000.
(ii)
We are further involved in the study of the catalytic and inhibition mechanisms of other enzymatic
reactions, some of them also thought to be involved in cancer, such as superoxide dismutase and farnesyl
transferase5,6,7.
(iii)
Continuation of the screening of 3,5 billion small molecules as potential inhibitors of 16 proteins,
directly involved in cancer, which are current targets of the pharmaceutical industry. This project is part of the
work carried out by the NFCR Centre for Computational Drug Design, based at Oxford, and directed by Prof.
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Graham Richard of which Maria João Ramos is an Associate Director, being financed by the National Foundation
for Cancer Research, U.S.A 8.
A.I.D.S.
(iv) The study of the problem of new potential therapeutic agents for the treatment of acquired
immunodeficiency syndrome (AIDS) by investigating the binding modes of different anti-AIDS chelators like
ATA, HPH and other analogs to achieve a better design of anti-HIV metal chelators, continued. Several
research works were published on this theme, as well as some reviews9,10,11,12. This project is being carried out
in close collaboration with Dr. Rakesh Sharma, an experimental organic chemist from Delhi University, in
India.
Hypertension
(v) To help on the modelling of mimetic modifications of bioactive peptides by inclusion of novel synthetic
amino acids, we have run extensive molecular dynamics on angiotensin II in both environments, water and
DMSO. This project is being worked on in close collaboration with an organic chemist (Prof. Hernâni Maia
from the University of Minho, Portugal), whom has agreed to syntethise the novel peptides, and being financed
by the Fundação para a Ciência e Tecnologia, Portugal, via project POCTI/35380/QUI/2000.
Malaria
(vi) Establishment of new antimalarial compounds based on electrostatic profiles of established drugs13. A
similar study has been performed on other systems, i.e. also based on the calculation and analysis of
electrostatic molecular potentials14. The establishment of new antimalarial compounds is being performed in
collaboration with Prof. Madalena Pinto from the Faculty of Pharmacy at the University of Porto, Portugal.
Drug Design Complementary Studies
(vii) Alanine scanning computational mutagenesis has been studied and a new methodology proposed. For
this reason, extensive molecular dynamics simulations of protein complexes has been carried out and
computational mutagenesis performed on the interface of these complexes, which has allowed the establishment
of the respective hot spots.
(viii) Continuation of the development of new formalisms to analyse the molecular interactions in association
processes15,16. This involves the partitioning of the physical observables of a molecular system into spatial and
physical meaningful components. These decomposition methodologies can be used as powerful tools for
molecular modelling and design of biological systems.
(ix) Efforts have also been made with imparting education on drug design17,18
Computational Genomics
Alzheimer’s disease
We have started a line of research on Computational Genomics with the goal of expanding the team’s research
experience on Bioinformatics, allowing the simultaneous integration of genomic19 and proteomic data. The
study of insertions of fragments of mtDNA into nuclear chromosomes is an important mechanism for the
evolution of genomes, and when targeted to gene loci, such insertions can be mutagenic, causing disease.
We have been studying this subject in more detail and additionally, we have been supervising research on the
evolution and comparative genomics of genes with important biological functions, namely the gene coding the
human enzyme ABAD that is linked to the mitochondrial toxicity in Alzheimer’s disease.
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References:
[1] S. Pereira; P. A. Fernandes; M. J. Ramos
Journal of Computational Chemistry, 2004, 25, 227-237
[2] S. Pereira; P. A. Fernandes; M. J. Ramos
Journal of Computational Chemistry, 2004, 25, 1286-1294
[3] N. M. F. S. A. Cerqueira; P. A. Fernandes; L. A. Eriksson; M. J. Ramos
Journal of Computational Chemistry, 2004, Vol. 25, 16, 2031-2037
[4] N. M. F. S. A. Cerqueira; P. A. Fernandes; L. A. Eriksson; M. J. Ramos
Journal of Molecular Structure (Theochem), 2004, Vol. 709, 53-65
[5] P. A. Fernandes; M. J. Ramos
Chemistry – A European Journal, 2004, 10, 257-266
[6] S. F. Sousa; P. A. Fernandes; M. J. Ramos
Biophysical Journal, in press
[7] S. F. Sousa; P. A. Fernandes; M. J. Ramos
Journal Biol. Inorg. Chem, in press
[8] http://www.chem.ox.ac.uk/cancer/ccdd.html
[9] P. A. Fernandes et al., Journal of Molecular Structure (Teochem), 2004, 685, 73-82
[10] R. K. Sharma; M. Otsuka; V. Pande; J. I. Inoue; M. J. Ramos
Bioorganic & Medicinal Chemistry Letters, 2004 Vol. 14, 6123-6127
[11] V. Pande; M. J. Ramos
Current Medicinal Chemistry, in press
[12] V. Pande; M. J. Ramos
Current Medicinal Chemistry, in press
[13] C. Portela; C. M. M. Afonso; M. M. M. Pinto; M. J. Ramos
Bioorganic & Medicinal Chemistry, 2004, Vol. 12, 3313-3321
[14] E. S. Henriques; N. Fonseca; M. J. Ramos
Protein Science, 2004, 13, 2355-2369
[15] A.R. F. Carvalho, A. Melo
Fundamental & Clinical Pharmacology, 2004, Vol.18 Suppl. 1, 23-126
[16] A. R. F. Carvalho; A. Melo
International Journal of Quantum Chemistry, in press
[17] M. J. Ramos; P. A. Fernandes; A. Melo
Journal of Chemical Education, 2004, 81, 72-75
[18] N. T. Brito, M. J. Ramos
Proceedings of International Conference on Education and Information Systems: Technology and Applications,
2004, 1, 79-82
[19] P. Gaubert, A. Antunes
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APPLIED BIOPHYSICS AND BIOCHEMISTRY
Staff Members:
Baltazar de Castro
Full Professor
José Costa Lima
Full Professor
Paula Gameiro
Assistant Professor
Maria Salette Reis
Assistant Professor
Eduarda Graça R. Fernandes
Assistant Professor
Carla Manuela S. Matos
Assistant Professor
Catarina Mansilha
Assistant Professor
Ph.D. Students:
Patrícia Neves, Sofia Costa Lima, Marlene Susana Dionísio Lucio, Helena Suzana da Costa Machado Ferreira,
David de Andrade Sousa Costa
M.Sc. Students:
Anabela Ferreira Gomes, Joana Moutinho da Veiga Marcelino, Ana Maria de Carvalhais Mendes Gomes
Number of articles in scientific journals: 12 (167,187,189, 212-220)
Biophysics of organized media
The interaction of the outer membrane protein OmpF in mixed micelles of o-POE/DMPC with fluoroquinolones
was studied by spetrophotometry and fluorimetry and the strength of the interaction increases with drug
“generation”, which is associated with a larger spectrum of activity and with smaller MIC values. The fluorimetric
results show the existence of to different binding sites for the drugs that can be related to the different location
of the protein tryptophanes. Supplementary support of the relationship between the structural properties of
this protein and the uptake of fluoroquinolones can be obtained in proteoliposomes. Different techniques were
used to prepare the proteoliposomes: dialysis, bio-beds and simple dilution. The conjunction of the two latter
shows to improve the homogeneity of the proteoliposomes as further extrusion allows obtaining very uniform
suspensions of unilamellar liposomes. Quenching studies are beginning to be performed in these
proteoliposomes and preliminary results show a great difference in protein conformation. Studies are being
conducted by other to better understand the role of OmpF in fluoroquinolones translocation.
Simultaneously, the minimum inhibitory concentration (MIC) of nalidixic acid, norfloxacin, ciprofloxacin and
moxifloxacin are being studied in several bacterial stains with series of porin mutants which lack one or
several major outer membrane proteins and the preliminary results show that OmpF seems very important for
the uptake of all the fluoroquinolones but not the nalidixic acid. Furthermore it seems that for ofloxacin and
norfloxacin OmpC can also be important. Synthesis of metal (Cu, Zn, Co, Ni) complexes with fluoroquinolonas
and ternary metal/fluoroquinolone/phenanthroline complexes are beginning to be performed and also some
studies to determine a possible activity of these new compounds in the bacteria stains. If these compounds
show to be active some toxicological studies will be performed.
For P-glycoprotein (Pgp) the use of its intrinsic Trp fluorescence to perform fluorescence quenching studies
with ATP and benzodiazepines showed the existence of independent binding sites these species and the use
of a mathematical treatment that allows the determinations of simultaneous equilibria in solution permitted to
obtain binding constants that showed that there is a precise requirement for ATP to alter benzodiazepines
bind, but ATP binding is independent of benzodiazepines presence. and these results seems to support the
alternating model described by Walmsley R. for Pgp transport mechanism. Studies with other drugs are being
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performed and some kinetics studies will be performed to try to obtain a better a«understanding of Pgp
transport mechanism.
The partition and location of non steroidal anti-inflamatory drugs (NSAIDs) within membranes, as well as the
overall physical perturbation they might induce to obtain as much information as possible about their interactions
with biomembranes, was been evaluated.
The partition coefficients (Kp) of NSAIDs between lipid bilayers of egg yolk phosphatidylcholine (EPC) unilamellar
liposomes (LUVs) and aqueous phase were calculated using derivative spectrophotometry and fluorescence
quenching.
The location of these NSAIDs within the bilayer was also determined by fluorescence quenching using a set
of n-(9-anthroyloxy) fatty acid probes (n=2, 6, 9 and 12) known as n-AS probes.
In addition, zeta-potential measurements were performed to evaluate changes in membrane surface resulting
from its interaction with NSAIDs.
Steady-state anisotropy measurements were made to determine the NSAIDs induced perturbation in membrane
structure at different depths of the bilayer.
To obtained a deeper insight into the anti-radical properties of NSAIDs, we have studied their antioxidant
activity in a membrane model which, when subjected to oxidative stress, is able to generate typical lipid
peroxidation cascade (oxy and lipid radicals) that takes place in tissues during the inflammatory reactions.
Thus, the study was undertaken in EPC liposomes where the peroxidative degradation of a fluorescence
probe (DPH-PA) was initiated by different types of radicals (hydroxyl and peroxyl radicals). The Fe2+/H2O2
system has been used to generate hydroxyl radicals and the water-soluble azo-compound AAPH to generate
carbon centered radicals by thermal decomposition. Peroxidation was simultaneously indicated by a decrease
in the probe’s fluorescence and by anisotropy measurements that detect changes in membrane rigidity. The
general screening DPPH and ABTS free radical methods, which evaluate the drugs’ radical scavenging
properties, also assessed antioxidant activity.
Apart from membranes, DNA is also a cellular component, which is particularly susceptible to oxidative
damage and may be involved in cancer and neurodegenerative diseases in which NSAIDs have been reported
to exert beneficial effects. Therefore, the inhibition of 2’deoxyguanosine oxidation to 8´-hydroxy-2’deoxyguanosine induced by Fe3+-EDTA/H2O2/ascorbic acid was accessed by HPLC.
During 2005 we propose the study of the NSAIDs interaction with membrane enzymes that are usually
involved in the inflammation processes using proteoliposomes. These proteoliposomes will be prepared using
the same kind of phospholipids and the enzyme under study. We also propose the study of the nature and
composition of liposome on the membrane interactions of NSAIDs. It can be done using liposomes prepared
with different kinds of phospholipids with or without cholesterol.
Scavenging activity studies for reactive oxygen and nitrogen species by non-steroidal antiinflammatory drugs (NSAIDs) and â-adrenergic antagonists
We have recently proposed that several NSAIDs may react with reactive oxygen species (ROS), namely
peroxyl radical (ROO.), hydroxyl radical (HO.), superoxide radical (O2.-), hydrogen peroxide (H2O2), and
hypochlorous acid (HOCl), and reactive nitrogen species (RNS), namely nitric oxide (.NO) and peroxynitrite
(ONOO-) produced at sites of inflammation. This type of effect has potential therapeutical relevance since the
antioxidant activity may strongly contribute for the final anti-inflammatory outcome to be attained by these
compounds. The scavenging activity is being evaluated using non-cellular in vitro methodologies, as well as
cellular systems, using Human neutrophils.
Several experimental and clinical evidences have linked an enhanced production of ROS and RNS to certain
diseases of the cardiovascular system. The involvement of enzymatic and non-enzymatic cellular and extracellular systems in the generation of these reactive species as well as in the antioxidant defense systems is
being evaluated. For this purpose, compounds known to induce cardiovascular diseases, like d-amphetamine
and 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), are being used in vivo, and the affected biological systems
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evaluated ex-vivo. In another approach, the antioxidant activities of â-adrenergic antagonists, which comprise
a group of drugs that are mostly used to treat cardiovascular disorders such as hypertension, ischemic heart
disease or cardiac arrhythmia, are also being studied. Today, there are evidences that ROS and RNS play an
important role in the pathology of cardiovascular diseases. On the other hand, the antioxidant properties
shown by some of the â-adrenergic antagonists have already been related to their therapeutic effects. Thus,
it seemed relevant to evaluate the scavenging activity for ROS and RNS by â-adrenergic antagonists. The
scavenging activity is being evaluated using non-cellular in vitro methodologies, for the ROS: O2.-, H2O2, .OH,
HOCl and ROO., and for the RNS: ONOO- and .NO.
During 2005, the reactions between NSAIDs with ROS and RNS will also be investigated using EPR tandem
mass spectrometry. Using the insights gained from the elucidation of the fragmentation patterns, the structures
of NSAIDs metabolites produced from in vitro reactions with chlorinating, oxidizing and nitrating reagents will
be tentatively deduced.
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BIOINORGANIC AND MEDICINAL INORGANIC CHEMISTRY
Head of Laboratory: Maria Rangel, Associate Professor
Staff Members:
Baltazar de Castro
Full Professor
Paula Gameiro
Assistant Professor
Eulália Pereira
Assistant Professor
Ph.D. Students:
Ana Claúdia Nunes
M.Sc. Students:
Maria João Amorim
Project Grantee:
Andreia Leite, Adelaide Miranda
Number of M.Sc thesis: 1
Design of orally active insulin-mimetic drugs. The design of these insulin-mimetic compounds involves
the synthesis of specific ligands, synthesis of the corresponding metal complexes and evaluation of its
chemical properties, toxicity and insulin-like performance. A set of chelators of the 3-hydroxy-4-pyridinone
type, with variable lipophilicity, and their corresponding metal complexes with VO(IV), Zn(II), Cu(II) and Cr(III)
have been synthesized and characterized in the solid state and in solution. Evaluation of the insulin-like
action of the synthesized complexes is being performed in collaboration with Prof. Hiromu Sakurai (Kyoto
University). This work is being developed under contract POCTI/QUI/35368/1999 (FCT, Portugal)
Design of functionalized siderophores to target infection processes. The design of new chelators is
crucial for treatment of diseases associated with iron overload and to prevent the growth of undesirable
bacteria associated to a great variety of infectious processes such as TUBERCULOSIS and AIDS. The work
that has been done in the current year and that will be pursued in the next one is focused on the design of
novel functionalized macromolecules to target infection processes and to act as intracellular fluorescent
probes. The work can be summarized as: (i) synthesis of new hexadentate chelators derived from 3-hydroxy4-pyrone and 3-hydroxy-4-pyridinone ligands; (ii) synthesis of fluorescent hexadentate ligands (ii)
functionalization of dextrans, with lipophilic substituents, siderophores and fluorophores.
Reactivity of B12 model compounds. The work in this project aims the establishment of structural factors
that may be determinant on the reactivity of photolysis products of B12 model compounds. We have identified
andcharacterized the photolysis products of cobaloximes and imino/oxime compounds with variable organic
radicals and nitrogen and phosphorus bases. The results obtained evidence that the primary products are
strongly dependent on the nature of the organic radical and are identical for the two different equatorial
moieties. This work is being developed under contract POCTI/QUI/46231/2002 (FCT, Portugal).
Metal complexes with cytotoxic effects. This work is performed in collaboration with Dr. Paula Marques
(University of Coimbra) and aims the synthesis of metal complexes of biogenic amines as potential anticancer drugs. The synthesis of palladium complexes was accomplished, and new synthetic methods for the
preparation of novel amines were developed in collaboration with Prof. J. E. Borges (CIQUP). The new amines
were used for the preparation of platinum complexes. All the new complexes are currently being studied
fortheir cytotoxic activity.
.
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COMPUTATIONAL STUDY OF METAL SURFACES
Head of Laboratory: José Ferreira Gomes, Full Professor
Staff Members:
Natália Cordeiro
Associate Professor
Alexandre L. Magalhães
Assistant Professor
Post-Doct Fellows:
Shuwen Yao
Ph.D. Students:
Hugo Santos, Akapong Suwattanamala
M.Sc. Students:
Ana Sofia Pinto
Number of Ph.D thesis: 1
COMPUTATIONAL STUDY OF METAL SURFACES
We are particularly interested in understanding the physical and chemical behaviour of heterogeneous systems
such as interfaces involving metals. The theoretical study of such systems is interesting on its own and is of
such difficulty that experimentalists find it very useful to complement their work with computational results;
many of our studies are thus being carried out in close collaboration with experimentalists. We have been
using mainly ab initio and DFT quantum methods. Representative of the studies performed in 2004 and to be
pursued in 2005, follows:
Modelling and interaction of species adsorbed on metal surfaces.
We have been studying the interaction of methoxy radical with copper and ruthenium surfaces using a cluster
model approach. The DFT results show that this approach gives a reasonable description of the clean and
pre-covered surfaces allowing a good prediction of the structural and energetics features of the adsorbate.
Furthermore, it shows that a reliable description of the experimental C-H stretching region of adsorbed methoxide
on Ru(0001) may be obtained.
This work is now being extended to new reactions and different metals, namely to the study of enantiomerically
pure metal catalysts, which can transmit chirality information and thereby be applied in enantioselective
synthesis/detection.
Calix[4]arenes are molecules that possess 4 aromatic moieties which can form π-electron-rich cavities and
behave as host compounds for ions and neutral species. Therefore, the adsorption of calix[4]arenes in electrode/
electrolyte interfaces is an interesting field to be explored to get more insight about the role and properties of
adsorbed molecules, as well as the nature of those interfaces and its applications to ion-selective electrodes
or potentiometric sensors. The work developed in this period has been concerned with the study of
thialcalix[4]arenes and their complexation with transition metal ions. We are further involved in the design of
new calixarene derivatives to help the experimental synthesis of potential host compounds of this type.
Selected References:
DJVA dos Santos, JANF Gomes, J. Phys. Chem. B, 2004, 108 (44): 17153-17159
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ASS Pinto, RB de Barros, MNDS Cordeiro, JANF Gomes, AR Garcia, LM Ilharco, Surf. Sci., 2004, 566-568:
965-970
A Suwattanamala, AL Magalhaes, JANF Gomes, Chem. Phys. Lett., 2004, 385 (5-6), 368-373
SM Fiuza, C Gomes, LJ Teixeira, MTG da Cruz, MNDS Cordeiro, N Milhazes, F Borges, MPM Marques,
Bioorg. Med. Chem., 2004, 12 (13): 3581-358
9HFP Martins, JP Leal, MT Fernandez, VHC Lopes, MNDS Cordeiro, J. Am. Chem. Soc. Mass Spectr.,
2004, 15 (6): 848-861
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NOVEL HETEROGENEOUS CATALYSTS
Head of Laboratory: Cristina Freire, Associate Professor / Baltazar de Castro, Full Professor
Staff Members:
Eulália Pereira
Assistant Professor
Uwe Pischel
Rita Ferreira
Assistant
Post-Doct Fellows:
Peter Eaton, Carla Alexandra Sousa, Ana Rosa Silva, Pankaj Das, Iwona K-Biernacka
Ph.D. Students:
Magda Martins, Andrea Carneiro, João Tedim
M.Sc. Students:
Rosa Bessada, Miguel de Sousa, Bruno Jarrais, Jorge Teixeira
Project Grantee:
Adelaide Miranda, Marek Kluciar
Novel catalysts by heterogenisation of metal complexes
We have pursed the preparation and characterisation of chiral and non-chiral manganese salen complexes.
The catalytic activity of the new complexes in the epoxidation of several alkenes was evaluated in CH2Cl2 and
CH3CN, using NaOCl, PhIO, terc-butylhydroperoxide, p-chlorometabenzoic acid as oxygen sources. The
experiments were done at room temperature and reaction time, the styrene conversion, chemical selectivity
of products (epoxide and benzaldehyde), respective yields and enantiomeric excesses were determined by
GC-FID. All the complexes showed catalytic activity in the experimental conditions used. The epoxidation of
alkenes was performed in a series of room temperature ionic liquids based on imidazolyl cation. The catalysts
were all active in these conditions, show a decrease in ee% but could be reused up to 3 times, although with
some decrease in ee% values. Complexes of Copper and Vanadium with acetylacetone have also been
prepared. Their catalytic properties in the azidination of styrene and epoxidation of alylic alcohols have been
evaluated.
The complexes of Mn (chiral and non-chiral) , Cu and V were anchored onto several supports: activated
carbons, pillared clays (PILCs), clays (Laponite) and mesoporous silica materials using several strategies
developed in our lab. All the materials were characterised by elemental analyses, XPS, SEM (EDS), XRD,
nitrogen adsorption, temperature programmed desorption (TPD), termogravimetry, EPR (Mn complexes), FTIR
and Uv-vis spectroscopies.
The catalytic properties of the new catalysts were determined in several heterogeneous reactions: (1) epoxidation
of several alkenes in CH2Cl2 and CH3CN, using the same oxygen sources, using equivalent experimental
conditions of those used in homogeneous phase, (2) aziridination of styrene and (3) epoxidation of allylic
alcohols. The same catalytic properties were evaluated (reaction time, styrene conversion, chemical selectivities
of products, epoxide and benzaldehyde and respective yields and enantiomeric excesses). For these catalysts
we have evaluated the leaching of the active phase and their reusabibility. All the catalysts showed catalytic
activity: the reaction times were larger than those observed in homogeneous phase, in some cases the
alkene conversion decreased, but chemical selectivities were quite similar to those of homogeneous media.
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Non-chiral Mn complexes immobilised in activated carbons and PILCs did not show leaching of Mn centres
and could be reused until 3 times without the loose of significant catalytic efficiency. For quiral Mn complexes
there is generally, a small decrease in ee%, when compared with those in
homogeneous media, and in some cases ee% did not decrease with reuse. All the catalysts of Cu and V
could be reused with a decrease in their catalytic properties.
FUTURE WORK
(1)
Design and preparation of new chiral complexes with homogeneous catalytic properties.
(2)
Catalytic reactions (epoxidation and aziridination of alkenes and epoxidation of allylic alcohols) will be
performed in ionic liquids.
(3)
Use of nanofiltration as a new method for the separation of homogeneous catalysts.
(4)
Anchoring/encapsulation of chiral Mn complexes onto several supports: mesoporous silica materials,
mesoporous carbons, nanotubes, mesoporous PILCs and clays.
(5)
The homogeneous and heterogeneous catalytic activity will be evaluated with the determination of
enantiomeric excesses. For the heterogeneous catalysts, reutilisation of the catalysts will be also evaluated.
Chemosensors based on transition metal complexes
We have pursed the preparation of nickel and copper complexes with Schiff base ligands functionalised with
groups that can act as coordination sites for representative and lanthanide cations and for anions (halides,
H2PO4-, HSO4-, NO3-) were prepared by methodologies developed in our laboratory. Several functionalities
were introduced in the aldehyde moiety and in the diimine bridge: crown ether and pseudo crown ethers
groups, pyrrole and morpholine derivatives. These complexes were characterised by the usual techniques:
elemental analyses, mass spectra, EPR, FTIR and UV-Vis spectroscopies, cyclic voltammetry.
The sensing properties of the complexes were performed in several solvents, using cyclic voltammetry, UV-vis
and conductimetry; for the complexes that can polymerise and give stable fims recognition studies were also
done by cyclic voltammetry, in-situ FTIR and UV-Vis, electroacoustic impedance and electrochemical
impedance.
In solution, it was possible to determine equilibrium constants for the complexation of lanthanides and alkalineearth cations for the majority of the metal complexes by Uv-vis spectroscopy. Cyclic voltammetry allowed the
semi-quantiative evalutation of the interaction of all the cations tested (+1, +2 and +3)
The polymeric films adsorbed all the metal cations tested (+1, +2 and +3), although they exhibited different
behaviour: stable films wre onle produced after addition of +2 cations. This technique also showed that
depending on the experimental conditions used, metal ion can be de-complexed and the film can be reused
for recognition of other metal ions. IR and UV-Vis spectroscopies indicated the film cation coordination sites
and the influence of their presence in the electronic structure of the films.
FUTURE WORK
(1)
Design, preparation and characterisation of new complexes with sensing properties for cations and
anions.
(2)
Preparation and characterisation of their corresponding films.
(3)
Evaluation of the association constants for the interaction of cations and anions with the complexes by
new techniques: fluorescence, calorimetric titration and conductimetry
(4)
Study of the interactions of cations and anions with the polymeric films by new techniques:
electrochemical quartz crystal microbalance and EXAFS.
(5)
Cation and anion selectivity studies and for polymeric films reutilisation studies.
Química Verde
71
Molecular materials with non-linear optical properties
The synthesis of materials with non linear optical properties (NLO) is a current research area due to the
potential application of these materials for the fabrication of high performance electro-optical switching elements
for telecommunication and optical information processing. In this context, transition metal complexes, which
show in some cases intrinsic NLO properties, are important building blocks for the preparation of material with
this type of properties. In this work we have synthesised nickel and copper complexes with salen type ligands
functionalised with donor-acceptor groups, designated generically as [M(DA-salen)]. The complexes were
electropolymerised by methods developed in our group and the 3d NLO properties of all compounds were
determined.
The 3d NLO properties were evaluated using the z-scan technique, which is based on the self-focusing or
defocusing of a converging beam of known direction (Nd:YAG laser, l=1064 nm). The technique permits the
evaluation of non linear refractive index (n2) and the non linear absorption coefficient (b); all the optical
measurements were made in-situ. The polymeric films show 3d NLO properties which have been strongly
enhanced when compared to the monomers in solution, and are dependent on the doped state of the film.
FUTURE WORK
(1)
Design, preparation and characterisation of new complexes with improved 3d NLO properties
(2)
Preparation and characterisation of their corresponding films.
(3)
Evaluation of 3d NLO properties for complexes and corresponding films.
(4)
Preparation and characterisation of new films by self-assembly layer-by-layer method.
(5)
Evaluation of 3d NLO properties for films prepared by self-assembly layer-by-layer method.
Metallosurfactants. The study of the p-A isotherms of Langmuir-Blodgett monolayers of the amphiphilic
complexes [Fe(RR’bpy)2(CN)2], where R and R’ are alkyl chains (C1-C17), was continued. Deposition of the
LB films in ITO electrodes allowed to characterize electrochemically the films formed. Ongoing studies and
future work includes the use of other substrates for the deposition of the LB films, AFM, SEM and RAIRS
characterization of these films. A collaboration with Prof. A. Lopes (ITQB) has started in order to determine
interfacial tension by the drop/bubble-shape analysis method.
In addition, new synthetic methodologies are being deveoped to obtain similar metalosurfactants with ruthenium
centers. Four novel metalossurfactants have been synthesized and characterized by NMR and FTIR. Future
work will be focused on the morphological characterization of the aggregates in aqueous solutions, LB films,
and in the development of synthetic and purification methods to obtain the enantiomericaly pure forms of
these compounds.
Metal Nanoassemblies. The preparation of nanotriangles of gold has been established, and dependence of
morphology upon reaction conditions is still under study. In addition, the method was applied to the preparation
of Pt and Pt/Pd nanoparticles with catalytic properties. The study of the catalytic properties is being undertaken
in collaboration with Prof. Isabel Fonseca (REQUIMTE/CQFB).
The preparation of silver nanoparticles, nanorods and nanowires for SERRS is also being pursued. The capping
agents selected are thiolated derivatives with a grafted funcionality that enhances its interaction with biological
molecules. The SERRS studies are being performed in collaboration with Prof. R. Franco (ITQB).
Photoactive compounds and solid state materials
In 2004 the research of the group concentrated on the realization of novel (supra)molecular devices able to
perform logic operations. In a second research line luminescent solid-state materials with promising applications,
for instance as optical blends have been realized. The first project made use of long-lived lanthanide
luminescence, which was sensitized by energy transfer from appropriate organic antenna chromophores. By
fine-tuning of the photophysical parameters of the involved chromophore and lanthanide it was possible to
Química Verde
72
obtain a molecular device, which showed the behavior of an INHIBIT-type logic gate. Such molecular entities
are of special interest in the context of functional supramolecular chemistry and the development of more
complex nanoscale devices to be used as computers or machines.
In continuation of a project which started in 2003 novel luminescent inorganic-organic materials based on
lanthanide-loaded zirconium organophosphonates have been prepared. They have been demonstrated to
possess exceptional properties like extremely long luminescence lifetimes (millisecond range) and highly
color-pure light emission. The conclusions, drawn from detailed investigations of the photophysical processes
within these materials, will be used to improve the already good performance of these materials. In 2005 the
research in these two lines will be continued, based on the promising results obtained in the last year.
Química Verde
ANNEXES
A - 2
ANNEX I
RESEARCH TEAM
Química Verde
A - 3
A
MEMBERS OF STAFF
Química Verde
A - 4
Alberto Sundaresan Prabhakar
Ana Maria F. T. Lobo
Baltazar Manuel Romão Castro
Fernando J. S. Pina
Hugo Gil Ferreira
Isabel Maria A. M. G. de Moura
Jose Alberto Nunes Ferreira Gomes
José J. G. de Moura
Jose Luis Fontes Costa Lima
Luis F. G. Sousa Lobo
Manuel M. Nunes da Ponte
Maria Lurdes Souteiro Bastos
Maria Conceição B. S. M Montenegro
Pedro Brito Correia
Professor Catedrático
Professora Catedrática
Professora Associada
UNL
UNL
FCUP
UNL
UNL
UNL
FCUP
UNL
FFUP
UNL
UNL
FFUP
FFUP
UNL
Abel José de Sousa Costa Vieira
Alberto Manuel Carneiro Sereno
Alberto Nova Araujo
Alexandra Bernardo
Ana Cristina Moreira Freire
Aquiles J. F.de Araújo Barros
Duarte José da Costa Pereira
João Paulo S. Goulão Crespo
Jose F. M. Magalhâes Cardoso
Karin Tonnies Gil Ferreira
Maria Conceição S. S. Rangel
Maria João Ribeiro Nunes Ramos
Maria João L. R. M. C. Romão
Maria Natália Dias Soeiro Cordeiro
Maria Pilar Figueroa Gonçalves
Maria Teresa Barros Silva
Rosa Maria Moreira Seabra Pinto
Susana Filipe Barreiros
Teresa Maria Fonseca de Moura
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Professora Assopciada
Professor Associado
UNL
FEUP
FFUP
Cristina Maria Delerue Alvim Matos
Maria Leonor Oliveira Madureira Pinto
Maria Carmo Veiga Fernandes Vaz
Rui Alves
Professora Coordenadora
ISEP
ISEP
ISEP
IPVC
Alberta Paula Gameiro Santos
Alexandre Lopes Magalhães
Ana I. N. M. Aguiar Ricardo
Ana M. F. C. Lourenço
Ana Maria Martelo Ramos
Andre Alberto Sousa Melo
Ângela M. S. Relva
António Gil de Oliveira Santos
António Jorge Parola
Carlos S. S. Pereira Lima
Eduarda Graças Rodrigues Fernandes
Elvira Maria M. Gaspar
Eulália Fernanda Carvalho Pereira
Eurico José da Silva Cabrita
Felix Dias Carvalho
Fernando Manuel Gomes Remião
Helena Maria Neto Ferreira
Helena Maria Vieira Monteiro Soares
Henrique J. R. Guedes
Isabel Borges Coutinho de Medeiro
Isabel Maria Ligeiro da Fonseca
Isabel Maria Viegas Oliveira Ferreira
Isabel Maria Rola Coelhoso
João Carlos S. B. Sotomayor
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
FFUP
FCUP
FCUP
UNL
UNL
UNL
FCUP
UNL
UNL
UNL
UNL
FFUP
UNL
FCUP
UNL
FFUP
FEUP
FFUP
FEUP
UNL
UNL
UNL
FFUP
UNL
UNL
FCUP
FCUP
FCUP
UNL
ICBAS-UP
UNL
ICBAS-UP
FCUP
UNL
FCUP
FEUP
UNL
FFUP
FFUP
UNL
UNL
Química Verde
A - 5
João Carlos Lima
João Luis Machado Santos
João M. Aires de Sousa
João Paulo C. Noronha
Joaquim Silvério Marques Vital
Jorge M. P. Lampreia Pereira
José António Maia Rodrigues
Jose Augusto Caldeira Pereira
Jose Oliveira Fernandes
José Paulo Barbosa Mota
Luisa M. S. P. Ferreira
Luísa Maria S. Vieira Peixe
Marco Diogo R.Gomes da Silva
Maria Alice S. Pereira
Maria Ascenção Reis
Maria Beatriz Prior Pinto Oliveira
Maria Beatriz Guerra Junqueiro
Maria Cristina O. Costa
Maria D’Anjos L. Macedo
Maria Gabriela Teles Cepeda Ribeiro
Maria João Melo
Maria Lúcia Sousa Saraiva
M. Madalena A.C.S.Dionísio de Andrade
Maria Manuela M. A.Pereira
Maria Margarida C.M. A. Cardoso
Maria Salete Reis Dias Rodrigues
Maria Teresa Avilés Perea
Miguel Freire de A. Cabral
Olivia Maria de Castro Pinho
Paula Cristina Branquinho Andrade
Paula Cristina S. Branco
Paulina M. E. N. Mata
Paulo Joaquim Ferreira Almeida
Pedro António Brito Tavares
Pedro Jorge Macedo Abreu
Pedro Manuel Alexandrino Fernandes
Pedro Miguel Calado Simões
Rui Manuel Freitas Oliveira
Sofia Pauleta
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
UNL
FFUP
UNL
UNL
UNL
UNL
FCUP
ICBAS-UP
FFUP
UNL
UNL
FFUP
UNL
UNL
UNL
FFUP
FFUP
UNL
UNL
FCUP
UNL
FFUP
UNL
UNL
UNL
FFUP
UNL
FFUP
FCNAUP
FFUP
UNL
UNL
FCUP
UNL
UNL
FCUP
UNL
UNL
UAl
Ermelinda Manuela Jesus Garrido
Jorge Manuel Pinto Jesus Garrido
Professora Adjunta
Professor Adjunto
ISEP
ISEP
Carla Manuela Soares Matos
Catarina Isabel Guerra Rodrigues
João Luís Tavares de Matos Gomes
Francisco Jorge Fernandes Caldeira
Lígia Maria da Silva Rebelo Gomes
Luísa Lima Gonçalves
Maria Alexandra Bernardo
Maria Gabriela Almeida
Maria da Graça Soveral Rodrigues
Maria Helena Reis Prado de Castro
Rita Isabel Lemos Catarino
Sara Isabel Xavier Candeias
Stephane Besson
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
UFP
UFP
ISCSN
ISCSN
ISCSN
UFP
ISCSS
ISCSN
ISCSS
ISCSS
ISCSS
FFUL
ISCSN
UFP
U. Lusófona
U. Lusófona
Ana Maria Philips
Maria Fernanda Cabral
Agostinho Pereira
Ana Bicho dos Santos
Ana Luísa Carvalho
Carlos Brondino
Investigadora Principal
Investigadora Principal
Investigador Auxiliar
Investigadora Auxiliar
UNL
FCUP
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
Química Verde
A - 6
Carlos Lodeiro Espino
Eurico Cabrita
Isabel Mafra
Maria Manuel Marques
Miguel Jorge
Loïc Hilliou
Owen Catchpole
Serguey Bursakov
Svetlozar Velizarov
Uwe Pischel
Manuel Rui Azevedo Alves
Maria Isabel Branco Alves Martins
Maria Teresa de Oliva Teles Moreira
Jorge Garrido
Helena Carmo
Luís Guilherme L. Ferreira Guido
Patrícia Carla Ribeiro Valentão
Susana Isabel Pereira Casal
Abel José Assunção Duarte
Florinda F. Martins
Hendrikus Petrus Antonius Nouws
Maria de Fátima de Sá Barroso
Maria Isabel Brito Limpo de Serra
Maria João D. Ramalhosa Ferreira
Maria Manuela Barbosa Correia
Olga Manuela Matos de Freitas
Rita Isabel Simões Ferreira
Salomé de Sousa Teixeira
Sónia Adriana R. C. Figueiredo
Valentina Maria FernandesDomingues
Maria Elisa A. M. Fernandes Soares
Eulalia Maria Bernardino Mendes
Maria Isabel Afonso da Rocha
Maria Isabel Almeida Cardoso
José Tomás Soares de Albergaria
Paula Paíga
Carla Rodrigues
Cecília Bonifácio
João Fraga
Nelson Brito
Rui Viegas
Maria Aurora Soares da Silva
Sérgio Cruz Monteiro de Morais
Professora Adjunta
Professora Adjunta
Professor Adjunto
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assessora Principal
Assessora
Assessora
Assessora
Técnico
Técnica
Téc. Sup. 2.ª Classe
Téc. Informática
Téc. Informática
Encarregada de trabalhos
Encarregado de trabalhos
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
IPVC
ISEP
ISEP
ISEP
FFUP
FCUP
FFUP
FFUP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
IPVC
ISEP
ISEP
ISEP
ISEP
FFUP
FFUP
FCUP
FFUP
ISEP
ISEP
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
ISEP
ISEP
KEY
UNL
FCUP
FFUP
FCNAUP
ISEP
ISCSN
UFP
Universidade Nova de Lisboa
Universidade do Porto - Faculdade de Ciências
Universidade do Porto - Faculdade de Farmácia
Universidade do Porto - Fac.Ciências da Nutrição
Instituto Superior de Engenharia do Porto
Instituto Superior de Ciências de Saúde (Norte)
Universidade Fernando Pessoa
FFUL
FEUP
ICBAS-UP
UAl
IPVC
ISCSS
U. Lusófona
Universidade de Lisboa - Faculdade de Farmácia
Universidade do Porto - Faculdade de Engenharia
Universidade do Porto - Inst. Ciências Biomédicas
Universidade do Algarve
Inst. Politécnico de Viana do Castelo
Instituto Superior de Ciências de Saúde (Sul)
Universidade Lusófona
Química Verde
A - 7
B
POST-DOCTORAL FELLOWS
Química Verde
A - 8
Ana Rosa Silva
Anders Thapper
Berta Covelo
Carla Sousa
Celina Santos
Cristina Timóteo
Damián Fernández
Dirk Boer
Elsa Henriques
Ewa Bogel Lukasik
Françoise Auchère
Gilda Carvalho
Iwona Biernacka
João Miguel Dias
José Trincão
Krasimira Petrova
Luísa Serafim
Lalit Sharma
Ludwig Kripahal
Maria Adília Lemos
Marta Isabel Machado da Silva
Patrícia Sousa
PauloLemos
Pankas Das
Pavel Izak
Pedro Rodrigues
Peter Eaton
Quingyou Zhang
Rakesh Kumar
Roeland Boer
Rui Duarte
Shabir Najmudin
Shuwen Yao
Snezhana Bakalova
Smilja Todorovic
Sunil Gupta
Susan Matthews
Svetlana Lyubchik
Svetlozar Velizarov
Teresa Casimiro
Teresa Ribeiro
Thierry Michaud
Thomas Schäfer
Vanya Bogdanova Kurteva
Vesna Nasdanovic-Visak
Yonh Hong Liu
Yuri Binev
Química Verde
A - 9
C
Ph. D. STUDENTS
Química Verde
A - 10
Akapong Suwattanamala
Alexandre Carvalho
Ana Nunes
Ana Martins
Ana Teixeira
Andrea Carneiro
Andreia Filipa Curto
António Rodrigo
António Nunes
Carina Machado
Carla Brazinha
Carla Portugal
Carla Ferreira
Carla Silva
Carlos Martins
Carmen Pinheiro
Célia Peres
Christophe Siquet
Cristina Alves
Cristina Cordas
Cristina Correia
Cristina Lopes
Cristina Matos
David Costa
Diogo Latino
Elizabete Machado
Fábio Larotonda
Fátima Lucas
Filomena Freitas
Filipe Duarte
Filipe Folgosa
Francisco SIlva
Gabriela Rivas
Gonçalo Carrera
Helena Ferreira
Isabel Esteves
Irina Moreira
Joana Amaral
Joana Raimundo
João Lourenço
João Araújo
João Prior
João Dias
João Rosa
João Capela
João Tedim
Jorge Dias
Jorge Rebelo
José Castanheiro
José dos Santos
Karine Marques
Laila Ribeiro
Liliana Guerreiro
Luís Magalhães
Luis Lopez
Luís Pinto
Luisa Serafim
Magda Martins
Márcia Carvalho
Márcia Guilherme
Margarida Moncada
Maria João Morgado
Maria Luisa Soares Silva
Maria M. Santos
Maria Teresa Plaza
Mariana Duarte
Mário Eusébio
Mário Gomes
Marlene Lucio
Marta Andrade
Marta Corvo
Marta Filipa Ribeiro
Marta Andrade
Miguel Faria
Nuno Cerqueira
Oriza Tavares
Pablo Gonzalez
Patrícia Antunes
Patrícia Neves
Patricia Oliveira
Patrícia Raleiras
Paula Cristina Jerónimo
Paula Gomes Pinto
Paula Silva
Paulo Glória
Pedro Manuel Santos
Pedro Miguel Pimenta Gois
Pedro Rodrigues Marques Maia
Pedro Vidinha
Raquel Fortunato
Ricardo Branco
Ricardo Oliveira
Rita Noronha
Rute Fonseca
Sara Cristina Silva Cunha
Sandra Quinteira
Sérgio Alves
Sérgio Sousa
Sílvia Garcia
Sofia Barata
Sofia A. Costa Lima
Sofia Gomes
Susana Pereira
Susana Gaudêncio
Teresa Alves
Teresa Casimiro
Teresa Santos Silva
Teresa Tiago
Vasco Bonifácio
Victor Alves
Victor Rosa
Vineet Pande
Wancheng Sittikijyothin
Zenaida Mourão
Química Verde
A - 11
D
M. Sc. STUDENTS
and
other research students
Química Verde
A - 12
M. Sc. Students
Anabela Gomes
Ana Margarida Fonseca
Ana Maria Gomes
Ana Sofia Pinto
Branca Teixeira
Bruno Jarrais
Joana marcelino
Jorge Teixeira
Luís Correia
Maria João Amorim
Maria Resende
Marta Sofia Pires
Miguel Sousa
Paula Rodrigues
Renata Silva
Rosa Bessada
Sergio Teixeira
Other Research Students
Adelaide Miranda
Alexandra Carvalho
Américo Duarte
Ana Brás
Ana Lopes
Andreia Barateiro
Andreia Leite
Ângela Machado
Artur Abreu
Carla Macedo
Carlota Macedo
Catarina Soares
Célia Silveira
João Dias
Márcio Tentem
Marek Kluciar
Marta Vilela
Miriam Sousa
Vanessa Cabral
Química Verde
A - 13
E
Profiles of the new researchers
contracted under the Associate
Laboratory Program
Química Verde
A - 14
Dr. Ana Isabel dos Santos was hired in order to strengthen the Biological Transport Research Group
of REQUINTE/CQFB. She has a PhD in Biochemistry, MPI for Biophysics and has a well-established knowledge
of different electrophysiological techniques. She has experience and is in charge of the maintenance of
cultured cell lines and in the development of protocols for the heterologous expression of membrane proteins.
Furthermore, she is also be involved in the electrophysiological characterization of these proteins and in
building mathematical models.
She is involved in several projects of the group and will supervise pre- and post-graduate students.
Dr. Jose-Luis Capelo-Martinez was chosen as member of the Bio-Organic and Analytic Group of
Requimte/CQFB because of the following items: i) high scientific production, ii) high impact index of published
manuscript, iii) research capability by his own. Furthermore, Dr. José-Luis Capelo-Martínez has a large
experience in developing new sample treatments for green chemistry based on Advanced Oxidation Processes
(AOP´s) for i) trace metal extraction (total and speciation) in biological and environmental samples, ii) organic
contaminants extraction such as PAHs from environmental samples and iii) protein digestion and identification
for proteomics. The skills of Dr. Capelo-Martinez include the following techniques: FI-CV-AAS, FI-HG-AAS,
ET-AAS, HPLC-ICP-MS, MALDI-TOF-MS, RF-HPLC-ESI-IT-MS/MS. His research is focused in developing
new sample treatments which must achieve the following items: 1.- Less chemical consumption. 2.- Low
chemical concentration. 3.- Easy implementation. 4.- Green chemistry. In addition, he has experience in
teaching (theory and laboratory, University of Vigo, Spain, and Instituto Superior Tecnico, IST, Portugal) in
Analytical and Environmental Chemistry and related disciplines. He leads a team of 5 students and heads the
European Group for Green Environmental Bio-Analytical Procedures( http://homepage.oniduo.pt/eggebap/).
Collaboarations with other groups includes: i) Group of Professor Maria de Lourdes Gonçalves, from the IST;
ii) Dr. C. Vaz, Laboratorio de Analisis do IST; iii) Grup of professor F. Pina, Chemistry Department, FCT/UNL;
iv) Group of Dr. Jesus Vazquez, Univesidad Autonoma de Madrid, Spain; v) Dr. Manuel Miro, Universidad de
las Islas Baleares, Spain.
Dr. Capelo-Martinez´s research has been published in the hottest Analytical Chemistry Journals: Anal
Chem, (3), Trac.Trend Anal. Chem, (2), J. Anal Atom Spectrom ( 3), Talanta (6), Fresen. J. Anal. Chem. (1),
Atom Spectrosc, (1), Ultrason. Sonochem (2), Z Anorg Allg Chem, (2), J Incl Phenom Macro (1), J AOAC Int,
(2), Current Analytical Chemistry (1). Total articles: 24.
Química Verde
A - 15
ANNEX II
PUBLICATIONS
Química Verde
A - 16
A
Scientific Articles
——
Book Chapters
——
Articles in Procedings
Química Verde
A - 17
Articles in Scientific Journals
1.
“Effect of immobilisation support, water activity and enzyme ionisation state on cutinase activity and
enantioselectivity in organic media”
P. Vidinha; N. Harper; N. M. Micaelo; N. M. T. Lourenço; M. D. R. Gomes da Silva; J. M. S. Cabral, C.
A. M. Afonso; C. M. Soares; S. Barreiros
Biotechnology and Bioengineering, 2004, Vol. 85 (4), 442-449
2.
“Potencialidades das técnicas uni e multidimensionais de GC-TOF-MS para a caracterização de óleos
essenciais”
Eduardo P. Mateus, M.D.R. Gomes da Silva, Jitka Zrostlíková, Higuinaldo Chaves das Neves & Maria
Rosa Paiva
Boletim da Sociedade Portuguesa de Química, 2004, Vol. 94, 49-56
3.
“Thermal and Photochemical Properties of 4’,7-dihydroxyflavylium in Water-Ionic Liquids Biphasic
Systems. A Novel Approach to Write-Read-Erase Molecular Switching System”
F. Pina, J. C. Lima. A. J. Parola, C. A. M. Afonso
Angew. Chem. Int. Ed. 2004, 43, 1525-1527
4.
“Multistate/Multifunctional Behaviour of 6-nitro,4’-hydroxyflavylium. A Write-lock/Read/Unlock/Enableerase/Erase Cycle Driven by Light and pH Stimulation”
M. C. Moncada, A. J. Parola, C. Lodeiro, F. Pina, M. Maestri, V. Balzani
Chem. Eur. J. 2004, 10, 1519-1526
5.
“Ground and Excited-State Electronic Interactions in Poly(propylene amine) Dendrimers Functionalized
with Naphthyl Units. Effect of Protonation and Metal Complexation”
F. Pina, P. Passaniti M. Maestri V. Balzani, F. Vögtle, M. Gorka, S.-K. Lee, J. van Heyst, H. Fakhrnabavi
Chem. Phys. Chem, 2004, 5, 473-480
6.
”Tuning of the Photochromic Properties of a Flavylium Compound by pH”
M. C. Moncada, F. Pina, A. Roque, A. J. Parola, M. Maestri, V. Balzani
Eur. J. Org. Chem. 2004, 2, 304 – 312
7.
“Protonation and coordination properties towards Zn(II), Cd(II) and Hg(II) of a phenanthroline-containing
macrocycle with an ethylamino pendant arm”
C. Bazzicalupi, A. Bencini, E. Berni , A. Bianchi , L. Borsari, C. Giorgi, B. Valtancoli , C. Lodeiro, J. C.
Lima, A. J. Parola, F. Pina
Dalton Transactions, 2004, 591-597
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“Selective fluoprescent Chemosensor for Zn(II). Exciplex formation in solution and in solid state”
Bencini, E. Berni, A. Bianchi, P. Fornasari, C. Giorgi, J. C. Lima, C. Lodeiro, M.J. Melo, J. S. Melo, A.
J. Parola, F. Pina. J. Pina, B. Valtancoli
9.
“Thermal and Photochemical Properties of 4’,-hydroxyflavylium in Water-Ionic Liquids Biphasic System”
D. Fernandez, F. Pina, A. J. Parola, C. A. M. Afonso, L. Branco
J. Photochem Photobiology Chemistry A: 2004, 168, 185-189.
10.
“Two Coupled Photochromic Systems of 3’,4’-(methylenedioxy)flavylium: Kinetic and Thermodynamic
Characterization”
D. Fernández, F. Folgosa, A. J. Parola, F. Pina
New. J. Chem. 2004, 28, 1221-1226
Química Verde
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“A new terpyridine-containing macrocycle for the assembly of dimeric Zn(II) and Cu(II) complexes coupled
by bridging hydroxide anions and ð-stacking interactions”
C. Bazzicalupi, A. Bencini, E. Berni, A. Bianchi, A. Danesi, C. Giorgi, B. Valtancoli, M. A. Bernardo, C.
Lodeiro, J. C. Lima, F. Pina
Inorg. Chem. 2004, 43, 5134-5146
12.
“Multistate properties of 7-(N,N-Diethylamino)-4’-hydroxyflavylium. An Example of an Unidirectional
Reaction Cycle Driven by pH”
M. C. Moncada, D. Fernández, J. C. Lima, A. J. Parola, C. Lodeiro, F. Folgosa, M. J. Melo, F. Pina
Org. Biomol. Chem., 2004, 2, 2802-2808
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“A Novel Binucleating Bis(florophoric) Bibrachial Lariat Aza-Crown”
M. P. Clares, J. Aguilar, C. Lodeiro, M. T. Albelda, F. Pina, J. C. Lima, A. J. Parola, J. S. de Melo, C.
Soriano, E. García-España
Inorg. Chem. 2004, 43, 6114-6122
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“Fluorescent Type II Materials from Naphthymethyl Polyamine Precursors”
J. Alarcó, R. Aucejo, M. T. Albelda, S. Alves, M. P. Clares. E. Garcia-España, C. Lodeiro, K. L. Marchin.
A. J. Parola, F. Pina. J. S. Melo, C. Soriano
Supramolecular Chemistry, 2004, 16, 573-580
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“Photophysical and Spectroscopic Studies of Indigo Derivatives in Their Keto and Leuco Forms”
J. Seixas de Melo; A. P. Moura; M. J. Melo
J. Phys. Chem. B., 2004, 108, 6975-6981
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“Metal ion interaction of two new tritopic N2O13 macrobicycles with B15C5 moieties in acetonitrile
solution”.
C. Lodeiro, J. L. Capelo
J. Inclu. Phem. Mac. Chem., 2004, 49, 249-258
17.
“Manganese(II), Cobalt(II) and Nickel(II) Perclhorate Complexes Containing N,O Donor Macrocyclic
Ligands”
C. Lodeiro, J. L. Capelo, E. Bértolo, R. Bastida
Z. Anorg. Allg. Chem., 2004, 630, 1110-1115
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“Síntesis and Spectroscopical charaterization of dinuclear Ca(II) complexes with oxa-aza macrocyclic
ligand.”
C. Lodeiro, E. Bértolo, J. L. Capelo, R. Bastida
Z. Anorg. Allg. Chem., 2004, 630, 914-920
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“Lanthanide(III) complexes of two novel pyridine-derived N3O4-donor Macrocycles”.
E. Bértolo, R. Bastida, C. Lodeiro, A. Rodríguez
J. Inclu. Phen. Macro. Chem.,2004, 48 (3-4)151-155.
20.
“A New Family of NxOy Pyridine-containing Macrocycles. Synthesis and Characterisation of their Y(III),
Ln(III), Zn(II) and Cd(II) Coordination Compounds.”
C. Lodeiro, R. Bastida, E. Bertolo, A. Rodríguez
Can. J. Chem., 2004, 82, 3, 437-447
21.
“Bis(2-diphenylglycolato-O)-bis(1,10-phenanthroline-N,N’)zinc(II)”
R. Carballo, B. Covelo, E. García-Martínez, E. M. Vázquez-López, A. Castiñeiras
Appl. Organomet. Chem., 2004, 18, 201-202
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“Solid State Coordination Chemistry of Mononuclear Mixed-ligand Complexes of Ni(II), Cu(II) and Zn(II)
with a-Hydroxycarboxylic Acids and Imidazole”
R. Carballo, A. Castiñeiras, B. Covelo, E. García-Martínez, J. Niclós, E. M. Vázquez-López.
Polyhedron, 2004, 23, 1505-1518
23.
“Intramolecular fluorescence quenching of tyrosine by the peptide alpha-carbonyl group revisited”
M. Noronha, J.C. Lima, P. Lamosa, H. Santos, C. Maycock, R. Ventura, A.L. Macanita
J. Phys. Chem.A, 2004, 108, 2155-2166
24.
“Unfolding of ubiquitin studied by picosecond time-resolved fluorescence of the tyrosine residue”
M. Noronha, J.C. Lima, M. Bastos, H. Santos, A. L. Macanita
Biophysical Journal, 2004, 87, 2609-2620
25.
“Unexpected Alkyne Transfer Between Gold and Rhenium Atoms and its Application to the Synthesis of
Alkynyl Rhenium(I)Compounds”
M. Ferrer, L. Rodríguez, O. Rossell, J. C.Lima, P. Gómez-Sal, A. Martín
Organometallics, 2004, 23, 5096-5099
26.
“Protein Stabilization by Osmolytes from Hyperthermophiles: Effect of Mannosylglycerate on the thermal
unfolding of recombinant Nuclease A from Staphylococcus aureus studied by picosecond time-resolved
fluorescence and calorimetry”
T. Q. Faria, J. C. Lima, M. Bastos, A. L. Macanita, H. Santos
J. Biol. Chem. 2004, 279, 48680-48691
27.
“Excited-State Electron Transfer in Anthocyanins and Related Flavylium Salts”
P. Ferreira da Silva, J. C. Lima, F. H. Quina, A. L. Macanita
J.Phys. Chem. A. 2004, 108, 10133-10140
28.
“A New Approach to N-protected Staurosporinones”
M. M. M. Santos, A. M. Lobo, S. Prabhakar, M. M. B. Marques
Tetrahedron Letters, 2004, 2347-2349
29.
“Identification of a Novel Small-Molecule Inhibitor of the Hypoxia-Inducible Factor 1 Pathway”
C. Tan; R. G. de Noronha; A. J. Roecker; B. Pyrzynska; F. Khwaja; Z. Zhang; H. Zhang; Q. Teng; A. C.
Nicholson; P. Giannakakou; W. Zhou; J. J. Olson; M. M. Pereira; K.C. Nicolaou; E. G. V. Meir
Cancer Res., 2005; Vol. 65, 605-612
30.
“Synthesis of Analogue Structures of the p-Quinone Methide Moiety of Kendomycin”
M. P. Green; S. Pichlmair; M. M. B. Marques; H. J. Martin; O. Diwald; T. Berger; J. Mulzer
Org. Lett., 2004, 18, 3131-3134
31.
“Natural Product Synthesis Special Feature:Toward the Synthesis of the Carbacyclic Ansa Antibiotic
Kendomycin”
J. Mulzer; S. Pichlmair; M. P. Green; M. M. B. Marques; H. J. Martin
PNAS, 2004, 101, 11980-11985
32.
“Palladium-Catalyzed Alpha-Arylation of Esters, Ketones, Amides and Imides”
M. M. B. Marques
Org. Chem. Highlights, 2004, November 25
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“Rearranged Jatrophane-Type Diterpenes from Euphorbia Species. Evaluation of their Effects on the
Reversal of Multidrug Resistance”
M. Madureira; M. J. U. Ferreira; N. Gyémánt; K. Ugocsai; J. R. Ascenso, P. M. Abreu, J. Hohmann, J.
Molnar
Planta Medica, 2004, Vol 70, 45-49
34.
“Pubescenes, Jatrophane Diterpenes, from Euphorbia Pubescens, with Multidrug Resistance Reversal
Activity on Mouse Lymphoma Cells”
C. Valente; M. J. U. Ferreira, P. M. Abreu, N. Gyémánt, K. Ugocsai, J. Hohmann, J. Molnar
35.
“Euphopubescenol and Euphopubescene, Two New Jatrophane Polyesters, and Lathyrane-Type
Diterpenes from Euphorbia Pubescens”
C. Valente; M. Pedro; J. R. Ascenso; P. M. Abreu: M. S. J. Nascimento; M. J. U. Ferreira
36.
“Effect of Cycloartanes on Reversal of Multidrug Resistance and Apoptosis Induction on Mouse Lymphoma
Cells”
M. Madureira; G. Spengler; A. Molnar; A. Varga; J. Molnar; P. M. Abreu; M. J. U. Ferreira
Anticancer Res., 2004, Vol 24 (2B), 859-864
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“Bioactive Diterpenoids, a New Jatrophane and two ent-Abietanes, and other Constituents from Euphorbia
Pubescens”
C. Valente; M. Pedro; A, Duarte; M. S. J. Nascimento; P. M. Abreu; M. J. U. Ferreira
Journal of Natural Products, 2004, Vol 67, 902-904
38.
“A New Sesquiterpene-Coumarin Ether and a New Abietane Diterpene and their Effects as Inhibitors of
P-Glycoprotein”
M. Madureira, A. Molnár, P. M. Abreu, J. Molnár, M. J. U. Ferreira
39.
“Structure d’un Coumarino-Lignane Isolé de la Partie Aérienne de la Plante Salsola Tetrandra”
M. M. Oueslati; H. Ben Jannet; P. J. Abreu; Z. Mighri
J. Soc. Alg. Chim., 2004, Vol 14, 181-187
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“Alcools Tetrahydropyraniques Polyacetyleniques Antibacteriens de la Plante Rantherium Suaveolens
Poussant dans le Sud Tunisien”
M. M. Oueslati; H. Ben Jannet; P. J. Abreu; Z. Mighri
J. Soc. Alg. Chim., 2004, Vol 14, 245-258
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“Structure-Based Predictions of 1H NMR Chemical Shifts Using Feed-Forward Neural Networks”
Y. Binev; J. Aires-de-Sousa
J. Chem. Inf. Comp. Sci., 2004, Vol. 44, 940-945
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“The Impact of Available Experimental Data on the Prediction of 1H NMR Chemical Shifts by Neural
Networks”
Y. Binev; M. Corvo; J. Aires-de-Sousa
J. Chem. Inf. Comp. Sci., 2004, Vol. 44, 946-949
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“Chirality Codes and Molecular Structure”
J. Aires-de-Sousa; J. Gasteiger; I. Gutman; D. Vidovi
J. Chem. Inf. Comput. Sci., 2004, Vol. 44, 831-836
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“Structure-based Predictions of 1H NMR Chemical Shifts of Sesquiterpene Lactones Using Neural
Networks”
F. B. da Costa; Y. Binev; J. Gasteiger; J. Aires-de-Sousa
Tetrahedron Lett., 2004, Vol. 45, 6931-6935
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“Cientistas de Palmo e Meio – Uma Brincadeira Muito Séria”
P. Mata; C. Bettencourt; M. J. Lino; M. Sousa Paiva
Análise Psicológica, 2004, Vol. XXII – 1, 169-174
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“Facile Conversion of O-Silyl Protected Sugars into their Corresponding Formates Using POCl3· DMF
Complex”
M. M. Andrade; M. T. Barros
Tetrahedron, 2004, Vol. 60, 9235-9243
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“Size- and Charge-State-Dependent Reactivity of Azidoacetonitrile with Anionic and Cationic Rhodium
Clusters Rh-n”
Balteanu; O. P. Balaj; B. S. Fox-Beyer; P. Rodrigues; M. T. Barros; A. M. C. Moutinho; M. L. Costa; M.
K. Beyer; V. E. Bondybey
Organometallics, 2004, Vol. 23, 1978-1985
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“Highly Stereoselective Aldol Reaction for the Synthesis of Gamma-Lactones Starting from Tartaric Acid”
M. T. Barros; A. J. Burke; J. D. Lou; C. D. Maycock; J. R. Wahnon
J. Org. Chem., 2004, Vol. 69, 7847-7850
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“Regioselective Copolymerization of Acryl Sucrose Monomers”
M. T. Barros; K. T. Petrova; A. M. Ramos
J. Org. Chem., 2004, Vol. 69, 7772-7775
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“Mass Spectrometric Studies of Azides: Reactions of Ar+ with 3-Azidopropionitrile, 2-Azidopropionitrile,
and Azidoacetonitrile in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer”
M. T. Barros; M. K. Beyer; M. L. Costa; M. F. Duarte; M. T. Fernandez; F. Martins; P. Rodrigues; P.
Watts
Int. J. Mass Spectrom., 2004, Vol. 237, 65-73
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“Improved Anomeric Selectivity for the Aroylation of Sugars”
M. T. Barros; C. D. Maycock; P. Rodrigues; C. Thomassigny
Carbohyd. Res., 2004, Vol. 339, 1373-1376
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“Novel Cyclic 1,2-Diacetals Derived from (2R,3R)-(+)-Tartaric Acid: Synthesis and Application as N,O
Ligands for the Enantioselective Alkylation of Benzaldehyde by Diethylzinc”
M. T. Barros; C. D. Maycock; A. M. F. Phillips
Eur. J. Org. Chem., 2004, 1820-1829
53.
“A Study of the Thermal Decomposition of 2-Azidoacetamide by Ultraviolet Photoelectron Spectroscopy
and Matrix-Isolation Infrared Spectroscopy: Identification of the Imine Intermediate H2NCOCHNH”
J. M. Dyke; G. Levita; A. Morris; J. S. Ogden, A. A. Dias, M. Algarra; J. P. Santos; M. L. Costa; P.
Rodrigues; M. T. Barros
J. Phys. Chem. A, 2004, Vol. 108, 5299-5307
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“Electron Ionization Mass Spectrometry in the Characterization of Azidonitriles”
F. Martins; M. F. Duarte; M. T. Fernandez; G. J. Lanley; P. Rodrigues; M. T. Barros; M. L. Costa
Rapid. Commun. Mass Sp., 2004, Vol. 18, 363-366
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“Imide-Amide Rearrangement of Oxazaphosphorimidates: Studies Towards the Application to the
Synthesis of Chiral Lewis Bases”
E. J. C. Cabrita; C. M. A. Afonso; A. G. Santos
Tetrahedron, 2004, Vol. 60, 11933-11949
56.
“A Computational Study of the Diels-Alder Reaction of Ethyl-S-Lactyl Acrylate and Cyclopentadiene.
Origins of Stereoselectivity”
S. M. Bakalova; A. G. Santos
J. Org. Chem., 2004, Vol. 69, 8475-8481
57.
“Electronic Absorption, Emission Spectra and Computational Studies of Some 2-Aryl, 2-Styryl and 2-(4’Aryl)Butadienyl Quinozolin-4-ones”
S. M. Bakalova; A. G. Santos; I. Timcheva; J. Kaneti; I. L. Filipova; G. M. Dobrikov; V. D. Dimitrov
J. Mol. Struct. (Theochem), 2004, Vol. 710, 225-230
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“Enantioselective Addition of Alkynes to Imines in Ionic Liquids”
J. N. Rosa; A. G. Santos; C. A. M. Afonso
J. Mol. Catal A: Chemical, 2004, Vol. 214, 161-165
59.
“Exploratory Applications of Diffusion Ordered Spectroscopy to Liquid Foods: An Aid Towards Spectral
Assignment”
M. Gil; I. Duarte; E. Cabrita; B. J. Goodfellow; M. Spraul; R. Kerssebaum
Anal. Chim. Acta, 2004, Vol. 506, 215-223
60.
“Microwave Accelerated Facile Synthesis of Fused Polynuclear Hydrocarbons in Dry Media by
Intramolecular Friedel-Crafts Alkylation”
V. B. Kurteva; A. G. Santos; C. A. M. Afonso
Org. Biomol. Chem., 2004, Vol. 2, 514-523
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“Phase Behaviour of the Catalyst Dicarbonyl(?5-cyclopentadienyl)-cobalt in Carbon Dioxide”
T.Casimiro; F. Montilla; S. Garcia; T. Avilés; S. Raeissi; A. Shariati; C.J. Peters; M. N. da Ponte; A.
Aguiar-Ricardo
Journal of Supercritical fluids, 2004, Vol 3, 1-8
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“Effect of Trimethylsilyl Substitution on the Chemical Properties of Triarylphosphines and their
Corresponding Metal-complexes: Solubilizing Effect in Supercritical Carbon Dioxide”
F. Montilla; A. Galindo; V. Rosa; T. Avilés
63.
“Regioselective Copolymerisation Of Acryl Sucrose Monomers”
M.T. Barros; K.T. Petrova; A.M. Ramos
Organic Chemistry, 2004, Vol. 69, 7772-7775
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“Plasma Torch Generation Of Carbon Supported Metal Catalysts”
H. Zea; C.K. Chen; K. Lester; A. Phillips; A. Datye; I.M. Fonseca; J. Phillips
Catalysis Today, 2004, Vol. 89, 237
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“Intercalation As An Approach To The Activated Carbon Preparation From Ukrainian Anthracites”
S.B. Lyubchik; L. Ya. Galushko; A.M. Rego; Yu. V. Tamarkina; O.L. Galushko; I.M. Fonseca
Journal of Physics and Chemistry of Solids, 2004, Vol. 65, 127
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“Kinetic Modeling Sudies Of Ethylene Polymerisation Reaction Using Supported Chromium Catalysts”
I. Matos; Y. Zhan; M.A.N.D.A. Lemos; F. Freire; I.F. Fonseca, M.M. Marques; F. Lemos
J. Polym. Sci. Part A Polym. Chem., 2004, Vol. 42, 3464
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“Kinetics And Thermodynamics Of The Cr(III) Adsorption On Activated Carbons From Co-Mingled Wastes”
S. Lyubchik; A. Luybchik; O. Galushko; L. Tikhonova; J. Vital; I.M. Fonseca; S. Lyubchik
J.Colloids and Surfaces A, PhysicoChemical Engineering Aspects, 2004, Vol. 242, 151-158
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“Properties Of Palladium Catalysts On Carbon Supports Prepared From Chemically Modified And Activated
Anthracites”
B.Kuznetsov; N.Chesnokov; N.M.Mikova; V.Drozdov; T.Shendrik; S.Lyubchik; I.M.Fonseca
Reaction Kinectics Catalysis Letters, 2004, Vol. 83, 361-367
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“Coupled pervaporation/mass spectrometry for investigating membrane mass transport phenomena”
T. Schäfer; J. Vital; J. Crespo
Journal of Membrane Science, 2004, Vol. 241, 197-205
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“A Visual Acoustic High-Pressure Cell for the Study of Critical Behaviour of Non Simple Mixtures”
Aguiar-Ricardo; M. Temtem; T. Casimiro; N. Ribeiro
Review of Scientific Instruments, 2004, Vol. 75, 3200-3202
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“Phase Behavior Studies of a Perfluoropolyether in High Pressure Carbon Dioxide”
T. Casimiro; A. Shariati; C.J. Peters; M. Nunes da Ponte; A. Aguiar-Ricardo
Fluid Phase Equilibria, 2004, Vol. 4224, 257-261
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“High Pressure Phase Behavior of the System Ethane + Orange Peel Oil”
A.R. Sampaio de Sousa; S. Raeissi; A.Aguiar-Ricardo; C.M.M. Duarte; C.J.Peters
J. Supercritical Fluids 2004, Vol. 29, 59-67
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“Solubility of Coenzyme Q10 in Supercritical Carbon dioxide”
A.M. Matias; A. V. M. Nunes; T. Casimiro; C. M.M. Duarte
J. Supercritical Fluids, 2004, Vol. 28, 201-206
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“Dynamic model of a countercurrent packed column operating at high pressure conditions”
R. Ruivo; A. Paiva; J.P.B. Mota; P. Simões
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Conditions”
R. Ruivo; A. Paiva; P. Simões
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“A novel non-intrusive microcell for sound-speed measurements in liquids. Speed of sound and
thermodynamic properties of 2-propanone at pressures up to 160 MPa”
R. Gomes Azevedo, J. Szydlowski, P.F. Pires, J.M.S.S. Esperança, H.J.R. Guedes, and L.P.N. Rebelo
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“A detailed thermodynamic analysis of [C4mim][BF4] + Water as a case study to model ionic liquid
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L.P.N. Rebelo, V. Najdanovic-Visak, Z.P. Visak, M. Nunes da Ponte, J. Szydlowski, C.A. Cerdeiriña, J.
Troncoso, L.Romani, J.M.S.S. Esperança, H.J.R. Guedes, H.C. de Sousa
Green Chemistry, 2004, Vol. 6, 369-381
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Biotechnol. Bioeng., 2004, Vol. 85, 442-449
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organic media”
S. Garcia; N.M.T. Lourenco; D. Lousa; A.F. Sequeira; P. Mimoso; J.M.S. Cabral; C.A.M. Afonso; S.
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Green Chemistry, 2004, Vol. 6, 466-470
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M. T. Viciosa, M. Dionísio
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“Molecular Motions in Chitosan Studied by Dielectric Relaxation Spectroscopy”
M. T. Viciosa, M. Dionísio, R. M. Silva, R. L. Reis, J. F. Mano
Biomacromolecules, 2004, vol. 5(5), 2073-2078
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“High Pressure Phase Equilibrium for d-Tocopherol + CO2”
P.J.A. Pereira, B. Coto, C.Menduiña, E.Gomes de Azevedo, M. Nunes da Ponte
Fluid Phase Equilibria 2004, 26, 53-57
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“Recent Advances in Chiral Resolution Using Membrane-based Approaches”
C. A.M. Afonso, J. G. Crespo
Angewandte Chemie Int. Ed., 2004, 43, 5293-5295
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“Membrane Bioreactors for the Removal of Anionic Micropollutants from Drinking Water”
J.G. Crespo; S. Velizarov; A.M. Reis
Current Opinion in Biotechnology, 2004, Vol. 15, 463-468
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“Bacterial Denitrification of Wastewater Stimulated by Constant Electric Field”
V. Beschkov; S. Velizarov; S.N. Agathos; V. Lukova
Biochemical Engineering Journal, 2004, Vol. 17, 141-145
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“Supported Liquid Membranes Using Ionic Liquids: Study Of Stability And Transport Mechanisms”
R. Fortunato; C.A.M. Afonso; M.A. Reis; J.G. Crespo
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V. D. Alves; I.M. Coelhoso
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V. Alves; B. Koroknai; K. Bélafi-Bakó; I. Coelhoso
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“Polyhydroxyalkanoates Production by Activated Sludge in a SBR Using Acetate and Propionate as
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P. C. Lemos; L. S. Serafim; M. A. M. Reis
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“Optimisation of Polyhydroxybutyrate Production by Mixed Cultures Submitted to Aerobic Dynamic
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L. S. Serafim; P. C. Lemos; R. F. Oliveira; M. A. M. Reis
Biotech. Bioeng., 2004, Vol. 87(2), 145-160
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“Mixing by Chaotic Advection in Three-Dimensional Periodic Flows”
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J.P.B. Mota; I.A.A.C. Esteves
Molec. Simul., 2004, Vol. 30, 387-396
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“Dynamic Modelling of an Adsorption Storage Tank Using a Hybrid Approach Combining Computational
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J.P.B. Mota; M. Rostam-Abadi
Comp. Chem. Eng., 2004, Vol. 28, 2421-2431
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R. Oliveira
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“Design of a stable adaptive controller for driving aerobic fermentation processes near maximum oxygen
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R. Oliveira, R Simutis, S. Feyo de Azevedo
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“Two azurins with unusual redox and spectroscopic properties isolated from the Pseudomonas chlororaphis
strains DSM 50083T and DSM 50135”
D. Pinho, S. Besson, C.D. Brondino, E. Pereira, B.Castro and I. Moura
J.Inorg.Biochem. 2004, 98, 276-86
99.
“Incorporation Of Either Molybdenum Or Tungsten Into Formate Dehydrogenase From Desulfovibrio
Alaskensis Ncimb 13491. Epr Assignment Of The Proximal Iron_Sulfur Cluster To The Pterin Cofactor In
Formate Dehydrogenase From Sulfate-Reducing Bacteria”
C.D.Brondino, M.C.G.Passeggi, J.Caldeira, M.J.Almendra, M.J.Feio, J.J.G.Moura, I.Moura
J.Biol.Inorg.Chem. 2004, 9, 145-151
100. “Antagonists Mo And Cu In A Heterometallic Cluster Present On A Novel Protein (Orange Protein-ORP)
Isolated From Desulfovibrio gigas”
S.Bursakov, O.Y.Gavel, G.DiRocco, J.Lampreia, J.Calvete, A.S.Pereira, J.J.G.Moura, I.Moura,
J. Inorg.Biochem. 2004, 98, 833-840
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101. “Direct Electrochemistry Of The Desulfovibrio gigas Aldehyde Oxidoreductase”
M.M. Correia Dos Santos, P.M. Sousa, M.L. Gonçalves, M.J. Romão, I.Moura, J.J. Moura
Eur J Biochem. 2004, 271, 1329-1338
102. “Structural Basis For The Mechanism Of Ca2+ Activation Of The Di-Heme Cytochrome C Peroxidase
From Pseudomonas Nautica 617”,
J.M.Dias, T.Alves, C.Bonifácio, A.S.Pereira, D.Bourgeois, I.Moura, M.J.Romão
Structure 2004, 12, 61-73
103. “Crystallization And Preliminary X-Ray Diffraction Analysis Of The 16-Haem Cytochrome Of Desulfovibrio
gigas”
T.Santos-Silva, J.J.M.Dias, G.Bourenkov, H.Bartunik, I.Moura, M.J.Romão
Acta Crystallogr D Biol Crystallogr. 2004, 60, 968-70
104. “Structural Stability Of Adenylate Kinase From The Sulfate-Reducing Bacteria Desulfovibrio gigas”
O.Y.Gavel, S.A.Bursakov, D.G.Pina, G.G.Zhadan, J.J.G.Moura, I.Moura, V.L.Shnyrov
Biophy. Chem. 2004, 110, 83-92
105. “Copper –Containing Nitrite Reductase From Pseudomonas Chloraphis Dsm 50135. Evidence For
Modulation Of The Rate Of Intramolecular Electron Transfer Through Nitrite Binding To The Type 2
Copper Center”
D.Pinho, S.Besson, C.D.Brondino, B.Castro, and I.Moura
Eur.J.Biochem. 2004, 271, 2361-2369
106. “Paracoccus pantotrophus Pseudoazurin is an electron donor to cytochrome c peroxidase”
S.R.Pauleta, F.Guerlesquin, C.F.Goodhew, B.Devreese, J.Van Beeumen, A.S.Pereira, I.Moura and
G.W.Pettigrew
Biochem. 2004, 43, 1114-11225
107. “Overexpression And Purification Of Treponema Pallidum Rubredoxin;Kinetic Evidence For SuperoxideMediated Electron Transfer With The Superoxide Reductase Neelaredoxin”
F.Auchere, R.Sikkink, C.Cordas, P.Raleiras, P.Tavares, I.Moura and J.J.G.Moura
J.Biol.Inorg.Chem. 2004, 9, 839-849
108. “A Copper Protein and a Cytochrome Bind at the Same Site on Bacterial Cytochrome c Peroxidase”
S.R.Pauleta, A.Cooper, M.Nutley, N.Errington, S.Harding, F.Guerlesquin, C.F.Goodhew, I.Moura,
J.J.Moura, G.W.Pettigrew
Biochem. 2004, 43, 14566-14576
109. “An Efficient Poly(pyrrole)-Nitrite Reductase Biosensor for the Mediated Detection of Nitrite”,
S. da Silva; S. Cosnier; M.G. Almeida; J.J.G. Moura
Electrochem. Comm. 2004, 6, 404-408
110.
“Biossensores: Modernas Ferramentas Para Monitorização E Controlo Analítico”
M.G. Almeida
Boletim de Biotecnologia 2004, 79, 12-23
111.
“Ligand K-edge X-ray absorption spectroscopy and DFT calculations on [Fe3S4]0,+ clusters:
delocalization, redox, and effect of the protein environment”
Dey A, Glaser T, Moura JJ, Holm RH, Hedman B, Hodgson KO, Solomon EI.
J Am Chem Soc. 2004, 126, 16868-78
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112.
“Decavanadate As A Biochemical Tool In The Elucidation Of Muscle Contraction Regulation”
T.Tiago; M.Aureliano ; JJ Moura,
J Inorg Biochem. 2004, 98, 1902-10
113.
“Mo And W Bis-MGD Enzymes: Nitrate Reductases And Formate Dehydrogenases”,
Moura JJ, Brondino CD, Trincao J, Romao MJ.
J Biol Inorg Chem. 2004, 9, 791-9
114.
“X-ray crystal structure and EPR spectra of “arsenite-inhibited” Desulfovibriogigas aldehyde dehydrogenase:
a member of the xanthine oxidase family”
Boer DR, Thapper A, Brondino CD, Romao MJ, Moura JJ.
J Am Chem. Soc. 2004, 126, 8614-5
115.
“EPR Spectroscopy Of Protein Microcrystals Oriented In A Liquid Crystalline Polymer Medium”
Caldeira J, Figueirinhas JL, Santos C, Godinho MH.
J Magn. Reson. 2004, 170, 213-9
116.
“The Family 11 Carbohydrate-Binding Module Of Clostridium Thermocellum Lic26A-Cel5E Accommodates
Beta -1,4 And Beta -1,3-1,4-Mixed Linked Glucans At A Single Binding Site.”
Carvalho AL, Goyal A, Prates JA, Bolam DN, Gilbert HJ, Pires VM, Ferreira LM, Planas A, Romao MJ,
Fontes CM.
J. Biol. Chem., 2004, Vol. 279, 34785-34793
117.
“Mutagenesis Study On Amino Acids Around The Molybdenum Centre Of The Periplasmic Nitrate
Reductase From Ralstonia Eutropha”
T. Hettmann, R. A. Siddiqui, C. Frey, T. Santos-Silva, M. J. Romão and S. Diekmann.
Biochem Biophys Res Commun., 2004, Vol. 320, 1211-1219
118.
“Ionic Transport In Tall Columnar Epithelial (TCE) Cells Obtained by Nasal Brushing From Non-Cystic
Fibrosis (CF) Individuals”
A.C. Maurício; D. Penque; M. D. Amaral; K.T.G. Ferreira
Acta Méd. Port., 2004, Vol 17: 427-434,
119.
“New chromosomal AmpC b-lactamase in Enterobacter cloacae”
T. Conceição; N. Faria; M. Pimentel; G. Soveral; A. Duarte; L. Lito; J. Melo Cristino; M. J. Salgado
Antimicrob. Agents Chemother, 2004, Vol. 48: 1437-1437
120. “Cristalografia e Função de Canais Membranares
T.F. Moura
Revista da Sociedade Portuguesa de Química, 2004 Vol. 92
121. “Sequential Injection Analysis of Chloride and Nitrate in Waters with Improved Accuracy using
Potentiometric Detection”
E. M. G. Santos; M. C. B. S. M. Montenegro; C. Couto; A. N. Araújo; M. F. Pimentel; V. L. Silva;
Talanta, 2004, Vol. 63, 721-727
122. “Ion Selective Electrodes for Penicillin-G Based on Mn (III)TPP-C1 and Their Application in Pharmaceutical
Formulations Control by Sequential Injection Analysis”
E. M.G. Santos; A. N. Araújo; C. M. C. M. Couto; M. C. B. S. M. Montenegro; A. Kejzlarová; P. Solich
J. Pharm. Biom. Anal., 2004, Vol. 36, 701-709
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123. “Determination of Gibberellic Acid by Sequential Injection Analysis Using a Potentiometric Detector
Based on Mn (III) – Porphyrin with Improved Characteristics”
E. M. G. Santos; C. M. C. M. Couto; A. N. Araújo; M. C. B. S. M. Montenegro; B. F. Reis
J. Braz. Chem. Soc., 2004, Vol. 15, 701-707
124. “Automatic Determination of Uric Acid in Urine in a FIA System with a Tubular Amperometric Detector”
M. B. Q. Garcia; J. L. F. C. Lima; M. L. Silva; J. P. Sousa
Port. Electrochim. Acta, 2004, Vol. 22, 249-262
125. “Chloride-Selective Membrane Electrodes and Optodes Based on an Indium (III) Porphyrin for the
Determination of Chloride in a Sequential Injection Analysis System”
M. Pimenta; A. N. Araújo; M. C. B. S. M. Montenegro; C. Pasquini; J. J. R. Rohwedder; I. M. Raimundo
Jr.
J. Pharm. Biom. Anal., 2004, Vol. 36, 49-55
126. “Direct Determination of Copper in Urine Using a Sol-Gel Optical Sensor Coupled to a Multicommutated
Flow System”
P. C. A. Jerónimo; A. N. Araújo; M. C. B. S. M. Montenegro; C. Pasquini; I. M. Raimundo Jr.
Anal Bianal Chem 2004, Vol. 380, 108-114
127. “Development of a Sol-Gel Optical Sensor for Analysis of Zinc in Pharmaceuticals”
P. C. A. Jerónimo; A. N. Araújo; M. C. B. S. M. Montenegro
Sens. Actuators B, 2004, Vol. 103, 169-177
128. “Colorimetric Bismuth Determination in Pharmaceuticals Using a Xylenol Orange Sol-Gel Sensor Coupled
to a Multicommutated Flow System”
P. C. A. Jerónimo; A. N. Araújo; M. C. B. S. M. Montenegro; D. Satinský; P. Solich
129. “Multi-pumping Flow Systems: An Automation Tool”
J. L.F.C. Lima; J. L. M. Santos; A. C. B. Dias; M. F.T. Ribeiro; E. A. G. Zagatto
Talanta, 2004, Vol. 64, 1091-1098
130. “Sequential Injection Chromatographic Determination of Paracetamol, Caffeine, and Acetylsalicylic Acid
in Pharmaceutical Tablets”
D. Šatínský; I. Neto; P. Solich; H. Sklenáøová; M. C. B. S. M. Montenegro; A. N. Araújo
J. Sep. Sci., 2004, Vol. 27, 529-536.
131. “Simultaneous Determination of pH, Chloride and Nickel in Electroplating Baths Using Sequential Injection
Analysis”
J. E. Silva; M. F. Pimentel; V. L. Silva; M. C. B. S. M. Montenegro; A. N. Araújo
Anal. Chim. Acta, 2004, Vol. 506, 197-202.
132. “Multi-Syringe Flow Injection System With In-Line Microwave Digestion for the Determination for the
Determination of Phosphorus”
M. I. G. S. Almeida; M. A. Segundo; J. L. F. C. Lima; A. O. S. S. Rangel
Talanta, 2004, Vol. 64, 1283-1289
133. “Between- Method Carryover in Flow Analysis”
E. P. Borges; J. A. V. Prior; S. Vicente; J. L. M. Santos; E. A. G. Zagatto; J. L. F. C. Lima
J. Flow Inj. Anal., 2004, Vol. 21, 29-32
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134. “Multicommutated Flow System for the Chemiluminometric Determination of Clomipramine in
Pharmaceutical Preparations”
K. L. Marques; J. L. M. Santos; J. L. F. C. Lima
135. “Determination of Aluminum (III) in Crytallized Fruit Samples Using a Multicommutated Flow System”
V. Tóth; A. O. S. S. Rangel; J. L. M. Santos; J. L. F. C. Lima
Agric. Food Chem. 2004, Vol. 52, 2450-2454
136. “Sequential Injection Analysis – Based Flow System for the Enzymatic Determination of Aspartame”
R. M. Peña; J. L. F. C. Lima; M. L. M. F. S. Saraiva
Anal. Chim. Acta., 2004, Vol. 514, 37-43
137. “Automatic Flow Systems Based on Sequential Injection Analysis for Routine Determinations in Wines”
M. A. Segundo; J. L. F. C. Lima; A. O. S. S. Rangel
Anal. Chim. Acta., 2004, Vol. 513, 3-9
138. “Sequential Injection Analysis of Lead Using Time-Based Colorimetric Detection and Preconcentration
on an Anionic – Exchange Resin”
N. Z. Aracama; A. N. Araújo; R. P. Olmos
Anal. Sci., 2004, Vol. 20, 679-682
139. “Gran Method for End Point Anticipation in Monosegmented Flow Titration”
E. V. Aquino; C. Pasquini; J. J. R. Rohwedder; I. M. Raimundo Jr.; M. C. B. S. M. Montenegro; A. N.
Araújo
Braz. Chem. Soc., 2004, Vol. 15, 111-115
140. “Sequential Injection System for Simultaneous Determination of Chloride and Iodide by a Gran’s Plot
Method”
N. Araújo; M. C. B. S. M. Montenegro; L. Kousalová; H. Sklenáøová; P. Solich; R. P. Olmos
141. “Reagente Generation for Chemical Analysis Assisted by Ultrasonic Irradiation”
M. Korn; S. S. Borges; P. R. M. Maia; J. L. F. C. Lima; R. A.S. Lapa
Ultrasonics, 2004, Vol. 42, 585-590
142. “Influence of Jam Processing Upon the Contents of Phenolics, Organic Acids and Free Amino Acids in
Quince Fruits (Cydonia oblonga Miller)”
B. Silva, P. Andrade, A. Gonçalves, R. Seabra, M. B. P.P. Oliveira, M. A Ferreira
Eur. Food Res and Technol, 2004, vol 218, 385-9
143. “Free Amino Acids Composition of Quince (Cydonia oblonga Miller) Fruit (Pulp, Peel and Jam)”
B. Silva, S. Casal, P. Andrade, R. M. Seabra, M. B. P. P. Oliveira, M. A Ferreira
J. Agric Food Chem, 2004, vol 52, 1201-6
144. “Protective Activity of Hypericum androsaemum Infusion Against tert-Butylhydroperoxide-induced Oxidative
Damage in Isolated Rat Hepatocytes”
P. Valentão, M. Carvalho, E. Fernandes, F. Carvalho, P. Andrade, R. Seabra e M. L. Bastos
Journal of Ethnopharmacology, 2004, vol 92, 79-84
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145. “Quince (Cydonia oblonga Miller) Fruit (Pulp, Peel, and Seed) and Jam: Antioxidant Activity”
B. Silva, P. Andrade, P. Valentão, F. Ferreres, R. Seabra, M. A Ferreira
146. “Phenolic Profile in the Quality Control of Walnut (Juglans regia L.) Leaves”
J. Amaral, R. Seabra, P. Andrade, P. Valentão, J. Pereira, F. Ferreres
Food Chemistry, 2004, vol 88, 373-9
147. “Hypericum androsaemum Infusion Increases tert-Butyl hydroperoxide-Induced Mice Hepatotoxicity in
Vivo”
P. Valentão, M. Carvalho, F. Carvalho, E. Fernandes, R. P. Neves, M. L. Pereira, P. Andrade, R. Seabra
e M. L. Bastos
Journal of Ethnopharmacology, 2004, vol 94, 345-351
148. “Triacylglycerol Composition of Walnut (Juglans regia L) Cultivars: Characterization by HPLC-ELSD and
Chemometrics”
J. Amaral, S. Cunha, R. Alves, J. Pereira, R. Seabra, B. P. P. Oliveira
149. “Evaluation of cheese authenticity and proteolysis by HPLC and urea-polyacrylamide gel electrophoresis”
A.C.A. Veloso; N. Teixeira; A.M. Peres; A. Mendonça; I.M.P.L.V.O. Ferreira
Food Chem., 2004, 87, 289-295
150. “Quince jam quality: microbiological, physicochemical and sensory evaluation”
I.M.P.L.V.O. Ferreira; N. Pestana; M.R. Alves; E.B. Proença; F.J.M. Mota; C. Réu; S.C. Cunha; M.B.P.P.
Oliveira
Food Control, 2004, Vol. 15, 291-295
151. “Effect of Olive Fruit Fly Infestation on the Quality of Olive Oil from Cultivars Cobrançosa, Madural and
Verdeal Transmontana”
J.A. Pereira; M.R. Alves; S. Casal; M.B.P.P. Oliveira
Italian J. of Food Science, 2004, Vol. 16, 355-365
152. “Discriminate analysis of the volatile fraction from “Terrincho” ewe cheese: correlation with flavour
characteristics”
O. Pinho; I.M.P.L.V.O. Ferreira; M.A.Ferreira
Int. Dairy J., 2004, 14, 455-464
153. “Evaluation of Some Carotenoids in Grapes by Reversed- and Normal-Phase Liquid Chromatography: A
Qualitative Analysis”
M.M. Mendes-Pinto; A.C. Silva Ferreira; M.B.P.P. Oliveira; P. Guedes de Pinho
J. Agric. Food Chem., 2004, Vol. 52, 3182-3188
154. “A novel Approach to the Quantification of Bovine Milk in Ovine Cheeses Using a Duplex Polymerase
Chain Reaction Method”
Mafra; I.M.P.L.V.O. Ferreira; M.A. Faria; B.P.P. Oliveira
155. “Free and Conjugated Biogenic Amines in Green and Roasted Coffee Beans”
S. Casal; E. Mendes; M.R. Alves; R.C. Alves; M.B.P.P. Oliveira; M.A. Ferreira
Química Verde
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156. “Predictive and Interpolative Biplots Applied to Canonical Variate Analysis in the Discrimination of Vegetable
Oils by their Fatty Acid Composition”
M.R. Alves; M.B.P.P. Oliveira
J. of Chemometrics, 2004, Vol. 18, 391-401
157. “Analysis of Heterocyclic Aromatic Amines in Foods by Gas Chromatography-Mass Spectrometry as
their tert.-butyldimethylsilyl Derivatives”
S. Casal; E. Mendes; J.O. Fernandes; M.B.P.P. Oliveira; M.A. Ferreira
J. of Chromatography A, 2004, Vol. 1040, 105-114
158. “Chemical, physical and sensorial characteristics of “Terrincho” ewe cheese: changes during ripening
and intravarietal comparison”
O. Pinho; E. Mendes; M.M. Alves; I.M.P.L.V.O. Oliveira
J. Dairy Sci., 2004, 87, 249-257
159. “Interrelationships among microbiological, physicochemical, and biochemical properties of Terrincho
cheese, with emphasis on biogenic amines”
O. Pinho; A.I.E. Pintado; A.M.P. Gomes; F. X. Malcata; I.M.P.L.V.O. Ferreira
J. Food Protection, 2004, 67, 2779-2785
160. “Quantification of Aflatoxins B1, B2, G1, and G2 in Pepper by HPLC/Fluorescence”
I.M.P.L.V.O. Ferreira; E. Mendes; M.B.P.P. Oliveira
J. Liq. Chromatogr. & Related Technol., 2004, Vol. 27, 315-324
161. “Enzymatic Hydrolysis of Whey Protein Concentrates: Peptide HPLC Profiles”
M.V.T. Mota; I.M.P.L.V.O. Ferreira; M.B.P.P. Oliveira; C. Rocha; J.A. Teixeira; D. Torres; M.P. Gonçalves
J. Liq. Chromatogr. & Related Technol., 2004, Vol. 27, 2625-2639
162. “Grapevine clones discriminated using stilbene synthase-chalcone synthase markers”
M.A. Faria; M. Beja-Pereira; A. Martins; M.A. Ferreira; M.E.S. Nunes
J. Sci. Food Agric., 2004, 84, 1186-1192
163. “Organismos Geneticamente Modificados e Alimentos Derivados: I. Legislação, Riscos Ambientais e
Segurança Alimentar”
Mafra; M.B.P.P. Oliveira
Alimentação Humana, 2004, Vol. 10, 131-145
164. “Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone
expressing an integron-borne OXA-30”
P. Antunes, J. Machado; J. C. Sousa; L. Peixe
J. Antimicrob. Chemother., 2004, Vol. 54, 429-434
165. “Antibiotic residues in edible tissues and antibiotic resistance of faecal E. coli in pigs from Portugal”.
Pena; C. Serrano; C. Réu; L Baeta; V. Calderón; I. Silveira; J. C. Sousa; L. Peixe
Food Additives and Contam., 2004, Vol. 21, 749-55
166. “Anodic Adsorptive Stripping Voltammetric Determination of Atrazine on Spiked Soil Samples with a
Gold Microelectrode”
S. Morais; O. Tavares; P. C. Baptista-Paíga; C. Delerue-Matos
Anal. Lett., 2004, Vol. 37, 3267-3281
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167. “Electrochemical Methods in Pesticides Control”
E. M. Garrido; C. Delerue-Matos; J. L. F. C. Lima; A. M. O. Brett
Anal. Lett., 2004, Vol. 37, 1755-1791
168. “Electrochemical Analysis of Opiates - An Overview”
J. M. P. J. Garrido; C. Delerue-Matos; F. Borges; T. R. A. Macedo; A. M. Oliveira-Brett
Anal. Lett., 2004, Vol. 37, 831-844
169. “Voltammetric Oxidation of Drugs of Abuse I. Morphine and Metabolites”
Electroanalysis, 2004, Vol. 16, 1419-1426
170. “Voltammetric Oxidation of Drugs of Abuse II. Codeine and Metabolites”
171. “Voltammetric Oxidation of Drugs of Abuse III. Heroin and Metabolites”
172. “Local genetic patterns within a Enterococcus faecalis clone in three hospitals in Portugal”
C. Novais; T.M., Coque; J. C. Sousa; F. Baquero; L. V. Peixe; Portuguese Resistance Study Group
Antimicrob. Agents Chemother., 2004, Vol. 48, 3613-3617
173. “In vitro activity of daptomycin against enterococci from nosocomial and community environments in
Portugal”
C. Novais; J. C. Sousa; T.M. Coque; L. V. Peixe; Portuguese Resistance Study Group
174. “Long term dissemination of an OXA-40 carbapenemase-producing Acinetobacter baumannii clone in
Iberian Peninsula”
G. J. da Silva; E. Bértolo; J. C. Sousa; S. Quinteira; L. Gallego; A. Duarte; L. Peixe
175. “Emerging of CTX-M â-lactamase producing Enterobacteriaceae in Portugal: report of an Escherichia
coli isolate harbouring BlaCTX-M-14”
E. Machado; T. Coque.; R. Cantón; J. C. Sousa; L. Peixe
Clinical Microbiol.Infect., 2004, Vol. 10, 755-757
176. “Challenges in modelling and optimisation of stability constants in the study of metal complexes with
monoprotonated ligands. Part III. A glass electrode potentiometric and polarographic study of Cu-DIPSOsystem”.
C.M.M. Machado; S. Sheerlinck; I. Cukrowski; H.M.V.M. Soares
Analytica Chimica Acta, 2004, Vol. 518, 117-126
177. “Simultaneous Determination of E-2-Nonenal and â-Damascenone in Beer by Reversed-phase Liquid
Chromatography using UV Detection”
L. F. Guido; J. R. Carneiro; J. R. Santos; P. J. Almeida; J. A. Rodrigues; A. A. Barros
Journal of Chromatography A, 2004, Vol. 1032, 17-22
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178. “The Impact of the Physiological Condition of the Pitching Yeast on Beer Flavour Stability: an Industrial
Approach”
L. F. Guido; P. G. Rodrigues; J. A. Rodrigues; C. R. Gonçalves; A. A. Barros
Food Chemistry, 2004, 87, 187-193
179. “Vitaltitration, a New Method for Assessment of Yeast Vitality Status”
P. G. Rodrigues; A. A. Barros; J. A. Rodrigues; A. A. Ferreira; C. Gonçalves, J. R. M. Hammond
Technical Quarterly, 2004, 41, 277-281
180. “An early formation of the nonenal potential in the malting process”
Luís F. Guido, Patrick Boivin, Nizar Benismail, Cristina R. Gonçalves and Aquiles A. Barros
European Food Research Technology, published on line: 7 December 2004
181. “Rheological Study of the Effect of Cassia Javanica Galactomannans on the Heat-set Gelation of a
Whey Protein Isolate at pH 7”
M.P. Gonçalves; D. Torres; C.T. Andrade; E.G. Azero; J.Lefebvre
Food Hydrocolloids, 2004, Vol. 18, 181-189
182. “A Study of the Effect of Locust Bean Gum on the Rheological Behaviour and Microstructure of a ?Lactoglobulin Gel at pH 7”
M.P. Gonçalves; W. Sittikijyothin; M.Vázquez; J.Lefebvre
Rheologica Acta, 2004, Vol. 43, 472 - 481
183. “Increasing the Force Torque Transducer Sensitivity of an RPA 2000 by a Factor 5-10 via Advanced Data
Acquisition”
L. Hilliou; D. van Dusschoten; M. Wilhelm; H. Burhin; E.R. Rodger.
Rubber Chemistry and Technology, 2004, Vol. 77, 192-200
184. “Modelling Shrinkage During Convective Drying of Food Materials: a Review”
L. Mayor; A.M. Sereno
Journal of Food Engineering, 2004, Vol. 61 (3), 373-386
185. “Prediction of the Solubility of Aromatic Components of Wine in Carbon Dioxide”
M. Vázquez da Silva; D. Barbosa
The Journal of Supercritical Fluids, 2004, Vol. 31, 9-25
186. “Simultaneous determination of Amphetamine derivatives in Human Urine After SPE Extraction and
HPLC-UV Analysis”
M.E. Soares; M. Carvalho; H. Carmo; F. Remião; F. Carvalho; M.L. Bastos
Biomedical Chromatography, 2004, Vol 18: 125-131
187. “Hepatotoxicity of 3,4-Methylenedioxyamphetamine and Á-Methyldopamine in isolated Rat Hepatocytes:
Formation of Glutathione Conjugates”
M. Carvalho; N. Milhazes; F. Remião; F. Borges; E. Fernandes; F. Amado; T. Monks; F. Carvalho; M.L.
Bastos,
Archives of Toxicology, 2004, Vol 78: 16–24
188. “d-Amphetamine-induced Hydrogen Peroxide Production in Skeletal Muscle is Modulated by Monoamine
Oxidase inhibition”
J.A. Duarte; F. Carvalho; E. Fernandes; F. Remião; M.L. Bastos; J. Magalhães; H. J. Appell
International Journal of Sports Medicine, 2004, Vol 25(6):446-449
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189. “Xanthine Oxidase inhibition by 1,3-Dipropyl-8-Sulfophenylxanthine (DPSPX), an Antagonist of Adenosine
Receptors”
T. Sousa; M. Morato; E. Fernandes; F. Carvalho; A. Albino-Teixeira
Journal of Enzyme inhibition and Medicinal Chemistry, 2004, Vol 19(1): 11-15
190. “Comparative Metabolism of The designer Drug 4-Methylthioamphetamine By Hepatocytes From Man,
Monkey, Dog, Rabbit, Rat and Mouse”
H. Carmo; J. G. Hegstler; D. de Boer; M. Ringel; F. Carvalho; E. Fernandes; F. Remião; L. Reys; F.
Oesch; M.L. Bastos
Naunyn-Schmiedeberg’s Archives of Pharmacology, 2004, Vol 369: 198-205
191. “Evaluation of Toxic/Protective Effects of The Essential Oil of Salvia officinalis on Freshly isolated Rat
Hepatocytes”
C. Lima; F. Carvalho; E. Fernandes; M.L. Bastos; PC Santos-Gomes; M. F. Ferreira; C. Pereira-Wilson
Toxicology in Vitro, 2004, Vol 18: 457-465
192. “Metabolism is Required for The Expression of Ecstasy-induced Cardiotoxicity in Vitro”
M. Carvalho; F. Remião; N. Milhazes; F. Borges; E. Fernandes; M. C. Monteiro; M.J. Gonçalves; V.
Seabra; F. Amado; F. Carvalho and M.L. Bastos
Chemical Research in Toxicology, 2004, Vol 17: 623-632
193. “The Toxicity of N-Methyl-Alpha-Methyldopamine to Freshly isolated Rat Hepatocytes is Prevented by
Ascorbic Acid and N-Acetylcysteine”
M. Carvalho; F. Remião; N. Milhazes; F. Borges; E. Fernandes; F. Carvalho and M. L. Bastos
Toxicology, 2004, Vol 200 (2-3): 193-203
194. “Implementation of a HPLC Methodology for the Quantification of Malondialdehyde in Cell Suspensions
and Liver”
M.E. Soares; M. Carvalho; F. Remião; F. Carvalho and M.L. Bastos
Journal of Chromatography B, Biomedical Aplications, 2004, Vol 27 (15): 2357-2369
195. “Strenuous Exercise Aggravates MDMA-induced Skeletal Muscle damage in Mice “
J.A. Duarte; A. Leão; J. Magalhães; A. Ascensão; M.L. Bastos; F. Amado; L. Vilarinho; D. Quelhas; H.
J. Appell; F. Carvalho
Toxicology, 2004, Vol 206 (3): 349-358
196. “Protective Activity of Hesperidin and Lipoic Acid Against Sodium Arsenite Acute Toxicity in Mice”
R.P. Neves; F. Carvalho; M. Carvalho; E. Fernandes; M.E. Soares; M.L. Bastos; M.L. Pereira
Toxicologic Pathology, 2004, Vol 32: 527–535
197. “Marine and Estuarine Algal Species as Test Organisms in Ecotoxicology”
B. Nunes; F. Carvalho; L. Guilhermino
Current Topics in Toxicology, 2004, Vol 1: 33-51
198. “Leucoisoprenochrome-O-Semiquinone Formation in Freshly isolated Adult Rat Cardiomyocytes”
F. Remião; D. Rettori; D. Han; F. Carvalho; M.L. Bastos and E. Cadenas
Chemical Research in Toxicology, 2004, Vol 17: 1584-1590
199. “Metabolism of The designer Drug 4-Bromo-2,5-Dimethoxyphenethylamine (2C-B) in Mice, After Acute
Administration”
H. Carmo; D. de Boer; F. Remião; F. Carvalho; L. A. Reys and M.L. Bastos
Journal of Chromatography B, 2004, Vol 811: 143-152
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200. “Acute and Chronic Effects of Clofibrate and Clofibric Acid on The Enzymes Acetylcholinesterase, Lactate
dehydrogenase and Catalase of The Mosquitofish, Gambusia Holbrooki”
B. Nunes; F. Carvalho; L. Guilhermino
Chemosphere, 2004, Vol 57: 1581-1589
201. “Design of 2-Cyclopentenone Derivatives with Enhanced NF-Kappa B: DNA Binding Inhibitory Properties”
P. A. Fernandes; A. I. S. Cruz; A. R. R. Maia; A. A. S. Almeida; A. M. N. Silva; B. F. B. Silva; C. M. S.
Ribeiro; C. F. B. Ribeiro; E. M. S. Cunha; F. R. N. C. Maia; J. A. C. Tedim; J. A. A. D. Ferreira; L. C.
Gomes; L. R. C. Matos; L. M. N. F. S. Cruz; M. A. B. P. Pinto; M. A. R. Encarnação; P. F. R. D. Teixeira;
R. S. G. R. Seixas; R. J. A. L. Quinta; S. S. Gomes; S. G. Patrício; S. D. S. Martins; T. F. Barros; T. S.
J. T. Selão; M V. Pande; M. J. Ramos
Journal of Molecular Structure (Teochem), 2004, Vol. 685, 73-82
202. “Theoretical Insights Into the Mechanism for Thiol/Disulfide Exchange”
P. A. Fernandes; M. J. Ramos
Chemistry – A European Journal, 2004, Vol. 10, 257-266
203. “Modeling Chemical and Biological Systems: A successful Course for Undergraduate Students”
M. J. Ramos; P. A. Fernandes; A. Melo
Journal of Chemical Education, 2004, Vol. 81, 1, 72-75
204. “Theoretical Study of Ribonucleotide Reductase Mechanism-Based Inhibition by 2’-Azido-2’Deoxyribonucleoside 5’-Diphosphates”
S. Pereira; P. A. Fernandes; M. J. Ramos
205. “Mechanism for Ribonucleotide Reductase Inactivation by the Anticancer Drug Gemcitabine”
S. Pereira; P. A. Fernandes; M. J. Ramos
206. “On the Modeling of Snake Venom Serine Proteinase Interactions with Benzamidine-Based Thrombin
Inhibitors”
E. S. Henriques; N. Fonseca; M. J. Ramos
Protein Science, 2004, Vol. 13, 2355-2369
207. “Evans Blue is an Inhibitor of nNuclear Factor-Kappa B (NF-kB)-DNA Binding”
R. K. Sharma; M. Otsuka; V. Pande; J. I. Inoue; M. J. Ramos
Bioorganic & Medicinal Chemistry Letters, 2004 Vol. 14, 6123-6127
208. “Definition of an Electronic Profile of Compounds with Inhibitory Activity Against Hematin Aggregation in
Malaria Parasite”
C. Portela; C. M. M. Afonso; M. M. M. Pinta; M. J. Ramos
209. “Ribonucleotide Activation by Enzyme Ribonucleotide Reductase: Understanding the Role of the Enzyme”
N. M. F. S. A. Cerqueira; P. A. Fernandes; L. A. Eriksson; M. J. Ramos
210. “New insights into a critical biological control step of the mechanism of Ribonucleotide reductase”
N. M. F. S. A. Cerqueira; P. A. Fernandes; L. A. Eriksson; M. J. Ramos
Journal of Molecular Structure (Theochem), 2004, Vol. 709, 53-65
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211.
“Natural Inhibitors of Proteases – Pharmacological Target for Destabilization/ Stabilization of the Protease/
Inhibitor Complex”
A.R. F. Carvalho, A. Melo
Fundamental & Clinical Pharmacology, 2004, Vol.18 Suppl. 1, 23-126
212. “Z-potential Measurements as a Tool to Quantify the Effect of Charged Drugs on the Surface Potential of
Liposomes”
B. Castro; P Gameiro; J. L. F. C. Lima; C. Matos; S. Reis
Langmuir, 2004, Vol. 20, 369 – 377
213. “Lanthanide Ion Recognition by Nickel(II) and Copper(II) Schiff Base Complexes Bearing Benzo-15crow-5 functionalities.
“C. Sousa; P. Gameiro; C. Freire; B. Castro.
Polyhedron, 2004, Vol. 23, 1401-1408
214. “Automatic Flow Procedure for the Determination of Glycerol in Wine Using Enzymatic Reaction and
Spectrophotometry”
E. N. Fernandes; M. N. C. Moura; J. L. F. C. Lima; B. F. Reis
Microchem. J., 2004, Vol. 77, 107-112
215. “In Vitro Scavenging Activity for Reactive Oxygen and Nitrogen Species by Nonsteroidal Anti-Inflammatory
Indole, Pyrrole, and Oxazole Derivative Drugs”
E. Fernandes; D. Costa; S. A. Toste; J. L.F.C. Lima; S. Reis
Free Rad. Biol. Med., 2004, Vol. 37, 1895-1905
216. “Noninvasive Methods to Determine the Cristical Micelle Concentration of Some Bile Acid Salts”
S. Reis; C. G. Moutinho; C. Matos; B. Castro; P. Gameiro; J. L. F. C. Lima
Anal. Biochem. 2004, Vol. 334, 117-126
217. “D-Amphetamin-Induced Hydrogen Peroxideproduction in Skeletal Muscle is Modulated by Monoamine
Oxidase Inhibition”
J.A Duarte; F. Carvalho; E. Fernandes; F. Remião; M. L. Bastos; J. Magalhães; H. J. Appell
Int. J. Sports Med. 2004, Vol. 25, 446-449
218. “Interaction of Anti-Inflammatory Drugs with EPC Liposomes: Calorimetric Strudy in a Broad Concentration
Range”
C. Matos; J. L. F. C. Lima; S. Reis; A. Lopes; M. Bastos
Biophysical Journal, 2004, Vol. 86, 946-954
219. “Influence of Some Anti-Inflammatory Drugs in Membrane Fluidity Studied by Fluorescence Anisotropy
Measurements”
M. Lúcio; H. Ferreira; J. L. F. C. Lima; C. Matos; B. Castro; S. Reis
Phys. Chem. Chem. Phys., 2004, Vol. 6, 1493-1498
220. “Zeta-Potential Measurements as a Tool to Quantify the Effect of Charged Drugs on the Surface Potenial
of Egg Phosphatidylcholine Liposomes”
C. Matos; B. Castro; P. Gameiro; J. L. F. C. Lima; S. Reis
Langmuir 2004, Vol. 20, 369-377
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221. “Fermi Resonance Coupling in the C-H Stretching Region of Methoxide Adsorbed on Clean Ru (001): a
Combined RAIRS and Theoretical Study”
S.S. Pinto; R. B. Barros; M. N. D. S. Cordeiro; J. A. N. F. Gomes; A. R. Garcia; L. M. Ilharco
Surface Science, 2004, Vol. 566-568, 965-970
222. “Structure and Conformational Equilibrium of New Thiacalix [4] Arene Derivatives”
Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes
Chemical Physics Letters, 2004, Vol. 385, 368-373
223. “Structure and Properties of Hexadecyltrimethylammonium Chloride Monolayers in Contact with Oil
Films with Different Thicknesses.”
D. J. V. A. Santos; J. A. N F. Gomes
J. Phys. Chem, B, 2004, Vol. 108, 17153, 2004
224. “Chain Length Effect on the Structure of Alkyltrimethylammonium Chloride Monolayers Between Two
Immiscible Liquids.”
D. J. V. A. Santos, J. A. N. F. Gomes
Prog. Colloid Polymer Sci., 2004, Vol. 126, 68-73
225. “Toward the Prediction of the Activity of Antioxidants: Experimental and Theoretical Study of the GasPhase Acidities of Flavonoids”
H. F. P. Martins; J. P. Leal; M. T. Fernandez; V. H. C. Lopes; M. N. D. S. Cordeiro
J. Am. Soc. Mass Spectrom, 2004, Vol. 15, 848-861
226. “Phenolic Acid Derivatives with Potential Anticancer Properties -A Structure-activity Relationship Study.
Part 1: Methyl, Propyl and Octyl Esters of Caffeic and Gallic Acids”
S. M. Fiuza; C. Gomes; L. J. Teixeira; M. T. G. Cruz; M. N. D. S. Cordeiro; N. Milhazes; F. Borges; M.
P. M. Marques
227. “Modulation of the Catalytic Activity of Manganese(III) salen Complexes in the Epoxidation of Styrene:
Influence of the Oxygen Source”
R. Silva, C. Freire, B. de Castro.
New J. Chem., 2004, Vol 28, 253 – 260
228. “Simultaneous Aluminium Oxide Pillaring and Copper (II) Complexes Encapsulation in a Montmorilonite”
P. Carvalho, C. Castanheira, B. Cardoso, J. Pires, A. R. Silva, C. Freire, B. de Castro, M. Brotas de
Carvalho
J. Mat. Chem., 2004, Vol. 14, 374-379
229. “Manganese(III) salen Complexes Anchored onto Activated Carbon as Heterogeneous Catalysts for the
Epoxidation of Olefins”
R. Silva, J. L. Figueiredo, C. Freire, B. de Castro.
Microp. Mesoporous Mat., 2004, Vol. 68, 83-89
230. “Development of Pillared Clays for the Encapsulation of Transition Metal Complexes”
J. Pires, J. Francisco, A. P. Carvalho, M. Brotas de Carvalho, A. R. Silva, C. Freire, B. de Castro.
Langmuir, 2004, Vol. 20, 2861-2866
231. “Anchoring of Copper(II) Acetylacetonate onto an Activated Carbon Functionalized with a Triamine”
R. Silva M. Martins, C. Freire, B. de Castro, J. L. Figueiredo.
Eur. J. Inorg. Chem., 2004, Vol. 10, 2027-2035.
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232. “Lanthanide Ion Recognition by Nckel(II) and Copper(II) Schiff Base Complexes Bearing Benzo-15-crown5 Functionalities”
C. Sousa, P. Gameiro, C. Freire, B. de Castro.
Polyhedron, 2004, Vol. 23, 1401-1408
233. “Epoxidation of Styrene by a Manganese(III) salen Complex Encapsulated in an Aluminium Pillared
Clay”
K. Biernacka, A. R. Silva, R. Ferreira, A. P. Carvalho, J. Pires, M. B. Carvalho, C. Freire, B. de Castro
New J. Chem. 2004, Vol. 28, 853-858
234. “Reactions of a Binuclear Nickel(II) Phosphine Complex Linked by a Bridged Bis(cyclopentadienyl)
Ligand with Isocyanides. X-ray Molecular Structure of [(CNtBu)(PPh3)Ni{?-(?-C5H4)CMe2(?C5H4)}Ni(PPh3)(CNtBu)][PF6]2”
F. G. Ribeiro, P. T. Gomes, A. R. Dias, J. L. Ferreira da Silva, M. T. Duarte, R. T. Henriques, C. Freire.
Polyhedron, 2004, Vol. 23, 2715-2724
235. “Jacobsen Catalyst Anchored onto an Activated Carbon as an Enantioselective Heterogeneous Catalyst
for the Epoxidation of Alkenes”
R. Silva, C. Freire, B. de Castro
Carbon, 2004, Vol. 42, 3027-3030
236. “Controlled Synthesis of 2-D and 3-D Dendritic Platinum Nanostructures”
Y. Song; Y. Yang; C. J. Medforth; E. Pereira; A. K. Singh; H. Xu; Y. Jiang; C. J. Brinker; F. van Swol; J.
A. Shelnutt
J. Am. Chem. Soc., 2004, Vol. 126, 635-645
237. “Cytotoxic Activity of Metal Complexes of Biogenic Polyamines: Polynuclear Platinum(II) Chelates”
L. J. Teixeira; M. Seabra; E. Reis; M. T. G. Cruz; M. C. Pedroso de Lima; E. Pereira, M. A. Miranda; M.
P. M. Marques
J. Med. Chem. 2004, Vol. 47, 2917-2925
238. “Theoretical and spectroscopic studies of the photochemistry of 3-(4-dimethylaminophenyl)-7methoxycylohepta-1,3,5-triene”
V. A. Kharlanov; W. Abraham; U. Pischel
J. Photochem. Photobiol. A, 2004, Vol. 162, 213-223
239. “Intramolecular singlet-singlet energy transfer in bichromophoric azoalkanes”
U. Pischel; F. Huang; W. M. Nau
Photochem. Photobiol. Sci., 2004, Vol. 3, 305-310
240. “An inhibit (INH) molecular logic gate based on 1,8-naphthalimide-sensitised europium luminescence”
M. de Sousa; M. Kluciar; S. Abad; M. A. Miranda; B. de Castro; U. Pischel
Photochem. Photobiol. Sci., 2004, Vol. 3, 639-642
241. “Zirconium organophosphonates as photoactive and hydrophobic host materials for sensitized
luminescence of Eu(III), Tb(III), Sm(III), and Dy(III)”
R. Ferreira; P. Pires; B. de Castro; R. A. Sá Ferreira; L. D. Carlos; U. Pischel
New J. Chem. 2004, Vol. 28, 1506-1513
242. “Photosensibilisierung durch Pharmaka”
U. Pischel
Nachr. Chem., 2004, Vol. 52, 1243-1246
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Books and books Chapters
1.
“Design Of Countercurrent Columns In Fractionation Processes With Supercritical Carbon Dioxide Application For Two Practical Examples”
in State of the Art Book on Supercritical Fluids, ed. AINIA, Centro Tecnológico Valencia, Spain, 193206 (2004)
M.N. Ponte; P.C. Simões
2.
“Mobilidade Molecular em Polímeros”
in Química de Polímeros, J. Seixas de Melo, M. J. Moreno, H. D. Burrows, M. H. Gil, Eds., Imprensa
da Universidade de Coimbra, (2004)
M. Dionísio, N. Alves, J. Mano
3.
“Bioplastics from Waste Materials”
in Resource Recovery and Reuse in Organic Waste Management, ed. P. Lens, B. Hamelers, H. Hoitink,
W. Bidlingmeier, IWA Publishing, 423- 440 (2004)
P. C. Lemos; L. S. Serafim; A. M. Ramos; M.A.M. Reis
4.
“Transport and adsorption in nanoporous materials”
in Nanoporous Materials - Science and Engineering, eds. G.Q. Max Lu and X.S. Zhao, Series on
Chemical Engineering, Vol. 4, Imperial College Press, 694-726 (2004)
J.P.B. Mota
5.
“Optimization of mixing protocol in a 3-D time-periodic Stokes flow”
in Computer Aided Chemical Engineering, eds. A. Barbosa-Póvoa, H. Matos, Vol. 18, Elsevier, 271-276
(2004)
A.J.S. Rodrigo; R.C.R. Rodrigues; N.F.C. Formiga; J.P.B. Mota; A. Lefevre; E. Saatdjian
6.
“Model-based bioreactor optimisation based on hybrid first principles/artificial neural network dynamical
models”
in Computer Aided Chemical Engineering, A. Barbosa-Póvoa & H. Matos (eds.), Vol. 18, 727-732,
Elsevier B.V., Amsterdam (2004)
R. Oliveira, A. Cunha, J. Clemente, M. J. T. Carrondo
7.
“Hybrid modelling of a PHA production process using modular neural networks”
in Computer Aided Chemical Engineering, A. Barbosa-Póvoa & H. Matos (eds.), Vol. 18, 733-738,
Elsevier B.V., Amsterdam (2004)
J. Peres, R. Oliveira, L. S. Serafim, P. Lemos, MA. Reis, S. Feyo de Azevedo
8.
“Hybrid modelling of fermentation processes using artificial neural networks: a study on identification
and stability”
in Computer Applications in Biotechnology, Marie-Noëlle Pons & Jan M. F. Impe (eds), Elsevier Ltd,
195-200 (2004)
R. Oliveira, J. Peres, and S. Feyo de Azevedo
9.
“Hybrid modelling of fermentation processes: a study on the use of modular neural networks for modelling
cells reaction kinetics”
in Computer Applications in Biotechnology, Marie-Noëlle Pons & Jan M. F. Impe (eds), Elsevier Ltd,
293-298 (2004)
J. Peres, R. Oliveira and S. Feyo de Azevedo
Química Verde
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10.
“Nutrition and heart disease - causation and prevention”
in Nutrition and heart disease - causation and prevention, eds. R.R. Watson and V.R. Preedy, CRC
Press, 119-136 (2004)
C. Lopes, S. Casal, B. Oliveira and H. Barros
11.
“Improving the texture of processed fruit: the case of olives”
in Texture in Food: volume 2 – Solid foods, ed. D.Kilcast, CRC Press, 410-431 (2004)
Mafra, M.A. Coimbra
12.
“Viscoelastic Characterization of Carrageenan Solutions and Gels Obtained from Portuguese Seaweeds”
in Progress in Rheology of Biological and Synthetic Polymer Systems, eds. A.C. Diogo, N.B. Alvarenga,
J. Canada, S. Ferro Palma and J.Dias, Instituto Politécnico de Beja, 9-15 (2004)
13.
“The Effect of the Degree of Hydrolysis and Concentration on the Heat Gelling Behaviour of Whey
Protein Concentrate Tryptic Hydrolysates”
D. Torres; C.R. Vicente; J.A. Teixeira; M.P. Gonçalves
14.
“Study of the Influence of Pre-heating Conditions and Mg2+ Concentration on Cold Gelation of a Whey
Protein Isolate”
M. Vázquez da Silva; D. Torres; M.P. Gonçalves
15.
“Mechanical Properties of Selected Biological Materials: Analysis of Experimental Data Using a New
Software Tool”
M. Vázquez da Silva; L. Mayor; M.A. Lemos; W. Sittikijyothin; M.P. Gonçalves; A.M. Sereno
16.
“Effect of Galactomannans on the Microstructure and Flow Properties of ?-Lactoglobulin Solutions at
pH 4.2”
W. Sittikijyothin; M.P. Gonçalves
17.
“Rheological and Interfacial Properties of Tara Gum as Additive of Reduced-calorie Mayonnaise”
J. Muñoz; M.C. Alfaro; M.Ruiz; J. De la Fuente; M. Martinez; D.P. Torres; M.P. Gonçalves
18.
“Antioxidant Compounds Extracted from Several Plant Materials”
R. Seabra, P.B. Andrade, P. Valentão, E. Fernandes, F. Carvalho, M.L. Bastos
in Biomaterials (Ed. M Fingerman), Science Publishers Inc, 2004
Química Verde
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Articles in Proceedings
“Polyhydroxyalkanoates Production by Activated Sludge in a SBR Using Acetate and Propionate as
Carbon Sources”
P.C. Lemos; L.S. Serafim; H. Santos; M.A.M. Reis
in Proceedings of the Third IWA Specialized Conference on Sequencing Batch Reactor Technology
(SBR3), Noosa (Queensland), Australia, 155-161, 2004.
“Removal of perchlorate and nitrate in an ion exchange membrane bioreactor”
S. Velizarov, C. Matos, J.C. Crespo, M.A.M. Reis.
in Proceedings of the European Symposium on Environmental Biotechnology (ESEB), 99-102, 2004.
“Removal of inorganic charged micropollutants in an ion exchange membrane bioreactor”
S. Velizarov, C. Matos, M.A.M. Reis, J.C. Crespo.
in Proceedings of the 6th conference “Membranes in Drinking and Industrial Water Production”, 15-17 de
Novembro de 2004, L’Aquila, Italia, 9 pp.
“Application Of Ion Exchange Membrane Bioreactor for Perchlorate and Nitrate Removal from Drinking
Water with High and Low Salinity”
Matos, C. Sequeira, A., Velizarov, S., C. Matos, J.C. Crespo, M.A.M. Reis. World Water Congress, 19- 24
de Setembro de 2004, 8 pp Marrakech, Marrocos.
“Biological organomercurial removal from vaccine production wastewaters by a pseudomonas putida strain“
R., Fortunato, J.C Crespo, M.A.M. Reis.
in Proceedings of the European Symposium on Environmental Biotechnology (ESEB),111-114, 2004.
“High Storage of PHB by Mixed Microbial Cultures Under Aerobic Dynamic Feeding Conditions”
L. S. Serafim; P. C. Lemos; R. Oliveira; A. M. Ramos; M. A. M. Reis
in Proceedings of ESBES 2004: European Symposium in Environmental Biotechnology, Oostende,
Belgium, 479-482, 2004.
“Hybrid Modelling of a PHA Production Process Using Modular Neural Networks”
J. Peres; R. Oliveira; L. S. Serafim; P. C. Lemos; M. A. M. Reis; S. Feyo de Azevedo
in Proceedings of ESCAPE-14: European Symposium on Computer Aided Process Engineering, Lisboa,
Portugal, 733-738, 2004
“Increased Oxidative Stress in Mesenteric Artery From Hypertensive Rats With Activation of The ReninAngiotensin System”
in Microcirculation. Proceedings of The 23rd Meeting of The European Society For Microcirculation. (Eds.
J.M. Silva, C. Saldanha, V. Oliveira, A. Prie, A. Shore), 2004, 137-140.
T. Sousa; M. Morato; D. Pinho; E. Fernandes; F. Carvalho; A. Albino-Teixeira
“Water Desorption Isotherms of Fresh and Partially Osmotic Dehydrated Pumpkin at Several
Temperatures”
L. Mayor; R. Moreira; A.M. Sereno; F. Chenlo.
in Drying - Proceedings of the 14th International Drying Symposium, eds. M.A. Silva; S.C.S. Rocha,
Ourograf Gráfica e Editora, 1481-1487 (2004)
“Effect of Drying on Cellular Structure of Apple Tissue”
L. Mayor; M.A. Silva; A.M. Sereno.
in Drying - Proceedings of the 14th International Drying Symposium, eds. M.A. Silva; S.C.S. Rocha,
Ourograf Gráfica e Editora, 1876-1883
Química Verde
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“Effect of Urea and Hydrolysed Waxy Starch on the Heat-Induced Gelation of Whey Protein Concentrate”
C.T. Andrade; G.K. Lopes; D.P. Torres; M.P. Gonçalves
in Proceedings of the 5th International Symposium on Natural Polymers and Composites, 330-332 (2004).
“Dehydration of Pumpkin Using Salt as Osmotic Agent: Evaluation of Water and Salt Coefficients of
Diffusion in Drying”
L. Mayor; R. Moreira; A.M. Sereno; F. Chenlo.
Proceedings of the 14th International Drying Symposium, eds. M.A. Silva; S.C.S. Rocha, Ourograf Gráfica
e Editora, 2157-2164 (2004)
“Purification et caractérisation de trois protéines impliquées dans le systeme respiratoire de la bactérie
sulfato-réductrice peptidolytique Desulfovibrio aminophilus”
López-Cortés, A., Bursakov, S., Figueiredo, A., Thapper, A.E., Todorovic, S., Moura, J.J.G., Ollivier, B.,
Moura, I. et Fauque, G.
Congres International de Biochimie. 3-6 Mai, 2004. Marrakech, Maroc 2004: 134-136
“On-line classroom, a way of collecting information about students’ skills”
N. T. Brito, M. J. Ramos
Proceedings of International Conference on Education and Information Systems: Technology and
Applications, 2004, Vol. 1, 79-82
Química Verde
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B
Dissertations
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Ph. D. Thesis
“Reacções envolvendo Clivagem da Ligação N—O em Compostos Orgânicos”
Duarte, M. S. P., Universidade Nova de Lisboa, 2004
Supervisor – A. M. Lobo, co-supervisor – S. Prabhakar
“Rearranjos 3-aza-Cope de N-Sililoxi-N-alil Enaminas”
Gomes, M. J. S., Universidade Nova de Lisboa, 2004
Supervisor - S. Prabhakar, co-supervisor – A. M. Lobo
“Physical and Thermodynamic Characterization of Environmental Friendly Substances. Ionic Liquids and
Alternative Refrigerants”
José Manuel Esperança, UNL, 2004
Supervisor: H. Guedes
“Phase Behaviour and Thermodynamic Properties of Ionic Liquid Solutions”
Vesna Najdanovic-Višak, UNL, 2004
Supervisors: M. Nunes da Ponte, L.P.N.Rebelo
“Prodution of Biopolymers By Mixed Cultures”
Luísa Alexandra Seuanes Serafim Martins Leal, UNL, Jul 2004
Supervisors: Maria D’Ascensão Miranda Reis, Ana Maria Ramos
“Hydration of á-Pinene in Polymeric Catalytic Membrane Reactors”
José Eduardo dos Santos Félix Castanheiro, UNL, Dec 2004
Supervisors: Joaquim Vital, Ana Maria Ramos
“Study and Optimization of nutrient removal by intermittent aeration”
Filomena Freitas, UNL, April, 2004
Supervisor(s): M.A.M.Reis
“Production of biopolymers by polyphosphate accumulating bacteria”
Luisa Serafim, UNL, July, 2004
Supervisor(s): M.A.M.Reis; A.Ramos
“Transport mechanisms in ionic liquid membranes and the study of thiomersal biodegradation”
Raquel Fortunato, UNL, December, 2004
Supervisor(s): M.A.M.Reis; J.G.Crespo
“Extraction and monitoring of biologically produced aromas”
Carmen Pinheiro, UNL, February, 2004
Supervisor(s): T. Schäfer; J.G.Crespo
“Study and Modelling of Extraction Processes with Facilitated Transport in Liquid Membranes”
Rui Viegas, UNL, February, 2004
Supervisor(s): I.Coelhoso; J.G.Crespo
“Concentration of Fruit Juices by Evaporation at Low Temperatures in Membrane Contactors”
Victor Alves, UNL, May, 2004
Supervisor(s): I.Coelhoso
Química Verde
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“Estudos Funcionais e Espectroscópicos na Redutase do Nitrato de Pseudomonas nautica 617”
Maria Cristina Miranda de Araújo Correia,UNL, March 2004
Supervisor(s): J.J.G.Moura e Jorge Lampreia
“Estudos Estruturais e Mecanísticos em Enzimas Multihémicas Isoladas de Bactérias desnitrificantes”
Isabel Cristina Timóteo, UNL, July 2004
Supervisor(s): I.Moura
“Estudos Estruturais e Mecanísticos da Redutase do Óxido Nitroso”
Inês Maria Cordeiro Gil Cabrito, UNL, October 2004
Supervisor(s): I.Moura e A.S.Pereira
“Crystallographic studies of molybdopterin-containing enzymes”
Jorge Rebelo, Technical University of Munich, Aug. 2004
Supervisors(s): Maria João Romão and Robert Huber
“Implementação de metodologias analíticas de reduzido impacto no ambiente para análise de queijo de
ovelha. Contributo para a caracterização do queijo Terrincho”,
Mestre Olívia Maria Castro Pinho, UP, Abr de 2004
Supervisor(s): M.A. Ferreira and I.M.P.L.V.O. Ferreira
“Compostos nitrogenados do café. Desenvolvimento de metodologias analíticas e sua aplicação na
discriminação de espécies e no controlo da intensidade da torra”
Susana Isabel Pereira Casal, UP, Jul de 2004
Supervisor(s): M.A. Ferreira and M.B.P.P. Oliveira
“Contaminação ambiental associada às areias residuais de fundição”
Maria Teresa Pereira de Oliva Teles Moreira, Universidade do Porto, Dezembro de 2004
Supervisor(s): Maria Conceição M. Alvim Ferraz
“Desenvolvimento de metodologias analíticas para a determinação de resíduos de produtos fitossanitários
em vinhos”
Maria Manuela Barbosa Correia, Universidade do Porto, Maio de 2004
Supervisor(s): Maria Arminda Costa Alves
“Mecanismos de oxidação electroquímica e quantificação de drogas de abuso”
Jorge Manuel Pinto de Jesus Garrido, Universidade de Coimbra, Março de 2004
Supervisor(s): Ana M. Oliveira-Brett
“Factors affecting beer flavour stability: studies on the whole process from barley to beer”.
Luís Guilherme de Lima Ferreira Guido, FCUP, 2004.
Supervisor(s): Aquiles Araújo Barros
Orientador: Ana M. Oliveira-Brett
“Estudos in vitro da hepatotoxicidade, nefrotoxicidade e cardiotoxicidade da metilenodioximetanfetamina
(“ecstasy”) e dos seus metabolitos conjugados com a glutationa, cisteína e n-acetilcisteína”
Márcia Cláudia Dias de Carvalho, FFUP, Nov de 2004
Supervisor(s): F. Carvalho
“Estudo Teórico do Mecanismo Catalítico da Enzima Piruvato Formato Liase”
Daniel Santos, FCUP, 2004
Supervisor(s): J. Ferreira Gomes
Química Verde
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M.Sc. Thesis
“Desenvolvimento de uma Metodologia Automática de Fluxo Continuo para a Determinação de Compostos
Fenólicos em Meios Micelares”
Ana Mafalda Lopes Coelho, UP, Nov de 2004
Orientador(es): M. A. Segundo e S. LReis
“Sistemas Baseados em Multi-seringa para Determinação de Micronutrientes em Extractos de Solos”
Delfina Maria Coelho Gomes Vasconcelos, UP, Nov de 2004
Orientador(es): M. A. Segundo e J. L. F. C. Lima
“Air Quality at Aberdeen Harber. An Overview and an Emission Inventory”
Célia Alves Meneses, UP, Dez de 2004
Orientador: J. L. F. C. Lima
“Desenvolvimento de um Sistema de Análise por Injecção Sequencial para a Determinação de Tensioactivos
Catiónicos em Agua”
Marieta Leite de Castro Passos, UP, Dez de 2004
Orientador: Lúcia Saraiva
“Desenvolvimento de uma Metodologia Automática de Fluxo Baseada em Multi-seringa com Pré-concentração
em Linha para a Determinação de Compostos Fenólicos”
Hugo Miguel Rodrigues Cunha Oliveira, UP, Dez de 2004
Orientador: M. A. Segundo e J. L. F. C. Lima
“Validação de um Método Analítico para a Quantificação Expedita de Carbamato de Etilo em Bebidas Alcoólicas”,
Susana Maria Ferreira de Melo Abreu, UP, Abr de 2004
Orientadores: M.B.P.P. Oliveira and P. Herbert
“Characterization of macrolides, lincosamides and streptogramines resistance genes in enterococci from
poultry”
Maria João da Silva Costa, Univ. Porto, Jun 2004
Coordinator: Luísa Peixe
“Extracção por solventes in-pulp de solos contaminados com compostos orgânicos”
Maria Aurora Soares da Silva, Eng do ambiente, Universidade do Porto, Março de 2004
Orientador: António Manuel Antunes Fíuza
“O professor como investigador – Uma experiência de investigação com materiais geossintéticos utilizados
em estruturas ambientais e desenvolvimento de um projecto no 3º Ciclo do Ensino Básico sobre a problemática
dos resíduos sólidos urbanos”.
Maria Fernanda Andrade Resende. FCUP, 2004.
Supervisors: Maria Gabriela Teles Cepeda Ribeiro and Paulo Joaquim Ferreira de Almeida
“Síntese, Caracterização de Complexos Metálicos com 3-Hidroxi-4-piridonas com Potencial Acção InsulinoMimética”
Maria João Lobo Loureiro de Amorim, FCUP, Junho, 2004
Supervisors: Maria Rangel and Baltazar de Castro
Química Verde
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C
Patents
Química Verde
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Patents
INPI 103143 C 2004/06/08
“Processo limpo para a Di-Hidroxilação ou Amino-hidroxilação de Olefinas em Líquidos Iónicos usando CO2
supercrítico”
Inventors: Luís C. Branco, Ana Serbanovic, M. Nunes da Ponte, Carlos A.M. Afonso
Brazilian Patent NI04051, submitted Nov 2004
“Process for Depolymerisation Of Acrylic Polymers By Catalytic Pyrolysis”
E. Falabella Souza –Aguiar; A. Figueiredo Costa; A.M. Ramos; I.M. Fonseca; J. Vital
Portuguese Patent, 103222 B, Granted 29/12/04.
“Process of Preparation Of Sulfonilureas”
M.F. Pereira; P.B. Correia
Patent nº WO04014436A1
“Cork product treatment system and apparatuses by extraction of compounds dragged in water vapor”
Cabral, M. F. 2004
Química Verde
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ANNEX III
RESEARCH PROJECTS
Química Verde
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Research Projects
International funding
“Molecular Level Devices and Machines”
HPRN-CT-2000-00029
Portuguese coordinator: Fernando Jorge da Silva Pina
“SuperGreenChem -a Marie Curie Research Training NetworK”
MCRTN-CT-2004-504005, 2004-08
HPRN-CT-1999-00084)
M.Nunes da Ponte (co-ordinator of the whole Network), Susana Barreiros, Ana Aguiar Ricardo
“The Mechanism, Specificity and Inhibition of Enzymes belonging to the Xantine Oxidase family: Medical
and Industrial Applications.”
Maria João Romão
“ A global study of the molecular genetics of pangolins, the scaly anteaters”
Project: Grant 7483-03
Organisation: National Geographic Society
Person in charge: Agostinho Antunes Pereira
“New approach to waste recovery into selective adsorbents of heavy metals”
Project NATO Science for Peace Program nº 977984, 2002-2006
Coordinator:
New Approach To Waste Recovery Into Selective Adsorbents Of Heavy Metals
Nato Science for Peace Proj. 977984, 2003-2006
Coordinator: José Paulo Mota
“Nanotechnology Network”
Luso-American Development Foundation (FLAD)
Principal Investigators: R. Franco and E. Pereira
Funded by FCT
“Hopanoid composition of sediments from Lower Tagus Basin”,
POCTI/QUI/45720/2002
Coordinator: Angela M. S. Relva
“New carbohydrate-based compounds from ethnopharmacological plants: bioactivity against pathogenic
yeasts”
POCTI/1999/FCB/35863
Coordinator:: Elvira Maria Mendes Sardão Monteiro Gaspar
“Volatile phenol yeast producers: a great concern in wine industry to be understood” POCTI/AGR/56771/
2004 - Ciências Agrárias e Florestais.
Project leader: Manuel Malfeito Ferreira, Departamento de Botânica e Engenharia Biológica, Instituto
Superior de Agronomia.
REQUIMTE participation: M.D.R. Gomes da Silva
Química Verde
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“Chemical and Sensorial Characterization of Malolactic Fermentation impact in Wines”
POCTI/AGR/55432/2004 - Ciências Agrárias e Florestais
Coordinator: : Ana Costa Freitas, Dep. Fitotecnia, U. Evora. REQUIMTE participation: M.D.R. Gomes da
Silva and Angela M.S. Relva
“Photophysics And Photochemistry Of Anthocyanins”
POCTI/QUI/33679/99
Coordinator: João Carlos dos Santos Silva e Pereira de Lima
“Building Blocks for Photoactive Molecular Cages”
Coordinator: António Jorge Dias Parola
“Randomly ordered DNA sensors based on direct questioning of immobilised probes with wavelength
shifting pairs”
POCTI/BIO/38922/2001
Member of team: João Carlos dos Santos Silva e Pereira de Lima
“Chemosensors Based on Fluorescent Poliamine Receptors”
Coordinator: Fernando Jorge da Silva Pina
“Nanostructures of noble metals in organized media: towards tecnhological application”
Member of team: João Carlos dos Santos Silva e Pereira de Lima
“A cor na iluminura portuguesa: uma abordagem interdisciplinar”
POCTI/EAT/33782/2000
Coordinator: Maria João Seixas de Melo
“New Synthetic methodology towards chiral gamma-amino acids useful in the design of new drugs”
Coordinator: Prof. Manuela Pereira.
‘Natural Alkaloids as Chiral Synthons’,
POCTI/QUI/44501/2002.
Coordinator: Prof. Ana Lourenço.
“Volatile phenol yeast producers: a great concern in wine industry to be understood.”
POCTI/AGR/56771/2004
Coordinator: Prof. M. Malfeito Ferreira (ISA)
Faculty of Science and Technology - New University of Lisbon, Researcher in charge of Chemistry: Prof.
Luísa Ferreira
“Saccharose as a Water Soluble Chiral Auxiliary and a Linker for Solid Phase Chemistry”
“Rationalising Stereoselectivity in Organic Chemistry: From Theory to Practice”
“High-Pressure NMR Spectroscopy of Polymers and biopolymers in CO2 Emulsions”
Química Verde
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Natural Vegetal Antioxidants
Coordinator: Abel José de Sousa Costa Vieira
Production and characterisation of a PDLC. New strategies for polymerisation and incorporation in
supercritical CO2
POCTI/CTM/47363/2002
Coordinator: Madalena Dionísio ;João Sotomayor - Collaborator
RenH2 – Stand-Alone Energy System Supported by Totally Renewable Hydrogen Production
POCTI/ENR/59623/2004
Coordinator: João Martins (ESTS/IPS) ;João Sotomayor - Collaborator
“Bacterial cytochrome c peroxidases- Activation, Enzymatic Mechanism And Structure”
Coordinator: Isabel Moura
“Enzimas Chave Da Reduçâo Do Nitrato.Redutase Do Óxido Nítrico E Redutase Do Óxido” Nitroso-As
Duas Enzimas Terminais”
POCTI/BME/42265/2001
Coordinator: Isabel Moura
“High Pressure NMR spectroscopy of polymers and biopolymers in CO2 emulsions”
Coordinator: Maria dos Anjos Macedo
“Estudos Electroquímicos Dinâmicos em Proteínas de Transferência Electrónica”
POCTI / QUI / 42277 / 2001
Coordinator:José J.G.Moura
“NMR structural studies of the active center of 5-aminolevulinate synthase, the first enzyme” of the
mammalian heme biosynthetic pathway”, 2002-2005
POCTI/BME/39184/2001
Coordinator. Maria dos Anjos Macedo
“Orientation of Proteins By Liquid Crystals”
Coordinator: Francisco Jorge Caldeira
“Structural and mechanistic studies of fatty acid desaturases. Looking for reactivity of diiron clusters”
Coordinator: Pedro Tavares
“Mechanistic and Structural Studies of Iron Oxidation and Storage by Fast Ferritins”
Coordinator: Alice S. Pereira
“Protease de Plasmodium chabaudi como alvo na terapia da malária”
POCTI/43637/BME/2000
Coordinator: Jorge Lampreia
Química Verde
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”As proteases de parasitas da malária como alvo na quimioterapia”
POCTI/ESP/42223/2001
Coordinator: Jorge Lampreia
“Mechanisms and Energetics of Electron Transport in Metalloproteins by Dynamic Voltammetry”
POCT/QUI/55743/2004
José J. G. Moura (FCT-UNL) and Margarida Santos (IST-UTL)
“Transient Protein Complexes – Soft Docking and NMR”
POCT/QUI/57741/2004 (66000•)
Coordinator:José J. G. Moura
Development of enzyme biosensors for multi-parametric control and monitoring of environmental samples.
Coordinator:M. Gabriela Almeida.
“New molybdo and copper cofactors in proteins isolated from sulphate reducers”
Coordinator:Isabel Moura
“Cobalt in metabolism of sulfate reducers. Noncorrin Co/Zn ATPS, AK, and a new Co-corrinoid protein”
Coordinator:Sergey Bursakov
“(Per)chlorate enzymatic reduction”
Coordinator:Cristina Costa
“Estudos Cinéticos e Mecanísticos da redução do Superóxido”
Coordinator:Alice S. Pereira
“Spectroscopy of Oriented Proteins on Conductive Polymers”
Coordinator:Francisco Jorge Fernandes Caldeira
“A crystallographic contribution to the understanding of the structure and function of Mo-enzymes”.
POCTI/1999/BME/35078
Coordinator:Maria João Romão
“Bacterial Cytochrome c Peroxidases- Activation, Enzymatic Mechanism and Structure”.
POCTI/QUI74230972001
Maria João Romão
“Structural and Functional Characterization of Nitrate Reductases and Formate Dehydrogenases”
Coordinator:Maria João Romão
“Molecular determinants of ligand specificity in carbohydrate-binding modules and cohesin-dockerin
complexes”
POCTI/BIA-PRO/59118/2004
Maria João Romão (with Prof Carlos Fontes and Prof. José Prates, FMV-UTL)
Química Verde
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“Efeitos biológicos dos isómeros do ácido linoleico conjugado (CLA) da dieta e de suplementação na
população portuguesa”
POCTI/2002/44750
Coordinator: Teresa Moura
“Development and Application of in Vitro Methodologies for Evaluation of Anti-Inflammatory and Antioxidant
Mechanisms of Non-Esteroidal Anti-Inflamatory Drugs”
POCTI/FCB/47186/2002
Coordinator :M. Salette Hipólito Reis
“Desenvolvimento e aplicação de metodologias expeditas de controlo e segurança no sector agroalimentar”
Ministério da Agricultura Desenvolvimento Rural e Pescas (AGRO/273)
Coordinator: José L. F. C. Lima
“Evaluation of membrane environment on modulation of drugs interactions with the P-glycoprotein multidrug
transporter”
Coordinator: M. Salette Hipólito Reis
“Assessing the dietary exposure of Portuguese consumers to acrylamide. Improvemrnt, development and
validation of rapide screening procedures and confirmatory analytical methodologies”
Coordinator: João L. M. Santos
“Study of geosynthetics durability in order to obtain a better definition of their safety factors”
POCTI/ECM/42822/2001
Proposing Institution – Faculty of Engineering of the University of Porto.
Principal Researcher – Maria de Lurdes Lopes (FEUP)
“Caracterização fitoquímica e actividade antioxidante de culturas in vivo e in vitro de Brassica oleracea var
costata”
Coordinator: Paula Andrade
“Valorização da Salvia officinalis, Melissa officinalis, Mentha piperita e Lavandula angustifolia para
obtenção de produtos de valor acrescentado, com actividade antimicrobiana e antioxidante”,
POCTI/AGR/43482/2001.
Coordinator: Manuel Ferreira (Universidade do Minho).
“Tecnologias para a valorização de bio-produtos da Salvia sp. (SalvaBiotech)”
Coordinator: Manuel Ferreira (Universidade do Minho)
Projecto de Investigação “Amphetamines and physical exercise. An hazardous combination? “
POCTI/ACT/43562/2001
Coordinator: Félix Dias Carvalho.
“Pharmacogenetic studies on the role of the metabolism and toxicity of amphetamine designer drugs”.
POCTI/SAL-FCF/57187/2004
Coordinator: Maria de Lourdes Bastos
Química Verde
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“Development and chemical, structural and rheological characterisation of protein-polysaccharide mixed
aqueous systems”
POCTI/QUI/ 36452/ 2000
Coordinator: Maria do Pilar Gonçalves
“Tecnologia para a obtenção de filmes e revestimentos comestíveis para alimentos a partir de recursos
nacionais de baixo valor”
POCTI/EQU/45595/2002
Coordinator: Alberto M. Sereno
“COMFOOD: Controling the microstructure of Food products by rheo-optical methods”
Coordinator: Loic Hilliou
“Tailored synthesis of biopolymers by mixed microbial cultures from molasses”
POCTI/BIO/55789/2004
Coordinator: Loic Hilliou
“Interaction between metal ions and buffers for the environmental and biological pH ranges”
Coordinator: Helena Soares
“Development of a clean technology for heavy metals removal and recovery from industrial effluents”
POCTI/CTA/47875/2002
Coordinator: Helena Soares
“Modelling inhibitor mechanisms in radical enzymes: a QM/CM approach”
POCTI/35376/QUI/2000
Coordinator: Maria João Ramos
“Investigation of Mimetic Modifications of Bioactive Peptides by Inclusion of Novel Synthetic Amino Acids”
POCTI/35380/QUI/2000
Coordinator: Hernâni Lopes da Silva Maia (Universidade do Minho)
REQUIMTE researchers involved: Maria João Ramos, André Melo
“Influence of Procyanidin Structures on their Ability to Complex with Anthocyanins - A Molecular
Interpretation of Colour”
Coordinator: Victor Armando de Freitas (Universidade do Porto)
REQUIMTE researchers involved: André Melo
“Experimental and Theoretical Studies on Model and Supported Catalysts: Catalytic Systems with
Industrial and Environmental Relevance”
POCTI/QUI/33765/99
Coordinator: Laura Maria Costa Iharco (Instituto Superior Técnico)
REQUIMTE researchers involved: José Ferreira Gomes, Maria Natália Cordeiro
“Electrocatalytic Reactivity of Gold Chiral Surfaces”
POCTI / QUI / 41704 / 2001
Coordinator: Ana Maria Teixeira Martins (Universidade do Porto)
REQUIMTE researchers involved: Maria Natália Cordeiro
Química Verde
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“A global study of the molecular genetics of pangolins, the scaly anteaters”
POCTI/BSE/47559/2002
Coordinator: Agostinho Antunes Pereira
“Nanostructures of nobel metals in organized media: technological applications”
Coordinator: Cristina Freire
“Design of novel metalosurfactants with catalytic properties: physico-chemical, morphological and
reactivity studies”
Coordinator:
“Nano estruturas de metais nobres em meios organizados: aplicações tecnológicas”
Coordinator
Sensores químicos: reconheciemnto molecular em interfaces sólidas (Chemosensors: molecular
recognition at solid interfaces)”
POCTI/CTM/ 46186/2002
Coordinator: Cristina Freire
“‘Desenvolvimento de membranas catalíticas poliméricas para a epoxidação de olefinas terpénicas
POCTI/CTM/ 45449/2002
Partcipants: Baltazar de Castro, Cristina Freire and Ana Rosa Silva.
‘’Aplicação de argilas com pilares funcionalizadas com complexos metálicos em catálise assimétrica
(Pillared Clays functionalised with transition metal complexes as new asymmetric catalysts)’
POCTI/QUI/ 42931/2001
Participants: Baltazar de Castro, Cristina Freire and Ana Rosa Silva.
“Síntese, estrutura, propriedades, especiação em solução e estudos biológicos de compostos de vanádio
com potencial para o tratamento de Diabetes”
POCTI /QUI/35368/99
Coordinator: João Costa Pessoa, Instituto Superior Técnico, Universidade técnica de Lisboa
Participantes: Maria Rangel, Lígia Gomes, João Gomes, Daniela Leite
“Factores Estruturais Determinantes na Reactividade dos Produtos de Fotólise de Compostos Modelo do
Sistema B12”
POCTI/QUI/ 46231/2002
Coordinator: Maria Rangel
“Phase-behaviour and microscopic characterisation of micro/macroemulsions in CO2. New strategies for
polymerisation and enzymatic catalysis.”
Coordinator: Ana Aguiar-Ricardo
“Production and Characterisation of a PDLC. New strategies for polymerization and incorporation in
scCO2.”
POCTI/CTM/47363/2002.
Coordinator: Madalena Dionísio
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“Changes in the molecular dynamics followed by dielectric spectroscopy during the formation of a polymer
dispersed liquid crystal”
Coordinator: Madalena Dionísio
“Computational fluid mechanics and process dynamics of supercritical fluid extraction columns with
structured packings and static mixer”
Coordinator: Pedro Simões
“Catalytic Depolymerisation Of Current Plastics Residues”
Mechanisms Of Drug Controlled Release From Microsphere
Tecnology For Obtention Of Edible Films And Coatings For Foods Obtained From Low Value National
Resources.
Transesterification Of Vegetable Oils. Application To The Production of Biodiesel
Development Of Catalytic Polymeric Membranes For The Oxidation Of Terpenic Olefins
“Static mixers as heat exchangers in supercritical fluid extraction processes - computational fluid
dynamics and process simulation studies”
POCTI/EME/61713/2004
Coordinator: José Paulo Barbosa Mota
“Process integration of supercritical fluid extraction and membrane separation to recover “vegetal” squalene
from olive oil residues”
Coordinator: Rui Manuel Pereira Ruivo
“Molecular Interactions on Liquids and Liquid Mixtures”
Coordinator: Pedro F. D. L. Pires
“Tecnology For Obtention Of Edible Films And Coatings For Foods Obtained From Low Value National
Resources”
Coordinator: Isabel Coelhoso
“Gas Separation By A Cyclic Processs Combining Membrane Permeation And Pressure Swing Adsorption”
“Anthocyanin separation by simulated moving bed chromatography
POCTIi/EQU/39391/2001, 2003-2005
Química Verde
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“Computational Fluid Dynamics And Process Smualtion Of Supercritical Fluid Extraction In StructuredPacking Columns And Static Mixers”
POCTI/EQU/349576/1999
Coordinator: Pedro Simões
“Mechanisms Of Drug Controlled Release From Microspheres”
Coordinator: Margarida Cardoso
Bilateral cooperations
“Molecular logic systems for information processing”
Acção Integrada Luso-Espanhola Nº E-38/02
Portuguese Coordinator: António Jorge Dias Parola
Spanish Coordinator: Prof. Enrique García-España
Departament de Química Inorgànica
Universitat de València, Spain
2002-2004
Acção Integrada Luso-Britânica Nº B-84/04
Portuguese Coordinator: Fernando Jorge da Silva Pina
English Coordinator: Prof. Amilra Prasanna de Silva
Queen’s University, Belfast, UK
2003-2004
Acção Integrada Luso-Espanhola Nº E-68/05
Portuguese Coordinator: António Jorge Dias Parola
Spanish Coordinator: Prof. Santiago V. Luis
Departamento de Quimica Inorganica y Organica
Universidade Jaume I de Castellón, Spain
2005-2006
PROYECTO X-10, “Proyecto Iberoamericano sobre Búsqueda y Evaluación de Nuevos Agentes activos en
patologías Digestivas” PIGASTRIN.
Portuguese Research coordinator: Prof. Ana Lourenço.
Project in collaboration with DGMEN
Faculty of Science and Technology - New University of Lisbon, Researcher in charge of Chemistry: Prof.
Luísa Ferreira.
“Computer Prediction of Biological Activity: Chirality in QSAR Applications”
Portuguese-German integrated Action A-11/04
Prof. João Aires de Sousa (Universidade Nova de Lisboa, Portugal)
Prof. Johann Gasteiger (University of Erlangen-Nürnberg, Germany).
“Analyse des interactions protéines-protéines par arrimage moléculaire sous contraintes RMN”
PROJECTO PICS Nº 1392
Entidade Financidadora: Centre National de la Recherche Scientifique / ICCTI
Coordinator: José J.G.Moura
Química Verde
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“Biochemical and Spectroscopy Studies on the Enzyme Nitrate Reductase (NAP) from the sulfatereducing organism Desulfovibrio desulfuricans”
Projecto De Cooperação Científica E Tecnológica
Portugal / Argentina, Programa 2003-2004 (J.J.G.Moura e Carlos Brondino)
“Study of a multihemic nitrite reductase by direct electrochemistry and electronic paramagnetic
ressonance spectroscopy (EPR)”
M. Gabriela Almeida and Bruno Guigliarelli.
CQFB, Dept. Química, FCT/UNL, Monte da Caparica and CNRS/Université de Provence, Aix-Marseille
I (France)
“Nanoemsambled based electroactive biointerfaces for environmental and food analysis of nitrates
and nitrites”
M. Gabriela Almeida and Serge Cosnier.
CQFB, Dept. Química, FCT/UNL, Monte da Caparica and Institut de Chimie Moléculaire de
Grenoble, UMR CNRS 5630, Université Joseph Fourier , Grenoble (France)
“Desenvolvimento de Sistemas Automatizados de Fluxo com Detecção Voltaamperométrica”
CRUP (Acção Integrada Luso-espanhola E-41/03)
Coordinator: M. Beatriz Q. G. Guerra Junqueiro
“Desenvolvimento de Mini-Sondas e de Sistemas Automáticos Visando Análise de Baixo Impacto
Ambiental”
FCT/GRICES/CAPES (Projecto bilateral Portugal/Brasil)
Coordinator: José L. F. C. Lima
Projecto de intercâmbio internacional, aprovado pela CAPES/ICCTI (092/02), em colaboração com a
Universidade Federal do Rio de Janeiro e com Faculdade de Engenharia da Universidade do Porto “Comportamento Reológico e Morfologia de Sistemas Mistos Proteínas/Polissacarídeos”
Bilateral cooperations
Rheological behaviour and morphology of protein-polysaccharide mixed systems
GRICES/ CAPES (Portugal/ Brazil)
Coordinator: Maria do Pilar Gonçalves
Frutas tropicais de alta humidade: processamento, embalagem sob atmosfera modificada e avaliação da
qualidade
GRICES/CAPES (Portugal/ Brazil)
Tecnologia de películas biodegradaveis para alimentos en ibero-américa” “Technology for food grade biodegradable
films in Ibero-America
CYTED project XI.20
International Co-ordinator: Dr. Paulo José do Amaral Sobral (Brazil)
“Acoustic wave sensor for metal ions based on poly[M(salen)crown] recognition chemistry”
Acções Integradas Luso-Britânicas entre o CEQUP/Departamento de Química/ICETA e o Department of
Chemistry, University of Leicester (Prof. A. R. Hillman) com o projecto intitulado: , [proj. B-6/03]; (2003 a Abril
de 2004).Participantes: Cristina Freire, Andrea Carneiro e Magda Martins.
Química Verde
A - 60
“Extraction Of Aminoacids And Peptides Through Liquid Membranes Using Ionic Liquids”
Acção Integrada E-62/03 Portugal- Espanha 2003 –2005.
Coordinator: Isabel Coelhoso
“Metabolism and toxicity of the designer drugs 4-MTA and 2-CB: identification of hypersensitive individuals
by pharmacogenetic studies”
Acções Integradas Luso-Alemãs
“Institut fur Toxikologie” da universidade de Mainz
Coordinator: Maria de Lourdes Bastos
“Production Of Biopolymers By Activated Sludge”
Cooperation Portugal/Italy, GRICES/CNR, 2003-2004.
Coordinator: M.A.M. Reis
Projects financed by “Agência de Inovação”
BEERVOLT (3 years project submitted to Agência de Inovação
Proposing Institution – Unicer, Bebidas de Portugal, SGPS S. A.
Other institutions involved: Faculty of Science of the University of Porto, Carlsberg S/A and CAI, Controle e
Automação Industrial, L.da.
Coordinator – Aquiles Barros.
“TCAexpress-Complementos de Amostragem e Metodologias Avançadas para a Detecção e Quantificação
Rápida de 2,4,6-tricloronisol “
Agência de Inovação, Programa IDEIA
Coordinator: Marcela Segundo
Química Verde

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