Annual Report 2003

Transcrição

Annual Report 2003

Laboratório Associado
para a Química Verde
Tecnologias e Processos Limpos
REPOR
T
REPORT
2003
ii
Executive Summary
REQUIMTE (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
CEQUP, has been focused on the topic GREEN CHEMISTRY œ CLEAN TECHNOLOGIES
AND 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 REQUIMTE: 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.
REQUIMTE can act as a promoter and is available to answer questions and problems placed
from outside. REQUIMTE 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 2003 was the second 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
more six 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 CQUP in
Fátima, January 9-10, 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 350
researchers, of which more than 130 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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REQUIMTE in 2003
2003 was the second 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.
2003 was the second year when the funding contract with FCT-MCES was operational and
REQUIMTE hired six new researchers and three technicians in order to carry out activities
included in the contract with the Ministry. Nine calls for application were open and more than
one hundred candidates have applied. The profiles of the six 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 209, much to the dedication of its graduate students, 19 of which have completed
their doctoral thesis and 11 their Master Thesis in 2003.
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People at REQUIMTE
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 Basto (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,WARSAW, 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
LABORATÓRIO ASSOCIADO
QUÍMICA VERDE – TECNOLOGIAS E PROCESSOS LIMPOS
REQUIMTE, is the bigest network in Chemistry Chemical Engenier stabihed in Portugal and results from the association between
CQFB (Universidade Nova de Lisboa) and CEQUP (Universidade do Porto). The large scientific experience and complementarity
of the available knowledge, allow us to deal with the topic “Green Chemistry – Technologies and clean processes” with a large
number of techniques and from different perspectives, and has the mission of coorporate in a continuous form, in a competent and
efficient in the prossocution of the specific aims of the national scientific and technologic politic of the following areas:
1
NATURAL 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
ENVIRONMENTAL CONTROL AND (BIO) REMEDIATION
Advanced analytical tools (AAS-EA, ICP-MS, GC-MS, HPLC-MS, AA 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
CATALYSTS, 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 2003 it occurred on the 20th 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. (the program is attached).
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 2003 a Forum of Chemistry
(see program attached) was organized by students and teachers of Chemistry.
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”.
Participation on the Program PULSE-public understanding of Life Sciences- 4 and 5 of
November 2003 on the Pavilhão do Conhecimento. 5 ateliers were organized to demonstrate
experiments related with Molecular Biology.(see program attached).
Monographic Courses (5 days) are ministrated in the themes:
Protein Purification, X-ray crystallography, EPR of Biological Materials, Structural Studies by
multidimensional NMR, Cleaner Processes with membranes, Chromatography, Safety in
Chemistry Laboratories.
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.
In the first day of the meeting the President of FCT and the Rectors of University of Porto and
New University of Lisbon attended the sessions. In the period before lunch the President of
FCT, Prof. Ramôa Ribeiro, addressed the audience informing on the organization, fund
management and future projects of FCT and a period for questions was allowed.
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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 2003.
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RESEARCH REPORT
2003
Part A
2
AREA 1
NATURAL PRODUCTS: SCREENING AND SYNTHESIS
OBJECTIVES OF THE LABORATÓRIO ASSOCIADO
• Screening of biologically active compounds of traditional plant-based Medicine and
support to certification of natural pharmaceuticals
• Synthesis of pharmacologically active compounds
• Chromatographic purification of new compounds and spectroscopic studies of
structure-function
The REQUIMTE Network has, from its inception, adopted a proactive attitude towards the field of Natural
Product research. This is, in fact, an area of intense worldwide interest, as these materials represent renewable
sources, their chemical structures have often complexity, including chirality, many have action, including
bio-action, and their study has uncovered scientific discoveries of enormous importance for mankind.
Different aspects of Natural Product Chemistry and Technology can be found in all five REQUIMTE’s areas
of activity. Area 1 involves those particularly focused on Screening and Synthesis of Natural Products.
ACHIEVEMENTS
OF THE
LABORATÓRIO ASSOCIADO 2002 / 03
• Implementation of a web version of a software program developed for predicting 1H
NMR chemical shifts of molecules, used for Natural Product screening and combinatorial
chemistry analysis - available at www.dq.fct.unl.pt/spinus , and widely used by researchers
in the field
• Implementation of a collaborative project in the area of abuse drugs. The first
metabolites of ecstsasy and related drugs were synthesized for toxicity studies.
• A patent on the use of terpenoids as chiral chromatography supports was filed in a
collaborative work with a national research laboratory
• A new antileishmanial tryptamine alkaloid was discovered
• A detail isoprenoid profiling of pine varieties cultivated in Portugal was used as an
indicator for insect attack in a collaborative program with environmental scientists
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Research highlights 2003
1. Screening
Access to screening of materials from foreign flora became possible through protocols of collaboration with
Guinea-Bissau, Tunisia and Argentina, and yielded a plethora of new bioactive compounds. These include
an antitumour antileishmanial diterpene form Holarrhena floribunda, antileishmanial catechins from Khaya
senegalensis and an antimalarial tryptamine indolic alkaloid from Sarcocephalus latifolius. Carbohydratebased natural compounds were also isolated and tested as potential new pharmaceutical drugs.
In a project aimed at protection of pine forests, several pine varieties cultivated in Portugal were differentiated
for their terpenoid contents, with ca. 254 different compounds identified. Special attention was focused on
the enantiomeric ratio of critical monoterpenes and its correlation with the severity of insect attacks on
trees.
Using different multidimensional chromatographic techniques, wine aroma changes during malolactic
fermentation were analysed and, in collaboration with external research groups, the olive oil flavour attributes
were chemically characterized in order to optimize the production process as well as the end product. An
interdisciplinary project for the study of potatoes (Solanum tuberosum) and their glycoalkaloid composition
was initiated.
A new software program developed for predicting 1H NMR chemical shifts of molecules was found particularly
useful for natural product screening and combinatorial chemistry analysis. The system, which is based on
neural networks, has a web version available in http://www.dq.fct.unl.pt/spinus, which is now widely
used by other researchers. Chirality codes were also developed to enable the prediction of the
chromatographic behaviour of a chiral molecule in a HPLC column.
2. Synthesis
The efforts in synthesis are three-fold: the first is to devise novel, simple, economic and non-polluting ways
of producing biomolecules or derivatives thereof with established pharmacological or industrial interest, the
second to explore natural products as renewable starting materials for synthesis, and the third to develop
new synthetic protocols mimeting green natural pathways. Mechanistic and theoretical studies supported
these efforts.
Along the first vector, indolo[2,3-a]carbazole alkaloids were selected because of their numerous important
biological actions. Among them, those of the staurosporine type are among the most highly researched due
to their dramatic effect as protein kinase C inhibitors. A simple, high yield synthesis of the aglycone
staurosporinone, using light at the key cyclisation step, was followed by the preparation of synthetically
advanced precursors en route to the final alkaloids. Similarly the synthesis of homopumilotoxins was pursued
using piperidines as model compounds. Derivatives of 3-piperidinemethanol were transformed on 3spiroepoxidepiperidine and 3-hydroxy derivatives with the objective of an enantioselective hydroxylation at
the position 3 of the ring. This simple transformation is one of the key steps on the hemisynthesis of
homopumiliotoxin enantiomers by a novel route. Coriandrin synthesis involved intermediates obtained by
using clean palladium catalysed reactions.
In the second area mentioned, ecologically friendly asymmetric synthesis using sugars was developed by
copolymerisation of a sucrose methacrylate monomer with styrene. The resulting polymeric material will be
next characterised. Sucrose was also used to prepare chiral auxiliaries for Diels-Alder reactions. Such
development was possible after a route for selective substitution at the 6’-position of the disaccharide was
found. Other new ligands containing donor N and O atoms were prepared. R,R-(+)-Tartaric acid derived
mono- and bis-oxazolines were used in the enantioselective addition of diethylzinc to enones.
Asymmetric aldol cyclisation was studied in relation to the reaction of meso-1,6-bis-aldehydes, derived from
meso-cyclohexene derivatives. The synthetic scope of the five membered ring carbocyclic molecules will
be evaluated for the obtention of biologically important cyclopentanols and aminocyclopentanols.
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The third area of interest, the development of new green synthetic protocols, saw its expression in the
studies related to 3,3-sigmatropic rearrangements. These reactions are a paradigm of atom economy.
Building on earlier work, the influence of heteroatom substituents, such as oxygen, in the energy barrier of
3-oxy-3-aza-Cope rearrangement was found to be of paramount importance, enabling reactions, which are
otherwise energetically prohibitive, to occur in good yield.
Lewis acids catalysts were also studied. For example, SbCl5 in moist acetonitrile was developed as a mild
new deketalisation reagent, and a comparison of BF3 , AlEtCl2 and TiCl4 reactivity towards Nacyloxazolidinones and N-acylimidazolidinones has surprisingly found no evidence for chelation in solution.
A detailed NMR study of Lewis acid promoted asymmetric addition to chiral unsaturated N-acyloxazolidinones
and N-acylimidazolidinones enabled a unified mechanism to be postulated that avoids the ambiguity of
Evans’ proposal.
The current studies on dirhodium(II) catalysed cyclization of α-diazo-α-phosphonoacetamides proved it to
be a high regio and stereocontroled procedure for the construction of α-phosphonolactams. Conformational
and electronic effects were studied in the γ-lactam formation. The steric effect of the N-substituent of the
amide was determinant in the stereoselectivity of the β-lactam formation. Preparation of α-diazo-αphosphonolactones was achieved, however moderate yields and reduce regio- and stereoselectivities were
observed.
Theoretical work using Hartree-Fock/ Moller-Plesset and density functional theories helped elucidate the
stereoselective behaviour of nucleophilic substitution reactions of haloacyl using a chiral auxiliary approach
based on ephedrine derived imidazolidinones under dynamic kinetic resolution conditions. A new model for
the reaction mechanism enabled the synthesis of second generation chiral auxiliaries that gave products of
higher diastereoisomeric excesses.
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AREA 1
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 quality control in food processing and development of
methodologies for fast pathogen detection
Despite the fact that food has never before been as safe as it is today, consumers are very uncertain and
increasingly critical about the safety and quality of their food. Safety is one of the product attributes that are
used by consumers in their evaluation of product alternatives and hence it determines purchase intentions
and choice. In today’s global economy, agribusinesses compete based not only on their capacity to lower
production costs, but also on their ability to offer safer and higher-quality products.
This REQUIMTE area involves different approaches focused in Food Quality Control, Authenticity and Safety
in order to answer consumer’s awareness, to help food industry improve quality and performance of the
produced foods, and to cooperate with authorities in order to prevent misrepresentation, mislabelling or
unfair trade.
ACHIEVEMENTS
OF THE
• Participation in International Collaborative study ISO/WD 15753:2001 about “Animal
and Vegetable fats and oils. Determination of Polycyclic Aromatic Hydrocarbons”.
• 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 consumer’s
awareness.
• 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.
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1. Food quality control
The development and implementation of analytical methodologies are one of the main working fields. In the
last year more attention has been devoted to the development of cleaner analytical procedures, sample
preparation and extraction/clean-up procedures. For example, a new headspace-SPME method for analysing
volatile compounds has been successfully developed and applied to traditional foods, like ewe cheese.
Sample preparation based on selective supercritical fluid extraction (SFE) and applied to food contaminants
and/or components is in implementation.
Biological techniques (PCR methodologies) were also implemented for food product authentication, namely
a duplex PCR method for the quantification of bovine milk in ovine cheeses. This methodology has been
tentatively applied to the detection of GMO crops and/or in food products containing these components.
Concerning quality control, several food matrices (conventional and traditional with Protected Denomination
of Origin - POD) have been evaluated, with special concern to milk and cheese, olives and vegetable oils,
nuts, coffee, alcoholic beverages and quince. Several chemical parameters were determined and statistical
treatment applied to the results allowing the discrimination of different varieties and/or geographical origins.
Some of the referred studies were solicited from consumer’s institutions or associations as well as from the
food industry.
An interdisciplinary project aiming at the discrimination of autochthonous Portuguese sheep breeds, and
the valorisation of the dairy derived products is in implementation. Besides the milk components of interest
it is of mention the presence of conjugated linoleic acid (CLA) with important physiological effects (e.g. anticarcinogenic) and considered a functional food component.
Some studies were also performed concerning food safety issues. The relationship between diet and health
stimulates public interest and awareness concerning the nutritional value of foods. Due to nutrient interactions
and alterations that can occur during cooking and industrial food processing, the studies also included the
analysis of processed diets (traditional Portuguese meals and fast food meals) as well as the respective raw
materials. As example of the work performed it is of mention the determination of food additives (sorbic
acid) and contaminants (PAH’s in vegetable oils, aflatoxins in spices as well as 4-(5)-methylimidazole and
furfural derivatives in roasted coffee and pesticides in olives and olive oils). The participation in an international
collaborative study of PAHs determination in oils and fats, which final report was published in August 2003,
was an example of the developed work. The implementation of methods aiming the determination of the
anisidine value in oils and fats is currently in progress.
In the ambit of a protocol established with REQUIMTE and Pharmaceutical Industry, several chromatographic
techniques were developed for evaluating the stability of products for veterinary use containing growth
promoter additives.
In order to transcribe some ISO and CEN documents concerning the analysis of food parameters to
Portuguese versions of European standards, a collaboration with technical committees has been maintained
and as results many NP, EN, and ISO documents are published and/or in press.
To manage food safety risks it is also very important to identify which foods, pathogens or situations lead to
illness, and the impact these have on human health. Priority was given to the incidence of Salmonella spp.
and Listeria monocytogenes in poultry carcasses. The high incidence of L. monocytogenes found in the
food products tested prompt us to adapt a multiplex PCR for a simple and rapid identification of Listeria
species.
Antibiotics have become an integral part of the livestock production industry and may be used therapeutically
to treat, or prophylatically to prevent disease. The occurrence of antibiotic residues in human food, arising
from its veterinary use, is a cause of apprehension to consumers worldwide. More recently, there is also a
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concern of acquisition of antibiotic-resistant bacteria by consuming food of animal origin. There is clear
evidence that an increase in the consumption of antibiotics by animals determinates a rise in the antibiotic
resistance in indicator bacteria in the faecal flora of animals and also in the pathogenic bacteria that can be
transmitted to humans via the food chain. Contamination of carcasses with faecal flora during slaughtering
usually occurs, and therefore edible products of animal origin may disseminate resistant bacteria and
resistance genes to humans. For that reason, surveillance of antimicrobial resistance in indicator and
pathogenic bacteria has been conducted as advised by several organizations, namely World Health
Organization (WHO). The high level of resistance especially for sulphonamides, tetracyclines and ampicillin
observed in E.coli from feacal samples of pigs raised also important questions about the relevance of
antibiotic residues detection as the only method for distinguish quality and safety of animals products for
human consumption.
Enterococcus are part of the natural feacal flora of several animals and of certain traditional cheeses,
playing an important role in the development of organoleptic characteristics during ripening. Once regarded
as a bacterial genus of little consequence to infectious disease, enterococci have rapidly emerged as
pathogens that frequently present considerable therapeutic challenge. It has been suggested that food
animals may be a reservoir of resistant enterococci and resistance genes capable of transferring to humans
through the food chain. Therefore the occurrence of resistance to different antibiotics in enterococci isolated
from food products (cheese, poultry and swines) was evaluated. Molecular characterization of the resistant
strains, resistance genes and genetic capture units was performed to assess their contribution to the increase
incidence of vancomycin resistant enterococci in clinical samples.
2. Safety evaluation
There are several toxicological assays internationally accepted for the safety evaluation of products and
compounds, almost all of them employing animal experiments. Beyond contributing for the universal aim of
reducing the number of animals used in the toxicological evaluation of compounds, in vitro models (both
chemical and biological) allow the establishment of the mechanisms of expression of toxicity/protection at
the cellular and molecular levels. Thus, studies of toxicity mechanisms of compounds, as well as the evaluation
of cytoprotection of plant extracts and isolated compounds, have constituted the main goals of this area.
Priority was given to the in vitro evaluation of the hepatoprotective activity of styrylchromones (compounds
of plant origin) and of the infusion of Hypericum androsaemu, which is a very popular infusion drink in
Portugal. The experimental model consisted in freshly isolated rat hepatocytes and some of the mechanisms
of the observed hepatoprotection were clarified. The hydroxyl radical and hypochlorous acid scavenging
activity of another infusion, obtained from Centaurium erythraea, was evaluated by non-cellular in vitro
chemical assays.
A Safety Report on a food additive, subject to an application submitted to the European Commission, Scientific
Committee on Food, to obtain Market Authorization, was elaborated
3. Beer ageing
One strong programme in this area is the consolidation of the growing research in the beer field. Particular
relevance is being devoted to studies related with the problem of beer ageing. The situation of this research,
which is being conducted in collaboration with some industrial and institutional partners, is described in the
following lines.
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— Cooperation with Unicer, Bebidas de Portugal SGPS, S A, also involving Carlsberg S/A and CAI
– There has been a strengthening of the good relationship already existing with Unicer, the main Portuguese
brewery. As a result of this cooperation, several papers and communications in congresses were produced
and a three-year joint project (project Beervolt) obtained external financial support, from AdI, the Portuguese
Agency for Innovation. The main objective of the work is the development of a voltammetric method for the
determination of diacetyl directly in the fermentation vessels, and it is based on a patent already approved
in Portugal1 and submitted to EPA. The project will involve two other companies: Carlsberg S/A and CAI.
Part of this work is also covered by another externally financed project “Determination of alpha-ketoacids
and alpha-diketones as products of cell metabolism”.
— Cooperation with Institut Français de la Brasserie et de la Malterie, I. F. B. M., Nancy, France – 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. It involves exchange of PhD students, in the
context of the collaboration with Unicer.
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AREA 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
Clean processes (based on alternative solvents, or membranes, or adsorption, or natural materials) have
been a focus of research of REQUIMTE groups for a long time. The Laboratorio Associado formation led to
a clear effort of Process Integration, using combinations of the Green Chemistry and Engineering approaches
that had been separately pursued in the past.
ACHIEVEMENTS
OF THE
• Extensive Clean Process development for agro-industrial applications, in collaboration
with industry and other LA, leading to:
i) Integrated Extraction-Membrane Separation of anti-oxidant compounds and
incorporation into food oils;
ii) Electronic nose on-line monitoring of wine fermentation and fruit juice production
• Participation in a recently awarded Network of Excellence – NanoMemPro - of the
NMP Area of the VI Framework Programme
• A workable adsorbed natural gas storage process, incorporating a guard bed for
reversibly filtering out trace contaminants and automatically re-odorise the natural gas
feed to the process, has been developed as part of a European project
• Novel hybrid systems coupling membrane permeation and pressure swing adsorption
have been developed for efficient gas separation.
• Scale-up of the supercritical CO2 extraction of a biologically-active substance from
cork industry residues for pharmaceutical applications (patents submitted)
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Processes using alternative solvents (supercritical fluids, ionic liquids, often combined with more conventional
GRAS solvents) have been developed. Integration with membrane separations has been a clear focus of
the research activity. Adsorption-based catalysis, storage and separations were studied, using either natural
or novel materials as adsorbents. Modelling, from molecular dynamics to CFD, was used for process
optimisation. On-line monitoring of processes was extensively studied.
1. Membranes
Design of clean membrane processes for recovery, separation and purification of high added-value products
focused on the understanding and mathematical modelling of the transport process of the species involved
across porous and non-porous membranes. Liquid membranes were also studied, including ionic liquidsupported ones, and selective carriers (chiral selectors).
Pervaporation was used as the separation method in integrated processes of chemical reaction / recovery
of solutes from dilute aqueous streams and from ionic liquids. On-line mass spectrometry allowed real-time
monitoring of the permeating stream. Concentration gradients of solutes, in particular ionic liquids, within
the membrane polymer was investigated by means of Raman Confocal Microscopy. Ultrafiltration was studied
for fractionation of proteins with pharmaceutical interest, and nanofiltration for recovery and fractionation of
antioxidant compounds from agro-industrial waste streams. Concentration of fruit juices by osmotic
evaporation was compared with membrane distillation (MD) in terms of water flux and aroma retention.
A different type of study involving membranes was the use of polymeric catalytic membranes, consisting of
molibdophosphoric acid (HPMo), immobilized in dense polymeric matrixes, such as polyvinyl alcohol (PVA)
and polydimethylsiloxane (PDMS).The tailoring of the hydrophilic/hydrophobic balance of the polymer matrix
was studied by esterifying the PVA OH groups with acetic anhydride, in successive extensions. The hydration
reaction of alpha-pinene was carried out.
2. Modelling of bioreactors
Modelling and control of processes included hybrid modelling of bioreactors, combining first principles with
artificial neural networks. Bounded Input Bounded Output (BIBO) stability conditions were derived and the
identification was studied in detail. A software package was developed implementing this technique. Hybrid
modelling with Mixture of Experts (MEs) networks was used for metabolic kinetic modelling. The ME network,
trained with the Expectation Maximisation (EM) algorithm, is able to detect different pathways with the
individual experts developing expertise in describing single pathways.
Precise Dissolved Oxygen control was achieved with an effective closed-loop control solution. Its economical
advantage in relation to the open-loop form of operation was shown and. the controller was implemented
and tested in a pilot 50 l fermenter, with a recombinant Pichia pastoris process.
3. Adsorption
Studies on the removal of chlorophenols from water used acidic and basic activated carbons at different pH
in batch and on column systems. Results have shown that clorophenols adsorb better in acidic carbons.
Adsorbed Natural Gas (ANG) on nanoporous carbon materials was studied as part of a European project,
completed by the end of 2003. A novel guard-bed system that is able to re-odorise the delivered gas to meet
European safety standards, has been developed.
Hybrid gas separation processes combining membrane permeation and pressure swing (PSA) adsorption
have been developed. A lab-scale unit of the hybrid process has been constructed and is scheduled to
operate during 2004.
Ab initio Process Modelling and Design involved the molecular simulation of adsorption processes. A new
method was developed to solve the governing equations for an isothermal adsorptive separation unit, under
equilibrium-controlled conditions. The technique has been thoroughly validated and its usefulness was
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demonstrated through application to a gas separation problem encompassing the major steps of practical
value to batch adsorption processes.
4. CFD and mixing
Viscous fluid mixing by chaotic advection in both time-periodic and spatially-periodic 3-D prototype flows
has been studied experimentally and theoretically. Numerical tools of analysis, such as stretching calculations
and tracer tracking methods, confirmed that an optimised protocol results in very effective mixing. A laboratoryscale chaotic mixer was built.
5. Supercritical carbon dioxide
Supercritical carbon dioxide extraction and fractionation of natural products at pilot plant scale has been an
area of intensive collaboration with agro-industrial, fishing and pharmaceutical companies. Two new processes
– extraction of a biologically-active substance from cork residues (patents submitted), and fractionation of
shark liver oil - were developed. Use of combinations of supercritical CO2 processes with extraction with
other GRAS (generally regarded as safe) solvents are in progress.
A dynamic model of a supercritical carbon dioxide packed fractionation column, incorporating information
on previously validated momentum and mass balances and using CFD, was developed.
Supercritical carbon dioxide was also used as the solvent medium for an integrated process of synthesis of
polymers polylactide and polyglycolide, and their subsequent impregnation with a model drug (salycilic
acid). Phase equilibrium measurements, DRS and DSC characterisation of synthesized polymers and
evaluation of drug release profiles were performed in this study.
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AREA 4
ENVIRONMENTAL CONTROL AND (BIO) REMEDIATION
• Advanced analytical tools (AAS-EA, ICP-MS, GC-MS, HPLC-MS, AA 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 recycling of materials.
Interdisciplinarity, ranging from Chemical Analysis to Bioreactors, and clean process expertise are the main
factors that allow REQUIMTE to contribute to this area. Development of sensors with diverse, environmentallyoriented applications has been pursued. Special problems of drinking-water contamination have been
addressed with novel concepts. Advanced analytical tools were applied to complex chemical systems.
ACHIEVEMENTS
OF THE
• A novel concept of membrane bioreactor to deal specifically with the problem of drinking
water contamination with ionic micropollutants was developed (international patent
granted). Know-how transfer to a multinational company of the water sector is under
negotiation.
• Development of a new process for biological production of biodegradable polymers
(PHA) from industrial wastes
• Development of sequence analysis (AA and DNA)
• Construction of transducers and (bio) sensors for chemical and clinical use:
i) Flow-through voltammetry electrodes – glucose, glycerol, herbicide detection
ii) Nafion-based Biosensor for nitrite detection
iii) Polyamine-based chemosensors for metal and anion detection
• Advanced analytical tools (AAS-EA, ICP-MS, GC-MS, HPLC-MS) were applied to complex
chemical problems - external service also. New automatic methods of analysis were
established and prototypes for laboratorial control and analytical detection were
developed (clean, rapid and reliable).
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1. Automation and Instrumentation
Continuous flow systems and dedicated equipment were developed and applied to automatic laboratory
measurements and to on-line control of industrial processes. Special attention has been dedicated to
procedures based on the multi-commutated flow methodologies and to the new concept of multi-pumping
flow analysis.
A new flow strategy exploits individual micro-pumps for liquid propelling, sample and reagent introduction
and component commutation, ensuring an effective and precise control of the sampled volume, either on a
time-based or on a pulse counting-based strategy.
Multi-pumping flow systems (MPFS) could become an advantageous alternative to other available procedures,
because they exhibit a high degree of automation, they are simple, fast, precise, accurate, and require low
reagent consumption and minor operator intervention.
2. (Bio)sensors and Transducers
New trends were investigated based on exploitation of different transducing schemes, such us optical and
electrochemical. Studies on immobilization techniques were used for monitoring low levels of drugs, food
and pesticides.
Studies were developed concerning the construction of flow-through voltammetry electrodes dedicated to
the implementation of manifolds with multi-side detection.
Applications: Glucose biosensor, Herbicide detection (CV and SWV), Voltammetric Flow systems, Periodate
selective electrodes for glycerol determination in spirits.
Chemosensors - Photochemical Transducers – metal and anion detection
In the framework of photochemistry and supramolecular chemistry, chemo sensors based on polyamine
receptors bearing a luminescent unit have been developed. Polyamine receptors are very versatile due to
their capacity to binding metals as well as anions. New chemosensors containing two fluorescent units were
prepared. They are capable of excimer formation, introducing another parameter whose appearance /
disappearance depends on the metal or anion to be analysed. Extension of this strategy to tripodal compounds
bearing three fluorescent units was carried out.
Development of a Nafion-Based Biosensor for Nitrite Determination
Cytochrome c nitrite reductase (NiR) isolated from D. desulfuricans ATCC 27774 membranes, catalyses
the direct reduction of nitrite to ammonia. It is highly stable and readily isolated in significant amounts. NiR
was immobilised on a glassy carbon electrode surface, by co-deposition with Nafion, a perfluorinated cationicexchange polymer.In these conditions, NiR is able to accumulate the chemical mediator (methyl viologen)
in the film. This system permits a reagentless and very specific analysis of nitrite, without interferences from
sulphite or nitrate. As nitrites are widely used in agricultural and technological practices and are also highly
toxic, this sensor should be of great interest.
3. (Bio) Remediation
In relation to biosensors – nitrite, particular emphasis was given to the denitrification cycle. Structural studies
on the enzymes involved in denitrification and applications to bioremediation are two different and
complementary aspects that were pursued. Further insights into nitrite and N(2)O reduction were obtained.
Nitrite Reductase
The cytochrome c nitrite reductase is isolated from the membranes of the sulphate-reducing bacterium D.
desulfuricans ATCC 27774 as a hetero-oligomeric complex composed by two subunits (61 kDa and 19 kDa)
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containing c-type hemes. The gene encoding cytochrome c nitrite reductase was sequenced and its crystal
structure determined to 2.3 Å resolution. In comparison to homologous structures, it presents structural
differences mainly at the regions surrounding the putative substrate inlet and product outlet, and includes a
well-defined second calcium site coordinated to two propionates and caged by a loop non-existent in the
previous structures.
N(2)O Reductase
Nitrous oxide reductase (N2OR) catalyzes the two-electron reduction of N2O to N2 and H2O in the last step
of the bacterial denitrification process. It is a dimeric protein. The recently solved crystal structure of N2OR
indicates that in each subunit there is a CuA center, which is the electron-transfer site, and a CuZ center,
which is the catalytic site. The neighbouring CuA and CuZ centers are from different subunits. The CuZ
center has a strikingly new structural motif consisting of a tetranuclear cluster. The CuZ cluster is coordinated
by seven His ligands.
4. Water Treatment
Membrane Bioreactors
Experimental studies and modelling of removal of polluting ions from drinking water streams according to
the Ion Exchange Membrane Bioreactor (IEMB) concept were performed for water contaminated with both
perchlorate and nitrate as well as for high salinity water (obtained from the Oceanarium of Lisbon) naturally
polluted by nitrate due to fish cultivation. Perchlorate and nitrate were reduced below the recommended
values of US EPA. A membrane-supported biofilm reactor was used.
Wastewater treatment using biological processes
Biological removal of nutrients (carbon, nitrogen and phosphorus) from wastewaters was studied in a
sequencing batch reactor (SBR) operated with short intermittent aeration. The complete cycle (feeding,
anaerobiosis, aerobiosis, settling and decanting) was only 36 minute long. The system proved to be robust
and dynamic.
The remediation of vaccine production wastewaters containing organomercurial compound was performed.
Kinetics of thiomersal degradation to metallic mercury, under aerobic conditions, by a pure culture of P.
putida spi3 strain was studied in a batch reactor and in a continuous stirred tank reactor.
5. Biomass conversion in high added value products
Production of Poly-Hydroxyalkanoates (PHA)
Optimization of PHAs production by activated sludge was studied in a Sequencing Batch Reactor (SBR)
operated under aerobic dynamic substrate feeding (ADF). The effect of several parameters, such as acetate,
nitrogen and phosphate concentration, pH and feed regimen, on the storage capacity of the mixed culture
was evaluated. The maximum yield of polymer was reached when operating the reactor with substrate
added by pulses. Indeed, the value obtained, 78% of PHB/cell dry weight, was not so far reached by mixed
cultures. A new strategy of reactor operation for PHA production, based on oxygen oscillations, was
implemented.
6. (Bio)Hydrogen – Bioenergy - Bioconversion - Biocorrosion
Hydrogen is a clean fuel and considered to be the fuel of the future. Bacterial systems contain elaborate
enzymatic systems for di-hydrogen production and consumption. Novel organometalic structures never
found in biology were revealed by complementary tools, such as Microbiology, Biochemistry, Molecular
Biology, Electrochemistry and Spectroscopy (EPR/ENDOR/X-Ray/EXAFS). [NiFe] (also [NiSeFe]) and [Fe]only Hydrogenases are typical examples of such sophisticated systems that carry out heterolytic cleavage
of dihydrogen (invoking hydride chemistry). 17O ENDOR detection of a solvent-derived Ni-(OH(x))-Fe bridge
that is lost upon activation of the hydrogenase was put in evidence and mechanistic implications deduced
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(Carepo et al. JACS 2003, 124, 281-286), supporting previous schemes proposed by our group and others.
Hydrogenases have been suggested to be involved in metal biocorrosion, a phenomenon with important
consequences in different economic sectors.
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AREA 5
CATALYSTS, SOLVENTS AND NON-TOXIC COMPOUNDS
• Green synthetic routes of chemicals and pharmaceuticals
• Alternative solvents and catalysis
• Enzymes in non-aqueous solvents
• Spectroscopic / computational techniques and molecular structure
The development of new green Synthetic Chemistry procedures has been pursued with strategies that
include (i) development of catalysts (ii) catalysis in non conventional solvents, such as ionic liquids and
supercritical fluids (iii) study of reaction mechanisms in classical and non classical reaction media; (iv) novel
techniques for product recovery, re-utilisation of solvents and reagents (recycling).
The main source of inspiration of Green Chemistry is Nature, especially in its living chemical reactors,
where selectivity, atom economy and mildness of reactions are at their utmost. In this respect, catalysis by
enzymes (biocatalysis) is also one of the most important research issues of REQUIMTE. A strong programme
of Structural Biology has been implemented. The catalytic activity, chemoselectivity and stability of enzymes
have been extensively studied, either in aqueous or non-aqueous solvents. Computational techniques,
using both molecular simulation and quantum calculations, have been used for molecular modelling of
chemical and biological systems.
ACHIEVEMENTS
OF THE
• A new family of ionic liquids was synthesised (patent submitted). Commercial
exploitation was started, via a spin-off company
• Combination of ionic liquids with supercritical carbon dioxide in catalytic cycles led to
a novel process (patent submitted).
• Co-ordination of a recently awarded Marie Curie Research Training Network –
SuperGreenChem – on reaction in supercritical fluids.
• The inhibition mechanism pathway of Gemzar®, an anti-cancer drug already in clinical
use, has been established for the first time.
• Structural determination of several components of the “Cellulosome” assembly, a
complex machinery responsible for plant cell wall degradation (e.g. the first dockerincohesin complex)
• Structural determination of a membrane bound cyt-c Nitrite Reductase, a crucial enzyme
involved in denitrification.
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1. Development of catalysts
Development of chiral catalysts (transition metal complex based catalysts) has been one of the important
topics of research. Both homogeneous and heterogeneous catalysts were prepared, characterized and
tested. Examples were: (i) improved solubility of organic cobalt and molybdenum catalysts was achieved by
the use of a trimethylsilyl group substitution in the ligand, enabling the clean epoxidation of cyclohexane
using molecular oxygen (ii) chiral and non-chiral manganese salen complexes have been synthesised and
their catalytic activity in the epoxidation of styrene evaluated (iii) the oxidation of limonene was carried out
with hydrogen peroxide, catalysed by cetylpiridinium peroxotungstophosphate, in a two-phase system (H2O2/
chloroform-limonene solution).
Novel catalysts by heterogenisation of metal complexes in several supports were also prepared and tested:
(i) cobalt acetylacetonate anchored on activated carbon via diamines and Mn(salen) and Cu(salen) complexes
was tested in the oxidation of several alkenes by t-butyl hydroperoxide (t-BHP), (ii) Mn(salen) complexes
immobilised in PILCs (pillared clays) were also tested in the heterogeneous epoxidation of styrene.
A new family of metallosurfactant-based catalysts was prepared, amphiphilic complexes of the type
[Fe(RR’bpy)2(CN)2], and their physical properties at water-air and water-metal interfaces were evaluated.
Metal-based catalysts were also prepared by using a photocatalytic method which enabled to prepare gold
sols with different nanoparticle morphology, namely triangular prisms, spheres and disks.
2. Catalysis in non conventional solvents
Non conventional solvents, such as ionic liquids and supercritical fluids, have been studied intensively
A new generation of ionic liquids (ILs), based on the tetraalkyl dimethylguanidinium unit, was prepared and
found to present complementary physical and chemical properties to those of earlier imidazolium based
salts. ILs were assessed as solvents in a variety of reactions such as biphasic nucleophilic displacement,
Sharpless asymmetric dihydroxylation and Baylis-Hillman reactions, with very promising results.
Combination of ILs with supercritical carbon dioxide as solvent media led to the development of novel
catalysis methodologies. Studies involved either phase switches that allowed single-phase catalysis and
biphasic removal of products or product withdrawal without any leaching of toxic catalysts (one patent
submitted).
Thermodynamic characterisation of new ionic liquids or of systems containing supercritical carbon dioxide
was also carried out extensively. Speed of sound, and density and phase equilibrium measurements were
performed, using newly developed techniques. Microscopic characterisation of micro/macro emulsions in
supercritical CO2 was carried out, including measurements of steady-state fluorescence emission,
fluorescence anisotropy and light scattering. High-pressure NMR techniques were used to provide structural
information on CO2-based systems including polymers and biopolymers.
3. Catalytic activity, and stability of enzymes in non aqueous solvents
Catalytic activity of enzymes in several media was tested and some mutagenic and inhibition studies completed
this issue. Several studies of serine hydrolases in non aqueous media were performed: (i) effects of water
activity, enzyme ionization and solvation on cutinase activity and enantioselectivity in supercritical fluids,
ionic liquids and organic solvents. (ii) effects of zeolites on lipase catalyzed esterification in supercritical
fluids and organic solvents. (iii) use of sol-gel immobilization technique to improve enzyme activity and
stability, and characterization of some of the materials produced.
4. X-ray crystallography studies of protein structures
Several enzymes have been structurally characterized: (i) membrane bound cytochrome c nitrite reductase
from D. desulfuricans ATCC 27774 - a second calcium site was found that is coordinated to two propionates
and caged by a loop non-existent in the previous structures. (ii) (CCP) the crystal structure of the di-heme
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Cytochrome c peroxidase from Pseudomonas nautica 617 was obtained in two redox states; (iii) High
molecular weight cytochrome from D. gigas has been purified and successfully crystallized and its structure
was solved by MAD methods (iv) the structures of several components of the “Cellulosome” assembly, a
complex machinery responsible for plant cell wall degradation, were also determined. Of particular importance
was the first three-dimensional structure of a dockerin-cohesin complex, which showed that protein-protein
recognition plays a pivotal role in the degradation of the plant cell wall by anaerobic microorganisms.
5. Molecular modelling of chemical and biological systems
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,
some molecular modelling of biological and chemical systems was performed: (i) establishment and/or
study of catalytic and inhibition mechanisms of enzymatic reactions, such as RNR (ribonuclease reductase,
a critical enzyme known to be involved in cancer), superoxide dismutase, farnesyl transferase and fumarate
reductase, was performed by both quantum mechanics and QM/QM or QM/MM methods; (ii) design of new
contrast agents for Magnetic Resonance Imaging, an important tool for clinical diagnosis (e.g. tumour therapy);
(iii) 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; (iv) 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; (v) modelling
of mimetic modifications of bioactive peptides by inclusion of novel synthetic amino acids; (vi) establishment
of new antimalarial compounds based on electrostatic profiles of established drugs; (vii) molecular simulations
on lanthanide(III) chelates and host-guest complexes with γ-cyclodextrins.
Footnotes)
Portuguese Patent 102608 – “Processo para a Determinação Voltamétrica, Célula Voltamétrica e Dispositivo
para Determinação em Fluxo”, Instituto Nacional da Propriedade Industrial, 03/03/2003.
1
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RESEARCH REPORT
2003
Part B
2
Analytical and Bioorganic Chemistry
Head of Laboratory: Ângela Relva, Assistant Professor
Research Team:
Elvira Maria M. Gaspar
Assistant Professor
Marco Diogo R.G. da Silva
Assistant Professor
João Paulo Noronha
Assistant Professor
Laila H. Ribeiro
PhD. Student
Number of articles in scientific journals: 2 (24, 25)
Number of M. Sc. Theses: 2
The research conducted by this group, which can be included in the wide field of analytical chemistry, uses
chromatography as its main tool. Modern chromatographic techniques are used to solve qualitative and
quantitative problems in the areas of safety and quality of Food, Natural Products, and Environmental
Chemistry. The on-going research includes the analysis of organic products by High Resolution
Chromatographic methods, such as Capillary Gas-Chromatography (CGC), Liquid Chromatography (LC),
Multidimensional Gas-Chromatography (MDGC), and their Hyphenated Techniques with spectroscopic
methods: i) determination of organoleptic properties of food products; ii) analysis of organic pollutants in
environmental samples; iii) study of biomolecules from plant origin and their activity as potential
biopharmaceuticals and/or green agrochemicals.
In the area of food related products, using different multidimensional chromatographic techniques, wine
aroma changes during malolactic fermentation was studied and, in collaboration with groups from the
Instituto Superior de Agronomia/Universidade Técnica de Lisboa, the olive oil flavour attributes were
chemically characterized in order to optimize the production process as well as the end product.
Several pine varieties, cultivated in Portugal, were differentiated for their terpenoid contents, with ca. 254
compounds identified. Special attention was focused on the enantiomeric ratio of critical monoterpenes
and their correlation with the severity of insect attacks on the trees. This work was done in cooperation with
Prof. Mosandl’s group in Frankfurt/Main, Germany. Comprehensive GC techniques were applied, GCxGCTOF (time of flight), for the complete identification of the compounds in pine trees’ simultaneous distillation
extracts. Natural carbohydrate-based compounds were also isolated and tested as potential new
pharmaceutical drugs.
Our interest in the monitorization of environmental pollutants included mapping the contamination in
sediments of river Sado’s estuary through the analysis of pesticides, PCB, PAH and heavy metals. Steroid
compounds in sediments from sewage effluents were also detected. Both studies also use atomic absorption
detection, and modern sample preparation techniques.
Production of course materials in the Chromatography area included a tutorial text “Glossary of
Chromatographic Separations” – in both a paper and a corresponding web (http://www.dq.fct.unl.pt/
cadeiras//glossario) versions. It includes a succinct compilation of mathematical and the general grammar
of chromatography for the needs of undergraduated chemistry students.
The year 2003 saw a reduced number of publications from this group due to disruption of research conditions,
namely the restructuring of the group, the reallocation of key laboratory equipment and the rebuilding of a
research laboratory which was consumed by fire.
Starting activities 2004: Geohopanoids constitute an important class of neutral compounds of bacterial
origin that are ubiquitous in the geosphere. We are starting the study of the hopanoid composition of
sediments from the lower Tagus basin, with the aim of characterising from a geochemical point of a view
such region.New chiral compounds will be used to prepare new stationary phases for enantiomeric GC,
which will be evaluated vis-à-vis similar existing market products.
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3
Photochemistry and Supramolecular Chemistry
Head of Laboratory: Fernando Pina, Full Professor
Research Team:
A. Jorge Parola
Assistant Professor
M. João Melo
Assistant Professor
J. Carlos Lima
Assistant Professor
Carlos Lodeiro
Assistant Researcher
Alexandra Bernardo
Associate Professor – ISCS-S
Number of articles in scientific journals: 18 (4, 26 to 41)
Number of articles in books: 1
During 2003, the Photochemistry and Supramolecular Chemistry Group developed scientific research based
on three basic lines: i) elementary molecular devices to write/read/erase, ii) chemical synthesis, and iii)
chemosensors, iv) Colour in art and nature.
Concerning the first item, a step towards the possible applications of photochromic systems based on
synthetic flavylium salts was achieved using water/ionic liquids biphasic systems. This work, done in
collaboration with Prof. Carlos Afonso from another group of Requimte, was accepted in Angew. Chem. Int.
Ed. and will appear in 2004.
The first synthetic multistate/multifuctional system in which a synthetic flavylium salt is encapsulated in a
solid polymeric matrix was accomplished and characterized. The matrix, a polymer hydrogel based on 2hydroxyethyl methacrylate, was choosen taking into account its permeation to aqueous acidic and basic
solutions. A manuscript is currently in preparation. Several synthetic flavylium systems were studied in
collaboration with the group of Prof. M. Maestri and Prof. V. Balzani from Bologna, Italy, the respective
papers accepted in J. Am. Chem. Soc. 2003, 125, 987-994; Chem. Eur. J. and Eur. J. Org. Chem. 2004, 304
- 312. In the future we are planning to improve the chemical response of the mentioned systems, pointing
out to the synthesis of new compounds.
In which concerns the second item – chemical synthesis – a project of hemicarceplexes (POCTI/QUI/
38637/2001, “Building Blocks for Photoactive Molecular Cages”, coordinator Prof. A. Jorge Parola) was
continued; two papers are in preparation concerning a) synthesis of metal complexing hemicarcerands
containing phenantroline units and, b) synthesis of substituted cavitands as building blocks for metal-induced
self-assembled hemicarcerands.
Synthesis of fluorescent ligands, namely linear or macrocyclic polyoxa-amines, and metal-complexing
flavylium salts as well as the complexes or adducts formed by interaction with cations and anions, is underway.
Chemosensors are a very active field of research within our group, third item.
A photophysical study of Dendrimers functionalized with naphthyl units was done in collaboration with Prof.
V. Balzani, Bologna, Italy, and Prof. Vögtle, Bonn, Germany, and accepted in Chem. Phys. Chem.
Several molecules whose behaviour can be considered as the basis of a fluorescent chemosensor were
developed in collaboration with Prof. Enrique Garcia-España, Valencia, Spain, and Prof. Antonio Bianchi
and Prof. Andrea Bencini, Florence, Italy.
A fluorescent temperature sensor based on the relative intensity of monomer/excimer emission was designed,
synthesised and characterised (M. T. Albelda, E. Garcia-España, L. Gil, J. C. Lima, C. Lodeiro, J. S. de Melo,
M. J. Melo, A. J. Parola F. Pina, C. Soriano, “Intramolecular Excimer Formation in a Tripodal Polyamine
Receptor Containing Three Naphthalene Fluorophores”, J. Phys. Chem. B. 2003, 107, 6573-6578). The
impact of the structural design on intramolecular excimer (J. S. Melo, J. Pina, F. Pina, C. Lodeiro, A. J.
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Parola, M. T. Albelda, M. P. Clares, E. Garcia-España, “Energetics and Dynamics of Naphthalene Polyamine
Derivatives. Influence of Structural Design in the Balance static vs. dynamic Excimer formation”, J. Phys.
Chem. 2003, 107, 11307-11318) or exciplex (Dalton Transations 2004 Accepted) formation was also explored
with other ligands. The fluorescent properties of these polyamine chemosensors can be used to detect
relevant biological analytes (M. T. Albelda, J. Aguillar, S. Alves, R. Aucejo, P. Diaz, C. Lodeiro, J. C. Lima, E.
Garcia-España, F. Pina, C. Soriano, “Potentiometric, NMR and Fluorescence Emission Studies on the Binding
of ATP by Open-Chain Polyamine Receptors containing Naphthyl and/or Anthrarylmethyl Groups”, Helv.
Chim. Acta, 2003, 86, 3118-3135), as well as negatively charged metal transition complexes (L. Rodríguez,
S. Alves, J. C. Lima, A. J. Parola, F. Pina, C. Soriano, M. T. Albelda, E. Garcia-España, “Supramolecular
Interactions of Hexacyanocobaltate(III) with Polyamine Receptors Containing a Terminal Anthracene Sensor,”
J. Photochem. Photobiol., A: Chem., 2003, 159/3, 251-256). Intrinsic fluorescent chemosensors, where the
complexing unit is itself fluorescent, can be achieved with macrocyclic ligands containing bipyridine units
(C. Anda; C. Bazzicalupi; A. Bencini; A. Bianchi, P. Fornasari; C. Giorgi; B. Valtancoli; C. Lodeiro; A. J.
Parola; F. Pina; “Cu(II) and Ni(II) Complexes with Dipyridine-Containing Macrocyclic Polyamines with Different
Binding Units”, Dalton Transations, 2003, 1299-1307). Several of these publications were done in collaboration
with Prof. Sérgio Melo, Coimbra, Portugal, in which photophysical studies are concerned.
Dr. Carlos Lodeiro has collaborations with the groups of Prof. Rufina Bastida, Santiago de Compostela,
Spain, concerning studies in Macrocyclic Chemistry, Lanthanides(III) and divalent metal ions. A new water
soluble zinc(II) macrocyclic sensor was developed (M. Vicente, R. Bastida, C. Lodeiro, A. Macías, A. J.
Parola, L. Valencia and E. S.-Spey, “Metal Complexes with a New N4O3 Amine Pendant-Armed Macrocyclic
Ligand. Synthesis, Characterization, Crystal Structures and Fluorescence studies.”, Inorg. Chem., 2003,
42, 6768-6779). Dr Carlos Lodeiro has also collaborations with the group of Professors Lluis Escriche and
Jaume Casabó, Barcelona, Spain, concerning the study of macrocycles with sulphur donor atoms, appropriate
for the detection of heavy pollutant metals (manuscripts in preparation).
Still in the field of fluorescent sensors, the application of energy transfer to the design of new probes to be
used in DNA chips is the subject of a project (POCTI/BIO/38922/2001 “Randomly ordered DNA sensors
based on direct questioning of immobilised probes with wavelength shifting pairs.”) in cooperation with Prof.
Guilherme Ferreira from Universidade do Algarve, that started in 2003.
Colour in Art and Nature is also investigated in our group, anthocyanins being a favourite subject. Blue
colour obtained through complexation with metal ions was studied and compared for natural anthocyanins
and synthetic flavylium salts (“Complexation of Aluminum (III) by Anthocyanins and Synthetic Flavylium
Salts. A Source of Blue and Purple Color” M. C. Moncada; S. Moura; M. J. Melo; A. Roque; C. Lodeiro, F.
Pina, Inorg. Chim. Acta, 2003, 356, 51-61). Anthocyanins are one of Nature favorite colour-molecules,
poppy red, cornflower blue, mauve violet, raspberry red, just to name a few. Synthetic flavylium salts are
molecules structurally similar. The ongoing project (POCTI/QUI/33679/99 “Photophysics and photochemistry
of anthocyanins”, coordinator Prof. João Carlos Lima) addresses the primary excited state processes in this
family of compounds (Paulo F. Moreira Jr., Leticia Giestas, Chang Yihwa, Carolina Vautier-Giongo, Frank H.
Quina, Antonio L. Maçanita and João C. Lima, “Ground and Excited Proton Transfer in Anthocyanins. From
Weak Acids to Super-Photoacids”, J. Phys. Chem.A. 2003, 107, 4203-4210) and the effect of heterogeneous
media in these processes (L. Giestas, C. Yihwa, J. C. Lima, C. Vautier-Giongo, A. Lopes, F. H. Quina, A. L.
Maçanita, “The Dynamics of Ultra-fast Excited State Proton Transfer in Anionic Micelles”, J. Phys. Chem., A,
2003, 107, 3263-3269).
The design of new molecules based on the above described family, to be used as blue natural food colorants,
“green” pigments and dyes will be initiated next year, in collaboration with the University of Porto.Chemistry
for the conservation of our cultural heritage is a recent area of research within our group (coordination Prof.
Maria João Melo). The stability of polymeric materials used as protective coatings for stone monuments has
been studied in collaboration with the ICVBC (Istituto per la Conservazione e la Valorizzazione dei Beni
Culturali) from Florence (“Correlating natural ageing and xenon irradiation of Paraloid® B72 applied on
stone”, S. Bracci, M. J. Melo, Polym. Degrad. Stab., 2003, 80, 533-541). The study of the materials and
techniques used in Portuguese medieval illumination is also being carried out, namely O apocalipse do
Lorvão from 1189. We are planning to publish the first results in 2004.
Química Verde
5
Organic Synthesis and Chemistry of Natural Products
Head of Laboratory: S. Prabhakar, Full Professor / Ana M. Lobo, Full Professor
Research Team:
Pedro Abreu
Paulina Mata
Manuela Pereira
Ana M. Lourenço
Paula S. Branco
Luisa M. Ferreira
João Aires de Sousa
Maria Manuel Marques
Rakesh Kumar
Yuri Binev
Susan Mathew
Sunil Gupta
Qingyou Zhang
Maria M. Santos
Mariana Duarte
Mário Gomes
Marta Corvo
Paulo Glória
Rita Noronha
Susana Gaudêncio
Vasco Bonifácio
Zulmira Gomes
Luís Pinto
Susana Nascimento
Carla Rosa
Iva Costa
Assistant Professor
Assistant Professor
Assistant Professor
Assistant Professor
Assistant Professor
Assistant Professor
Assistant Professor
Assistant Researcher (from May 2003)
Pos-doc (until September 2003)
Pos-doc
Pos-doc
Pos-doc (from Dez. 2003)
Pos-doc (from Nov. 2003)
PhD student
PhD student
PhD student
PhD student
PhD student
PhD student (with Prof. K. Nicolau, USA)
PhD student
PhD student
Ph.D. student
Research Training
Research Training
Research Training
Research Training
Number of articles in scientific journals: 11 (1 to 2, 5 to 13)
Nº of articles published in books: 3
Nº of Ph.D. Theses: 3
Nº of Patents: 1
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.
1
The area of natural products which continues to attract the interest of research workers of this
group includes:
a) The study of medicinal plants from the Portuguese, Tunisian and Argentina flora.
b)
An interdisciplinary project aiming at the study of potatoes (Solanum tuberosum) for their
glycoalkaloid composition.
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. Among
the indolo[2,3-a]carbazoles,the alkaloids of the staurosporine type are among the most highly
researched ones in view of their dramatic effect as PKC inhibitors. Advanced precursors based on
the aglycone of staurosporin, staurosporinone, were synthesised by a novel route.
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6
Studies on the synthesis of homopumilotoxins were pursued using piperidines as model compounds.
Derivatives of 3-piperidinemethanol were transformed on 3-spiroepoxidepiperidine and 3-hydroxy
derivatives with the objective of an enantioselective hydroxylation at the position 3 of the ring. This
transformation is one of the key steps on the hemisynthesis of homopumiliotoxin enantiomers.
In the development of a new deketalisation reagent SbCl5 in moist acetonitrile was found to be
selective and efficient.
3
As a paradigma of atom economy, rearrangements continued to be studied, and the influence of
substituents assessed.Thus, in the mechanistic area, the influence of heteroatom substituents
such as an oxygen in the energy barrier of 3-oxy-assisted 3-aza Cope rearrangements was found to
be of paramount importance, enabling reactions which are otherwise prhibitive from an energy point
of view. A full account of novel sigmatropic rearrangements of enehydroxylamine derivatives was
published and a mechanistic study, involving cross-over experiments, points towards an intramolecular
mechanism.
4
Computational methods were developed in response to practical chemical problems related to
quantitative structure-property relationships. Thus a system was developed for the automatic prediction
of the 1H NMR chemical shifts of molecules. The system is based in neural networks and a web
version is available in http://www.dq.fct.unl.pt/spinus . Since August 2003 the system was accessed
by more than 350 different computers. Chirality codes were also developped to enable the prediction
of the chromatographic behaviour in a chiral HPLC column of a chiral molecular structure.
5
Pedagogic tools
Given the academic activities of the majority of the elements of this group, teaching materials were
also produced.
The history of chemical technology was pursued in a collaborative effort involving other research
groups from the Faculty of Science and Technology of the New University of Lisbon (FCT-UNL) and
an involvement with secondary schools (‘Ciência Viva’ Program) for the introduction in the curricula
of experimental chemistry has continued.
Química Verde
7
Selective Synthesis and Structural Chemistry
Head of Laboratory: Maria Teresa Barros
Research team:
Maria Teresa A. Perea
Assistant Professor
Carlos Alberto M. Afonso
Assistant Professor
António Gil O. Santos
Assistant Professor
Ana Maria M. M. F. Phillips
Principal Researcher
Eurico J. Cabrita
Sara Isabel X. Candeias
PhD. Associate Member
Krasimira Petrova
Pos-Doc
Thierry Michaud
Pos-Doc
Vanya B. Kurteva
Pos-Doc
Snezhna Bakalova
Pos-Doc
João Miguel R. A. N. Rosa
PhD student
Jorge Alexandre S. Pereira
PhD student
Marta Morais S. de Andrade
PhD student
Pedro Miguel P. Góis
PhD student
Luís Alexandre A. F. C. Branco
PhD student
Paula Alexandra C. Rodrigues
MsD. student
Nuno M. M. Mateus
Graduate Project Researcher
Number of articles in scientific journals: 13 (14 to 23, 43 to 44)
Ecologically friendly asymmetric chemistry using sugars and non-polluting solvents Under this topic
directed toward the valorisation of sucrose by incorporating it in polymers, its conversion to natural compounds
and its possible application as a chiral auxilliary, we have developed a selective derivatisation of sucrose In
order to prepare usefull substrates for asymmetric synthesis we have obtained derivatives of saccharose
regioselectively formylated and transformed them into chiral auxiliairies which could control asymmetric
synthesis, as for example Diels-Alder reactions. Another goal was to contribute to the synthesis of
macromolecules with biological interest. Sugars are an important resource for the development of new
materials such as water-soluble and/or biocompatible polymers owing to their low price, various
applications, and potential biodegradability. Since sucrose has eight chemically active hydroxyl groups,
regio-selective derivatisation is important for the selective synthesis of sucrose-containing linear polymers.
A route for selectively substituting of the 6'-position of the sucrose by protecting-deprotecting strategy was
developed in our laboratory. Thus, we were able to obtain a monofunctional sucrose esters, which monomers
could be converted into pure linear polymers, avoiding the presence of mixtures of di- and higher substituted
vinyl esters, which results in cross-linked polymerisation.We have also prepared novel benzyl protected
vinyl sucrose monomers in a four step sequences from sucrose.
A study on the copolymerisation of these sugar monomers with styrene and methylmethacrylate, as well
as some physical properties of the resulting polymer materials is also being carried out.
OR
RO
RO
OR
O
O
RO
RO
R2
O
R=Bn, H
RO
O
R1
O
Química Verde
8
Asymmetric synthesis and homogeneous catalysis New chiral ligands containing donor N and O
atoms were prepared for asymmetric synthesis and their ability to catalyse a model reaction, the
enantioselective alkylation of benzaldehyde by diethylzinc, was investigated. R,R)-(+)-Tartaric acid was
used as a chiral synthon for the synthesis of novel cyclic 1,2-diacetals. New chemistry of these compounds
was studied, and they were used in the development of novel ligands for asymmetric catalysis. Monooxazoline carbinols 1 containing N,O-donor atoms which can bind to metals were synthesized. The same
basic structural unit was used in the synthesis of bis-oxazolines 2, N,N-donors for metals, which were
applied as chiral ligands in the copper-catalyzed enantioselective addition of diethylzinc to enones. This
research is still under way.
OMe
R1
R3
*
1
O
O
O
*
R2
HON
R1
OMe
2
R , R = H or Ph
R3 = i- Pr, Me, Ph
NN
R1
OMe
O
*
O
O
O
OMe
R1 = i-Pr, Ph, tert-Bu
Using palladium catalysed reactions we have been able to synthesise important intermediates in a proposed
synthesis of coriandrin and also complete syntheses of some polyoxygenated natural products which have
both saturated and unsaturated side chains
Stereoselective behavior of chiral a-haloacyl compounds in nucleophilic substitution reactions,
under dynamic kinetic resolution conditions. We studied, at ab initio level, using Hartree-Fock/MøllerPlesset and Density Functional Theories, with 6-31G** basis set, the stereoselective behavior of nucleophilic
substitution reactions of a-haloacyl compounds, using a chiral auxiliary approach, based on ephedrine
derived imidazolidinones under dynamic kinetic resolution conditions. With this study we were able to propose
a new model for the reaction mechanism, and to propose new chiral auxiliaries with expected better
performance. Some of the theoretical proposed structures were synthesised and their performance
experimentally estimated, confirming the theoretical previsions of expected high final diastereoisomeric
excesses.
Lewis acids interactions with N-acyloxazolidinones and N-acylimidazolidinones. The use of Lewis
acids to enhance the reactivity and stereoselectivity of a number of reactions involving N-acyloxazolidinones
and N-acylimidazolidinones is a routine procedure in many transformations in asymmetric synthesis. However,
for some transformations the stereoselectivity induced by the complexes formed can’t be entirely explained
by the normal accepted models. In order to be able to understand the structure and reactivity of these
complexes we have undertaken a NMR and molecular modeling study of the complexation of Nacyloxazolidinones and N-acylimidazolidinones with different Lewis acids. Since the molecules in question
have two key Lewis base sites, two 1:1 and one 1:2 solution complexes are possible. Depending on the
nature of the Lewis acid, chelates have also be considered. Besides the compounds mentioned before, we
have also prepared and studied several model compounds. Their complexation with the non-chelating BF
3
Lewis acid (in its etherate form) was studied and compared with the complexation with AlEtCl and TiCl .
2
4
Until now no evidence was found for the formation of the chelated forms in solution. This information can
have a tremendous impact in the accepted mechanisms for all reactions where N-acyloxazolidinones and
N-acylimidazolidinones are used and prompt us to the proposal of new mechanism pathways.
Lewis acid promoted asymmetric additions to chiral a,b-unsaturated N-acyloxazolidinones and Nacylimidazolidinones. Chiral-auxiliary based reactions retain a position of central importance in chiral
additions to double bonds. During the last year, our interest in this subject was mainly related with DielsAlder reactions and the addition of electrophilic species as, for instance, amines. The use of unsaturated Nacyloxazolididinones and N-acylimidazolidinones is based in the mechanism proposed by Evans, more
than 20 years ago. Nevertheless, the reactions are still very useful and the wide affordable information is
becoming more and more difficult to interpret, due to many unexpected experimental results. Based in our
Química Verde
9
previous work on DKR reactions, using N-imidazolidinones as chiral auxiliaries, we started the development
of a full theoretical and experimental study, this one mainly 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 the proposal of a new mechanism, which can explain
the observed stereochemistries, both in Diels-Alder and in electrophilic addictions, avoiding some of the
most unclear aspects of the Evans proposal.
Ionic liquids; We are currently performing research in the subject of ionic liquids, mainly in two complementary
areas: development of new ionic liquids, including some of their physical properties and their use mainly as
a reaction media in catalytic synthetic transformations. In the subject of the development of new ionic
liquids, we have been successful in the synthesis of new generation of ionic liquids based on the tetra-alkyldimethyl-guanidinium cation unit. Our first exploratory studies demonstrated that those ionic liquids presents
peculiar solubility properties and high stability under thermal, basic, acid, nucleophilic and oxidative conditions.
These properties clearly complements the ones known for the ionic liquids generation based on the
imidazolium unit. Our research efforts were also focused on the study of the physico-chemical behaviour of
imidazolium ionic-liquids by NMR, namely to gather information about rotational motion, transport properties,
molecular structure and molecular properties. He have made diffusion measurements (DOSY), 1H,1H-NOESY
and 19F,1H-HOESY studies of 1-buthyl-3-methylimidazolium (BMIM) with several counter ions (e.g. PF6-,
BF4-). These studies allow the determination of proton distances and the strength of ion pair association in
the pure ionic liquids. In another area, we have been studying some applications of ionic liquids in separation
processes as a supported membrane and in catalytic reactions were the catalyst is immobillised in the ionic
liquid as in the Baylis-Hillman reaction, biphasic nucleophilic displacement, Sharpless catalytic asymmetric
dihydroxylation and dirhodium(II) catalysed cyclization of a-diazo-a-phosphonoacetamides.
Intramolecular asymmetric aldol reaction; We are currently studying the development of efficient catalysts
for the asymmetric aldol cyclization of linear meso-1,6-bis-aldehydes, readily obtained from meso-cyclohexene
derivatives by olefin oxidation. This transformation is synthetically powerful because allows the formation of
five member ring carbocyclic molecules, with stereocontrol and with the introduction of the 2,3-unsaturatedformyl functionality, which is extremely useful for further functional group transformation. The carbocyclic
unit, obtained by this approach, is structurally very similar to carbocylic analogues of cyclopentanols and
aminocyclopentanols, which are an important biological class of compounds. In this research, the efforts is
mainly on the catalytic asymmetric key step and on the study of the synthetic scope of the resulting chiral
aldol adducts, by well known synthetic methodologies.
Application of NMR diffusion and NOE techniques. The usefulness of the application of diffusion ordered
spectroscopy (DOSY) to complex liquid foods such as fruit juices and beer, as a complementary aid to
spectral assignment based on the hydrodynamic volume, and hence diffusivity of different components,
was shown mainly for aliphatic and aromatic compounds found in these drinks. Application to the study of
interactions in supercritical CO2 Preliminary studies for the application of NOE methods and diffusion ordered
spectroscopy to the study of the mechanism of polymerization of methyl metacrylate in supercritical CO2 in
the presence of fluorinated surfactants were undertaken, the main goal is to understand the nature of the
interactions that lead to the stabilization of the polymer in the supercritical CO2.
Studies on C-H insertion of Rh(II) catalysed decomposition of a-diazo-a-phosphonoamides; The
current studies on dirhodium(II) catalysed cyclization of a-diazo-a-phosphonoacetamides proved to be a
high regio and stereocontroled procedure for the construction of a-phosphonolactams. Conformational and
electronic effects were studied in the g-lactam formation. The steric effect exerted by the N-substituent of
the amide was determinant in the stereoselectivity of the b-lactam formation. Preparation of a-diazo-aphosphonolactones was also achieved however moderate yields and reduce regio- and stereoselectivities
were observed.
In Organometallic compounds the effect of the trimethylsilyl (TMS) as solubiliser group of ligands and
their corresponding metal-complexes in supercritical carbon dioxide (scCO2) has been analysed using the
easily available TMS-substituted complexes (h5-Me3SiC5H4)MoO2Cl , (h5-Me3SiC5H4)2ZrCl2 , and (h5Me3SiC5H4)Co(CO)I2 . For comparison purposes, the solubility measurements were performed in parallel
Química Verde
10
with the analogous non-substituted derivatives. In all cases an increase in the solubility of the TMS-substituted
complexes was observed, being the influence of the TMS group in the chemical behaviour of the complexes
small. Complex (h5-Me3SiC5H4)MoO2Cl has been used as catalyst for two homogeneous catalysed processes
in scCO2: the oxidation of PPh3 using molecular oxygen as oxidant and the epoxidation of cyclohexene
using t-butylhydroperoxide (TBHP).
The molecular structure of (h5-Me3SiC5H4)Co(CO)I2 has been determined by X-ray crystallography.( see
figure )
C9
Si
C7
C8
Co
O
C
I3'
2.74
I3
I2
3.41
I1
Química Verde
11
Physical Organic Chemistry / Radical Chemistry
Head of Laboratory: Abel J. Vieira, Associate Professor
Research team:
Pedro Manuel C. C. P. dos Santos
João Adalberto A. Lourenço
PhD student
PhD student (staff from INETI)
Number of articles in scientific journals: 1 (23)
Radical reactions of DNA bases and other biologically relevant purine bases. Effect of antioxidants.
Radical oxidation of caffeic acid and cinnamic acid. Identification of final products.
Relative antioxidant properties of xanthine derivatives.Study of superoxide elimination from hydroxyl-adducts
to purine bases. Effect of molecular oxygen and substituents.
The group, which established itself recently, has interests centred in the radical reactions of DNA bases and
other biologically relevant compounds, namely oxygen derived radical induced alterations. Thus the antioxidant
properties of xanthine derivatives, and the superoxide elimination, leading to hydroxyl-adducts of purine
bases, was studied and the structure of transient radicals elucidated. In parallel, the radical oxidation of
caffeic and cinnamic acids was investigated, with identification of the final products.
Química Verde
12
B6
Head of Laboratory: Joao Carlos Sotomayor, Assistant Professor
Number of articles in books: 1
Solar Energy Conversion
The approach here is to combine the light harvesting properties associated with the high surface roughness
of Grätzel films, utilise the rectifying nature of the wide bandgap semiconductors and the ability to spatially
separate the donor and acceptor molecules on an unsupported TiO2 membrane to prevent fast back-electron
transfer. Already described is the process of charge separation in one molecule by means of visible light
irradiation: by the light absorption in the ruthenium centre, an electron transfer is obtain from this centre to
the viologen component and the characteristic colour of the viologen reduced form is obtain, in absence of
O2. This turns possible long-lived charge separation in the molecule and, due to the appearance of colour,
several write-read-erase cycles.
The next thing to do is to use the extra energy storage of the reduced viologen component as a reactant in
the water reduction reaction to produce H2, in the presence of a suitable catalyst, such as platinum. Samples
on TiO2 supported films were already made and a special cell for solar irradiation is under construction.
Polymer impregnation and photochemical aging
The study of polymer impregnation and photochemical aging is starting now. The main goal of this project is
to find a new strategy to produce a polymer dispersed liquid crystal. Supercritical carbon dioxide will be
used to incorporate the liquid crystal in the polymer matrix. Different experimental approaches will be
attempted: the incorporation of the liquid crystal in the end of the polymerisation process and during the
polymerisation process. These methods will result in different morphologies, which will determine different
electro-optical properties.
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Chemical Engineering Science
Head of Laboratory: Manuel Nunes da Ponte, Full Professor
Research Team:
18 Members of staff
Manuel Nunes da Ponte
Full Professor
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
Owen Catchpole
5 PostDoc
Svetlana Lyubchik, Paulo Lemos, Pavel Izak, Thomas Schäfer, Teresa Casimiro
21 PhD students
Rui Ruivo (PhD July 2003), Gundula Wolf (PhD Oct 2003), Mário Eusébio, José Esperança, António Rodrigo,
Carla Portugal, Carmen Pinheiro, Cristina Matos, Filomena Freitas, Isabel Esteves, José Luís Santos,
Luísa Serafim, Raquel Fortunato, Víctor Alves, Eugénia Nogueiro, Sílvia Garcia, Célia Peres, Pedro Vidinha,
Joao Dias (from Nov 2003), Ana Teixeira (from Nov 2003), Carla Brazinha (from Nov 2003).
6 project grantees
Marta Lopes, Pedro Nunes, Tânia Costa, Maria Teresa Viciosa Plaza, Márcio Tentem; João Paulo Lavrado
Number of articles in scientific journals: 32 (46 to 77)
Number of book chapters and papers in proceedings: 16
Number of patents: 5
Number of Ph.D thesis: 6
Scientific interests
The scientific interests of the Chemical Engineering Science Area span a wide range of subjects:
Polymeric Catalytic Membranes & Heterogeneous Catalysts
Polymeric membranes are used in catalytic membrane reactors. Heterogeneous catalysts based on materials
such as zeolites, activated carbons and mesoporous silicas are developed for the use in fine chemistry
reactions such as hydration and oxidation of terpenic olefins and esterification/transesterification reactions,
as prepared or dispersed in polymeric matrixes (catalytic membranes).
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14
Environmental catalysis and adsorption.
Tailored activated carbon is used as adsorbent to enhance removal of organic compounds ( phenols,
dioxins,dyes) and heavy metals from liquid phase .
The catalytic conversion of NO, N2O, CO2, SO2, VOCs and Dioxins is carried out using tailored activated
carbon and zeolites as adsorbents or catalyst support.
Molecular mobility associated with the glass transition
Molecular mobility associated with the glass transition of both polymer and glass former materials studied
by calorimetry and dielectric relaxation spectroscopy in bulk and constrained conditions (crystallization,
impregnation).
Monitoring of both molecular mobility and kinetical behaviour in glass forming monomers upon polymerization.
Depolymerisation studies and characterisation of biopolymers
Studies on catalytic depolymerisation of plastic wastes in order to recover pure monomers to be further
polymerised, to produce first quality products.Characterisation of biopolymers by size exclusion
chromatography (SEC, determination of average molecular weights and polydispersity) and differential
scanning calorimetry (DSC, determination of transition temperatures and cristallinity).
Membrane Processes for Improved Reaction and Separation
Development of membrane processes for intensification of (bio)catalytic reactions and recovery of target
compounds from complex media. Emphasis on process monitoring using in-vivo, real-time, non-invasive
techniques, and modelling of integrated reaction-separation processes.
Membrane Separation Processes in Biotechnology and Water Treatment
The current research focus is on development of membrane bioreactors for removal of emerging charged
micropollutants from drinking water supplies. Furthermore, nanofiltration is being studied as a tool for
concentration and separation of valuable compounds and/or reaction products from complex mixtures. In
both research lines, process modelling and design are emphasized.
Selective transport of bioproducts in membrane contactors
Membrane extraction of bioproducts using selective carriers and green solvents (ionic liquids). Modelling of
heat and mass transfer in osmotic evaporation using membrane contactors. Fixed carrier films for food
packaging.
Biosynthesis and Bioremediation Processes
Development of biological processes for production of biodegradable plastics from different substrates :
mechanisms and process optimization. Biological processes for inorganic and organic pollutants removal
from water and wastewater: microbiology, metabolism and process engineering.
Bioprocess modelling, monitoring, optimisation and control
Structured and unstructured modelling of biological processes, hybrid modelling employing mechanistic
models and knowledge engineering techniques. Dynamic optimisation of fermentation processes. Adaptive
control and physiological state control based on intracellular information. On-line optimising control.
Colloidal systems for slow release and separation of biological products
Colloidal systems for the controlled release of substances of interest for food and pharmaceutical applications:
mass transport studies. Biosynthesis and separation of biological products.
Biocatalysis in nonaqueous solvents
Development of rationales for the design of more efficient biocatalysts (from the standpoint of enzyme
activity, selectivity and stability) and biocatalytic processes (focus on practical strategies for green chemistry
and green processing) in non-aqueous media.
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Thermodynamics of Ionic Liquids
Measurement of the density and speed of sound of room temperature ionic liquids, and the consequent
calculation of the thermodynamic and thermophysical properties as a function of the temperature and the
pressure.
Process and product design in supercritical CO2
New applications of scCO2 , especially in separation processes, polymer synthesis and processing. The
ongoing research applies newly designed high pressure cells for phase equilibrium studies, acoustic and
spectroscopic techniques for better description of the phase behaviour and microscopic characterization of
target systems in scCO2.
Chemical reaction and supercritical carbon dioxide
Study of the influence of phase equilibrium on chemical reaction rates in systems containing supercritical
carbon dioxide. Combinations of ionic liquids and supercritical CO2 as alternative reaction media.
Supercritical fluid extraction of liquid mixtures
Phase equilibria of multicomponent systems at high-pressure conditions: Hydrodynamics and Mass Transfer
kinetics of countercurrent packed columns in supercritical fluid processes; Dynamic simulation models of
supercritical fluid processes.
Adsorption processes: molecular simulation, natural gas storage, separation processes
Key issues of large-scale stationary Adsorbed Natural Gas technology. Hybrid gas separation processes
combining membrane permeation and pressure swing (PSA) adsorption. Molecular simulation: hybrid
techniques, with characteristics of continuum modelling and Monte Carlo conducted in the grand canonical
ensemble.
Viscous fluid mixing. Experiments and CFD
Viscous fluid mixing by chaotic advection in both time-periodic and spatially-periodic 3-D prototype flows:
experimental and theoretical studies.
Research Activities in 2003
The interplay of these multiple research interests resulted in a research effort leading, as an ultimate,
strategic goal, to the study of Process Integration. During 2003, the main areas of research can be grouped
under three headings: Clean Solvents and Clean Processes, Process Simulation and Polymeric Materials
Clean Solvents and Clean Processes
Developments in this area resulted from the combination of long-standing interests in supercritical fluids as
alternative solvents, membranes in clean processes, adsorption for gas storage, and heterogeneous catalysts,
with newly developed research in ionic liquids, non-invasive monitoring techniques and hybrid gas separation
processes.
Clean Solvents – Supercritical Carbon Dioxide
1) Acoustic techniques were applied to study the critical behaviour of the binary systems CO2 + perfluorinated
surfactant (Krytox) using a new visual acoustic high-pressure cell. Comparison of visual and acoustic data
enabled the evaluation of the applicability of the acoustic technique to study the critical behaviour of mixtures
of CO2 with a polymeric material.
2) High-pressure phase equilibrium of systems containing supercritical carbon dioxide involved surfactants
and polymers: CO2 + Fluorlink C, CO2 + DEGDMA and CO2 + DEGDMA + Krytox:, CO2 + MMA + stabilisers
Química Verde
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HGAoct and HGAhex:. Modelling of the experimental phase equilibrium compositions of CO2 + Krytox was
performed using the Sanchez-Lacombe equation of state.
3) Microscopic characterisation of micro/macro emulsions in supercritical CO2 was carried out using a new
high pressure spectroscopic cell. Measurements of steady-state fluorescence emission, fluorescence
anisotropy and light scattered at 90º were obtained at 313,15 K and pressures up to 200 bar, for several
solutions: pyrene in CO2; pyrene + krytox + CO2; pyrene -butanoic acid in CO2 and pyrene-butanoic acid +
krytox + CO2.
4) High pressure NMR techniques provided structural information on polymers + surfactants + supercritical
CO2. (Krytox + CO2; Krytox + MMA + CO2). These experiments were undertaken between 303,15 - 333,15
K and pressures up to 15 MPa.
Clean Solvents –Ionic Liquids
1). Speed of sound and density measurements of several ionic liquids was carried out within broad ranges
of pressure and temperature,.
2) Density measurements contributed to the study of the influence of both anion and cation on excess
volumes of mixtures of C2 to C10MIM PF6 and NTf2.
3) Studies of the influence of solvent deuteration on phase equilibria of [C4mim][PF6] + ethanol and
[C4mim][PF6]+H2O, phase equilibria of the ternary system [C4mim][N(Tf)2]+ isobutanol+ H2O, and preliminary
studies of phase equilibria of phosphonium ionic liquids with both organic solvents and ionic liquids were
carried out.
Clean Solvents –Ionic Liquids and Supercritical Carbon Dioxide
Combinations of ionic liquids, supercritical carbon dioxide and other GRAS solvents (water, ethanol) were
used as Green Chemistry media to perform homogeneous catalysed oxidations, using pressure and
concentration as phase equilibrium switches.
Membrane Processing and Monitoring
1 - Recovery of aroma compounds from diluted aqueous streams by pervaporation. was focused on the
understanding of the molecular interactions between target solutes and the membrane material. Real-time
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.
2 - Development of integrated pre-enrichment techniques for sample discrimination by electronic sensorial
systems (electronic nose) in 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.
3 - Design of clean membrane processes for recovery, separation and purification of high added-value
products focused on the understanding and mathematical modelling of the transport process of the species
involved across porous and non-porous membranes, with a special emphasis on water transport in liquid
membranes, transport of solutes through ionic liquid supported liquid membranes, transport of hydrophobic
molecules through non-porous membranes and transport of electrically charged species in ion-exchange
membranes.
4 - Study of membrane processes for clean and selective recovery of biological products from dilute streams
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.
5 - Development of non-invasive, on-line monitoring techniques. used on-line mass spectrometry, 2Dfluorescence, Confocal Laser Scanning Microscopy and Raman Confocal Microscopy. Confocal Laser
Scanning Microscopy in combination with molecular probes was employed for investigating pH-gradients
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above dense membranes used in the dialytic mode (collaboration with the Group of Plant Biology, Universidade
de Lisboa). 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).
6 - Extraction of amino acids and derivatives using ionic liquids was studied using two different membrane
configurations. The transport mechanism that regulates solute transport, diffusion through water clusters
inside the ionic liquid, was compared for both configurations.
7 - Concentration of fruit juices by osmotic evaporation was compared with membrane distillation (MD) in
terms of water flux and aroma retention. Heat and mass transport models were developed, allowing the
description of the evaporation process in both cases.
Bioremediation
1- Membrane Bioreactors were used in the removal of polluting ions from drinking water streams, according
to the Ion Exchange Membrane Bioreactor (IEMB) concept, for water contaminated with both perchlorate
and nitrate, as well as for high salinity water (obtained from the Oceanarium of Lisbon) naturally polluted by
nitrate due to fish cultivation. Perchlorate and nitrate were reduced below the recommended values of
USEPA.
2-Conventional biological processes., such as the biological removal of nutrients (carbon, nitrogen and
phosphorus) from wastewaters, were studied in a sequencing batch reactor (SBR) operated with short
intermittent aeration. The complete cycle, comprising feeding, anaerobiosis, aerobiosis, settling and decanting
were only 36 minutes long.. The metabolism for propionate utilization by phosphorus accumulating organisms
(PAOs) in activated sludge was elucidated by in vivo NMR.
A mixed culture able to degrade molinate (a systemic thiocarbamate herbicide used worldwide to control
weeds in rice paddies) was obtained, and it was able to grow with molinate as sole carbon and nitrogen
source. The metabolic pathway for mineralization of the herbicide molinate by a defined bacterial consortium
was elucidated.
The remediation of vaccine production wastewaters containing organomercurial compound was performed.
Kinetics of thiomersal degradation to metallic mercury, under aerobic conditions, by a pure culture of
Pseudomonas putida spi3 strain was studied in a batch reactor and in a continuous stirred tank reactor
(CSTR) fed with the synthetic medium was studied.
Gas Separation and Storage by Adsorption
1 - Adsorbed Natural Gas (ANG). on nanoporous carbon materials is the storage technology of natural gas
that achieves the best compromise between compression costs and storage capacity. We have looked at
some of the key issues of large-scale stationary ANG technology, such as the efficient management of heat
effects and the development of filtering systems to protect the main gas storage vessel by trapping undesirable
components during the filling phase and releasing them in a controlled manner on the emptying part of the
cycle. A novel guard-bed system that achieves these goals, and is able to re-odorise the delivered gas to
meet European safety standards, has been developed. This work is has been carried out as part of a
European project that was finished by the end of 2003.
Adsorption processes
1- Studies on the removal of chlorophenols from water were continued, using acidic and basic activated
carbons at different pH in batch and on column systems. The obtained results showed that clorophenols
adsorb better in acidic carbons. Models based on the Langmuir, BET, Toth, Radke-Prausnitz isotherms
were applied to those experimental results. The adsorbent materials were characterised by XPS, TPD and
N2 adsorption (77K).
2-Studies on the removal of chromium Cr (III) and Cr (VI) from water by adsorption were performed on novel
activated carbons at different pH in column and batch systems. The activated carbon was prepared by
blending petroleum waste, biomass and low grade coal. The experimental conditions of activation of this
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novel carbon were optimized by changing the heating rate, temperature of activation and soaking time. The
kinetics and thermodynamics of the Cr (III) adsorption on carbon from co-mingled natural organic wastes
have been evaluated through sets of equilibrium and time-based experiments under varying temperature,
initial chromium concentration and carbon loading. The data obtained showed that the novel carbon is a
better adsorbent compared to a Norit commercial activated carbon.
Polymeric Catalytic Membranes
Polymeric catalytic membranes consisting of molibdophosphoric acid (HPMo) immobilized in dense polymeric
matrixes, such as polyvinyl alcohol (PVA) and polydimethylsiloxane (PDMS), were prepared. The tailoring of
the hydrophilic/hydrophobic balance of the polymer matrix was studied by esterifying the PVA OH groups
with acetic anhydride, in successive extensions. The hydration reaction of alpha-pinene was carried out
over those membranes and kinetic modelling was performed. The hydration of alpha-pinene was also studied
in a membrane reactor using tubular PDMS membranes loaded with USY/HPMo, in order to achieve complete
elimination of the solvent. The membrane hydrophobicity was decreased by loading zeolite NaY, in order to
increase membrane activity and selectivity to alpha-terpineol. Kinetic modelling was also performed.
Heterogeneous Catalysis & Fine Chemistry
The epoxidation reaction of limonene was studied using several types of catalysts.Cobalt acetylacetonate
anchored on activated carbon catalysts were prepared by using diamines as linking agents. The effect of
the chain length of the linking agent was studied, as well as that of the hydrophilic/hydrophobic balance of
the carbon support. The reactions were carried with t-butyl hydroperoxide (t-BHP) as oxidant.
Manganese Salen catalysts, prepared by another REQUIMTE group were used to study the influence of
ligands and metals (Mn and Cu. A kinetic model taking into account the decomposition of the oxidant reagent
and the oxidation of limonene was developed.
The oxidation of limonene was carried out with hydrogen peroxide, catalysed by cetylpiridinium
peroxotungstophosphate, in a two-phase system (H2O2/chloroform-limonene solution).
Process Simulation and Modelling
Modelling of biological processes
1- Hybrid modelling. In this work a bioreactor dynamical hybrid structure was developed that combines first
principles modelling with artificial neural networks: the bioreactor system is described by a set of mass
balance equations, and the cell population system is represented by an adjustable mixture of neural network
and mechanistic representations. Bounded Input Bounded Output (BIBO) stability conditions were derived
and the identification was studied in detail. A software package was developed implementing this technique.
2- Hybrid modelling with Mixture of experts (MEs) networks. The main objective of this work was to
demonstrate the applicability of complex modular network architectures, and in particular the Mixture of
Experts (ME) network, for metabolic kinetic modelling. It was concluded that the ME network, trained with
the Expectation Maximisation (EM) algorithm, is able to detect different pathways with the individual experts
developing expertise in describing single pathways. Also, in terms of accuracy, the ME network outperformed
the multi layer perceptron network in its ability to describe metabolic switches.
3- Non-mechanistic modelling of membrane-supported biofilm reactor. A membrane-supported biofilm reactor
was used for the treatment of industrial effluents contaminated 1,2-dichloroethane and 3-chloro-4methylaniline. Three modelling approaches were used, i.e. autonomous static modelling, non-autonomous
static modelling, as well as a novel approach termed dynamic modelling with embedding of ANN inputs.
They were compared with regard to their ability to infer process performance for 1,2-dichloroethane and 3chloro-4-methylaniline.
Process control
1-Precise DO control. In many industrial fermentation processes oxygen availability is the main limiting
factor for product production. Typically the dissolved oxygen (DO) concentration decreases continuously at
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the beginning of the batch until it reaches a critical level where the oxygen transfer rate is very close to the
vessel’s maximum transfer capacity. The process may be further driven close to this sensitive operating
point with a controller that manipulates the carbon source feed rate. The main purpose of this study was to
derive an effective closed-loop control solution and to demonstrate its economical advantage in relation to
the open-loop form of operation. The controller was implemented and tested in a pilot 50 l fermenter with a
recombinant Pichia pastoris process.
Ab initio Process Modelling and Design
1 - Molecular simulation of adsorption processes. A new molecular simulation method was developed to
solve the governing equations for an isothermal adsorptive separation unit, under equilibrium-controlled
conditions. The technique is formulated in the Gibbs ensemble, but is more appropriately viewed as a hybrid
of a molecular simulation and continuum modelling. If an analytical equation of state for the fluid phase is
known, the simulation procedure is considerably simplified and acquires many characteristics of a Monte
Carlo simulation conducted in the grand canonical ensemble. The technique has been thoroughly validated
and its usefulness was demonstrated through application to a gas separation problem encompassing the
major steps of practical value to batch adsorption processes.
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. For the case of
periodically forced flows we have shown that there is an optimum modulation frequency that leads to better
mixing. The use of numerical tools of analysis, such as stretching calculations and tracer tracking methods,
confirmed that the optimised protocol does result in very effective mixing. A laboratory-scale chaotic mixer
was built jointly with LEMTA (France) to experimentally validate the theoretical models.
Process Simulation And Modelling / Clean Solvents
A dynamic model of a SCF packed extraction column incorporating information on momentum and mass
balances previously validated was used to evaluate the effect of small perturbations of the macro variables
pressure, temperature, and solvent to feed throughput on the composition profiles of the column’s effluent
streams.
Polymeric Materials
Drug controlled-release systems
Microspheres, made of a biocompatible polymeric material, offer enormous potential to be used as carriers
for anti-inflammatory drugs as they have shown a high adhesion to anti-inflammatory tissues when compared
with the free drug and show high resistance and durability properties. This capacity of adhesion has been
shown to increase when microspheres are coated with chitosan.
Microspheres containing anti-inflammatory drugs were produced using two polymeric systems: one using
ethylcellulose and the solvent evaporation method and one using polyphosphate/chitosan and the
agglomeration/aggregation method. For each system the drug load capacity and the association drug/polymer
were determined. Kinetic studies of drug release from these polymeric systems under different operating
conditions were evaluated.
Production of Biopolymers
Optimization of polyhydroxyalkanoates (PHAs) production by activated sludge was studied in a Sequencing
Batch Reactor (SBR) operated under aerobic dynamic substrate feeding (ADF). The effect of several
parameters was evaluated. The maximum yield of polymer was reached when by operating the reactor with
substrate added in pulses wise. Indeed, the value obtained, 78% of PHB/cell dry weight, was not so far
reached by mixed cultures. A new strategy of reactor operation for PHAs production, based on oxygen
oscillations, was implemented. The SBR operation cycles were implemented using a rule-based supervisory
controller. The acetate pulses were implemented with a DO-based feed controller.
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The characterisation of the polyhydroxyalkanoates was performed. Glass transition and melting temperatures,
melting enthalpy and cristallinity degree, were measured by differential scanning calorimetry (DSC). The
determination of average molecular weights and polydidpersity was carried out by GPC/SEC.
Polymer characterization / Thermodynamics
i) The bio-polymer chitosan with both different water contents and protonation state was characterized by
dielectric relaxation spectroscopy and by thermogravimetry .
Polymer processing in clean solvents
i) The dispersion polymerisation of diethylene glycol dimethacrylate (DEGDMA) was undertaken in SC CO2
using HGAoct and HGAhex, as a stabilizer, and AIBN as initiator. The effect of the initial concentration of
surfactant, initiator, monomer and reaction pressure on the morphology of the resulting polymer was studied.
The molecular mobility and fragility behaviour of the monomers n-ethyleneglycol dimethacrylate (with n
ranging from 2 to 4) were studied by both DRS and DSC. DSC techniques (Univ. Valencia) were applied to
monitor the heat release upon polymerization of the 3-ethyleneglycol dimethacrylate monomer +AIBN mixture.
The kinetic behaviour of this free radical induced process was also investigated.
ii) Extraction of biopolymers (polyhydroxy alkanoates) from microbial cultures using SC CO2.
Depolymerisation and monomer recovering
Under a cooperation project with Petrobrás, Brasil, the catalytic depolymerisation of polymethylmethacrylate
(PMMA) was studied in order to obtain the pure monomer for further polymerisation. Different temperature
conditions were tested, over six industrial FCC catalysts with very different acidity and accessibility properties.
These catalysts are prepared from NaY zeolite, modified with rare earth metal oxides, aiming at to maximize
conversion at lower temperatures than those commonly used in simple pyrolysis.
Integration of Processes
The above-described research activities led to collaborative efforts that joined different groups, in order to
develop integrated processes. In 2003, the main achievements in this area were:
Use of Polymeric Catalytic Membranes in solventless reactions
The hydration of alpha-pinene was also studied in a membrane reactor using tubular PDMS membranes
loaded with USY/HPMo, in order to achieve complete elimination of the solvent. The membrane hydrophobicity
was decreased by loading zeolite NaY, in order to increase membrane activity and selectivity to alphaterpineol. Kinetic modelling was also performed
Development of reaction/impregnation processes in clean solvent media.
Synthesis of biopolymers in SC CO2, namely, polylactide, polyglycolide and poly(lactide-glycolide), and
subsequent impregnation with anti-inflammatory drug substances. Phase equilibrium measurements of the
systems CO2 + drug, CO2 + monomer, CO2 + drug + monomer and CO2 + drug + polymer; NMR and
emission spectroscopy characterization of the micro/macro emulsions of (surfactant+ monomer + CO2) and
(surfactant+ peptides+ CO2); DRS and DSC characterization of the synthesized polymers; evaluation of the
drug release profiles of impregnated biopolymers
Hybrid gas separation processes combining membrane permeation and pressure swing (PSA) adsorption
have been developed. The coupled process increases the efficiency of the pressurization and high-pressure
adsorption steps, thereby improving separation performance as compared to a stand alone PSA. A labscale unit of the hybrid process has been constructed and is scheduled to operate during 2004.
Pervaporation was used as the separation method in integrated processes of chemical reaction / recovery
of solutes from dilute aqueous streams and from ionic liquids. On-line mass spectrometry allowed real-time
monitoring of the permeating stream.
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Biochemistry and Biophysical of Proteins
Bioinorganic Chemistry and Protein Engineering
Head of Laboratory: Isabel Moura, Full Professor and José J. G. Moura, Full Professor
Research Team:
Jorge Lampreia
Assistant Professor
Cristina Costa
Assistant Professor
Anjos Macedo
Assistant Professor
Jorge Caldeira
Associate Professor – ISCS-S
Pedro Tavares
Assistant Professor
Alice Pereira
Assistant Professor
Carlos Brondino
Stephane Besson
Assistant Professor
Rui Duarte
Post-doc
Serguey Bursakov
Post-doc
Anders Thaper
Post-doc
Sofia Pauleta
Post-doc
Patricia Sousa
Post-doc
Gabriela Almeida
Post-doc
Cristina Timóteo
PhD Student
Teresa Alves
PhD Student
Cristina Correia
PhD Student
Inês Cabrito
PhD Student
Patricia Raleiras
PhD Student
Cristina Cordas
PhD Student
Gabriela Rivas
PhD Student
Pablo Gonzales
PhD Student
Jorge Dias
PhD Student
Ana Martins
Research Student
Filipe Folgosa
Research Student
Carlos Martins
Research Student
Number of Ph. D. Thesis:: 5
Cytochrome c Peroxidase - CCP
Peroxidases play an important metabolic pathway in desintoxification. We have characterized in the past
CCP according to a model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma,
N., Krippahl, L., and Moura, J. J. (1999)]. The structure of an electron-transfer complex containing a
cytochrome c and a peroxidase was described. Electron transfer complexes of cytochrome c peroxidase
from Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550)
at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation,
molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be
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accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme
methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can
be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse
cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c
peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome
c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary
solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550)
show that the enzyme can accommodate both cytochromes at the same time on adjacent sites.
Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse
cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of
cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome
c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome
c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required
for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.
The production of cytochrome c peroxidase (CCP) from Pseudomonas (Ps.) stutzeri (ATCC 11607) was
optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was
isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms.
The activity of Ps.stutzeri CCP was studied using two different ferrocytochromes as electron donors:
Ps.stutzeri cytochrome c(551) (the physiological electron donor) and horse heart cytochrome c. These
electron donors interact differently with Ps.stutzeri CCP, exhibiting different ionic strength dependence. The
CCP from Paracoccus (Pa.) denitrificans was proposed to have two different Ca(2+) binding sites: one
usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The
Ps.stutzeri enzyme was purified in a form with tightly bound Ca(2+). The affinity for Ca(2+) in the mixed
valence enzyme is so high that Ca(2+) returns to it from the EGTA which was added to empty the site in the
oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration
chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans
CCP which requires added Ca(2+) for formation of the dimer and also for activation of the enzyme. This is
consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium
and the transition to the active form of the enzyme. Additional Ca(2+)does affect both the kinetics of oxidation
of horse heart cytochrome c (but not cytochrome c(551)) and higher aggregation states of the enzyme. This
suggests the presence of a superficial Ca(2+)binding site of low affinity.
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.
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.
Formate Dehydrogenase
Incorporation of Molybdenum or Tungsten
We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase
(FDH) from the sulfate-reducing organism Desulfovibrio (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
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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 D.alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variabletemperature 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.
Aldehyde oxidoreductase
We report the kinetic behavior of the enzyme aldehyde oxidoreductase (AOR) from the sulfate reducing
bacterium Desulfovibrio gigas (Dg) encapsulated in reverse micelles of sodium bis-(2-ethylhexyl)
sulfosuccinate in isooctane using benzaldehyde, octaldehyde, and decylaldehyde as substrates. Dg AOR is
a 200-kDa homodimeric protein that catalyzes the conversion of aldehydes to carboxylic acids.
Ultrasedimentation analysis of Dg AOR-containing micelles showed the presence of 100-kDa molecular
weight species, confirming that the Dg AOR subunits can be dissociated. UV-visible spectra of encapsulated
Dg AOR are indistinguishable from the enzyme spectrum in solution, suggesting that both protein fold and
metal cofactor are kept intact upon encapsulation. The catalytic constant (k(cat)) profile as a function of the
micelle size W(0) (W(0)=[H(2)O]/[AOT]) using benzaldehyde as substrate showed two bell-shaped activity
peaks at W(0)=20 and 26. Furthermore, enzymatic activity for octaldehyde and decylaldehyde was detected
only in reverse micelles. Like for the benzaldehyde kinetics, two peaks with both similar k(cat) values and
W(0) positions were obtained. EPR studies using spin-labeled reverse micelles indicated that octaldehyde
and benzaldehyde are intercalated in the micelle membrane. This suggests that, though Dg AOR is found in
the cytoplasm of bacterial cells, the enzyme may catalyze the reaction of substrates incorporated into a cell
membrane.
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.
Nitrite Reductase
The cytochrome c nitrite reductase is isolated from the membranes of the sulfate-reducing bacterium
Desulfovibrio desulfuricans ATCC 27774 as a heterooligomeric complex composed by two subunits (61
kDa and 19 kDa) containing c-type hemes, encoded by the genes nrfA and nrfH, respectively. The extracted
complex has in average a 2NrfA:1NrfH composition. The separation of ccNiR subunits from one another is
accomplished by gel filtration chromatography in the presence of SDS. The amino-acid sequence and
biochemical subunits characterization show that NrfA contains five hemes and NrfH four hemes. These
considerations enabled the revision of a vast amount of existing spectroscopic data on the NrfHA complex
that was not originally well interpreted due to the lack of knowledge on the heme content and the oligomeric
enzyme status. Based on EPR and Mossbauer parameters and their correlation to structural information
recently obtained from X-ray crystallography on the NrfA structure [Cunha, C.A., Macieira, S., Dias, J.M.,
Almeida, M.G., Goncalves, L.M.L., Costa, C., Lampreia, J., Huber, R., Moura, J.J.G., Moura, I. & Romao, M.
(2003) J. Biol. Chem. 278, 17455-17465], we propose the full assignment of midpoint reduction potentials
values to the individual hemes. NrfA contains the high-spin catalytic site (-80 mV) as well as a quite unusual
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high reduction potential (+150 mV)/low-spin bis-His coordinated heme, considered to be the site where
electrons enter. In addition, the reassessment of the spectroscopic data allowed the first partial spectroscopic
characterization of the NrfH subunit. The four NrfH hemes are all in a low-spin state (S = 1/2). One of them
has a gmax at 3.55, characteristic of bis-histidinyl iron ligands in a noncoplanar arrangement, and has a
positive reduction potential.
N(2)O Reductase
Nitrous oxide reductase (N2OR) catalyzes the two-electron reduction of N2O to N2 and H2O in the last step
of the bacterial denitrification process. It is a dimeric protein. The recently solved crystal structure of N2OR
indicates that in each subunit there is a CuA center, which is the electron-transfer site, and a CuZ center,
which is the catalytic site. The neighboring CuA and CuZ centers are from different subunits. The CuZ
center has a strikingly new structural motif consisting of a mð4-sulfide bridged tetranuclear cluster. The CuZ
cluster is coordinated by seven His ligands with weakly bound water at the CuI/CuIV, which is the substrate
access site. CuZ center in dithionite-reduced N2OR (the resting form) is a partially delocalized S=1/2, 1CuII/
3CuI cluster. On the basis of the structural similarity with the CuA of COXs and the fact that mutant containing
only this cluster showed no activity toward N2O, CuA is assumed to be an electron transfer center whereas
CuZ is believed to be the active center of the enzyme. This cluster provides a mechanism for overcoming the
reaction barrier of N2O reduction by a simultaneous two-electron reduction pathway to the substrate bound
in a mð-1,3-bridging mode. We demonstrate now that the redoxes active form of the CuZ cluster in enzymatic
turnover is the all reduced 4CuI form by using a combination of activity determinations and EPR spectroscopy.
This is the first demonstration that the S =1/2 form of CuZ can be further reduced. DFT calculations were
performed to provide insight into the nature of N2O binding to and activation by this all-reduced 4CuI form
relative to the 1CuII/3CuI resting form of the CuZ site. CuZ is a one-hole system (1CuII/3CuI) and the
gradual decrease of the EPR signal on incubating with methyl viologen and dithionite indicates that the CuZ
cluster is reduced to the 4CuI form.
Simple metal sites - Superoxide reductases
Superoxide reductases (SORs) catalyze the monovalent reduction of superoxide anion to hydrogen peroxide.
Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K(3)Fe(CN)(6) oxidation
of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio
vulgaris was reported. Oxidation with K(3)Fe(CN)(6) reveals a band in the near-IR with lambda(max) at
1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy
provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-)(1), assigned
to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance
(EPR) spectra of oxidized TpNlr reveal at least three different species indicating structural heterogeneity in
the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active
site Fe(3+) ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model
of a hexacoordinate
[Fe-4S] containing proteins, Rubredoxin (Rd) and Desulforedoxin (Dx), present related structures and
activemetal sites to SOR. The differences in geometry at the metal centres in Rd and Dx are postulated to
be a result of the different spacing of the C-terminal cysteine pair in the two proteins. In order to address this
question, two mutants of D. gigas Dx with modified cysteinyl spacing were prepared and NMR has determined
their solution structures. Mutant-1 of Dx (DxM1) has a single glycine inserted between the adjacent cysteines
(C28 and C29) found in the wild type Dx sequence. Mutant-3 (DxM3) has two amino acid residues, -P-V-,
inserted between C28 and C29 in order to mimic the primary sequence found in Rd from D. gigas. The
solution structure of DxM1 exists, like wild type Dx, as a dimer in solution although the single glycine inserted
between the adjacent cysteines disrupts the stability of the dimer resulting in exchange between a dimer
state and a small population of another, probably monomeric, state. For DxM3 the two amino acid residues
inserted between the adjacent cysteines results in a monomeric protein that has a global fold near the metal
centre very similar to that found in Rd.
Broad Temperature Range Spectroscopy of Two Centre Modular Redox Metalloprotein Desulfoferrodoxin
was also reported.
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Protein-protein interactions
Transient Electron Transfer Protein Complexes
Multinuclear NMR - Soft Docking
BIGGER and CHEMERA are two software packages developed by us (available to the Scientific Community)
for the study of macromolecular interactions. Computational and spectroscopic studies on protein interactions
were extensively applied. We demonstrate a combination of docking algorithms under development with
multinuclear NMR data to study protein-protein interactions. NMR is also used in conjunction with X-ray
data. Restriction to docking solutions will also use site directs mutagenesis data as well as prediction of
electron pathways.
Protein – Protein transient complexes studies were extended to Cytochrome b5-Cytochrome c (and a large
complexe formed between Ferredoxin NADP-reductase – plant type Ferredoxin were undertaken, under
preparation). The method was also applied in the CCP section described above.
The interaction of reduced rabbit cytochrome b(5) with reduced yeast iso-1 cytochrome c has been studied
through the analysis of (1)H-(15)N HSQC spectra, of (15)N longitudinal ( R(1)) and transverse ( R(2))
relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the
adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated
with docking calculations. The result is that cytochrome b(5) has two negative patches capable of interacting
with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture,
two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct
is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison
with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence
of cytochrome b(5).
Under this topic we also participate in the Critical Assessment of PRediction of Interactions (CAPRI)
experiment, using the protein docking program BiGGER (Bimolecular complex Generation with Global
Evaluation and Ranking) (Palma et al., Proteins 2000;39:372-384). Of five target complexes (CAPRI targets
2, 4, 5, 6, and 7), only one was successfully predicted (target 6), but BiGGER generated reasonable models
for targets 4, 5, and 7, which could have been identified if additional biochemical information had been
available.
Metallothionein
Metallothioneins (MT) were obtained after purification from metal-exposed clams (Ruditapes decussatus)
using gel-permeation and ion-exchange chromatography. Four cadmium-metallothioneins (CdMTs) were
resolved by ion-exchange chromatography and they all had similar molecular weights, high cadmium content
and an absorption spectra indicative of the presence of characteristic Cd-S aggregates. The NH(2)-terminal
sequence suggests the presence of at least two class I clam MT isoforms. For the other two putative clam
CdMTs isolated, the results of the amino acid determination were inconclusive. One was slightly contaminated
and the other one had a blocked NH(2)-terminal. These clam metalothioneins contain glycine, which seems
to be a common feature of molluscan MT family and exhibited more similarity to oysters than to mussels.
Further investigation on the inducibility of these isoforms will be necessary if clams are to be used as
biomarkers of metal exposure.
Apotosis
Bax is a potent pro-apoptotic member of the Bcl-2 protein family that localizes to the mitochondrial membrane
during apoptosis. Tauroursodeoxycholic acid (TUDCA) modulates the apoptotic threshold, in part, by
preventing Bax translocation both in vitro and in vivo. The mechanisms by which Bax induces and TUDCA
inhibits release of cytochrome c are unclear. We show here that recombinant Bax protein induced cytochrome
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c release in isolated mitochondria without detectable swelling. Co-incubation with TUDCA prevented efflux
of mitochondrial factors and proteolytic processing of caspases in cytosolic extracts. Spectroscopic analyses
of mitochondria exposed to Bax revealed increased polarity and fluidity of the membrane lipid core as well
as altered protein order, indicative of Bax binding, together with loss of spin-label paramagnetism,
characteristic of oxidative damage. TUDCA markedly abrogated the Bax-induced membrane perturbation.
In conclusion, our results indicate that Bax protein directly induces cytochrome c release from mitochondria
through a mechanism that does not require the permeability transition. Rather, it is accompanied by changes
in the organization of membrane lipids and proteins. TUDCA is a potent inhibitor of Bax association with
mitochondria. Thus, TUDCA modulates apoptosis by suppressing mitochondrial membrane perturbation
through pathways that are also independent of the mitochondrial permeability transition.
(In collaboration with the Faculdade de Farmacia, UL).
EPR of partially oriented molecules
The EPR study of paramagnetic molecules (proteins or organometalic model compounds) in a partially
oriented sample has important advantages: i- additional resolution of the resonances by direct spectral
orientation. ii- increased sensitivity in different regions of the spectra due to alignment of the “spin-packets”.
Complexes of Cu(D,L-Ala)2.H2O and Cu(L-Tyr).H2O were, synthesized and crystallized. Pseudoazurin from
T. pantotropha were overexpresed and crystallized for single crystal/aligned multicrystal EPR studies.
Samples were prepared from solutions of hydroxypropylcellulose in water and several copper protein/
complexes micro crystals. The samples were casted to produce thin films.
The EPR spectra display a large variation upon angle rotation film plane relative to the applied magnetic
field caused by the non-random molecular distribution in the sample.
FUTURE RESEARCH PLAN (2004)
Single crystal versus aligned multicrystal EPR spectroscopy study.
Comparison with of single crystal and variable aligned multicrystal can describe this system in a tunable
degree of order
Future software development will include of hyperfine interactions; different orientation distribution functions;
integration of structural and crystallographic information with EPR spectroscopy.
The search for new methodologies for bulk protein orientation will be tested with a HPC/Protein/ionic exchange
cellulose resin.
NMR STUDIES OF METAL ION BINDING OF THE COLICIN E9 DNASE DOMAIN
Olicins E2, E7 and E9 are microbial toxins that kills bacteria through random degradation of chromosomal
DNA. The central and N-terminal regions of these toxins are responsible for receptor binding to susceptible
cells and translocation of the killing domain into its cytoplasm, respectively, while the C-terminal regions
contain the 15 kDa toxic domain housing the DNase activity which can be over-expressed and purified in
isolation from the rest of the toxin.
The DNase domain of colicin E9 comprises 134 amino acids, has a monomeric molecular mass of 15,070
Da and contains a 32 residue zinc-finger-like H-N-H motif. This motif displays a variety of metal ion binding
properties, some of which are central to its ability to hydrolyze DNA. His102 and His127 are both ligands to
the Ni2+ and Zn2+ in X-ray structures of the DNase domains from colicins E7 and E9 but His 131 is only a
clearly defined ligand in the Zn2+-bound E7 DNase crystal structure, though NMR studies show that His 131
does bind to the Ni2+ in the E9 DNase. Mutagenesis has been used to test the importance of the conserved
H-N-H amino acids in catalysis and metal ion binding and four amino acids have been identified that are
essential for Mg2+ and Ni2+ dependent activities: Glu100, His102, His103 and His127. When these are
individually changed to alanines, only the H103A variant retains the ability to bind transition metals.
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A project in collaboration with Prof. Dr. Geoffrey Moore from School of Chemical Sciences & Pharmacy,
University of East Anglia, Norwich NR4 7TJ, UK.
The murine 5-Aminolevulinate Synthase, the first enzyme of the heme biosynthesis
5-aminolevulinic acid synthase, ALAS, catalysis the condensation of glycine and succinyl-CoA to yield ALA.
NMR Spectroscopy As Been Used As A Tool To Probe Structural Changes In The Active Center And The
Enzymatic Mechanism Of An Active Truncated Form Of Alas (From Q109 To V465, 43kda Mm). 1h-15n
Hsqc Spectra Of The 15n Labelled Protein Show Very Few Discernable Peaks Due To The Size Of The
Protein And Also Probably To Aggregation. In Order To Improve Spectral Resolution Perdeuteration In
Conjuction With Trosy And Crinept-Trosy Experiments (At 600mhz With Cryoprobe, And 800mhz) Has
Been Attempted. The Results Point To A More Detailed Study Using These Techniques, To Detect And
Identify The Amino Acids Involved In The Active Center. Ms Spectrometry Is Used To Evaluate The Degree
Of Deuterarion.
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. Heteronuclear nOe experiments will be performed in the
systems under study (including solubilized small peptides in scCO2) detecting molecular interactions and
understand polymer and surfactant solubility in CO2
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Protein Crystallography
Head of Laboratory: Maria João Romão, Associate Professor
Research Team:
Ana Luisa Carvalho
Associated Laboratory Researcher
Roeland Boer
Post-doc (EU Project)
José Trincão
Post-doc (FCT)
João Miguel Dias
Post-doc (FCT)
Carlos C. Cunha
PhD student (FCT)
Jorge Rebelo
PhD student (FCT)
Teresa Santos Silva
PhD Student (FCT)
Cecília Bonifácio
Associated Laboratory Technician
Number of Ph. D. Thesis: 1
One of the main targets of our research have been metalloproteins and/or proteins involved in electron
transfer processes (containing Fe/S centers, Mo, W, hemes). An important achievement has been the
structure solution of the membrane bound cytochrome c nitrite reductase.
In parallel we have conducted research in enzymes involved in plant cell wall degradation.
1- Metalloproteins - Heme containing enzymes
Multi-heme, membrane bound nitrite reductase
The gene encoding cytochrome c nitrite reductase from D. desulfuricans ATCC 27774 was sequenced and
its crystal structure determined to 2.3 Å resolution. In comparison to homologous structures, it presents
structural differences mainly at the regions surrounding the putative substrate inlet and product outlet, and
includes a well defined second calcium site coordinated to two propionates and caged by a loop nonexistent in the previous structures. The main role of this calcium ion may not be electrostatic but structural,
namely in the stabilization of the conformation of the additional loop that cages it and influences solvent
accessibility. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme
distal side nearly in the same location as occurs in peroxidases.
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 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.
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
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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 to trace
most of the polypeptide chain and refinement is in progress.
2- Metalloproteins - Molybdopterin-containing enzymes
Periplasmic nitrate reductase (NAPA) of Ralstonia eutropha –mutagenesis studies
The catalytic subunit of the periplasmic nitrate reductase (NAPA) of Ralstonia eutropha contains a lysine
residue (K85), highly conserved in periplasmic nitrate reductases. It is located between an [4Fe-4S] centre
and one of the molybdopterin cofactors, mediating the through bonds electron flow. To examine the role of
K85 it was replaced by site-directed mutagenesis, yielding K85R and K85M, respectively. The specific
nitrate reductase activity of the mutant enzyme carrying K85R showed 23% of the wild-type activity, whereas
K85M resulted in complete loss of the catalytic activity. The nitrate reductase activity detected was not due
to different quantities of the expressed gene products, as controlled immunologically.
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.
Protein-protein recognition plays a pivotal role in the degradation of the plant cell wall by anaerobic
microorganisms. These organisms synthesize an extensive repertoire of glycoside hydrolases and esterases
that physically associate to form a high molecular weight complex termed the “Cellulosome”.
We have solved the first three-dimensional structure of a dockerin-cohesin complex (CohDD). The dockerin
domain is folded into a loop helix motif followed by a helix-loop-helix motif, connected by a six-residue
segment. The ²-barrel topology of the cohesin domain did not undergo significant conformational change in
complex with its dockerin ligand. The structure of the complex shows that protein-protein recognition is
mediated mainly by hydrophobic interactions; there are relatively few direct hydrogen bonds between the
two protein molecules. The structure challenges the view that the highly conserved tandem hydroxy amino
acid motif in the N- and C-terminal segment of the dockerin domain both play a pivotal role in cohesin
recognition. The structure of the CohDD provides an explanation for the lack cross-species recognition
between cohesin-dockerin pairs, and will direct and inform future strategies designed to engineer novel
specificity into the dockerin-cohesin interaction.
Planned research activities for 2004
Metalloproteins
Formate dehydrogenase from D. vulgaris - Crystallization conditions will be optimized.
Periplasmic nitrate reductase (NAPA) from 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
Research Team:
Hugo Gil Ferreira
Invited Full Professor (ICBAS)
Karin Tonnies Gil Ferreira
Invited Associate Professor
Maria da Graça Soveral Rodrigues
Assistant Professor (Fac. Farmácia Lisboa)
2.1. Toxicological Studies (In collaboration with Faculdade de Farmácia de Lisboa)
2.1.1. Analysis of metals and trace elements
The most consumed beverages by the Portuguese population were identified and an extensive screening of
the total amount of metals and trace elements in these beverages was performed using the ICP (Induced
Coupled Plasma) technique.
2.1.2. Inhibition of the mithocondrial pyruvate carrier by valproate and cetovalproate
The effect of valproic acid and its metabolite ceto-valproate on the pyruvate transport is being studied using
purified vesicles preparations of the inner membrane from rat mitochondria. These techniques make use of
a rapid filtration system with 14C–pyruvate as a marker.
2.2. Mathematical models (In collaboration with Prof. Robert Macey from University of California Berkeley)
Mathematical models for cells and epithelial systems are being developed and the numerical simulations
are implemented in the Berkeley Madonna package (http://www.kagi.com/authors/madonna).
2.2.1. Regulation of the Cellular Proton Balances in an Epithelial System (In collaboration with Prof. Augusta
Rebelo da Costa ICBAS/UP)
All animal cells produce protons at rates depending on their functional state. The main sources of protons
are CO2 and non-volatile acids (organic, sulfuric and organic acids). Despite this constant acidification the
cytoplasm pH exhibits very small fluctuations as a result of the operation of three mechanisms: the action of
intracellular buffers, mainly proteins; the operation of transport mechanisms that move protons across the
cell membrane: proton channels, a Na+/H+ counter-transport and at least two proton pumps; the operation
of transport systems that move bicarbonate across the cell membrane in exchange for chloride. These
systems can be identified using specific inhibitors but although intracellular pH can be monitored using
fluorescent dyes methods for studying the balance between production and extrusion of protons in isolated
cells are not available.
Several epithelia (the gastric mucosa, the kidney distal tubule, the outer mantle epithelium (OME) of the
mollusk Anodonta sygnea) may provide useful experimental models for the study of this problem.
Of these the OME is particularly useful since: it can be studied as a large (several cm2) diaphragm where
fluxes can be measured comfortably; under short-circuit conditions it produces an electric current that is a
direct measure of the flux of protons across one of its faces; this current is sensitive to a variety of inhibitors
which indicate the presence of specific transport systems.
A mathematical concentrated parameter model of the OME is being built in order to simulate the results
obtained in vitro with this preparation. The model 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.
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2.2.2. Tubular organ
A mathematical model describing the behavior of the thick ascending limb of the Henle´s loop of mammalian
kidney was developed. Numerical simulations for the salt reabsorption of the filtrate entering the thick
ascending limb (TAL) of the Henle´s loop of the mammalian kidney were done. At the entrance of TAL the
filtrate is considered isotonic with the basolateral space and becomes hypotonic at the exit of TAL by the
active reabsortion of salt. 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.
Future work in 2004:
3.1 Toxicological Studies
3.1.1. Analysis of metals and trace elements
The same elements already studied in the beverages (2.1) will be tested in milk samples from two groups of
breast feeding mothers subjected to a nutritional assessment: (i) one group living in Lisbon, used as control,
(ii) and the second group living in another geographic region where high levels of mercury in fish were
reported (risk group).
3.1.2. Inhibition of the mithocondrial pyruvate carrier by valproate and cetovalproate
The characterization of the effect of valproic acid and its metabolites on the pyruvate transport will be
continued in the inner mitochondrial membrane (inverted sub mitochondrial particles) in order to identify the
inhibition profile of these drugs. The effects of these drugs will also be studied in intact mitochondria.
3.2. Mathematical models (in collaboration with Prof. Robert Macey from University of California Berkeley)
3.2.1. Regulation of the Cellular Proton Balances in the human body
Since the model written for the regulation of the cellular proton balances in an epithelial system is in a
modular form it can be expanded or adapted to other cell systems.
We plan to expand it so as to include the regulation of the proton balances in the human body
3.2.2. Yeast cell (In collaboration with Prof. M.C. Loureiro Dias, Centro de Botânica Aplicada à Agricultura)
A mathematical model of a yeast cell will be developed taken into account the different transport systems
described in the literature. In Saccharomyces cerevisiae the whole genome has been sequenced and functions
have been assigned to almost all putative membrane proteins. This provides a solid framework to integrate
classic physiological and kinetic data that can be coherently put together within a mathematical model.
3.3. Detection and role of aquaporins in the halotolerant yeast Debaryomyces hansenii
The presence of active aquaporins will be investigated in D. hansenii. Genes coding for aquaporins have
been detected in the genome of this yeast. The functionality of proteins coded by these genes will be
investigated by measuring energy of activation of water fluxes in protoplasts. Strains of S. cerevisiae lacking
genes AQY1 and AQY2 will be used as negative controls and strains over expressing these genes as
positive controls.
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DEVELOPMENT OF TRANSDUCERS
Head of Laboratory: José Luís Costa Lima, Full Professor
Research Team
José Luís Fontes da Costa Lima
Full Professor
Alberto Nova Araújo
Associate Professor
Rui Alexandre Santos Lapa
Assistent Professor
Maria da Conceição B. S. M. Montenegro
Associate Professor
Agostinho Almiro Almeida
Assistent Professor
João Luís Machado Santos
Assistent Professor
Maria Lúcia M. F. Sousa Saraiva
Assistent Professor
Marcela Alves Segundo
Associate Laboratory Reseracher
Ivone Valente Oliveira
Assistent Professor
João Alexandre Velho Prior
Ph. D Student
Paula Cristina de Azevedo Gomes Pinto
Ph. D Student
Cristina Manuela Pinto Vieira
Ph. D Student
Pedro Rodrigues Marques Maia
Ph. D Student
Karine Lopes Marques
Ph. D Student
Marta Filipa Teixeira Ribeiro
Ph. D Student
Luis Miguel Andrade de Magalhães
Ph. D Student
Ana Coelho
M. Sc, Student
Delfina de Vasconcelos
M. Sc, Student
Hugo Miguel Rodrigues Cunha Oliveira
M. Sc, Student
Célia Clarisse Carnapete Alves Menezes
M. Sc, Student
Marieta Leite de Castro Passos
M. Sc, Student
Number of Ph. D Thesis: 1
New trends were investigated based on exploitation of different transducing schemes such us optical and
electrochemical. Studies on immobilization techniques were used for the monitoring low levels of drugs,
food and pesticides.
Studies were developed concerning the construction of flow-through voltametric electrodes dedicated to the
implementation of manifolds with multi-side detection.
In more detail it can be referred the following results:
- Development of a voltametric detector with tubular configuration able to move in a flow manifold followed
the concept of multi-side detection. The flow system with amperometric detection was applied to a great
variety of pharmaceuticals that are known to lead the rapid poisoning of the working electrode surface. With
the movement of the detector after each measurement the conditioning of the electrode was possible through
the passage by the surface of a regeneration solution without implying the alteration of the carrier that
flowed in the analytical channel of the manifold.
- It was been constructed, evaluated a selective electrodes to gibberellate anion for the determination of
gibberellic acid in agricultural products. Several types of PVC membrane electrodes without internal reference
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solution were prepared using manganese (III) complex of meso-tetraphenylporphyrin as ionofore and dibutyl
phthalate as plasticizer. The incorporation of lipophilic chemical species as additives was also carried out
aiming the evaluation of the response characteristics of the electrode. This potentiometric unit presented a
linear response between 10-4 and 10-1 mol L-1 and a reproducibility of about + 1 mVday-1.
- The preparation of a biosensor based on the enzymatic immobilization in polypyrrole polymer for the
detection of antidepressant drugs was been developed. The enzyme monoamine oxidase was immobilized
by electropolymerization of pyrrole around a platinum electrode, at constant potential of +0.75 V (vs Ag/
AgCl) in a such way to obtain a membrane thickness, which was constant and equal to 100mC/cm2. The
biosensor was adapted to a continuous flow injection analysis system with amperometric detection of hydrogen
peroxide produced by reaction carried out at a potential of +0.7V (vs. Ag/AgCl), pH 7.4 and temperature of
37ºC. The analytical use of the biosensor developed was evaluated through analysis of commercial
pharmaceutical products containing fluoxetine on the Portuguese market.
- An enzymatically modified carbon paste electrode constituted by polyphenol oxidase obtained from Annona
Muricata L. tissue graphite, silicone and 7,7,8,8 tetracyanoquinodimethane, was used as flow-through detector
in a flow injection analysis manifold dedicated to the amperometric determination of dopamine in
pharmaceutical formulations. The developed biosensor showed good stability and reproducibility, enabling
up to 500 determinations in 60 days, without considerable loss of enzymatic activity.
- A tubular electrode for dipyrone, comprising a polymeric membrane containing tetraoctylammonium as an
electroactive material, dibutylphtalate as solvent mediator and PVC directly applied to condutive graphite
support was been developed. This unit was incorporated in a flow monochannel manifold and applied to the
analysis of pharmaceutical preparations (oral and injectable) containing dipyrone.
- Electrochemical oxidation of the herbicide propanil in deuterated solutions was studied by cyclic, differential
pulse, and square wave voltametry using glassy carbon microelectrode. The oxidation of propanil in deuterated
acid solutions occurs at the nitrogen atom of the amine at a potential of +1.15 V vs Ag/AgCl. It was also
found that, under the experimental conditions used, proponation at the oxygen atom of propanil occurs,
leading to the appearance of another species in solution which oxidizes at +0.60 V. The anodic peack found
a +0.79 V in deuterated basic solutions is related to the presence of an anionic species in which a negative
charge is on the nitrogen atom. The electrochemical data were confirmed by the identification of all species
formed in acidic and basic deuterated solutions by means of NMR spectroscopy.
In 2004, 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
Research Team
JJosé Luís Fontes da Costa Lima
Full Professor
Alberto Nova Araújo
Associate Professor
Assistent Professor
Maria da Conceição B. S. M. Montenegro
Associate Professor
Assistent Professor
João Luís Machado Santos
Assistent Professor
Maria Lúcia M. F. Sousa Saraiva
Assistent Professor
Associate Laboratory Reseracher
Assistent Professor
Ph. D Student
Ph. D Student
Cristina Manuela Pinto Vieira
Ph. D Student
Ph. D Student
Ph. D Student
Ph. D Student
Luis Miguel Andrade de Magalhães
Ph. D Student
Ana Coelho
M. Sc, Student
M. Sc, Student
M. Sc, Student
Célia Clarisse Carnapete Alves Menezes
M. Sc, Student
M. Sc, Student
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:
- Development of an automated multicommutated flow methodology was implemented for the
spectrophotometric determination of trimipramine in pharmaceutical preparations by oxidation with ammonium
monovanadate in acid medium. The developed procedure exploits a new approach for sample/reagent
intermixing by combining binary sampling with flow-reversal.
- A procedure for the determination of bopindolol using a FIA technique, with spectrophotometric detection
was been developed. The method was based on the production of green, water-soluble complex with ferric
ions in acid medium. The automated lad-made FIA system was used for the direct determination of bopindolol
in tablets. Bopindolol was adsorbed onto the solid phase in mini-column, which was intercalated directly into
the flow system.
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- The development of a pre-concentration procedure based on sequential injection analysis with flame
atomic absorption spectrometry for the determination of Mn in water samples presenting a concentration
between 4.5 and 200.0 mð. - Easy to implemented FIA system with potentiometric detection for L-glutamate
determination in food samples. The procedure is based on measurements of carbon dioxide produced by
decarboxylation of L-glutamate catalysed by L-glutamate decarboxylase from Cucurbita maxima. The flow
potentiometric system includes an enzymatic reactor with length of 8 cm and thickness of 5 mm packed with
200 mg of Cucurbita maxima outer layern cut in small pieces.
- A novel flow-based procedure involving the multi-pumping approach was developed for the
spectrophotometric determination of bromhexine in pharmaceutical preparation. The method is based on
the reaction with 3-methyl-2-benzothiazolinone hydrazona and Ce(IV). Several solenoid micro-pumps are
present in the manifold in order to provided system control. Critical tasks in continuous flow analysis, samplereagent introduction and solution propelling are then efficiently carried out.
- A sequential injection analysis flow system, for the determination of nitrates and nitrites in human serum
was developed. The exact timing of the fluidic manipulations and the small volumes used in the automated
SIA systems provide exquisite control of the reaction conditions and the economy in the biological fluid and
enzymes used. The automatic developed method is a good alternative for rotine implementation since is
four time faster and it requires one third of the sample and half of the nitrate reductase than the batch
procedure.
- A novel flow system for the spectrophotometric determination of dipyrone with p-dimethylaminobenzaldehyde
exploiting the multi-pumping approach was developed. The proposed methodology utilises several micropumps for propelling the involved fluids under improved mixing conditions introducing sample-reagent aliquots
and providing commuting facilities. As consequence the multi-pumping systems presents high versatility
and manifold simplicity, as well as a straightforward operational control and enhanced analytical capabilities.
- A new procedure for cetylpyridinum chloride determination in oral disinfectants, based on a FIA system
with potentiometric detection. The determination was based on the measurement of picrate concentration
decrease as result of ion-pair reaction with the analyte present in the injected sample. The procedure
enables the determination of cetylpyridinium at a sampling rate of 60 samples per hour.
- An automatic procedure for the determination of free and total potassium in wines that combines sequential
injection analyses with potentiometric detection. The sequential injection manifold was coupled to a microwave
system to conduct the on-line digestion of the sample, thereby making the injection of the sample possible
without prior treatment.
- An automated flow potentiometric titration procedure for the determination of chloride exploiting the
monosegmented flow approach was developed. A tubular selective electrode and a Ag/AgCl electrode were
employed as indicator and reference, respectively. An algorithm based on the potential difference between
two subsequent titration additions was developed allowing to reach the end point in less than 10 attempts
with a precision better than 1%. The proposed system was evaluated by determining chloride in milk and
wine using a standard solution of AgNO3 as titrant.
- An flow procedure based on the multicommutation concept comprising three-way solenoid valves for the
spectrophotometric determination of 3-hydroxybutyrate in animal serum and plasma is proposed. The 3hydroxybutirate was enzymatically converted to acetoacetate with the reduction of NAD+ to NADH monitored
at 340 nm. It was possible to carry out up to 600 determinations without a significant decrease in the
analytical signal, with 5 mg of 3-hydroxybutyrate dehydrogenase immobilized on porous silica beads and
packed in a column. The system enabled 60 determinations per hour of 3-hydroxybutyrate in the range of
10-150 mgL-1 with a consumption of 0.9 mg of NAD+ and 200 mðL of sample per determination.
- New strategies were exploited to implement multi-pumping flow systems relying on the utilization of multiple
devices that acts simultaneously as sample-insertion, reagents-introduction and solution-propelling units.
The solenoid micro-pumps that were initially used as the only active elements of the multi-pumping systems,
and which were able to produce pulses of 3 to 25 mðL, were replaced by syringe pumps with the aim of
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producing pulses between 1 and 4 mðL. The performance of the developed flow system was assessed by
using distinct sample-insertion strategies like single volume, merging zones and binary sampling in the
spectrophotometric determination of isoniazied in pharmaceutical formulations upon reaction with 1,2naphthoquinone-4-sulfonate, in alkaline medium.
- Development of an on-line SIA system using amperometric detection for the simultaneous monitoring of
glucose and ethanol in fermentation media. The automatic analytical procedure is based in a sequential
injection analysis strategy and uses catalytic reactors of oxidase enzymes immobilized on controlled-pore
glass. The system was applied to monitor glucose and ethanol in beer fermentation with a sampling rate of
50 samples per hour. In a way to adjust the levels of ethanol present in the broth to the characteristics of the
proposed system a dialysis unit was used between the fermenter and the SIA manifold
As activities during 2004 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 degradation of CCl4.
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QUALITY CONTROL AND AUTHENTICITY OF FOOD PRODUCTS
Head of laboratory: Rosa Seabra, Associate Professor, Beatriz Oliveira, Assistant Professor and Isabel
Ferreira, Assistant Professor
Research Team
Rosa Seabra
Associate Professor
Margarida Ferreira
Full Professor
Beatriz Oliveira
Assistant Professor
Isabel Ferreira
Assistant Professor
Paula Andrade
Assistant Professor
José Fernandes
Assistant Professor
Patrícia Valentão
Assistant
Susana Casal
Assistant
Rui Alves
Professor Coordenador
Olívia Pinho
Assistant
Sara Cunha
Ph. D. Student
Miguel Faria
Ph. D. Student
Joana Amaral
Ph. D. Student
Virgínia Mota
M. Sc. Student
Carla Veiros
M. Sc. Student
Number of Ph. D. Thesis: 1
Our group has large experience in the field of food quality control and, more recently in the authenticity
concerns. Some MSc 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 and validation of analytical methodologies
One of the main working fields of our team is related to the development and implementation of analytical
methodologies, mostly applied to food products. Techniques such as high-performance liquid chromatography
(with UV/VIS, diode-array, fluorimetric, refractive index and light scattering detectors), and gas chromatography
(with FID, NPD and Mass spectrometric detectors) are commonly used. Different derivatization techniques
for HPLC and GC analytical purposes are also optimised and implemented. In the last two years more
attention has been devoted to the development of cleaner analytical procedures.
Some developments were made with this objective, especially in sample preparation and extraction/cleanup procedures. A new headspace-SPME method for analysing volatile compound has been successfully
developed.
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We are now beginning the development of sample preparation procedures based on selective supercritical
fluid extraction (SFE) for the determination of pesticides (and perhaps other kind of compounds) in different
matrixes.
Biological techniques (PCR methodologies) are being implemented for food product authentication. As an
example, a duplex PCR method was recently developed for the quantification of bovine milk in ovine cheeses.
The development of PCR based methods to detect GMOs crops, namely soybean and corn, as well as
derived food and feedstuffs available in Portuguese market, are also of interest for our investigation.
2. Food quality and authenticity studies. Main guidelines.
Dairy products
Milk composition
Progressive attempts have been made by the industry to bring the composition of infant formulae closer to
that of human milk, not only with regard to major components, but also to minor compounds that may be
involved in the newborn development. In an attempt to know the actual situation, non-protein nitrogen
components
free amino acid, taurine, free nucleotides, orotic acid, free and total L-carnitine) of infant formulae and follow
up milks were quantified and compared with cow and human milk. Global statistic treatment of the results by
multivariate analysis indicated great similarities in the content of N-compounds under study in all the studied
formulations but showed significant differences with regard to human milk composition. Human milk
composition
concerning protein and fatty acid profiles, variation in composition, individual variability, differences between
beginning and end of feeding, and day-to-day variation will be studied.
Cheese
Physico-chemical characterization of cheeses with protected denomination of origin (PDO) was performed,
including lipolytic and proteolytic products characterization, and correlation between volatile components
(determined by SPME/ GC/MS) and sensory characteristics. Determination of free amino acids and biogenic
amines were also aim of study for hygienic and food safety reasons.
Proteolysis and authenticity criteria based in lactoglobulins HPLC analyses for identification of homologous
proteins in milk mixtures from different species were also evaluated.
Milk percentages of fresh and ripened cheeses made from binary mixtures of bovine, ovine or caprine raw
milks were studied by RP-HPLC separation and quantification of homologous caseins from bovine, ovine
and caprine milks.
Antibiotic resistancein comensal bacteria
Ocurrence of enterococci resistant to antibiotics was evaluated in food products namely cheese, poultry
carcasses and faecal samples of suines. Molecular characterization of the resistant strains, resistance
genes an genetic capture units was performed to assess their contribution to the increase incidence of
vancomycin resistant enterococci in clinicla samples.
Olives and vegetable oils
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Olive oil has a social and economic relevance enhanced by the recognized excellent nutritive qualities of
this product. 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 application of organophosphorus
pesticides (dimethoate, fenthion, phosmet) to control olive pests and its detection, in trace amounts, in
olives and olive oils is reported in literature. The pesticides are known to accumulate in the lipophilic tissue
of the plant were they can be metabolized into more toxic compounds.
A GC/NPD methodology is in implementation for the specific determination and quantification of the referred
pesticides and metabolites, and will be applied to olives and virgin olive oils from organic and conventional
agriculture.
Several quality parameters were determined in varietal olive oils in order to characterize some cultivars of
Olea
uropaea (fatty acids, sterols, tocopherols, triglycerides) and will be applied to POD olive oils “Azeite de Trás
os-Montes”.
Coffee
Some important coffee constituents were determined in both green and roasted coffee and their behaviour
with roasting evaluated and discussed. The studies performed included major coffee constituents as well as
compounds formed during the roasting process, which are claimed to be toxic or of unknown activity.
The last years have been devoted essentially to the development and validation of HPLC and GC analytical
methodologies applied to coffee roasting and authenticity assessment, namely: D/L amino acids, trigonelline,
caffeine, nicotinic acid, hydroxycinamic acids, cis/trans fatty acids, heterocyclic aromatic amines, 4-(5
)methylimidazole, D-amino-acids, and furfural derivatives.
In a near future it is expected to extend these coffee studies to other compounds and roasting techniques.
Biological studies with coffee are also programmed, including in vitro studies of coffee extracts on isolated
rat hepatocytes and cardiomyocytes.
Alcoholic beverages
Beer
Studies about the beer foam stability by characterization of protein profiles using reversed phase and size
exclusion HPLC, were conducted. The contribution of other components as iso-alpha acids, organic acids,
sugars, amino acids and others compounds is under study. In a near future a compositional comparison
between two types of beer (with and without alcohol) will be performed.
Wine
Authenticity studies in wines and grape musts and grapevine cultivar/clone characterisation using PCR
based techniques are under study. The results already obtained showed the possibility to quantify the relative
level of different grape varieties in musts and to completely discriminate grapevine cultivars using microsatellite
markers. Grapevine clone discrimination using stilbene synthase (StSy) – chalcone synthase (CHS) 5’
untranslated genomic regions markers has also been achieved.
A study about the origin and evolution of biogenic amines in Port wines was concluded. The results showed
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40
some interesting statistically significant correlations between the levels of some of the amines, namely
pyrrolidine, cadaverine and putrescine, and the type and the age of the wines.
The non-coloured phenolic composition of red wine obtained from Touriga Nacional grapes, growing in Dão
region, was determined by HPLC/DAD and 16 compounds were identified and quantified. The effect of 9
different Dekkera bruxellensis strains, contaminating yeast, on the levels of phenolic acids and flavonoids of
the wine was also evaluated. In all samples (inoculated and non- inoculated) phenolic acids were predominant
relatively to flavonoids. In inoculated samples a considerable rise in the amounts of gallic acid was observed
while the amounts of t-CAFTA, t-COUTA, caffeic and p-coumaric acids were statistically decreased.
Quinces
A GC/FID methodology for the separation and identification of 21 free amino acids was developed and
applied to quince fruit (pulp, peel and seed) and jam, in order to define the qualitative and quantitative
profiles of these food products. The analyses showed some differences between quince pulps, peels and
seeds. The total free amino acid content was higher in seeds than in pulps and peels. Generally, higher
content in total free amino acids and in glycine was found in peels than in pulps. This study suggests that the
free amino acid analysis can be useful for the evaluation of quince jam authenticity.As part of a continuing
study of quince, the qualitative and quantitative composition of quince seeds, in termof
phenolic compounds, organic acids and free amino acids, was established. Quince seeds presented a
phenolic profile composed by 3-, 4- and 5-O-caffeoylquinic acids, 3,5-dicaffeoylquinic acid, lucenin-2, vicenin2, stellarin 2, isoschaftoside, schaftoside, 6-C-pentosyl-8-C-glucosyl chrysoeriol and 6-C-glucosyl-8-Cpentosyl chrysoeriol.
All samples had a similar organic acid profile composed of 6 identified organic acids: citric, ascorbic, malic,
quinic, shikimic and fumaric acids. The free amino acid profile of quince seeds was composed by 21 identified
free amino acids, being the three most abundant glutamic and aspartic acids and asparagine. The influence
of jam processing upon the contents of phenolics, organic acids and free amino acids in quince fruit was
also evaluated.
During phenolic analysis of quince fruit and jam, several unidentified compounds, that seem to be epicatechin
dimers bounded to sugars, were detected. We have already proceeded to the isolation of these compounds
and now we will try to identify them by spectroscopic means (nuclear magnetic resonance, mass spectroscopy
and UV).
Quince fruit (pulp, peel and seed) and jam antioxidant activity is being evaluated by a micro assay using 2,2’
diphenyl-1-picrylhydrazyl (DPPH).
Related to the above mentioned research, there are projects for the determination of phenolic compounds
and organic acids in Brassica oleracea var. costata and in 6 edible wild mushrooms species (Amanita
caesarea, Boletus edulis, Hydnum rufescens, Gyroporus castoneus, Lactarius deliciosus and Xerocomus
chrysenteron) are in course.
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. The relationship between diet and health
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stimulates public interest and awareness concerning the nutritional value of foods. Additionally, food
globalisation has contributed to the alteration of the population food behaviours. 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 ingredients used. 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”.
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 being conducted.
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
anti/carcinogenic, anti-atherogenic, lean body mass promoting, and anti-diabetic. The results suggest that
this milk can be considered an interesting source of CLA. 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.
Contaminants
Besides interest for several nutrient compounds, the evaluation of different contaminants in foodstuffs has
also been in the scope of our group. Methodologies were optimized and implemented for the determination
polycyclic aromatic hydrocarbons (PAH’s) in vegetable oils, and aflatoxines in nuts. Above-mentioned works
with biogenic amines, heterocyclic aromatic amines and pesticides can also be classified in this field.
In this moment we are developing a GC-MS method for screening acrylamide in different glucidic foodstuffs,
such as potato crisps, French fries, biscuits and bread. Acrylamide is a known carcinogen and the
determination of its levels in the Portuguese foods, and consequent daily intake, is a main priority.
Plants of possible biological interest
A project regarding the valorisation of Salvia officinalis, Melissa officinalis, Mentha piperita and Lavandula
angustifolia is in course. The extracts for definition of the phenolic profiles of in vitro material (calli, shoots,
plantlets) and in vivo material (at 4 different vegetative stages) of each species were prepared and will be
analysed by HPLC/DAD.
We intend to start with the isolation and structural identification of some phloroglucinol derivatives found in
the extract of Hypericum androsaemum, which might have some biological interest.
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Environmental Control and Remediation
Head of laboratory: Cristina Delerue Matos, Prof. Coordenador and Helena Soares, Assistant Professor
Research Team
Cristina Maria F. Delerue Alvim de Matos
Maria Leonor Madureira Pinto
Maria do Carmo Veiga Fernandes Vaz
Helena Maria Vieira Monteiro Soares
Assistant Professor
Ermelinda Manuela Pinto de Jesus Garrido
Professor Adjunto
Simone Barreira Morais
Assistant
Maria Goreti Ferreira Sales
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
Carina Machado
Ph.D Student
Cristina Maria Rodrigues Ferreira Alves
Ph.D Student
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
Oriza Paula Guedes Tavares
Student
Number of articles in scientific journals: 11 (131 to 133, 138 to 142, 144 to 146)
Number of M. Sc. Thesis: 3
Environmental Control
Pesticide Analysis
Present environmental concerns at the analytical chemistry field suggest the establishment of experimental
procedures that enable the emission of low toxicity effluents. It is also important to decrease the consumption
of reagents, not only in terms of cost, but also regarding sustainability concerns. Combining these perspectives
and in order to simplify the samples pretreatment several methodologies for the control of pesticides in
environment were established using electroanalytical detection or chromatographic techniques.
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The FIA systems coupled with potentiometric and amperometric detectors are suitable for pesticide analysis.
The former is distinguished for its ability in performing analytical readings of electrochemically active species
in a wide variety samples and they can be emphasized by its selectivity if regarding ion selective electrodes
or potential applied. Natural waters and commercial samples were analysed.
In soils the analysis of pesticides involves an extraction step. The employment of microwave-assisted solvent
extraction (MASE) has many advantages over other classical extraction techniques like reduction of extraction
time and solvent consumption, as well as, the possibility of running multiple samples, etc. The use of MASE
for the determination of herbicides from soil samples was investigated. Alternatively solid phase extraction
and microextraction have been developed to determine pesticide residues in grapes and wine using GCMS or GC-ECD.
Laboratory Waste Management
Discussion of green chemistry materials leads today’s students to the concepts associated with developing
environmentally benign processes and products and towards reducing the amounts of reagents and solvents
used in laboratory work. Consistently, current and future chemists and engineers are now being trained to
design, develop, and apply chemical processes (and products) to reduce or eliminate the use, and generation,
of substances hazardous to human health and the environment.
Despite, it is still difficult to define what should be done with the wastes generated by our society, even when
the quantity is small… One example of this reality is the analytical laboratory in both research and educational
institutions, inherently generators of small quantities of hazardous wastes even after a greening approach.
No matter the quantity, if mismanaged these wastes have the same potential for harm as do fully regulated
waste from larger sources.
Thus, combining environmental education with chemistry, a waste management program has been
implemented. The main activities concern the establishment and practical implementation of proper strategies
for the disposal of the wastes generated. Following alterations in experimental procedures (to achieve an
immediate reduction in the quantity of generated wastes), a properly selective collection of wastes was
implemented. All wastes collected enter either a reutilization/recycling process or suitable chemical treatment
followed by analytical control.
Soils characterization and remediation
It is widely known that human activities have promoted a constant and increasing degradation of our habitat.
Industries play here a major role, emitting repeatedly highly toxic pollutants to the atmosphere and to superficial
waters. Though the existence of these industries is fundamental, significant efforts have been made through
the legal restriction of these emissions. Yet, there are still contaminated soils that need to be taken care...
Considering the complexity of the concerned matrices, with a wide variety of organic compounds, separative
techniques are here an important tool. Specifically, chromatographic analytical procedures have been
developed to quantify total petroleum hydrocarbons in refinery soils and some resin components (phenol,
furfuryl alcohol and formaldehyde) in foundry waste sands.
Once soils are characterized, an adequate clean-up technology, such as soil vapor extraction and solvent
extraction, may be established. Specifically, the effect of soil water and organic matter content in the efficiency
of soil vapor extraction of cyclohexane, benzene, toluene and ethylbenzene, have been studied. The selection
of operating conditions and the influence of soil characteristics at the efficiency of solvent extraction (mixture
of ethyl acetate/propanone/water) have been considered as well.
Metal ion control
In the framework of Project 6, “Interaction between metal ions and buffers for the environmental and biological
pH ranges”, the influence of DIPSO on solutions containing copper(II) and of TAPSO on solutions containing
cadmium(II), lead(II) or zinc(II) was studied by glass electrode potentiometry (GEP) and direct current
polarography (DCP), at fixed total ligand to total metal ion concentration ratios and various pH values, at
25.0±0.1ºC and ionic strength 0.1 M KNO3. Several complexes were identified for each ligand-metal ion pair
and their global stability constants were determined.
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Plan of activities for 2004:
The work will pursue with the complexation study of additional ligand-metal ion pairs. Metal-pH buffers
stoichiometric stability constants and the structure of some of the complexes will be determined when
complexation occurs.
In the framework of the Project “Recycling of coagulant agent present in the water treatment plant sludges”,
recently financed by the Investigação Científica na pré-graduação program, we started to develop a clean
methodology in order to reduce the amount of solids present in the sludges and to allow the reuse of the
recovered alum as coagulant.
We started the control of heavy metal pollution in involving environments of abandoned mines (Ph.DThesis
of Cristina Alves). Total and speciation of several heavy metals will be performed in water, sediments and
soils collected in different sampling stations along the environmental systems potentially polluted.
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ANALYTICAL METHODOLOGIES
Head of laboratory: Aquiles de Barros, Associated Professor
Research Team:
Aquiles José Ferreira de Araújo Barros
Associated Professor
José António Maia Rodrigues
Assistant Professor
Paulo Joaquim Ferreira de Almeida
Assistant Professor
Luís Guilherme de Lima Ferreira Guido
Assistant
Pedro Miguel Gonçalves Rodrigues
Ph. D. Student
Maria Isabel Afonso Rocha
Assesora
Maria Fernanda Rocha M. L. O. Cabral
Associate Researcher
Andreia Filipa da Silva Curto
M. Sc. Student
Marta Sofia Roma Pires
M. Sc. Student
Maria Fernanda Andrade Resende
M. Sc. Student
Sérgio José Pinto Teixeira
M. Sc. Student
Cristina Maria F. Delerue Alvim de Matos
Assistant
Number of publications: 5 (143, 183 to 186)
Number of Ms.C. thesis: 4
Number of patents: 1
Development of new methodologies
Activity in 2003
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 will be devoted to the studies related with the problem of beer
ageing, no doubt the main particular topic to be considered in the development of the project. Nevertheless,
when possible the application of voltammetric techniques will continue to be investigated, as these techniques
have been a distinctive characteristic of this group.
In the context the growth of the presence of this group in the research related with beer field, which is the
principal objective of the project, the main activities under development during 2003 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 it was possible to propose, to the “Agência de Inovação”,
a three years joint research which was approved. The objective of the work, based on a patent meanwhile
submitted (already approved in Portugal and in phase of EC approval), is the development of a voltammetric
method for the determination of diacetyl directly in the fermentation vessels and will involve 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.
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— The cooperation with Institut Français de la Brasserie et de la Malterie, I. F. B. M., Nancy, France –
During part of 2003, the Ph. D. student Luís Guido was sent to IFBM, where he made part of his research
work, in the context of a collaboration also involving Unicer; another Ph. D. student (Andreia Curto) will go
there by the end of 2004 and part of 2005, to continue the work of Luís Guido, 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 specially intense degradation conditions.
As a result of the investigation activity of the group in 2003, 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 4 M. Sc. theses. For 2004 a similar production is expected.
Plan for 2004
For 2004, the objective of the group is to continue the activity of 2003, 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.
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).
Electroanalysis
In recent years the interest in the study and analysis of biomolecules has raised, particularly due to increased
demand of understanding the mechanisms and molecular interactions occurring in vivo and to its quantification
in biological samples.
Electrochemistry is nowadays one of the main techniques used for the determination of these species
mainly due to its selectivity, sensitivity and low operating costs.
Selective serotonin reuptake inhibitors (SSRIs) is one group of compounds studied using AdSV. In recent
years, members of the SSRI class have been administered to treat anxiety disorders, obsessive compulsive
disorder and post traumatic stress disorder and its consume have increasingly.
Electrochemical techniques are especially attractive for the study of biotransformation oxidative mechanism
of drugs of abuse (morphine, codeine, dihydrocodeine, heroin, apomorphine and codeine). Different methods
were developed based on SWV and FIA with amperometric detection for the quantification of drugs in
pharmaceutical preparations and seizure samples.
Ion-selective electrodes (ISEs) are of easy construction and enable the selective reading of a wide range of
inorganic and organic ions. When good response characteristics are reported, ISEs may turn out an important
tool at the analytical chemistry field, allowing quick readings and avoiding inaccurate results. Several
compositions of ISE’s must be tested and the best one shall proceed to the analysis of active principles in
pharmaceutical preparations.
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Physicochemical characterization of food products
Head of laboratory: Maria do Pilar Gonçalves, Associate Professor and Alberto Sereno, Associate
Professor
Research Team
Maria do Pilar Figueroa Gonçalves
Associate Professor
Alberto Manuel Carneiro Sereno
Associate Professor
Loïc Hilliou
Associate Laboratory Researcher
Maria Adília Costa Leite Lemos
Post-Doc Student
Marta Isabel de Glória V. M.da Silva
Post-Doc Student
Luis Mayor Lopez
PhD Student
Wancheng Sittikijyothin
PhD Student
Duarte Paulo Martins Torres
Research Fellow (BIC)
We proceeded with on-going studies on micro-structural and rheological characterisation of biopolymer
(proteins and polysaccharides) mixed systems, in aqueous media.
1. Development and chemical, structural and rheological characterisation of protein/
polysaccharide mixed aqueous systems
The effect of locust bean gum, LBG, on the heat induced gelation of a pepsin whey protein hydrolysate
(WPH) was studied by small deformation rheology: an enhancement of the aggregation rate and of the
strength of the protein gel was observed, the magnitude of these effects depending on the WPH/LBG
ratio. Light microscopy showed that the final gels were two-phase, with a continuous matrix of WPH
enriched phase. (Project POCTI/QUIM/36452/2000).
Work plan for 2004
- characterisation of the peptides resulting from hydrolysis with trypsin (amino-acid composition);
- essays in gels of the trypsin hydrolysates (with different degrees of hydrolysis) alone and in a mixture
with LBG. Microstructure of the gels will be studied by confocal laser scanning microscopy.
2. Rheological behaviour and morphology of protein/polysaccharide mixed systems
We pursued on-going studies on experimental measurement of viscoelastic properties and
microstructure of whey protein concentrate (Wb)
Work plan for 2004
The rheological study will pursue testing other working parameters (pH, mixing ratio, shear effects).
3. Use of galactomannan/starch mixtures in low-oil food emulsions stabilisation
Rheological studies were carried out on the continuous phase - formulated with a highly crosslinked starch
pasted in the presence of tara gum - of low fat O/W food emulsions. It was concluded that the presence of
tara gum decisively influence the gelation (pasting) process of the modified starch used. (project CRUP, E19/02, with University of Sevilla, Spain).
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4. Cold gelation of globular proteins
Bibliographic research about cold gelation of whey protein isolates was done.
A computational question was also explored through the implementation of several computer programs that
enable the use of Plazek, Kaschta and Cole-Cole methods in the analysis of experimental rheological data.
A software tool that makes possible the analysis of experimental textural data - compression with / without
rupture, relaxation and TPA - was also improved.
In the last trimester, some preliminary tests were done with the objective of establishing the experimental
protocol that would be correct to improve. The first experiments were made in order to study the effect of
pre-heating conditions (temperature and holding time) on Mg2+ - induced gelation of a whey protein isolate
(WPI).
Work plan for 2004
The studies on the effect of pre-heating conditions (temperature and holding time) on Mg2+ - induced gelation
of WPI will be concluded.
Once established the optimal pre-heating conditions, it will be analyzed the effect of salt concentration,
protein concentration and pH on the cold gelation of WPI.
A similar study will be carried out over other protein / polysaccharide systems.
5. Effect of ohmic heating on the rheological properties of model food systems
During this period, three main steps were performed:
(i) A literature search related to ohmic heating in different systems and also the effect of this
treatment on the rheological properties of proteins; (ii) Preliminary studies with the ohmic heating
equipment in order to gain experience of using the apparatus and to adjust the conditions required for the
whey protein solutions; (iii) Rheological analysis of the ohmic heated samples.
Work plan for 2004
The work plan for 2004 has the following steps/aims:
Optimisation of the process conditions, in the ohmic heating system, for different concentrations of whey
protein solution;
Determination of electrical conductivity of the whey protein solutions at different temperatures;
Study of the different factors that can affect the rheological properties of the whey protein solutions under
different ohmic heating conditions, such as voltage and the time that this is applied to the solutions.
6. Besides these lines, research activity will be also devoted to the possible implementation of advanced
data analysis procedures and Fourier Transform Rheology concepts to existing apparatus in the laboratory,
namely a texture meter and a stress rheometer. As a result of these studies, an increase in instruments
sensitivity and the emergence of a new food sample characterization technique are foreseen. Finally, the
possibility of applying rheo-optical techniques to the study of structural changes in food samples undergoing
shear will be tested.
7 Studies in food structure
The quality of porous products is dependent on their porosity but its determination for materials with
high moisture contents is not an easy task since it is mandatory the knowledge of the true volume of the wet
solid matrix. To measure this volume successfully a new gas pycnometer, specially designed for high moisture
foods, was built and tested by means of a carefully designed set of experiments to validate the technique.
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The reproducibility obtained of 0.018% is excellent when compared to similar commercial instruments. Further
development of on-going studies concerning the effect of osmotic dehydration on the properties and quality
factors of fruits and vegetables, with particular emphasis on shrinkage, mechanical properties and on changes
of the microstructure of the cellular tissue.
Workplan for 2004
To finish experimental programme of osmotic dehydration of fresh fruits; develop and validate mathematical
models describing the effect of osmotic dehydration on food structure and properties.
8 Technology for edible biodegradable films and coatings for foods
This constitutes a new line of research on the production and characterisation of edible biodegradable films
and coatings, obtained from national low value resources namely biopolymers from marine macroalgae,
starches from several types of oak acorns and pectin from residues from the fruit industry. This research
activity was started within the scope of CYTED project XI.20, an international project co-ordinated by FZEA/
USP/Brazil, and project POCTI/45595/EQU/2002.
Workplan for 2004
To identify and select raw material sources (macro-algae and fruits residues); to design, implement and test
environmently friend methods of biopolymer extraction; to start-up biopolymer characterisation.
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TOXICITY AND PROTECTION STUDIES
Head of Laboratory: Maria de Lourdes Bastos, Full Professor
Research Team
Maria Lourdes Bastos
Full Professor
Felix Carvalho
Assistant Professor
Fernando Remião
Assistant Professor
Helena Carmo
Assistant
Márcia Carvalho
Ph. D. Student
Maria Elisa Soares
Assessor Principal
Eulália Mendes
Assessor Principal
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 one of its metabolites, ±-methyldopamine, as well as
the formation of glutathione adducts of this last toxic compound, were evaluated.
The validated Daphnia magna in vitro model was used for the evaluation of environmental contamination by
acethylcholinesterase inhibitors.
An in vivo experimental model was used for the evaluation of the toxic effects of the designer drug of abuse
4-methythioamphetamine (4-MTA), at the physiological level. The hyperthermic effect of this drug and the
pathways involved in its thermogenic effect were evaluated in mice.
Also in mice, the toxic effects elicited by d-amphetamine at the skeletal muscle 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.
Studies in rat cardiomyocyte suspentions 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.
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-
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bromo-2,5-dimethoxyphenethylamine (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 hydroxyl radical and hypochlorous acid scavenging activity of Centaurium erythraea infusion was
evaluated by non-cellular in vitro chemical assays.
The hepatoprotective activity of polyhydroxylated 2-styrylchromones against tert-butylhydroperoxide-induced
toxicity was evaluated in freshly isolated rat hepatocytes. The high protective action showed by some of
these compounds can make them potential medicines.
The Hypericum androsaemum infusion was also evaluated for its hepatoprotective effect against t-BHPinduced toxicity in isolated rat hepatocytes, and in vivo studies, in mice. In vitro assays showed that the preincubation of the cells with the infusion prevented t-BHP induced loss in cell viability and lipid peroxidation.
The 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.
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BIOMIMETIC SYSTEMS AND SIMULATION
Head of Laboratory: Maria João Ramos, Associate Professor
Research Team
Maria João Ramos
Associate Professor
Alexandre Magalhães
Assistant Professor
André Melo
Assistant Professor
José Augusto Pereira
Assistant Professor
Pedro Fernandes
Invited Assistant Professor
Agostinho Antunes Pereira
Assistant Researcher, REQUIMTE
Elsa Henriques
Post-Doctoral Student
Susana Pereira
Ph.D. Student
Rute Fonseca
Ph.D. Student
Nuno Cerqueira
Ph.D. Student
Fátima Lucas
Ph.D. Student
Akapong Suwattanamala
Ph.D. Student
Vineet Pande
Ph.D. Student
Sérgio Sousa
Ph.D. Student
Ricardo Branco
Ph.D. Student
Zenaida Mourão
Ph.D. Student
Irina Moreira
Ph.D. Student
Alexandre Carvalho
Ph.D. Student
Alexandra Teresa Carvalho
Research Student
Number of publications: 12 (152 to 163)
Molecular Modelling and Simulation
We are generally interested in the molecular modelling of biological and chemical systems. Accordingly,
we have been working on systems such as proteins, cyclodextrins, lanthanide (III) chelates and small
peptides. Many of these studies are carried out in close collaboration with experimentalists.
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, to perform these studies. A more detailed description concerning our main present interests,
which have derived from years 2002/2003 and will continue in 2003/2004, follows:
I.
Disease Related Research
Cancer
(i)
Study of the catalytic and inhibition mechanisms of enzymatic reactions, such as P450
1A2 (an enzyme involved in the metabolic pathway of carcinogenesis) and RNR 2,3 (ribonuclease reductase,
an enzyme thought to be involved in cancer), resorting to both quantum mechanics, QM/QM and QM/MM
methods. These studies are 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.
1
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(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, farnesyl
transferase, vascular endothelial growth factor and fumarate reductase.
(ii)
Molecular simulations on lanthanide (III) chelates and host-guest complexes with g-cyclodextrins:
the aim is to design new contrast agents for Magnetic Resonance Imaging, an important tool for clinical
diagnosis (e.g. tumour therapy). This project is part of the COST Programme, having been financed by the
Fundação para a Ciência e Tecnologia, Portugal, project PRAXIS/PCEX/C/QUI/67/96 4,5.
(iii)
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. Graham
Richard of which Maria João Ramos is an Associate Director, being financed by the National Foundation for
Cancer Research, U.S.A 6.
A.I.D.S.
(iii) We are studying 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 7. This project is being
carried out in close collaboration with Dr. Rakesh Sharma, an experimental organic chemist from Delhi
University, in India.
Hypertension
(iii) Modelling of mimetic modifications of bioactive peptides by inclusion of novel synthetic amino
acids. 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 8.
Malaria
(iv) Establishment of new antimalarial compounds based on electrostatic profiles of established drugs.
This study is being performed in collaboration with Prof. Madalena Pinto from the Faculty of Pharmacy at
the University of Porto, Portugal 9,10.
II.
Drug Design Complementary Studies
(v) The natural environment of molecules with biological and pharmacological activity is the aqueous
physiological medium. The study of the solvation effect in their physico-chemical behaviour is, obviously,
of crucial importance. The simulation of vibrational spectra of several molecules in solution constitutes an
excellent test for the solvation models employed. Such theoretical models can then be used to simulate
drug molecules in their natural environment 11.
(vi) Development of new formalisms to analyse the molecular interactions in association processes.
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.
Selected References:
1
R. Da Fonseca; M. C. Menziani; A. Melo; M. J. Ramos, Molec. Phys., 2003, Vol. 101, 2731-2741
2
PA Fernandes, MJ Ramos, J. Am. Chem. Soc., 2003, Vol. 125, 6311-6322
3
PA Fernandes, MJ Ramos, Chem. Eur. J., 2003, Vol. 9, 5916-5925
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4
P.A. Fernandes; A. T. P. Carvalho; A. T. Marques; A. L. F. Pereira; A. P. S. Madeira; A. S. P. Ribeiro; A. F. R.
Carvalho; E. T. A. Ricardo; F. J. V. Pinto; H. A. Santos; H. D. G. Mangericao; H. M. Martins; H. D. B. Pinto; H.
R. R. Santos; I. S. Moreira; M. J. V. Azeredo; R. P. S. Abreu; R. M. S. Oliveira; S. F. M. Sousa; R. J. A. M.
Silva; Z. S. Mourao; M. J. Ramos, J. Computer-Aided Molec. Design, 2003, Vol. 17, 463-473
5
E. S. Henriques; C. F. G. C. Geraldes; M. J. Ramos, Molec. Phys., 2003, Vol. 101, 2319-2333
6
http://www.chem.ox.ac.uk/cancer/ccdd.html
7
V. Pande; M. J. Ramos, Curr. Med. Chem., 2003, Vol. 10, 1603-1615
8
M. A. C. Preto; A. Melo; S. P. G. Costa; H. L. S. Maia; M. J. Ramos, J. Phys. Chem. B, 2003, Vol. 107,
14556-14562
9
C. Portela; C. M. M. Afonso; M. M. M. Pinto; M. J. Ramos, Febs Lett., 2003, Vol. 547, 217-222
10
C. Portela; C. M. M. Afonso; M. M. M. Pinto; M. J. Ramos, J. Computer-Aided Molec. Design, 2003, Vol.
17, 583-595
11
AL Magalhães and AS Soares Pinto, Theor. Chem. Acc. (2003) 110, 70-78
Artificial Chemistry
The work being developed is an artificial chemistry program inspired in the general principles of Lewis acidbase reactions. This kind of reactions is the base of nucleophilic additions and substitutions for the synthesis
of the most important biological molecules using ATP. In the formal system being developed the agents
have therefore different virtual donor and acceptor properties and interact using the well stirred reactor
abstract dynamics. Bonding occurs, with some probability, when a donor finds an acceptor in the well stirred
reactor, forming polyagents, the equivalent to molecules.
This basic formal system did not display properties like catalysis and molecular recognition and a more
elaborate definition of molecules was necessary, namely, the definition of internal coordinates for each
molecule and the definition of inductive effects between agents. These features were added to the program
in 2003. Another important feature was a modification on the dynamics to positively discriminate intramolecular
interactions. With these improvements the system is now capable of reproducing phenomena like SN2
mechanisms and general acid-base catalysis. Molecular recognition was an emergent characteristic of the
system due to the 2D definition of molecules.
The main work in 2004 will be to explore the capabilities of the system, as implemented in the program.
Since the system is now able to mimetize the most basic features of carbon chemistry we hope to design
some transformation pathways with biochemical resemblance.
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APPLIED BIOPHYSICS AND BIOCHEMISTRY
Head of laboratory: Baltazar de Castro, Full Professor and José Costa Lima, Full Professor
Research Team:
Baltazar de Castro
Full Professor
José Luís Fontes da Costa Lima
Full Professor
Paula Gameiro
Assistant Professor
Maria Salette F. F. H. Reis Dias Rodrigues
Assistant Professor
Eduarda Graça Rodrigues Fernandes
Assistant Professor
Carla Manuela S. Matos
Assistant Professor
Catarina Mansilha
Assistant Professor
Patrícia Neves
Ph.D Student
Sofia Costa Lima
Ph.D Student
Marlene Susana Dionísio Lucio
Ph.D Student
Helena Suzana da Costa Machado Ferreira
Ph.D Student
David de Andrade Sousa Costa
Ph.D Student
Anabela Ferreira Gomes
M. Sc. Student
Joana Moutinho da Veiga Marcelino
M. Sc. Student
Ana Maria de Carvalhais Mendes Gomes
M. Sc. Student
Number of articles in scientific journals: 5 (92, 103 to 105, 137)
Biophysics of organized media
The histidine-tagged mouse MDR3 P-glycoprotein was expressed in metanotrophic yeast Pichia pastoris.
Plasma membrane proteins were solubilised with two surfactants, n-dodecyl-b-D-maltoside and 3-[(3cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid, and further purified using affinity
chromatography on a Ni-NTA agarose resin. Reconstitution of P-glycoprotein in lipid bilayer vesicles was
performedby by three reconstitutions techniques in Escherichia coli lipids bilayer vesicles using surfactant
removal by dialysis, adsorption on polymeric beads and gel filtration chromatography. The resulting
proteoliposomes displayed constitutive and drug-stimulated ATPase activity dependent on the reconstitution
technique and detergent used. The highest verapamil-stimulated ATPase activity was observed on
proteoliposomes reconstituted by gel filtration chromatography. Detergent dialysis and gel filtration
chromatography produced uniform and homogenous LUVs populations of vesicles. Gel filtration
chromatography appears to be the more effective and functional technique to reconstitute Escherichia coli
lipids- mouse P-glycoprotein proteoliposomes.
The interaction of the outer membrane protein OmpF in o-POE 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.
Biochemistry of organized media
The effect of several non steroidal anti-inflammatory drugs (NSAIDs) on biological membranes was studied
using liposomes as membrane mimetic models as they are generally accepted as suitable models for the
study of membrane structure and properties due to their structural similarity to the lipidic matrix of cell
membranes. In these studies we have evaluated the partition coefficient of NSAIDs, the location of that
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drugs in membrane, their effect on membrane fluidity and their anti-peroxidation properties. The effect of
NSAIDs on surface potential of the liposomes was also determined.
The determinations of partition coefficients were performed by derivative spectrophotometry and by fluorescence.
The effect of NSAIDs on surface potential was assessed by measure the zeta potential in the presence of
different drug concentrations. This methodology was applied to the study of the NSAIDs concentration
effects. Results revealed an intense membrane charging that was proportional to the amount of negatively
charged drug in media. A mathematical formalism was adapted and an analytical expression derived to
calculate directly surface potentials from zeta-potential data. The membrane loading state, expressed as
the number of molecules per unit area was calculated for the negative and for the neutral forms of the
drugs. An approach was also developed which allows the determination of the maximum number of molecules
per unit area. The calculation of the maximum mole lipid:drug ratio was also estimated and related to the
binding stoichiometry, as well as to the maximum lipid loading capacity. The concentration profiles for drugs
were established in terms of distance to the liposome surface.
Location and membrane fluidity were evaluated by fluorescence using fluorescent probe molecules. These
probes offer several distinct advantages over other techniques: (a) they yield information about the polarity
and microviscosity of the environment (b) different probes are available to monitor specific membrane
regions, and (c) they can be used at low concentrations, which minimizes membrane perturbations by the
probe. Quenching fluorescence studies were performed to assess drug location using a set of n-(9anthroyloxy) fatty acid probes (n=2,6,9 and 12) whose fluorophores locate at a graded series of positions in
the transverse plan of the bilayer. These probes are capable of sensing “fluidity” gradient through the bilayer
leaflet.
To determine changes in membrane fluidity anisotropy fluorescence studies were performed using the
same n-AS probes that permit to examine gradients in fluorescence polarization through bilayers. The
fluorophores of these probes report the environment at a graded series of depths from the surface to the
centre of the bilayer structure. Results were analysed according to the Perrin equation that relates measured
polarization to the rotational relaxation time of the fluorophore. Although this equation applies only to isotropic
rotation of a fluorophore and is not strictly applicable to the anisotropic rotation of the probes in lipid bilayers
it does offer some correction for the variation of lifetime. The results obtained show that NSAIDs increase
the fluidity of the membranes. This effect can be related with the anti-oxidant properties of NSAIDs namely
in their role on peroxidation process which develops in biological membranes, primary involves
polyunsaturated fatty acids and results in an alteration of dynamic and structural properties of the membrane
which could be the main reason for the impairment of cell and organism functioning. The 3-(4-(6-phenyl)1,3,5-hexatrienyl) phenylpropionic acid (DPH-PA) was also used as fluorescent probe to monitoring not only
the antiperoxidation activity of the NSAIDs but also the changes in membrane fluidity during the peroxidation.
These studies were developed to evaluate if NSAIDs can act by another synergic mechanism that involve
changes on membrane fluidity and not only as free radical scavenging as the mechanism of protective
action by alteration of cell membranes is manifest in several experimental studies where compounds that
are not free radical scavengers, or inhibitors of xantine oxidase or chain-breaking antioxidants, were shown
to protect still against peroxidative injury. The results obtained suggest that the anti-oxidant properties of the
NSAIDs are not only due to their scavenger properties against several free radical species but also to their
effect on membranes properties.
During 2004 and after the study of the effect of NSAIDs on the membrane liposomes referred we propose
the study of their 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.
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Scavenging activity studies for reactive oxygen and nitrogen species by non-steroidal antiinflammatory drugs (NSAIDs)
Among the various reactive species that are known to be produced in excess during the inflammatory
reactions, the reactive oxygen species (ROS): peroxyl radical (ROO.), hydroxyl radical (HO.), superoxide
radical (O2.-), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) and the reactive nitrogen species
(RNS): nitric oxide (.NO) and peroxynitrite (ONOO-) play important roles in inflammatory sites, which makes
them potential targets for the chemotherapy of inflammation.
The non-steroidal anti-inflammatory drugs (NSAIDs) block prostaglandin synthesis by cyclooxygenases
(COX-1 and/or COX-2). However, it has been suggested that the anti-inflammatory activity of NSAIDs may
be also partly due to their ability to scavenge ROS and RNS. Therefore, the scavenging activity of various
NSAIDs against the above-mentioned ROS and RNS is being evaluated. For this purpose, chemiluminimetric,
spectrophotometric and fluorimetric methodologies are being implemented and adapted to a microplate
reader.
The use of the implemented microanalysis methods allows the reduction of the amount of
reactants and working compounds, which is economical and environmental advantageous. Another advantage
of this kind of methodologies is the improvement of experimental conditions as they allow the execution of
simultaneous and rapid screening assay of different compounds at various concentrations and replicates.
This minimizes the interference of time-dependent variations in the conditions of the assays, like environmental
temperature and the biological and chemical reactivity of the assay mixtures.
We have recently proposed that several NSAIDs may react with ROS and RNS 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 has been evaluated using non-cellular in vitro methodologies.
During 2004, the aforementioned reactions will be investigated using tandem mass spectrometry (MS/MS).
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
Research Team:
Baltazar de Castro
Full Professor
Paula Gameiro
Assistant Professor
Eulália Pereira
Assistant Professor
Ana Claúdia Nunes
Ph.D Student
Maria João Amorim
Ms.C Student
Andreia Daniela Leite
Research fellow (BI-FCT)
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 and their corresponding metal complexes with Zn(II), Cu(II), Ni(II) and Co(II) have been synthesized
and characterized in the solid state and in solution. Stability constants were determined by potentiometric
and spectrophotometric methods and speciation diagrams have been established. Evaluation of the toxicity
of the prepared compounds has been evaluated in terms of cell viability and effect in oxidative stress in
collaboration with the toxicology group (REQUIMTE). Evaluation of the insulin-like action of the synthesized
complexes is being performed in collaboration with Prof. Hiromu Sakurai (Kyoto University).
2 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 can be summarized as:
(i) establishment of synthetic pathways to obtain hexadentate chelators derived from 3-hydroxy-4-pyridinone
and cathecol ligands that can be anchored in macromolecules; (ii) functionalization of macromolecules,
with lipophilic substituents, siderophores and fluorescent groups in order to obtain molecules able to target
infection processes and to act as agents iron depletion.
3 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. In addition to platinum complexes, we are currently carrying the synthesis of palladium
complexes.
A project focused on the study of Structural factors determinant on the reactivity of photolysis products
of B12 model compounds was initiated in late 2003 and will be developed through the years 2004 to 2006.
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COMPUTATIONAL STUDY OF METAL SURFACES
Head of Laboratory: José Ferreira Gomes, Full Professor
Research Team
José Ferreira Gomes
Full Professor
Natália Cordeiro
Associate Professor
Shuwen Yao
Post-Doctoral Student
Hugo Santos
Ph.D. Student
Ana Sofia Pinto
M.Sc. Student
Number of Ph.D.thesis : 1
The understanding of the physical and chemical behaviour of metal surfaces is of such difficulty that
experimentalists find it very useful to complement their work with computational results. Furthermore, the
theoretical study of metal surfaces and their interaction with adsorbed molecular species is interesting on
its own and is now a very active field of research.
Adsorption of methoxy radical and other species on metal surfaces.
The group has now accumulated ten years of experience on the application of DFT computational methods
to the interaction of metal surfaces with adsorbed and non-adsorbed neighbouring species. Extensive use
of the cluster models for the metal surface has shown the potential and the limitations of this strategy for the
calculation of interactions.
Representative of the studies done in recent months and to pursue in 2004 are the studies of the adsorption
of methoxy radical on copper and ruthenium, the comparative study of different adsorption sites and their
vibrational spectra.
The interaction of methoxy radical with the metallic surfaces has been studied using a cluster model approach.
The density functional theory results show that this approach gives a reasonable description of the surface
allowing a good prediction of structural and energetics features of the adsorbate.
Selected References:
CGPM Bernardo, JANF Gomes, J Mol Struct-THEOCHEM, 2003, 629, 251-261
DJVA dos Santos, JANF Gomes, Langmuir, 2003, 19 (3), 958-966
E Martinez-Nunez, A Fernandez-Ramos, MNDS Cordeiro, SA Vazquez, FJ Aoiz, L Banares, J Chem
Physics, 2003, 119 (20): 10618-10625
SW Yao, VHC Lopes, F Fernandez, X Garcia-Mera, M Morales, JE Rodriguez-Borges, MNDS Cordeiro,
Bioorganic & Med Chem, 2003, 11 (23): 4999-5006
MNDS Cordeiro, E Martinez-Nunez, A Fernandez-Ramos, SA Vazquez
Chem Phys Letters, 2003, 381 (1-2): 37-44
M Frankowski, BS Fox, AM Smith-Gicklhorn, MK Beyer, VE Bondybey, M Algarra, ML Costa, P
Rodrigues, MT Barros, MNDS Cordeiro, Low Temp Phys, 2003, 29 (9-10): 870-875
MNDS Cordeiro, Mol Simulat, 2003, 29 (12): 817-827
MNDS Cordeiro, E Martinez-Nunez, A Fernandez-Ramos, SA Vazquez
Chem Phys Letters, 2003, 375 (5-6): 591-597
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NOVEL HETEROGENEOUS CATALYSTS
Head of Laboratory: Cristina Freire, Associate Professor and Baltazar de Castro, Full Professor
Research Team
Baltazar de Castro
Full Professor
Ana Cristina Freire
Associate Professor
Eulália Pereira
Assistant Professor
Uwe Pischel
Associate Laboratory Researcher
Rita Ferreira
Assistant
Carla Alexandra Sousa
Post-Doctoral Fellow
Ana Rosa Silva
Pankaj Das
Magda Martins
Ph.D Student
Andrea Carneiro
Ph.D Student
Elsa Pereira
Ms.C Student
Mário Cardoso
Ms.C Student
Adelaide Miranda
Research Fellow
Marek Kluciar
Research Fellow
Miguel de Sousa
Ms.C Student
Number of Ms.C. thesis: 3
1
Novel catalysts by heterogenisation of metal complexes
Chiral and non-chiral manganese salen complexes have been synthesised by methodologies developed in
our lab and characterised by several techniques: elemental analyses, mass spectra, cyclic voltammetry,
EPR, FTIR and UV-Vis spectroscopies. The catalytic activity of these complexes in the epoxidation of
styrene was evaluated in CH2Cl2 and CH3CN, using NaOCl, PhIO and p-chlorometabenzoic acid as oxygen
sources. The experiments were done at room temperature and reaction time, the styrene conversion, chemical
selectivities 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 complexes were anchored onto activated carbons and were encapsulated and pillared clays (PILCs)
using several strategies already developed in our lab. The complexes were anchored onto microporous
activated carbons (obtained from NORIT) using linking agents such as cyannuric chloride or by direct reaction
between the groups on the ligands and the surface carbon groups. The complexes were encapsulated in
PILCs by the flexible ligand method and by simultaneous/pillaring procedure. 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 the heterogeneous epoxidation of styrene in CH2Cl2 and
CH3CN, using the same oxygen sources, using equivalent experimental conditions of those used in
homogeneous phase. 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
Química Verde
61
the catalysts showed catalytic activity: the reaction times were larger than those observed in homogeneous
phase, the styrene conversion decreased, but chemical selectivities were quite similar to those of
homogeneous media. 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, but with the several reuses the ee% start to decrease significantly.
FUTURE WORK
2
(1)
Design and preparation of new chiral complexes with catalytic properties.
(2)
Synthesis of room temperature ionic liquids to be used as green solvents in catalytic reactions.
(3)
Anchoring/encapsulation of chiral Mn complexes onto several supports: mesoporous silicious
nanotubes, PILCs and clays.
(4)
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.
(5)
Use of new catalytic reactions: aziridination of alkenes and oxidation of alcohols.
Chemosensors based on transition metal complexes
Nickel and copper complexes with Schiff base ligands functionalised with groups that can act as coodination
sites for representative and lanthanide cations and for anions (halides, H2PO4-, HSO4-, NO3-) were prepared
by methodologies developed in our laboratory. The functionalities, crown ether and pseudo crown ethers
groups, flexible pendent arms bearing oxygen and nitrogen atoms and polyamines, were introduced in the
aldehyde moiety and imine bridge of the Schiff base. These complexes were characterised by several
techniques: elemental analyses, mass spectra, EPR, FTIR and UV-Vis spectroscopies and cyclic voltammetry.
Cation and anion recognition studies were performed in several solvents, using cyclic voltammetry, UV-vis,
NMR and conductimetry and for the complexes that polymerise in CH3CN, recognition studies were also
done in the respective films using cyclic voltammetry, in-situ FTIR and UV-Vis and electroacoustic impedance.
In solution, it was possible to determine equilibrium constants for the complexation of lanthanides and
alkaline-earth 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 adsorded all the metal cations tested (+1, +2 and +3), although they exhibited different
behaviour. 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. Equilibrium constants for the adsorption of Barium were determined by electroacoustic impedance.
FUTURE WORK
(1)
Design, preparation and characterisation of new complexes with properties to recognise cation
and anions.
2)
Preparation characterisation of films that can recognise cation and anions
(3)
Evaluation of the equilibrium constants for the interaction of cations and anions with the
complexes or polymeric films with the appropriate techniques.
(4)
Cation and anion selectivity studies and for polymeric films reutilisation studies
(5)
Preparation of metal complexes that will exhibit non linear optical properties.
Química Verde
62
3
Metallosurfactants. The study of the amphiphilic complexes [Fe(RR’bpy)2(CN)2], where R and R’
are alkyl chains (C1-C17), was focused on the films at water-air and water-metal interfaces. p-A isotherms
of Langmuir-Blodgett monolayers of the complexes and ligands were determined. Cyclic voltammetry was
used to study the electrochemical behavior of self-assembled films of the complexes. Ongoing and future
work include Brewster Angle Microscopy of Langmuir-Blodgett monolayers, determination of pKa of the
ligands by an electrochemical method, characterization of LB films by spectroscopy (UV-vis, FTIR) and
XRD.
4
Metal Nanoassemblies. Application of a photocatalytic method (previously developed in our laboratory
in collaboration with Prof. John Shelnutt, Sandia Nat. Lab. EUA) to gold reduction enabled us to prepare
gold sols with different nanoparticle morphology, namely triangular prisms, spheres and disks. This method
provides remarkable homogeneous morphology. Ongoing studies aim the optimization of the method in
order to minimize size dispersion and to understand the main factors controlling the morphological
characteristics of the nanoparticles.
5
Photoactive Compounds and Solid-State Materials
In 2003 the research of the group concentrated on two main lines: (a) investigation of stereoselectivity in
the quenching of excited states (collaboration with the Polytechnical University of Valencia, Spain), and
(b) design of novel luminescent compounds and solid-state materials based on lanthanide ions.
In the first project various bichromophoric dyads based on 2-arylpropionic acids were synthesized and
photophysically characterized. It has been demonstrated that the excited chromophores are quenched with
different efficiency, depending on the chiral information contained in the dyads. Marked diastereoselectivity
has been noted, including quenching by hydrogen transfer and electron transfer. Further research will deal
with the tailored synthesis of bichromophoric dyads in order to elaborate the recognition principles behind
the noted diastereoselectivity effects.
In the second project novel luminescent inorganic-organic materials based on lanthanide ions have been
prepared. They have been demonstrated to possess exceptional properties like extremely long luminescence
lifetimes (microsecond range) and highly color-pure light emission. In 2004 the research within this project
will concentrate on the further development and use of those photoactive materials as chemosensors.
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
Margarida Alice Ferreira
Maria Lurdes Souteiro Bastos
Pedro Brito Correia
Professor Catedrático
Professora Catedrática
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
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 B. S. M Montenegro
Maria Conceição S. S. Rangel Gonçalves
Maria João Ribeiro Nunes Ramos
Maria João L. R. M. C. Romão
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
Professora Associada
Professor Associado
Professor Associado
Professor Associado
Professor Associado
UNL
FEUP
FFUP
FCUP
FCUP
FCUP
UNL
ICBAS-UP
UNL
FFUP
ICBAS-UP
FCUP
UNL
FEUP
UNL
FFUP
UNL
UNL
Cristina Maria Delarue Alvim Matos
Maria Leonor Oliveira Madureira Pinto
Maria Carmo Veiga Fernandes Vaz
Professora Coordenadora
ISEP
ISEP
ISEP
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
Felix Dias Carvalho
Fernando Manuel Gomes Remião
Helena Maria Vieira Monteiro Soares
Henrique J. R. Guedes
Isabel Maria Ligeiro da Fonseca
Isabel Maria Viegas Oliveira Ferreira
Isabel Maria Rola Coelhoso
João Carlos S. B. Sotomayor
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
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
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
FFUP
FCUP
FCUP
UNL
UNL
UNL
FCUP
UNL
UNL
UNL
UNL
FFUP
UNL
FCUP
FFUP
FFUP
FEUP
UNL
UNL
FFUP
UNL
UNL
UNL
FFUP
UNL
UNL
UNL
UNL
Química Verde
A - 5
José António Maia Rodrigues*
Jose Augusto Caldeira Pereira
Jose Oliveira Fernandes
José Paulo Barbosa Mota
Luisa M. S. P. Ferreira
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 Natália Dias Soeiro Cordeiro
Maria Salete Reis Dias Rodrigues
Maria Teresa Avilés Perea
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
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora 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
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
FCUP
ICBAS-UP
FFUP
UNL
UNL
UNL
UNL
UNL
FFUP
FFUP
UNL
UNL
FCUP
UNL
FFUP
UNL
UNL
UNL
FCUP
FFUP
UNL
FFUP
UNL
UNL
FCUP
UNL
UNL
FCUP
UNL
FFUP
UNL
Ermelinda Manuela Jesus Garrido
Jorge Manuel Pinto Jesus Garrido
Professora Adjunta
Professor Adjunto
ISEP
ISEP
Adriana Martins Pimenta
Carla Manuela Soares Matos
Catarina Isabel Guerra Rodrigues
Cristina Maria C. Morais Couto
João Luís Tavares de Matos Gomes
Francisco Jorge Fernandes Caldeira
Lígia Maria da Silva Rebelo Gomes
Luísa Lima Gonçalves
Maria Alexandra Bernardo
Maria da Graça Soveral Rodrigues
Maria Helena Reis Prado de Castro
Rita Isabel Lemos Catarino
Sara Isabel Xavier Candeias
Stephane Besson
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor 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
FFUL
ISCSN
UFP
U. Lusófona
U. Lusófona
Ana Maria Philips
Maria Fernanda Cabral
Agostinho Pereira
Ana Lu]isa Carvalho
Carlos Brondino
Carlos Lodeiro Espino
Eurico Cabrita
Isabel Mafra
Maria Manuel Marques
Loïc Hilliou
Owen Catchpole
Serguey Bursakov
Svetlozar Velizarov
Uwe Pischel
Investigadora Principal
Investigadora Principal
Investigador Auxiliar
Investigadora Auxiliar
UNL
FCUP
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
Química Verde
A - 6
Manuel Rui Azevedo Alves
Maria Isabel Branco Alves Martins
Maria Teresa de Oliva Teles Moreira
Luís Guilherme L. Ferreira Guido
Patrícia Carla Ribeiro Valentão
Olivia Maria de Castro Pinho
Susana Isabel Pereira Casal
Abel José Assunção Duarte
Hendrikus Petrus Antonius Nouws
José Tomás Soares de Albergaria
Maria de Fátima de Sá Barroso
Maria Goreti Ferreira Sales
Maria Isabel Brito Limpo de Serra
Maria João Dantas Ramalhosa Ferreira
Maria Manuela Barbosa Correia
Olga Manuela Matos de Freitas
Rita Isabel Simões Ferreira
Salomé de Sousa Teixeira
Valentina Maria Fernandes Rodrigues
Maria Elisa A. M. Fernandes Soares
Eulalia Maria Bernardino Mendes
Maria Isabel Afonso da Rocha
Maria Isabel Almeida Cardoso
Carla Rodrigues
Cecília Bonifácio
João Fraga
Nelson Brito
Rui Viegas
Maria Aurora Soares da Silva
Sérgio Cruz Monteiro de Morais
*
Professora Adjunta
Professora Adjunta
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assessora Principal
Assessora
Assessora
Assessora
Téc. Sup. 2.ª Classe
Téc. Informática
Téc. Informática
Encarregada de trabalhos
Encarregado de trabalhos
IPVC
ISEP
ISEP
FCUP
FFUP
FCNAUP
FFUP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
ISEP
IPVC
ISEP
ISEP
ISEP
FFUP
FFUP
FCUP
FFUP
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
ISEP
ISEP
Members that joined REQUIMTE in December 2003
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 Ferbando Pessoa
FFUL
FEUP
ICBAS-UP
Universidade de Lisboa - Faculdade de Farmácia
Universidade do Porto - Faculdade de Engenharia
Universidade do Porto - Inst. Ciências Biomédicas
IPVC
Inst. Politécnico de Viana do Castelo
ISCSS
Instituto Superior de Ciências de Saúde (Sul)
U. Lusófona Universidade Lusófona
Química Verde
A - 7
B
POST-DOCTORAL FELLOWS
Química Verde
A - 8
Ana Rosa Silva
Anders Thapper
Carla Sousa
Dirk Boer
Elsa Henriques
Françoise Auchère
Iwona Biernacka
João Miguel Dias
José Trincão
Krasimira Petrova
Lalit Sharma
Maria Adília Lemos
Maria Gabriela Almeida
Marta Isabel Machado da Silva
PauloLemos
Pankas Das
Pavel Izak
Pedro Rodrigues
Quingyou Zhang
Rakesh Kumar
Roeland Boer
Rui Duarte
Shuwen Yao
Snezhana Bakalova
Smilja Todorovic
Sofia Pauleta
Sunil Gupta
Susan Matthews
Svetlana Lyubchik
Svetlozar Velizarov
Teresa Ribeiro
Thierry Michaud
Thomas Schäfer
Vanya Bogdanova Kurteva
Yuri Binev
Química Verde
A - 9
C
Ph. D. STUDENTS
Química Verde
A - 10
Akapong Suwattanamala
Ana Cláudia Barreira Nunes
Andrea Carneiro
Andreia Filipa Curto
António Rodrigo
Branca Maria Cardoso Monteiro da Silva
Bruno Mendes
Carina Madalena Martins Machado
Carla Portugal
Carla Ferreira
Carmen Pinheiro
Célia Peres
Christophe Siquet
Cristina Cordas
Cristina Correia
Cristina Manuela Pinto Vieira Lopes
Cristina Matos
Daniel Antunes dos Santos
David de Andrade Sousa Costa
Emilia Maria Gonçalves Santos
Eugénia Nogueiro
Francisco SIlva
Helena Carmo
Inês Cabrito
Isabel Cristina Timóteo
Isabel Esteves
Joana Brito
João Adalberto Amaral Lourenço
João Miguel Reis de Aguiar Navarro y Rosa
Jorge Alexandre S. Pereira
Jorge Dias
Jorge Rebelo
José Eduardo dos Santos Félix Castanheiro
José Esperança
José Luis dos Santos
José Ricardo Carneiro
Luís Magalhães
Luís Alexandre Fernandes Cobra Branco
Luis Mayor Lopez
Luisa Serafim
Magda Basto
Manuela Frasco
Márcia Cláudia Dias Carvalho
Marco Preto
Margarida Moncada
Maria de Fátima Assunção Lucas
Maria Filomena Freitas
Maria Gabriela Rivas
Maria Luisa Soares Silva
Maria Teresa Alves
Maria Teresa Viciosa Plaza
Mariana Duarte
Mário Gomes
Marlene Susana Dionísio Lucio
Marta Andrade
Marta Corvo
Marta Morais Saraiva de Andrade
Miguel Faria
Nestor Aracama
Nuno Cerqueira
Nuno Milhazes
Nuno Mateus
Nuno Lourenço
Olga Gavel
Patrícia Neves
Patricia Oliveira
Patrícia Raleiras
Paula Cristina de Almeida Jerónimo
Paula Garcia
Paulo Glória
Pedro Manuel da Cunha Catalão Pires dos
Santos
Pedro Miguel Pimenta Gois
Pedro Vidinha
Raquel Fortunato
Rita Noronha
Rui Costa
Rui Ruivo
Rute Fonseca
Sara Cristina Silva Cunha
Sérgio Alves
Sérgio Sousa
Sílvia Garcia
Sofia A. Costa Lima
Sofia Gomes
Susana Gaudêncio
Teresa Alves
Teresa Casimiro
Teresa Maria de Jesus Teixeira
Teresa Santos Silva
Vasco Bonifácio
Victor Alves
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
Luis Neto
Maria Manuela Mendes Pinto
Maria Virgínia Gomes Mota
Mário Cardoso
Marta Sofia Pires
Marta F. Resende
Paula A. C. Rodrigues
Sergio Teixeira
Susana M: F. de M. Abreu
Ana Coelho
Ana Isabel Farinha
Ana Vinha
Angélica Alves da Silva
Carla Beatriz Rodrigues Veiros
Joana Maria Fernandes Meireles
Luís Carlos Matos
Luis Miguel do Carmo
Other Research Students
Alexandra Teresa Pires Carvalho
Alexandre Rodrigues Faria de Carvalho
Ana Claúdia Vaz Gonçalves
Ana Isabel Alves Pinto Lopes da Silva
Ana Sofia Soares Pinto
Carina Madalena Martins Machado
Celina Maria Lemos dos Santos
Duarte Paulo Martins Torres
João Paulo Martins Ferreira Lavrado
Jorge da Silva Dias
Marcio Milton Nunes Temtem
Maria Adelaide Carvalho Miranda
Maria de Fátima Assunção Lucas
Maria João Lobo Loureiro de Amorim
Mário Joaquim dos Santos Cardoso
Nuno Manuel F.S. de Azevedo Cerqueira
Olga Manuela Oliveira Nunes de Carvalho
Oriza Paula Guedes Tavares
Paula Alexandra Carvalho Rodrigues
Sara Raquel Rodrigues Santos Madaíl
Susana Rodrigues Pinto
Valdemar Jorge Borges da Costa Figueira
Ana Barreiros
Ana Cristina Vinha
Ana Maria de Carvalhais Mendes Gomes
Anabela Ferreira Gomes
Andreia Daniela Leite
António Manuel Esteves Nunes
Branca Sofia Teixeira
Carmen Maria dos Santos Fernandes de Sousa
Célia Clarisse Carnapete Alves Meneze
Cristina Manuela Pinto Vieira Lopes
Elsa Sofia Farinha Soares
Iva Costa
Joana Moutinho da Veiga Marcelino
Jorge Alexandre Simões Durães Pereira
Jorge Manuel Ferreira Lages
José Luís de Freitas Andrade
Laila Hassane Ribeiro
Lara Alexandra Martins Marques Magalhães
Luís Carlos Fernandes Neto
Luís Carlos Matos
Luís Miguel do Carmo Correia
Margarida Moncada
Maria Carlota Soares Martinez Veiga de Macedo
Soraia Patrícia Pinto Santos
Vítor Hugo da Costa Gomes Teixeira
Química Verde
A - 13
E
Profiles of the new researchers
contracted under the Associate
Laboratory Program
Química Verde
A - 14
Dr. Ana Luísa Carvalho joined the ReQuimTe - Laboratório Associado in November 2003, as Assistant
Researcher in Protein Crystallography. She graduated in Applied Chemistry in 1995 in Faculdade de Ciências
e Tecnologia, Universidade Nova de Lisboa, and in 1998, she was awarded the Masters degree in Biophysics
from the Faculdade de Ciências (Universidade de Lisboa). In November 2002, Ana Luísa Carvalho was awarded
the Ph.D. degree in Protein Crystallography in Faculdade de Ciências e Tecnologia.
As a protein crystallographer, Ana Luísa Carvalho worked in close collaboration with several research
groups in Europe. In several interships, she joined the groups of Prof. Robert Huber of the Max-Planck Institüt
für Biochemie in Germany, Dr. Antonio Romero of the Centro de Investigaciones Biologicas in Madrid, and Dr.
Juan Calvete of the Instituto de Biomedicina de Valencia (CSIC, Spain). Her research interests include the
structural analysis of macromolecules involved in diverse biological processes, and recently her research is
focused in understanding the structure-function relationship of several modules from the Cellulosome, a
megaDalton complex involved in cellulose degradation.
As a collaborator of the Protein Crystallography Group of the CQFB, Dr. Ana Luísa Carvalho is also
dedicated to the co-organization and teaching of the Monographic Courses and is a member of the Portuguese
Block Allocation Group, from the European Synchrotron Radiation Facility.
Dr. Carlos Lodeiro Espiño is a Spanish citizen, born in 1966 in Caracas, Venezuela, and obtained
his MsC degree in Chemistry in 1995 and a doctorate in Inorganic Chemistry in 1999 from the University of
Santiago de Compostela, Spain, with the Highest Honors (Sobresaliente Cum laude), working in synthesis
and characterization of several new macrocyclic compounds and its metal complexes, specially with
lanthanide(III) ions.
In September 1999 he took a postdoctoral position in the Photochemistry and Supramolecular Chemistry
group of the Chemistry Department in the New University of Lisbon, Portugal. Since October 2000 until
December 2003 he was supported by a prestigious Research Postdoctoral Fellowship of the European
Community, integrated in the European Network “Molecular Level Devices and Machines”.
He has a large experience in developing new Synthesis of Organic and Inorganic Supramolecular devices.
At the end of 2003 Dr. C. Lodeiro joined the Requimte-Laboratório Associado-Centro de Química Fina e
Biotecnologia where he is currently an Auxiliary Research in Photochemistry and Supramolecular systems.
He is involved in several projects and he is author/co-author of more than 34 full articles and 65 communications.
His scientific interests are: Synthesis of Supramolecular Devices, Fluorescent Chemosensors, Photochemistry
and Photophysics of Supramolecular systems, Lanthanide(III) Chemistry and Green synthetic methods.
Dr. Carlos D. Brondino was born in Santa Fe, Argentina, where he obtained a degree in Biochemistry
and a Ph. D. in Biological Sciences by Universidad Nacional del Litoral.
In 1996 he was awarded by the Argentinean Association of Physical Chemistry for his doctoral thesis work in
the field of the Physical Chemistry. He has produced original investigation in Bioinorganic, Biophysics,
Biochemistry, and Solid State Physical-Chemistry. He is author/co-author of about 30 articles in peer reviewed
publications and two review papers.
The present research/professional activities are concerned with the role of transition metals ions in
biology (molybdenum, tungsten, nickel, iron, copper, etc), inorganic systems as models for biological
compounds, and application of magnetic resonance techniques such as Electronic Paramagnetic Resonance
(EPR) applied to the study of biological compounds.
Dr. Svetlozar Velizarov was born on October 16, 1962 in Plovdiv, Bulgaria. He received his Ph.D.
degree in Chemistry from the “M.V. Lomonossov” Moscow State University, Moscow, Russia in 1993. From
1993 to 1999, he was a Professor in Chemical and Biochemical Engineering in the Institute of Chemical
Engineering at the Bulgarian Academy of Sciences in Sofia. During that period, he performed an 18 months’
post-doctoral fellowship at the Institute for Molecular Biotechnology in Jena, Germany and later was an
Invited Scientist for 4 months at the University of Aarhus in Denmark. In 1999, he accepted the invitation of
Prof. J. Crespo to join the Biochemical Engineering and Separation Processes Group in the Department of
Chemistry of the New University of Lisbon (UNL). In the educational years 2002/03 and 2003/04, Dr. Velizarov
was an Invited Professor at the same Department teaching undergraduate courses in “Mass Transfer” and
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“Separation Processes” (Diploma in Chemical Engineering) and in “Food Processing” (Diploma in Food
Technology and Safety). From the beginning of 2004, he is an Investigator in the Associated Laboratory REQUIMTE, hosted by the Department of Chemistry at UNL. His current research interests are focused on
membrane separation processes, especially nanofiltration, and novel membrane bioreactors for removal of
emerging micropollutants from drinking water. Dr. Velizarov has been a Coordinator and/or a Member of a
number of Research Projects. He is currently a co-supervisor of two PhD students and several Diploma and
Erasmus students. He has published 27 articles and has given 23 oral and 35 poster presentations at
scientific conferences.
Dr. Loïc Hilliou studied the physico-chemistry of molecular and macromolecular materials at the
Université Louis Pasteur of Strasbourg (France). The topic of his PhD thesis (1996) was the design of a microrheometer to study the mechanical properties (at the micron scale) of some liquid crystalline polymers and
elastomers (now called model artificial muscles). From 1997 to 1999, he stayed in the group of Prof. G. Fytas
at the FO.R.T.H. (Heraklion, Crete) where he developed a rheo-optical technique to probe the structure of
complex fluids undergoing shear. From 1999 to 2001 he was a post-doc fellow at the Universidade Nova de
Lisboa, where he worked with Prof. A. Farinha Martins using rheo-NMR: a technique that couples rheology
and NMR tools to elucidate the nano-scale and macro-scale structure of flowing materials. He then was hired
by TotalFinaElf company and BASF-AG company as a post-doc scientist and consultant for the transfer of
technologies from the Max Planck Institute for Polymer Research in Mainz, Germany. Since December 2003,
he serves as an Associate Researcher at REQUIMTE. His current research activities include the study of the
interplay between the structure and the flow properties of biodegradable polymers synthesized from Portuguese
industrial wastes, which could be potential candidates for edible films technology.
Dr. Agostinho Pereira completed his degree in Biology at the University of Porto in 1994. Three years
later obtained a Master in Science, within the field of Animal Genetics, at the same University. In january of
2000 he got a PhD in Biology at the University of Porto and in collaboration with the INRA, in Paris under the
scope of Molecular Genetics and Evolution. In February of that same year started as a Postdoctoral Research
Fellow at the Laboratory of Genomic Diversity from the National Cancer Institute, National Institute of Health,
USA, where he stayed till becoming an Auxiliary Investigator of REQUIMTE in 2004. His current research
interests center in the field of computational and comparative genomics, phylogenomics, population genomics
and evolution.The recent international research effort to determine the DNA sequence of the entire human
genome, as well as of several other genome organisms of biological interest opened new windows of knowledge
for Genomic Research. Despite the voluminous genome datasets generated, the recovering of information is
still surprisingly poor, as the ability to produce molecular genetic data is largely outstripping our ability to
analyze it — computationally or experimentally. The challenge to genomics now is to turn to functional tools,
most importantly bioinformatics (computational biology), allowing the simultaneous integration of genomic,
transcriptomic and proteomic data.Our main goal is to narrow down the bridge between Genomics and
Proteomics by obtaining conceptual bases and practical methods for detecting systemic functional behaviours
of the cell and the organism. A principal focus of our investigation concerns the interaction and evolution of
mammalian cellular protein coding genes operating in the immune system, retroviruses, and cancer onset;
and to determine the comparative mammalian genetic principles that participate in these processes.
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ANNEX II
PUBLICATIONS
Química Verde
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A
Scientific Articles
——
Book Chapters
——
Articles in Procedings
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Articles in Scientific Journals
1.
“Chiral Synthesis of N-Aryl Aziridines”, Aires-de-Sousa, J.; Prabhakar, S.; Lobo, A. M.; Rosa, A. M.;
Gomes, M. J. S.; Corvo, M. C. (in part); Williams, D. J.; White, A. J. P.; Tetrahedron Assymmetry, 2002,
12, 3349-3365.
2.
“Studies in 3-oxy-assisted 3-aza Cope rearrangements”, Gomes, M. J. S.; Sharma, L.; Prabhakar, S.;
Lobo, A. M.; Gloria, P. M. C.; Journal of the Chemical Society, Chemical Communications, 2002, 746747.
3.
“Supported Liquid Membranes using Ionic Liquids: study of transport mechanisms and stability”, Raquel
Fortunato, Luís; C. Branco; Carlos A. M. Afonso; M. A. Reis; João G. Crespo, Membranes News, 2002,
60, 35.
4.
“Zinc and cadmium complexes with an achiral symmetrical helicand. Crystal structure of an
enantiomerically pure (M)-Zn(II) monohelicates”, Manuel R. Bermejo; Miguel Vázquez; Jesús Sanmartín;
Ana M. García-Deibe; Matilde Fondo; Carlos Lodeiro, New J. Chem., 2002, 26 (10), 1365-1370
5.
“3,3-Sigmatropic Rearrangements Involving N—O Bond Cleavage of Enehydroxylamines Derivatives”,
Reis, L. V.; Lobo, A. M.; Prabhakar, S.; Duarte, M. P. European Journal of Organic Chemistry, 2003,
190-208.
6.
“A Short Synthesis of Staurosporinone (K-252c)”, Gaudêncio, S. P.; Santos, M. M. M.; Lobo, A. M.;
Prabhakar, S, Tetrahedron Letters, 2003, 44, 2577-2578.
7.
“Synthesis of Bioactive Indole Alkaloids”, Lobo, A. M.; Prabhakar, S.; Branco, P. S.; Cardoso, A. S.;
Gaudêncio, S. P. ; Marques, M. M. B.; Santos, M. M. M.; Santos, P. F. Acta Phytotherapeutica (Italia),
2003, 16-23.
8.
“SbCl5-wet Acetonitrile: a New System for Chemoselective O-Desilylation”, Glória, P. M. C.; Prabhakar;
S.; Lobo, A. M.; Gomes, M. J. S. (in part) Tetrahedron Letters, 2003, 8819-8821.
9.
“A Mechanistic Reinvestigation of tris(p-Bromophenyl)aminium Hexachloroantimonate Induced
Deketalisation”, Glória, P. M. C.; Prabhakar, S.; Lobo, A. M.; Bulletin of the Korean Chemical Society,
2003, 1841-1842.
10.
“Three New Jatrophane-Type Diterpenes from Euphorbia pubescens”, Claudia Valente, Maria José U.
Ferreira, Pedro M. Abreu, Madalena Pedro, Fátima Cerqueira, Maria São José Nascimento, Planta
Medica, 2003, 69, 361-366.
11.
“Natural product-like combinatorial libraries”, Pedro M Abreu and Paula S. Branco, J. Braz. Chem.
Soc., 2003, 14 (5), 675-712.
12.
“Differentiation of Isomeric C8-Substituted Alkylaniline Adducts of Guanine by Electrospray Ionization
and Tandem Quadropole Ion Trap Mass Spectrometry”, L. Li, P. S. Branco; A. M. Antunes; M. Matilde
Marques; L. L. Gonçalves, F; A. Beland; M. P. Chiarelli, J. Am. Soc. Mass Spectrometry, 2003, 14
(12), 1488-1492.
13.
“Representation of DNA sequences with virtual potentials (SEQREP) and their processing by Kohonen
self-organizing maps”, J. Aires-de-Sousa; L. Aires-de-Sousa, Bioinformatics, 2003, 19 (1), 30-36.
14.
“Matrix-isolation FTIR study of azidoacetone and azidoacetonitrile”, Marcin Frankowski; Manuel Algarra;
Paula Rodrigues; Maria T. Barros; M. Natália D.S. Cordeiro; Brigitte S. Fox; Alice M. Smith-Gicklhorn;
Martin K. Beyer; Maria L. Costa; Vladimir E. Bondybey, Fizika Nizkikh Temperatur, 2003, 29 (9/10).
15.
“Mass Spectrometric Study of a-carbonyl Azides”, M. Filomena Duarte; Filipa Martins; M.Tereza
Fernandez; G. John Langley; Paula Rodrigues; M. Teresa Barros; M. Lourdes Costa, Rapid
Communications in Mass Spectrometry ,2003, 17, 957-962.
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16.
“Trimethylsilyl-substituted ligands as solubilizers of metal complexes in supercritical carbon dioxide”.
F. Montilla; V. Rosa; C. Prevett; T. Avilés; M. Nunes da Ponte; D. Masi; C. Mealli, Journal of the
Chemical Society, Dalton Trans., 2003, 2170.
17.
“Studies on the Preparation of 4-Ethoxyalkyliden and 4-Aminoalkyliden-5(4H)-oxazolones”, Marta R. P.
Norton Matos; Pedro M. P. Gois; Maria L. E. N. Mata; Eurico J. Cabrita; Carlos A. M. Afonso; Synthetic
Comm., 2003, 33,1285-1299.
18.
“Ionic Liquid as an Efficient Promoting Media for Two-Phase Nucleophilic Displacement Reactions”;
Nuno M. T. Lourenço; Carlos A. M. Afonso, Tetrahedron, 2003, 59, 789-794.
19.
“Glass Transition Relaxation and Fragility in Two Room Temperature Ionic Liquids”, Joaquim J. Moura
Ramos; Carlos A. M. Afonso; Luís C. Branco J. of Thermal Analysis and Calorimetry, 2003, 71, 659666.
20.
“Synthesis and Properties of tetra-alkyl-guanidinium Salts as a Potential New Generation of Ionic
Liquids”, Nuno M. M. Mateus; Luís C. Branco; Nuno M. T. Lourenço; Carlos A. M. Afonso, Green
Chem. 2003, 5, 347-352.
21.
“Dirhodium(II)-catalysed C-H Insertion on a-Diazo-a-phosphono-acetamides in an Ionic Liquid”, Pedro
M. P. Gois; Carlos A. M. Afonso; Tetrahedron Letters 2003, 44, 6571-6573.
22.
“Regio- and Stereoselective Dirhodium(II)-catalysed C-H Insertion on a-Diazo-a-phosphono-acetamides
and Acetates, Pedro M. P. Gois; Carlos A. M. Afonso, Europ. J. Org. Chem. 2003, 3798-3810.
23.
“Transient Spectroscopy of Ninhydrin”, Leinman, M.H.; Telo, J.P.; Vieira, A.J.S.C.; Bhone, C.; NettoFerreira, I.C., Photochem. Photobiol. 2003, 77 (1), 10
24.
“Different multidimensional chromatographic approaches applied to the study of wine malolactic
fermentation”, L. Fernandes; A.M. Relva; M.D.R. Gomes da Silva; A.M. Costa Freitas, J. Chromatogr.
A, 2003, 995, 161-169.
25.
“Water Activity Effects on Geranyl acetate Synthesis Cathalized by Novozym in Supercritical Ethane
and in Supercritical Carbon Dioxide”, Celia Peres; Marco D.R. Gomes da Silva; S. Barreiros, J. Agric.
and Food Chemistry, 2003, 51 (7), 1884-1888.
26.
“Correlating natural ageing and xenon irradiation of Paraloid® B72 applied on stone”, S. Bracci; M. J.
Melo; Polym. Degrad. Stab., 2003, 80, 533-541.
27.
“Multistate/multifunctional systems. A thermodynamic, kinetic and Photochemical Investigation on the
4’-Dimethylaminoflavylium Compound”, A. Roque; C. Lodeiro; F. Pina; M. Maestri; S. Dumas; P.
Passaniti; V. Balzani, J. Am.Chem. Soc. 2003, 125, 987-994.
28.
“Intramolecular Excimer Formation in a Tripodal Polyamine Receptor Containing Three Naphthalene
Fluorophores”, M. T. Albelda; E. Garcia-España; L. Gil; J. C. Lima; C. Lodeiro; J. S. de Melo; M. J.
Melo; A. J. Parola; F. Pina; C. Soriano, J. Phys. Chem. B. 2003, 107, 6573-6578.
29.
“Complexation of Aluminum (III) by Anthocyanins and Synthetic Flavylium Salts. A Source of Blue and
Purple Color,” M. C. Moncada; S. Moura; M. J. Melo; A. Roque; C. Lodeiro; F. Pina, Inorg. Chim. Acta,
2003, 356, 51-61.
30.
“Supramolecular Interactions of Hexacyanocobaltate(III) with Polyamine Receptors Containing a Terminal
Anthracene Sensor”, L. Rodríguez; S. Alves; J. C. Lima; A. J. Parola; F. Pina; C. Soriano; M. T.
Albelda; E. Garcia-España, J. Photochem. Photobiology, A: Chem., 2003, 159/3, 251-256
31.
“Cu(II) and Ni(II) Complexes with Dipyridine-Containing Macrocyclic Polyamines with Different Binding
units”, C. Anda; C. Bazzicalupi; A. Bencini; A. Bianchi; P. Fornasari; C. Giorni; B. Valtancoli; C.
Lodeiro; A. J. Parola; F. Pina, Dalton Trans., 2003, 1299-1307
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32.
“Linear ditopic acetylide gold or mercury complexes: synthesis, photophysic studies and potential use
as building blocks for molecular polygons”, M. Ferrer; L. Rodríguez; O. Rossell; F. Pina; M. F. Bardia;
X. Solans, J. Organomet. Chem. 2003, 678, 82-89
33.
“Potentiometric, NMR and Fluorescence Emission Studies on the Binding of ATP by Open- Chain
Polyamine Receptors containing Naphthyl and/or Anthrarylmethyl Groups”, M. T. Albelda; J. Aguillar;
S. Alves; R. Aucejo; P. Diaz; C. Lodeiro; J. C. Lima; E. Garcia-España; F. Pina; C. Soriano, Helv.
Chim. Acta, 2003, 86(9), 3118-3135.
34.
“The Dynamics of Ultra-fast Excited State Proton Transfer in Anionic Micelles”, L. Giestas; C. Yihwa;
J. C. Lima; C. Vautier-Giongo; A. Lopes; F. H. Quina; A. L. Maçanita, J. Phys. Chem., A 2003, 107,
3263-3269.
35.
“Ground and Excited Proton Transfer in Anthocyanins. From Weak Acids to Super-Photoacids”, Paulo
F. Moreira Jr.; Leticia Giestas; Chang Yihwa; Carolina Vautier-Giongo; Frank H. Quina; Antonio L.
Maçanita and João C. Lima, J. Phys. Chem.A. 2003, 107, 4203-4210
36.
“Linear ditopic acetylide gold or mercury complexes: synthesis and photophysic studies. X-ray crystal
structure of PPh4[Au(CºCC5H4N)2]”, Montserrat Ferrer; Laura Rodríguez; Oriol Rossell; Fernando
Pina; João C. Lima; Mercè Font Bardia; Xavier Solans, J. Organomet. Chem. 2003, 678, 82-89
37.
“Metal Complexes with a New N4O3 Scorpionand Macrocyclic Ligand. Synthesis, Characterization,
Crystal Structures and Fluorescence Studies”, M. Vicente; R. Bastida; C. Lodeiro; A. Macías; A. J.
Parola; L. Valencia; S. E. Spey, Inorg. Chem., 2003, 42(21), 6768-6779.
38.
“Co-ordination chemistry of Copper(II) with oxo-aza macrocyclic ligands. Crystal structure of a dinuclear
tetramer copper(II) complex”, Carlos Lodeiro; Rufina Bastida; Emilia Bértolo; Alejandro Macías and
Adolfo Rodríguez, Polyhedron, 2003, 22, 1701-1710.
39.
“Synthesis and characterization of four novel NxOy-Schiff-Base macrocyclic ligands and their metal
complexes”, Carlos Lodeiro; Rufina Bastida; Emilia Bértolo; Alejandro Macias; Adolfo Rodríguez, Trans.
Metal Chem., 2003, 28, 388-394.
40.
“Lanthanide(III) Coordination Compounds with Pyridine Head Macrocyclic Ligands”, Emilia Bértolo;
Rufina Bastida; David E. Fenton; Carlos Lodeiro; Alejandro Macias; Adolfo Rodríguez, J. Inclu. Phen.
Macro. Chem., 2003, 45 (1-2): 155-160,
41.
“Metal complexes with four macrocyclic ligands derived from 2,6-bis(2-formylphenoxymethyl)pyridine
and 1,7-bis(2’-formylphenyl)-1,4,7-trioxaheptane”, Carlos Lodeiro; Rufina Bastida; Emilia Bértolo; Alejandro
Macías; Adolfo Rodríguez; Inorg. Chim. Acta, 2003, 343, 133-140.
42.
“An Expedient Synthesis of Cationic Rhodamine Fluorescent Probes Suitable for Conjugation to Amino
Acids and Peptides”, Carlos A. M. Afonso; V. Santhakumar; Alan Lough, Robert A. Batey, Synthesis
2003, 2647-2654.
43.
“Aziridines as a Protecting and Directing Group. Stereoselective Synthesis of (+)-Bromoxone”, M. T.
Barros; P. M. Matias; C. D. Maycock; M. R. Ventura; Org. Letters, 2003, 5, 4321-4323
44.
“The Total Enantioselective Synthesis of (+)-Nephrosteramic Acid” M. T. Barros; C. D. Maycock; M. R.
Ventura; Org. Letters, 2003, 5, 4097-4099
45.
“Energetics and Dynamics of Naphthalene Polyaminic Derivatives. Influence of Structural Design in
the Balance Static vs. Dynamic Excimer Formation”, J. Seixas de Melo; J. Pina, F. Pina; C. Lodeiro; A.
J. Parola; J. C. Lima; M. Teresa Albelda; M. Paz Clares; Enrique Garcia-España and Conxa Soriano.
J. Phys. Chem. A, 2003, 107, 11307-11318.
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46.
“Salt hydrates for in-situ water activity control have acid-base effects on enzymes in nonaqueous
media”, N. Fontes; N. Harper; P.J. Halling; S. Barreiros Biotechnol. Bioeng., 2003, Vol. 82, 802-808.
47.
“Water activity effects on geranyl acetate synthesis catalyzed by Novozym in supercritical ethane and
in supercritical carbon dioxide”, C. Peres; M.D.R. Gomes da Silva; S. Barreiros, J. Agric. Food Chem.,
2003, Vol. 51, 1884-1888.
48.
“Control of enzyme ionization state in supercritical ethane by sodium/proton solid-state acid-base
buffers”, N. Fontes; P.J. Halling; S. Barreiros Enzyme Microb. Technol., 2003, Vol. 33, 938-941.
49.
“Removal of Trace Mono-valent Inorganic Pollutants in an Ion Exchange Membrane Bioreactor: Analysis
of Transport Rate in a Denitrification Process”, S. Velizarov; M.A. Reis; J.G. Crespo J. Membrane Sci.,
2003, Vol. 217, 269 - 284
50.
“Water Treatment Using the New Concept of Ion Exchange Membrane Bioreactor”, S. Velizarov; M.A.
Reis; J.G. Crespo Polish J. Chem. Technol., 2003, Vol. 5, 40-43
51.
“Comparison of Various Models for Transport of Binary Mixtures through Dense Polymer Membrane”, P.
Izák; L.Bartovská; K. Friess; M. Šípek; P. Uchytil Journal of Polymer, 2003, vol.44, 2679-2687
52.
“Description of Binary Liquid Mixtures Transport through Non-porous Membrane by Modified MaxwellStefan Equations”, P. Izák, L.Bartovská, K. Friess, M. Šípek, P. Uchytil Journal of Membrane Science,
2003, vol.214, 293-309
53.
“Mass Transport Phenomena during Pervaporation”, T. Schäfer; J. G. Crespo Polish Journal of Chemical
Technology, 2003, Vol. 6, 14-18
54.
“Metabolic Pathway for Propionate Utilisation by Phosphorus Accumulating Organisms in Activated
Sludge: 13C-Labelling and In vivo NMR”, P.C. Lemos; L.S. Serafim; M.M. Santos; M.A.M. Reis; H.Santos
Appl. Environ. Microbiol., 2003, Vol. 69, 241-251
55.
“Production of Polyhydroxyalkanoates by Mixed Microbial Cultures”, M.A.M. Reis; L.S. Serafim; P.C.
Lemos; A.M. Ramos; F.R. Aguiar; M.C.M. van Loosdrecht Bioproc. Biosys. Eng., 2003, Vol. 23, 377385.
56.
“Production if Polyhydroxyalkanoates by a Mixed Culture in a Sequencing Batch Reactor: the Use of
Propionate as Carbon Source”, P.C. Lemos; L.S. Serafim; H. Santos; M.A.M. Reis.
57.
Com. Agric. Appl. Biol. Sc., 2003, Vol. 68, 109-114.
58.
“Produção de Bioplásticos por Culturas Microbianas Mistas”, L.S. Serafim; P.C. Lemos; M.A.M Reis
Boletim da Sociedade Portuguesa de Biotecnologia, 2003, Vol. 76, 15-20.
59.
“Monitoring of Biofilm Reactors Using Natural Fluorescence Fingerprints”, C.G. Wolf.; J.G. Crespo;
M.A.M. Reis Water Science and Technology, 2003, Vol. 47, 161-167.
60.
“Hydrogen Metabolism Production in Desulfovibrio desulfuricans strain New Jersey NCIM8313Comparative Study with D. vulgaris and D. gigas Species”, M. Carepo; J. F. Baptista; A. Pamplona; G.
Fauque; J.J.G. Moura; M.A.M. Reis The Anaerobe , 2003, Vol.8, 325-332.
61.
“A Novel Pathway for Mineralization of the Thiocarbamate Herbicide Molinate by a Defined Bacterial
Consortium”, L. Barreiros; B. Nogales, C. M. Manaia; A. C. S. Ferreira; D. H. Pieper, M. A.M. Reis, O.
C.Nunes Environmental Microbiology , 2003, Vol.5(10), 944-953.
62.
“Enantioselective Hydrolysis of a meso-Diester Using Pig Liver Esterase in a Two Phase Stirred Tank
Reactor”, H. A. Sousa; C. A. M. Afonso; J. P. B. Mota; J.G. Crespo Ind. Eng. Chem. Res., 2003, Vol.
42, 5516-5525
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63.
“On the optimization of mixing protocol in a certain class of 3-D Stokes flows”, A. J. S. Rodrigo; J. P.
B. Mota; E. Saatdjian; A. Levefre Physics of Fluids, 2003, Vol. 15, 1505-1516
64.
“Chaotic advection in a 3-D Stokes flow”, A. J. S. Rodrigo; J. P. B. Mota; A. Lefevre; J.C. Leprévost; E.
Saatdjian AIChE J., 2003, Vol. 49, 2749-2758
65.
“Chaotic advection and heat transfer enhancement in Stokes flows”, A. Lefevre; J. P. B. Mota; A. J. S.
Rodrigo; E. Saatdjian Int. J. Heat Fluid Flow, 2003, Vol. 24, 310-321
66.
“Polymerisation of Pinenes Using Vanadium Supported on Activated Carbon”, A.C. Encarnação; A.
Flores; S.I. Mota; C. Palma; A.M. Ramos; J. Vital; I. Fonseca Catal. Today 2003, Vol. 78, 197-201
67.
“Adsorption of SO2 Using Vanadium and Vanadium-Copper Supported on Activated Carbon”, S. A.
Carabineiro; A.M. Ramos; J. Vital; J.M. Loureiro; J.J.M. Orfão; I. Fonseca Catal. Today 2003, Vol. 78,
203-210.
68.
“The acid-catalysed reaction of ?-pinene over molibdophosphoric acid immobilized in dense polymeric
membranes”, J.E. Castanheiro; A.M. Ramos; I. Fonseca; J. Vital Catal. Today 2003, Vol. 82, 187-193.
69.
“NO conversion using binary vanadium mixtures supported on activated carbon”, S. A. Carabineiro; F.
B. Fernandes; J. Vital; A. M. Ramos; I. Fonseca Appl. Catal. B-Environ., 2003, Vol. 44, Nr. 3, 227.
70.
“Activated carbons with immobilised manganese(III) salen complexes as heterogeneous catalysts in
the epoxidation of olefins: influence of support and ligand functionalisation on selectivity and reusability”,
A. R. Silva, C. Freire, B. de Castro, J. Vital, J. L. Figueiredo New J. Chem. 2003, Vol. 27, 1511-1517.
71.
“Oxidation of pinane over phthalocyanine complexes supported on activated carbon. Effect of the
support surface treatment”, A. Valente; C. Palma; I. Fonseca; A.M. Ramos, J. Vital Carbon 2003, Vol.
41, 2793-2803.
72.
“Ethylene polymerisation over transition metals supported catalysts; 2-chromium on zeolite, silica and
charcoal characterization and activity studies”, Y. Zhan; I. Matos, M. Lemos, F. Freire, T.G. Nunes ,
A.M. Botelho do Rego, R.T. Henriques, I. Fonseca, M.M. Marques, F. Lemos J. Polym. Sci., 2003,
Vol. 41, 3768.
73.
“Interactions of omeprazole and precursors with beta-cyclodextrin host molecules”, SS Braga, P RibeiroClaro, M Pillinger, IS Goncalves, AC Fernandes, F Pereira, CC Romao, PB Correia, JJC Teixeira-Dias
J. Incl. Phenom. Macro, 2003, Vol. 47, 47.
74.
“Encapsulation of sodium nimesulide and precursors in beta-cyclodextrin”, SS Braga, P Ribeiro-Claro,
M Pillinger, IS Goncalves, F Pereira, AC Fernandes, CC Romao, PB Correia, JJC Teixeira-Dias Org.
Biomol. Chem., 2003, Vol. 1, 873.
75.
“Supercritical CO2 – Induced Phase Changes in (Ionic Liquid + Water + Ethanol) Solutions. Application
to Biphasic Catalysis”, Vesna Najdanovic-Visak; Ana Serbanovic; José M. S. S. Esperança; Henrique
J. R. Guedes; Luís P. N. Rebelo; Manuel Nunes da Ponte, Chem. Phys Chem., 2003, 4, 520-522
76.
“Pressure, Isotope, and Water Co-solvent Effects in Liquid-Liquid Equilibria of (Ionic Liquid + Alcohol)
Systems” Vesna Najdanovic-Visak; José M. S. S. Esperança; Luís P. N. Rebelo; Manuel Nunes da
Ponte; Henrique J. R. Guedes; Kenneth R. Seddon; Hermínio C. de Sousa; Jerzy Szydlowski J. Phys.
Chem. B, 2003, 107, 12797-12807
77.
“An Apparatus for high-pressure VLE measurements using a static mixer. Results for
(CO2+limonene+Citral) and (Co2+limonene+linalool)”, J.Fonseca; P.C.Simões; M.Nunes da Ponte J.
Supercritical Fluids, 2003, 25, 7-17.
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78.
“Ca2+ and The Bacterial Peroxidases: The Cytochrome C Peroxidase From Pseudomonas Stutzeri”,
C.G.Timóteo; P.Tavares; C.F.Goodhew; L.C.Duarte, K.Jumel, F.M.F.Gírio, S.Harding, G.W.Pettigrew
and I.Moura, J.Biol.Inorg.Chem. 8, 29-37 (2003)
79.
“The NMR Solution Structures of two Mutants of Desulforedoxin”, B.J.Goodfellow, F.Rusnak, I.Moura,
C.S.Ascenso and J.J.G.Moura, J.Inorg.Biochem. 93, 100-108 (2003)
80.
“Formation of a Stable Cyano-Bridged Dinuclear Iron Cluster Following Oxidation of the Superoxide
Reductases from Treponema pallidum and Desulfovibrio vulgaris with K(3) Fe(CN)(6)”, F.Auchere;
P.Raleiras; L.Benson; S.Y.Venyaminov; P.Tavares; J.J.G.Moura; I.Moura and F.Rusnak Inorg.Chem.
42, 938-940 (2003)
81.
“Broad Temperature Range Spectroscopy of Two Centre Modular Redox Metalloprotein Desulfoferrodoxin”,
N.H.Andersen; S.E.Harnung; I.Trabjerg; I.Moura; J.J.G.Moura and J.Ulstrup, Dalton Transactions 2003
82.
“The Isolation And Characterization Of Cytochrome C Nitrite Reductase Subunits (Nrfa And Nrfh)”,
M.G.Almeida, S.Macieira, L.L.Goçalves, R.Huber, C.A.Cunha, M. J.Romão, C.Costa, J.Lampreia,
J.J.G.Moura, I.Moura Eur.J.Biochem. 270, 3904-3915 (2003)
83.
“Electron Transfer Complexes Of Cytochrome Cperoxidase From Paracoccus Denitrificans Containing
More Than One Cytochrome”, G.W.Pettigrew, S.R.Pauleta, C.F.Goodhew, A.Cooper, M.Nutley, K.Jumel,
S.E.Harding, C.Costa, L.Krippahl, I.Moura, J.J.G.Moura, Biochem. 42, 11968-11981 (2003)
84.
“N(2)O Reduction by the Fully Reduced ?-Sulfide Bridged Tetranuclear Cu(Z) Cluster in Nitrous Oxide
Reductase”, Ghosh S, Gorelsky SI, Chen P, Cabrito I, Moura JJ, Moura I, Solomon EI., J.Am. Chem.Soc.
125, 15708-15709 (2003)
85.
“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. 2003, in press.
86.
“Cytochrome C Nitrite Reductase From Desulfovibrio Desulfuricans ATCC 27774. Part I: The Relevance
Of The Two Calcium Sites In The Structure Of The Catalytic Subunit (Nrfa).”, C. A. Cunha; S. Macieira;
J. M. Dias; G. Almeida; L. L. Gonçalves; C. Costa; J. Lampreia; R. Huber; J. J. G. Moura; I. Moura; M.
J. Romão J. Biol. Chem., 2003, Vol. 278, 17455-17465
87.
“Mammalian Molybdo-Flavoenzymes, An Expanding Family Of Proteins: Structure, Genetics, Regulation,
Function And Patho-Physiology”, E. Garattini; R. Mendel; M. J. Romão, R. Wright; M. Terao Biochem.J.,
2003, Vol. 372, 15-32
88.
“Crystallization And Preliminary X-Ray Diffraction Analysis Of The Di-Haem Cytochrome C Peroxidase
From Pseudomonas Stutzeri”, C. Bonifácio; C.A. Cunha; A. Müller; C.G. Timóteo; J. M. Dias; I. Moura;
M.J. Romão Acta Crystallogr. D Biol. Crystallogr, 2003, Vol. 59, 345-347
89.
“A Positively Charged Amino Acid Is Required To Enable Electron Transport In Periplasmic Nitrate
Reductases”, T. Hettmann; R. A. Siddiqui; J. v. Langen; C. Frey; M. J. Romão; S. Diekmann Biochem
Biophys Res Commun. 2003, Vol. 310, 40-47
90.
“Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex”, A. L.
Carvalho; F. M. V. Dias; J. A. M. Prates; L. M. A. Ferreira; H. J. Gilbert; G. J. Davies; M. J. Romão; C.
M. G. A. Fontes, Proc. Natl. Acad. Sci. USA, 2003, Vol. 100, 13809-13814
91.
“A Practical Method for the Detection of Macroprolactinaemia using Ultrafiltration”, S. Prazeres; M.A.
Santos; H.G. Ferreira; L.G. Sobrinho Cli. Endocrinol. 2003 (Ox.) Vol. 58: 686.
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92.
“Involvement of reactive oxygen and nitrogen species in inflammatory processes. Implications for the
potential therapeutic use of NSAIDs with antioxidant activity” E. Fernandes; J.L.F.C. Lima; S. Reis
Current Topics in Medicinal Chemistry, 2003, Vol. 3, 221-236.
93.
“Trimipramine Determination in Pharmaceutical Preparations With an Automated Multicommutated
Reversed-Flow System” J.A.V. Prior; J.L.M. Santos; J.L.F.C. Lima J. Pharm. Biom. Anal., 2003, Vol.
33, 903-910.
94.
“Flow–Injection Amperometric Determination of Dopamine in Pharmaceuticals Using a Polyphenol
Oxidase Biosensor Obtained From Soursop Pulp” V.S. Bezerra; J.L.L. Filho; M.C.B.S.M. Montenegro;
A.N. Araújo; V.L. Silva J. Pharm. Biom. Anal., 2003, Vol. 33, 1025-1031.
95.
“Electroimmobilization of MAO Into a Polypyrrole Film and its Utilization for Amperometric Flow Detection
of Antidepressant Drugs” M.H. Vela; D.S. Jesus; C.M.C.M. Couto; A.N. Araújo; M.C.B.S.M. Montenegro
Electroanalysis., 2003, Vol. 15, 133–138.
96.
“Determination of Bopindolol Using the Flow Injection Technique Coupled with Solid Phase Extraction”
J. Meireles; H. SklenáYová; D. ‘atínský; P. Solich; A.N. Araújo; M.C.B.S.M. Montenegro J. Pharm.
Biom. Anal., 2003, Vol. 33, 1149-1153.
97.
“Amperometric Biosensor Based on Monoamine Oxidase (MAO) Immobilized in Sol-Gel Film for
Benzydamine Determination in Pharmaceuticals” D.S. Jesus; C.M.C.M. Couto; A.N. Araújo; M.C.B.S.M.
Montenegro J. Pharm. Biom. Anal., 2003, Vol. 33, 983–990.
98.
“Solid Phase Retention With Reversed Elution in a SIA System With Detection by Flame Atomic
Absorption Spectrometry” R.C.C. Costa; A.N. Araújo; M.C.B.S.M. Montenegro; M.F. Pimentel; V.L.
Silva Can. J. Anal. Sc. Spect., 2003, Vol. 48, 157.
99.
“Simple and Inexpensive Flow L-Glutamate Determination Using Pumpkin Tissue”, N.J.M. Arruda;
J.L.L. Filho; M.C.B.S.M. Montenegro; A.N. Araújo; V.L. Silva J. Agric. Food Chem., 2003, Vol. 51,
6945-6948.
100. “Flow Amperometric Determination of Pharmaceuticals With On-Line Electrode Surface Renewal”,
R.I.L. Catarino; A.C.L. Conceição; M.B.Q. Garcia; M.L.S. Gonçalves; J.L.F.C. Lima; M.M. C. Santos
J. Pharm. Biom. Anal., 2003, Vol. 33, 571-580.
101. “Multi-Pumping Flow System for Spectrophotometric Determination of Bromhexine”, A.C.B. Dias; J.L.M.
Santos; J.L.F.C. Lima; E.A.G. Zagatto Anal. Chim. Acta., 2003, Vol. 499, 107-113.
102. “Sequential Injection Analysis of Nitrites and Nitrates in Human Serum Using Nitrate Reductase”,
P.C.A.G. Pinto; J.L.F.C. Lima; M.L.M.F.S. Saraiva Clin. Chim. Acta., 2003, Vol. 337, 69-76.
103. “The Metabolism of Sulindac Enhances its Scavenging Activity Against Reactive Oxygen and Nitrogen
Species”, E. Fernandes; S.A. Toste; J.L.F.C. Lima; S. Reis Free Rad. Biol. Med., 2003, Vol. 35, 10081017.
104. “Application of a Potentiometric System With Data-Analysis Computer Programs to the Quantification
of Metal-Chelating Activity of Two Natural Antioxidants: Caffeic Acid and Ferulic Acid”, F. Borges;
J.L.F.C. Lima; I. Pinto; S. Reis; C. Siquet Helv. Chim. Acta., 2003, Vol. 86, 3081–3087.
105. “Partition and Location of Nimesulide in EPC Liposomes: a Spectrophotometric and Fluorescence
Study”, H. Ferreira; M. Lúcio; B. Castro; P. Gameiro; J.L.F.C. Lima; S. Reis Anal Bioanal Chem., 2003,
Vol. 377, 293-298.
106. “Multi-Pumping Flow System for the Spectrophotometric Determination of Dipyrone in Pharmaceutical
Preparations”, J.L.F.C. Lima; S.M.O. Sá; J.L.M. Santos; E.A.G.J. Zagatto J. Pharm. Biom. Anal.,
2003, Vol. 32, 1011-1017.
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107. “Determinação Potenciométrica em Fluxo de Cloreto de Cetilpiridinio em Desinfectantes Bucais”, P.C.S.
Batista; A.N. Araújo; M.C.B.S.M. Montenegro Química Nova., 2003, Vol. 26, 475-478.
108. “Determination of Dipyrone in Pharmaceutical Products by Flow Injection Analysis With Potentiometric
Detection”, J.S. Alburqueque; V.L. Silva; F. Lima; A.N. Araújo; M.C.B.S.M. Montenegro Anal. Sci.,
2003, Vol. 19, 691-694.
109. “Ion-Selective Electrodes Based on Metalloporphyrins for Gibberellic Acid Determination in Agricultural
Products”, E.M.G. Santos; C.M.C.M. Couto; M.C.B.S.M. Montenegro; M.G.P.M.S. Neves; S.L.H.
Rebelo; J.A.S Cavaleiro; B.F. Reis Anal. Bioanal. Chem., 2003, Vol. 375, 511-516.
110.
“Sequential Injection Analysis of Free and Total Potassium in Wines Using Potentiometric Detection
and Microwave Digestion”, N. Zárate; A.N. Araújo; M.C.B.S.M. Montenegro; R.P. Olmos Am.J.Enol.
Vitic., 2003, Vol. 54, 46-49.
111.
“Monosegemented Flow Potentiometric Titration for the Determination of Chloride in Milk and Wine”,
J.A. Vieira; I.M.R. Jr.; B.F. Reis; M.C.B.S.M. Montenegro; A.N. Araújo J. Braz. Chem.Soc., 2003, Vol.
14, 259–264.
112.
“Determination of Hydrogen Peroxide by Near Infrared Spectroscopy”, A.M. Pimenta, S.H.F. Scafri; C.
Pasquini; I.M.R. Jr.; J.J.R. Rohwedder; M.C.B.S.M. Montenegro; A.N. Araújo J. Near Infrared Spect.,
2003, Vol. 11, 49-53.
113.
“An Automatic Flow Procedure for the Determination of 3-Hidroxybutyrate in Animal Serum and Plasma”,
C.K. Pires; P.B. Martelli; B.F. Reis; J.L.F.C. Lima; M.L.M.F.S Saraiva J. Agric. Food Chem., 2003, Vol.
51, 2457-2460.
114.
“Sampling Strategies Exploiting Multi-Pumping Flow Systems”, J.A.V. Prior; J.L.M. Santos; J.L.F.C.
Lima Anal Bioanal Chem., 2003, Vol. 375, 1234-1239.
115.
“Development of a Sequential Injection Analysis System for the Simultaneous Biosensing of Glucose
and Ethanol in Bioreactor Fermentation”, R.A.S. Lapa; J.L.F.C. Lima; I.V.O.S. Pinto Food Chem.,
2003, Vol. 81, 141–146.
116.
“Electrochemical and Spectroscopic Studies of the Oxidation Mechanism of the Herbicide Propanil”,
E.M. Garrido; J.L.F.C. Lima; C.D. Matos; F. Borges; A.M.S. Silva; J.A.P. Piedade; A.M.O. Brett J.
Agric. Food Chem., 2003, Vol. 51, 876-879
117.
“Hydroxyl Radical and Hypochlorous Acid Scavenging Activity of Small Centaury (Centaurium erythraea)
Infusion. A Comparative Study with Green Tea (Camellia sinensis)”, P. Valentão; E. Fernandes; F.
Carvalho; P. B. Andrade; R. M. Seabra; M.L. Bastos Phytomedicine, 2003, Vol. 10, 517-522
118.
“Variability in Phenolic composition of Hypericum androsaemum”, P. Valentão; A. Dias; M. Ferreira; B.
Silva; P.B. Andrade; M.L. Bastos; R.M. Seabra Natural Products Research, 2003, Vol.17, 135-140
119.
“Solid-phase Microextraction of Volatile Compounds in “Terrincho”, Ewe Cheese. Comparison of Different
Fibers” O. Pinho; C. Pérès; I.M.P.L.V.O. Ferreira. J. Chromatography A., 2003, Vol. 1011, 1-9.
120. “Quantification of Non-Protein Nitrogen Compounds of Infant Formulae and Follow-up Milks: Comparison
with Cow’s and Human Milk” I.M.P.L.V.O. Ferreira. British Journal of Nutrition, 2003, Vol. 90, 127-133.
121. “Optimization of Extraction Procedures for Analysis of Benzoic and Sorbic acids in Foodstuffs” F. J.
M. Mota; I.M.P.L.V.O. Ferreira; S.C. Cunha; M. B. P.P. Oliveira Food Chemistry, 2003, Vol. 82, 469473.
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122. “Contribution of FA Profile Obtained by High-Resolution GC/Chemometric Techniques to the Authenticity
of Green and Roasted Coffee Varieties”, M. R. Alves; S. Casal; M.B.P.P. Oliveira; M.A. Ferreira J.
AOCS, 2003, Vol 80(6), 511-517.
123. “Detection and Quantification of Bovine, Ovine, and Caprine Milk Percentages in Protected Denomination
of Origin Cheeses by Reversed-phase High-Performance Liquid Chromatography of Beta-Globulins”, I.
M.P.L.V.O. Ferreira; Helena Caçote J. Chromatography A., 2003, Vol. 1015, 111-118.
124. “Discrimination Between Arabica and Robusta Coffee Species on the Basis of Their Amino Acid
Enantiomers”, S.Casal; M. R. Alves; E. Mendes; M. B. P.P. Oliveira, M.A. Ferreira J.Agric. Food
Chem., 2003, Vol. 51, 6495-6501.
125. “Determination of Caseinomacropeptide by an RP-HPLC Method and Monitoring of the Addition of
Rennet Whey to Powdered milk”, I. M.P.L.V.O. Ferreira; M.B.P.P.Oliveira J. Liq. Chrom. & Rel. Technol.,
2003, Vol 26(1), 99-107.
126. “Quantification and Variability of Conjugated Linoleic Acid Levels in Sheep Milk of Two Autochthonous
Portuguese Breeds”, E. Barbosa; C. Oliveira; S. Casal; L. Soares; A.P. Vale; J.C. Lopes; B. Oliveira;
N.V. Brito Electronic Journal of Environmental, Agricultural and Food Chemistry, 2003, 2 (4).
127. “Determination of Sterol and Fatty Acid Compositions, Oxidative Stability, and Nutritional Value of Six
Walnut (Juglans regia L.) Cultivars Grown in Portugal”, J. S. Amaral; S. Casal; J. A. Pereira, R. M.
Seabra; M. B. P.P. Oliveira J.Agric. Food Chem., 2003, Vol. 51, 7698-7702.
128. “Development and Evaluation of a GC/FID Method for the Analysis of Free Amino Acids in Quince Fruit
and Jam”, B. M. Silva; S. Casal; P.B. Andrade; R.M. Seabra; M.B.P.P. Oliveira; M.A. Ferreira Analytical
Sciences, 2003, Vol. 19, 1285-1290.
129. “Interpolative Biplots Applied to Principal Component Analysis and Canonical Analysis”, M. R. Alves,
M. B. Oliveira Journal of Chemometrics, 2003, Vol. 17, 1-9.
130. “Quantification of Short-chain Free Fatty Acids in “Terrincho” Ewe Cheese: Intravarietal Comparison”,
O. Pinho; I.M.P.L.V.O. Ferreira; M.A Ferreira Journal of Dairy Science, 2003, Vol. 86, 3102-3109
131. “Heavy metals induce leakage of compounds with complexing properties in starved cells of
Saccharomyces cerevisiae: relationship to toxicity and bioavailability”, E.V. Soares; K. Hebbelinck;
H.M.V.M. Soares Can. J. Microbiol., 2003, Vol. 49, 336-343
132. “Challenges in modelling and optimisation of stability constants in the study of metal complexes with
monoprotonated ligands. Part I: a glass electrode potentiometric and DPP study of Cu-TAPSO-system”,
C.M.M. Machado; I. Cukrowski; P. Gameiro; H.M.V.M. Soares Analytica Chimica Acta, 2003, Vol. 493,
105-119
133. “Challenges in modelling and optimisation of stability constants in the study of metal complexes with
monoprotonated ligands. Part II. A glass electrode potentiometric and polarographic study of CuAMPSO-system”, C.M.M. Machado; I. Cukrowski; H.M.V.M. Soares, Helvetica Chimica Acta, 2003,
Vol. 86, 3288-3304.
134. “The enhancement of the cellulolytic activity of Cellobiohydrolase I and Endoglucanase by the addition
of Cellulose Binding Domains derived from Trichoderma reesei”, M.A. Lemos; J.A. Teixeira; M. Mota;
F.M. Gama Enzyme Microbial Technology, 2003, Vol. 32, 35-40.
135. “Thermal transitions of osmotically dehydrated tomato by modulated temperature differential scanning
calorimetry”, A. Baroni; A.M. Sereno; M.D. Hubinger Thermochimica Acta, 2003, Vol. 395, 237 – 249.
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136. “Evaluation of mass transfer coefficients and volumetric shrinkage during osmotic dehydration of apple
using sucrose solutions in static and non-static conditions”, R. Moreira; A.M. Sereno Journal of Food
Engineering, 2003, Vol. 57, 25 – 31.
137. “Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies”,
C. Rodrigues, P.Gameiro, M. Prieto, B. Castro Biochem. Biophys. Acta, 2003, 1620, 151-159.
138. “Chlomequat selective electrodes: construction, evaluation and application at FIA systems”, M. Goreti
F. Sales, Nuno F. M. C. Lino, Paula C. B. Paiga Intern. J. Environ. Anal. Chem., 2003, 83 (4), 295-305.
139. “Development of electrochemical methods for determination of tramadol - analytical application to
pharmaceutical dosage forms”, E. M. P. J. Garrido, J. M. P. J. Garrido, F. Borges, C. Delerue-Matos
Journal of Pharmaceutical and Biomedical Analysis, 2003, 32, 975-981.
140. “Sustainable use of resources and waste management in chemical laboratories”, Isabel Serra, Aurora
Silva, Sérgio Morais, M. Goreti Sales, Isabel Branco Martins EJEAFChe - Electron. J. Environ. Agric,
Food Chem, 2003, ISSN 1579-4377.
141. “Electrochemical determination of dihydrocodeine in pharmaceuticals”, J. M. P. J. Garrido, C. DelerueMatos, F. Borges, T. R. A. Macedo, A. M. Oliveira-Brett Analytical Letters, 2003, 36 (3), 577-590.
142. “Flow injection electrochemical determination of apomorphine”, J. M. P. J. Garrido, C. Delerue-Matos,
F. Borges, T. R. A. Macedo, A. M. Oliveira-Brett Analytical Letters, 2003, 36 (10), 2199-2210.
143. “Adsortive stripping voltammetric determination of venlafaxine in urine with a mercury film microelectrode”,
Simone Morais, Christine P. M. C. A. Ryckaert, Cristina Delerue-Matos Analytical Letters, 2003, 36
(11), 2515-2526.
144. “Amperometric and spectrophotometric determination of carbaryl in waters and commercial formulations”,
Dionísia C. Portela, Isabel M. F. Pereira, Paula Paíga, Cristina Delerue-Matos, M. Carmo V. F. Vaz
Anal. Bioanal. Chem., 2003, 377, 356-361.
145. “Construction and evaluation of cysteine selective electrodes for FIA analysis of pharmaceuticals”, M.
Goreti F. Sales, Annouschka Pille, Paula C. B. Paíga Analytical Letters, 2003, 36 (14), 2925-2940.
146. “Electrochemical and spectroscopic studies of the oxidation machanism of herbicide propanil”, E. M.
Garrido, J. L. F. C. Lima, C. Delarue-Matos, F. Borges, A. M. S. Silva, A. M. Oliveira-Brett J. Agric.
Food Chem., 2003, 51 (4), 876-879.
147.
“Synthesis and Analysis of Aminochromes by HPLC- Photodiode Array. Adrenochrome Evaluation in
Rat Blood”, F. Remião; N. Milhazes; F. Borges; F. Carvalho; M.L. Bastos; F. Lemos-Amado; P.
Domingues; A. Ferrer-Correia Biomedical Chromatography, 2003, Vol. 17, 6-13
148. “Hepatoprotective Activity of Polyhydroxylated 2-Styrylchromones Against tert-Butylhydroperoxide-Induced
Toxicity in Freshly Isolated Rat Hepatocytes”, E. Fernandes; M. Carvalho; F. Carvalho; A.M.S. Silva;
C.M.M. Santos; P.C.G.A. Pinto; J.A.S. Cavaleiro; M.L. Bastos Archives of Toxicology , 2003, Vol. 77,
500-505
149. “4-Methylthioamphetamine-Induced Hyperthermia in Mice: Influence of Serotonergic and
Catecholaminergic Pathways”, H. Carmo; F. Remião; F. Carvalho; D. de Boer; L.A. Reys; M.L. Bastos
Toxicology and Applied Pharmacology, 2003, Vol. 190, 262-271
150. “Use of Atropine-Treated Daphnia Magna Survival for Detection of Environmental Contamination by
Acethylcholinesterase Inhibitors”, F. Carvalho; I. Machado; M. Sánchez Martínez; A. Soares, L.
Guilhermino Ecotoxicology and Environmental Safety, 2003, Vol. 53, 43-46.
151. “The Toxicological Potential of Khat”, F. Carvalho Journal of Ethnopharmacology, 2003, Vol. 87, 1-2
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152. “Parametrization of Synthetic Amino Acids”, M. A. C. Preto; A. Melo; S. P. G. Costa; H. L. S. Maia; M.
J. Ramos J. Phys. Chem. B, 2003, Vol. 107, 14556-14562.
153. “Theoretical Studies On The Mode Of Inhibition Of Ribonucleotide Reductase By 2 ‘-Substituted
Substrate Analogues”, P. A. Fernandes; M. J. Ramos Chem. Eur. J., 2003, Vol. 9, 5916-5925
154. “A Theoretical Study Of Radical-Only And Combined Radical/Carbocationic Mechanisms Of Arachidonic
Acid Cyclooxygenation By Prostaglandin H Synthase”, P. J. Silva; P. A. Fernandes; M. J. Ramos Theor.
Chem. Acc., 2003, Vol. 110, 345-351
155. “Computational Studies Of New Potential Antimalarial Compounds - Stereoelectronic Complementarity
With The Receptor” C. Portela; C. M. M. Afonso; M. M. M. Pinto; M. J. Ramos J. Computer-Aided
Molec. Design, 2003, Vol. 17, 583-595
156. “Modelling The Metabolic Action Of Human And Rat CYP1A2 And Its Relationship With The
Carcinogenicity Of Heterocyclic Amines” R. da Fonseca; M. C. Menziani; A. Melo; M. J. Ramos Molec.
Phys., 2003, Vol. 101, 2731-2741
157. “New Designs For MRI Contrast Agents”, P.A. Fernandes; A. T. P. Carvalho; A. T. Marques; A. L. F.
Pereira; A. P. S. Madeira; A. S. P. Ribeiro; A. F. R. Carvalho; E. T. A. Ricardo; F. J. V. Pinto; H. A.
Santos; H. D. G. Mangericao; H. M. Martins; H. D. B. Pinto; H. R. R. Santos; I. S. Moreira; M. J. V.
Azeredo; R. P. S. Abreu; R. M. S. Oliveira; S. F. M. Sousa; R. J. A. M. Silva; Z. S. Mourao; M. J.
Ramos J. Computer-Aided Molec. Design, 2003, Vol. 17, 463-473
158. “Studies Of Inclusion Complexes Between Cyclodextrins And Polyazamacrocyclic Chelates Of
Lanthanide(III) Ions”, E. S. Henriques; M. Bastos; C. F. G. C. Geraldes; M. J. Ramos J. Chem.
Thermod., 2003, Vol. 35, 1717-1735
159. “Modelling Studies In Aqueous Solution Of Lanthanide (III) Chelates Designed For Nuclear Magnetic
Resonance Biomedical Applications”, E. S. Henriques; C. F. G. C. Geraldes; M. J. Ramos Molec.
Phys., 2003, Vol. 101, 2319-2333
160. “Nuclear Factor Kappa B: A Potential Target For Anti-HIV Chemotherapy”, V. Pande; M. J. Ramos
Curr. Med. Chem., 2003, Vol. 10, 1603-1615
161. “Receptor-Drug Association Studies In The Inhibition Of The Hematin Aggregation Process Of Malaria”,
C. Portela; C. M. M. Afonso; M. M. M. Pinto; M. J. Ramos Febs Lett., 2003, Vol. 547, 217-222
162. “Pyruvate Formate Lyase: A New Perspective”, M. F. Lucas; P. A. Fernandes; L. A. Eriksson; M. J.
Ramos J. Phys. Chem. B, 2003, Vol. 107 5751-5757
163. “Theoretical Studies On The Mechanism Of Inhibition Of Ribonucleotide Reductase By (E)-2 ‘Fluoromethylene-2 ‘-Deoxycitidine-5 ‘-Diphosphate”, P. A. Fernandes; M. J. Ramos J. Am. Chem.
Soc., 2003, Vol. 125, 6311-6322
164. “A Direct Classical Trajectory Study Of The Acetone Photodissociation On The Triplet Surface”, E.
Martinez-Nunez; A. Fernandez-Ramos; M. N. D. S. Cordeiro; S. A. Vazquez; F. J. Aoiz; L. Banares J.
Chem. Phys., 2003, Vol. 119, 10618-10625
165. “Synthesis and QSAR Study of The Anticancer Activity Of Some Novel Indane Carbocyclic Nucleosides”,
S. W. Yao; V. H. C. Lopes; F. Fernandez; X. Garcia-Mera; M. Morales; J. E. Rodriguez-Borges; M. N.
D. S. Cordeiro Bioorg. Med. Chem., 2003, Vol. 11, 4999-5006
166. “Direct Dynamics Study Of The Photodissociation Of Triplet Propanal At Threshold”, M. N. D. S.
Cordeiro; E. Martinez-Nunez; A. Fernandez-Ramos; S. A. Vazquez Chem. Phys. Lett., 2003, Vol. 381,
37-44
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167. “Matrix-Isolation FTIR Study Of Azidoacetone And Azidoacetonitrile”, M. Frankowski; B. S. Fox; A. M.
Smith-Gicklhorn; M. K. Beyer; V. E. Bondybey; M. Algarra; M. L. Costa; P. Rodrigues; M. T. Barros;
M. N. D. S. Cordeiro Low Temp. Phys., 2003, Vol. 29, 870-875.
168. “Interfacial Tension Behaviour Of Water/Hydrocarbon Liquid-Liquid Interfaces: A Molecular Dynamics
Simulation”, M. N. D. S. Cordeiro Molec. Simulation, 2003, Vol. 29, 817-827
169. “A Direct DFT Dynamics Study Of The Photodissociation Of Triplet Acetaldehyde”, M. N. D. S. Cordeiro;
E. Martinez-Nunez; A. Fernandez-Ramos; S. A. Vazquez Chem. Phys. Lett., 2003, Vol. 375, 591-597
170. “Cluster Model DFT Study Of Acetylene Adsorption On The Cu (100) Surface”, C. G. P. M. Bernardo; J.
A. N. F. Gomes J. Molec. Struc.-THEOCHEM, 2003, Vol. 629, 251-261
171. “Molecular Dynamics Study Of A Hexadecyltrimethylammonium Chloride Monolayer At The Interface
Between Two Immiscible Liquids”, D. J. V. A. dos Santos; J. A. N. F. Gomes Langmuir, 2003, Vol. 19,
958-966
172. “Vibrational Analysis Of Small Species In The Liquid Phase By A Combined Density Functional Theory
And Polarizable Continuum Method”, A. L. Magalhães; A. S. S. Pinto Theor. Chem. Acc., 2003, Vol.
110, 70-78
173. “Isolation and Structural Characterization of New Acylated Anthocyanin-Vinyl-Flavanol Pigments
Occurring in Aging Red Wines”, N. Mateus, E. Carvalho, A.R.F. Carvalho, A. Melo, A. M. GonzálezParamás, C. Santos-Buelga, A. M. S. Silva, V. Freitas J. Agric. Food Chem., 2003, Vol. 51, 277-282
174. “Acoustic wave sensor for barium based on poly[Ni(salen)(crown)] recognition chemistry“, M. Martins,
C. Freire, A. R. Hillman Chem. Comm 2003, 434-435.
175. “Immobilization of Mo(IV) complex in hybrid matrix obtained via sol-gel technique“, C. Marques, A. M.
Sousa, C. Freire, I. C. Neves, A. M. Fonseca, C. J. R. Silva J. Alloys Compounds, 2003, 360, 272-278.
176. “Electrochemical behaviour of a new precursor for the design of high performance poly[Ni(salen)] based
modified electrodes“, Miguel Vilas Boas, Isabel C. Santos, Mark J. Henderson, Cristina Freire, A.
Robert Hillman and Eric Vieil Langmuir, 2003, 19, 7460-7468
177. ”Synthesis and characterization of benzo-15-crown-5 ethers with appended N2O Schiff bases.” Carla
Sousa, Cristina Freire e Baltazar de Castro Molecules 2003, 8, 894-900.
178. “Chiral recognition in quenching of excited states by hydrogen donors”, U. Pischel; S. Abad; L. R.
Domingo; F. Boscá; M. A. Miranda Angew. Chem. Int. Ed., 2003, Vol. 42, 2531-2534
179. “Stereoselective fluorescence quenching by photoinduced electron transfer in naphthalene-amine dyads”,
U. Pischel; S. Abad; M. A. Miranda J. Chem. Soc., Chem. Commun., 2003, 1088-1089
180. “Selective fluorescence quenching of 2,3-diazabicyclo[2.2.2]oct-2-ene by nucleotides”, C. Marquez;
U. Pischel; W. M. Nau, Org. Lett., 2003, Vol. 5, 3911-3914
181. “Activity and Location of Olive Oil Phenolic Antioxidants in Liposomes” F. Paiva-Martins; M. H. Gordon;
P. Gameiro Chemistry and Physics of Lipids, 2003, 124, 23 - 36
182.
“Study of the oxidation products of the VO(dmpp)2 complex in aqueous solution under aerobic conditions:
comparison with the vanadate-dmpp system”, M. Margarida C. A. Castro, Fernando Avecilla, Carlos
F.G.C. Geraldes, Baltazar de Castro and Maria Rangel Inorg. Chim. Acta , 2003, 356, 142-154.
183. “Determination of E-2-Nonenal by High Performance Liquid Chromatography with UV Detection: an
Assay for the Evaluation of Beer Ageing” J. R. Santos, J. R. Carneiro, L. F. Guido, P. J. Almeida, J. A.
Rodrigues, A. A. Barros, Journal of Chromatography A, 2003, 985, 395-402.
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184
“Voltammetric Determination of Free and Total Sulphur Dioxide in Beer” P. J. Almeida, J. A. Rodrigues,
L. F. Guido, J. R. Santos, A. A. Barros and A. G. Fogg, , Electroanalysis, 2003, 15, 587-590.
185
“Free Sulphur Dioxide in Beer as the Difference between Total Sulphur Dioxide and Acetaldehyde: A
Voltammetric Approach”, P. J. Almeida, J. A. Rodrigues, L. F. Guido, J. R. Santos, A. A. Barros and A.
G. Fogg, Journal of the American Society of Brewing Chemists, 2003, 61(4):191-195.
186
“A voltammetric assay for the aging of beer”, L. F. Guido, J. R. Santos, N. A. Fortunato, J. A. Rodrigues
and A. A. Barros, Journal of Agricultural and Food Chemistry, 2003, 51, 3911-3915.
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Books and Book Chapters
1
João Sotomayor
Cinética Química, Lidel, Lisboa, 2003.
2
J. Aires-de-Sousa
Chirality Descriptors, in Chemoinformatics - A Textbook, Johnann Gasteiger and Thomas Engel (Eds.),
Wiley-VCH, 418-427, 2003.
3
M. Hemmer, J. Aires-de-Sousa
Structure-Spectra Correlations, in Chemoinformatics - A Textbook, Johnann Gasteiger and Thomas
Engel (Eds.), Wiley-VCH, 515-541, 2003.
4
J. Aires-de-Sousa
Representation of Molecular Chirality, in Handbook of Chemoinformatics, Johnann Gasteiger (Ed.),
Wiley-VCH, 1062-1078, 2003.
5
F. Pina, M. Maestri, V. Balzani
Photochromic systems based on synthetic flavylium compounds and their potential use as molecularlevel memory devices, in Handbook of Photochemistry and Photobiology, ASP, , 411-449 Chapter 9,
Vol. 3, 2003
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Articles in Proceedings
1
A.A. Ricardo; C.M.M. Duarte; M. Nunes da Ponte
“Properties of Mixing. Properties of Gas Mixtures” in IUPAC Experimental Thermodynamics, Vol.6:
Measurement of the Thermodynamic Properties of Single Phases, Goodwin A.R.H., Marsh K.N.,
Wakeham W.A. (Ed.), Elsevier, Amesterdam, pp 388-403 (2003)
2
S. Lyubchik; R. Melo; C.Palma; I. Fonseca
“Adsorption Equilibrium in the System Cr(III)-Activated Carbon in Role of Interfaces in Environmental
Protection”, ed. S. Barany, Nato Science Series, Vol. 24, Kluwer Academic Publishers, pp. 355-377
Dordrecht, 2003.
3
M.A.M. Reis; J.S.Almeida; E.J. Zungalia
“Bioremediação”, In Biotecnologia Fundamentos e Aplicações, M. Mota, N. Lima (Eds) , Lidel-Edições
Técnicas Lda, pp. 285-300 (2003)
4
R. Ruivo; A. Paiva; P. C. Simões
“Dynamic studies on a SCF countercurrent extraction process”, Proceedings of the 6th International
Symposium on Supercritical Fluids, Versailles, France, G. Brunner, I. Kikic, M. Perrut eds. (ISBN 2905-267-37-02), pp. 617-621, 2003.
5
C.M.M. Duarte; ANA M. Matias; Ana V.M. Nunes; M. Rosário Bronze, H.I. Mota Veiga, M. Nunes da
Ponte
“Separation of Antioxidant Components from White Grape Skin and seeds using Compressed Carbon
Dioxide”, Proceedings of the 6th International Symposium on Supercritical Fluids, Versailles, France,
G. Brunner, I. Kikic, M. Perrut eds. (ISBN 2-905-267-37-02), pp. 135-140, 2003.
6
A. Banet Osuna, A. Milewska, I. Fonseca, M. Nunes da Ponte
“Biphasic Hydrogenation and Oxidation of Terpenes in Supercritical Carbon Dioxide”, Proceedings of
the 6th International Symposium on Supercritical Fluids, Versailles, France, G. Brunner, I. Kikic, M.
Perrut eds. (ISBN 2-905-267-37-02), pp. 1107-1110, 2003.
7
P.C. Lemos; L.S. Serafim; H. Santos; M.A.M. Reis
“Production if Polyhydroxyalkanoates by a Mixed Culture in a Sequencing Batch Reactor: the Use of
Propionate as Carbon Source”, Com. Agric. Appl. Biol. Sc., 2003, Vol. 68/2(a), 109-114.(ISSN 13791176)
8
V. D. Alves; I. M. Coelhoso
“Osmotic Evaporation: A new process to obtain juice concentrates”, In Ingenería de Alimentos. Nuevas
Fronteras en el Siglo XXI, ed. P. Fito, A. Mulet, A. Chiralt, A. Andrés, Editorial de la UPV, vol III,13-18
(2003). (ISBN 84-9705-399-0)
9
R.M.C. Viegas; C. M. Afonso; J.P.S.G. Crespo; I.M. Coelhoso
“Using Membrane Contactors for Chiral Separations”, Proceedings 4º CITEM, 2003, 575-579. (ISBN
85-903547-1-7)
10
V. D. Alves; I.M. Coelhoso
“Fruit Juice Concentration by Osmotic Evaporation Using Hollow-Fibre Membrane Contactors”,
Proceedings 4º CITEM, 2003, 580-584.(ISBN 85-903547-1-7)
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11
12
R. Fortunato; C.A.M. Afonso; J.G. Crespo; M.A. Reis
“Organomercurial Removal from Vaccine Production Wastewaters in a Supported Liquid Membrane
Bioreactor” Com. Agric. Appl. Biol. ScVol. 68/2(a), 41-46, 2003. (ISSN 1379-1176)
F. Freitas, M. Temudo, J.S. Almeida, M.A.M.Reis
“Optimisation of nutrient removal in a sequencing batch reactor operated with high frequency oxygen
oscillations”, Com. Agric. Appl. Biol. Sc, 68/2 (a), 85-92, 2003, (ISSN 1379-1176) .
13
J. P. B. Mota, A. J. S. Rodrigo, I. A. A. C. Esteves, M. Rostam-Abadi
“Dynamic modelling of an adsorption storage tank using a hybrid approach combining computational
fluid dynamics and process simulation”, in Computer Aided Chemical Engineering, A. Kraslawski & I.
Turunen (eds.), Vol. 14, 797 802, Elsevier Science B.V., Amsterdam (2003).
14
M. F. J. Eusébio, A. M. Barreiros, R. Fortunato, M. A. M. Reis, J. G. Crespo, J. P. B. Mota
“On-line monitoring and control of a biological denitrification process for drinking-water treatment”, in
Computer Aided Chemical Engineering, A. Kraslawski & I. Turunen (eds.), Vol. 14, 1079-1084, Elsevier
Science B.V., Amsterdam (2003).
15
A. Lefevre, J. P. B. Mota, A. J. S. Rodrigo, E. Saatdjian
“Transfert thermique chaotique 3-D entre deux cylindres de section elliptique”, In Actes du Congres
SFT 2003, P. Marty et al. (eds.), Elsevier, Paris, 169-174 (2003).
16
J.P.B. Mota
“Towards the atomistic description of equilibrium-based separation processes. 1. Isothermal stirredtank adsorber”, in Computer Aided Chemical Engineering, A. Kraslawski & I. Turunen (eds.), Vol. 14,
791-796, Elsevier Science B.V., Amsterdam (2003).
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B
Dissertations
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Ph. D. Dissertations
“Aziridinação de Olefinas por Catálise de Paládio(II)”
Alexandra M. M. Antunes, PhD. in Organic Chemistry, Faculdade de Ciências e Tecnologia da Universidade
Nova de Lisboa, March 2003, Supervisor: Paula C. S. Branco.
“Síntese de Alcalóides Indolizidínicos Biologicamente Activos”
Marta C. C. Corvo, PhD. in Organic Chemistry, Faculdade de Ciências e Tecnologia da Universidade Nova de
Lisboa, December 2003, Supervisor: Manuela A. Pereira.
“Studies on Critical Factors Affecting Enzyme Activity in Nonaqueous Media: Supercritical Fluids and
Organic Solvents”
Nuno Miguel Deodato Fontes, UNL, Apr 2003
Supervisor(s): S. Barreiros
“Dynamic Study of a Supercritical Extraction Process”
Rui Ruivo, UNL, July 2003
Supervisor: Pedro Simões
“Estudo de Reactores de Biofilmes Suportados em Membranas para Remoção de Poluentes” Anabela
Fonseca, UNL, Jan 2003
Supervisor(s): M.A.M.Reis; J.S.Almeida
“Monitorização e Controlo de Reactores de Biofilme Suportado sobre Membranas no Tratamento de
Efluentes Industriais”
Christine Gundula Wolf, UNL, Out. 2003
Supervisor(s): M.A.M.Reis; J.G.Crespo
“Vapour-Liquid Equilibrium Measurement and Emulsion Formation in SC CO2”
Joana Fonseca, UNL, Jul 2003
Supervisor(s): Manuel Nunes da Ponte and Pedro Simões
“Chemical Synthesis and Phase Behaviour in Supercritical Carbon Dioxide”
Teresa Casimiro, UNL, July 2003
Supervisor: Manuel L.M. Nunes da Ponte and Ana Aguiar Ricardo
“The Crystal Structure of Cytochrome c Nitrite Reductase from Desulfovibrio desulfuricans and Modeling
Studies of Electron Transfer between Flavodoxin and Mono-heme cytochromes”
Carlos M. Costa Cunha, UNL, Nov 2003
Supervisor(s): M. João Romão e Cláudio Soares
“Electron Transfer Complexes between Paracoccus CCP and its electron donors”
Sofia Rocha Pauleta, UNL, Nov 2003
Supervisor(s): I.Moura e Graham Pettigrew
“Estudos estrurais e funcionais da redutase do nitrito de Desulfovibrio desulfuricans”
Maria Gabriela Machado de Almeida, UNL, Oct 2003
Supervisor(s): I.Moura e Jorge Lampreia
“Estudos Estruturais por RMN de Proteínas contendo Centros Ferro-Enxofre [Fe-4S]”
Sofia Homem de Gouveia Costanzo Nunes, UNL, Jun 2003
Supervisor(s): I.Moura e Brian Goodfellow
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“Integrating Protein Structural Information”
Ludwig Krippahl, UNL, Dec 2003
Supervisor(s): J.J.G.Moura e Pedro Barahona
“Proteinas de Ferro-Enxofre Complexas-Hidrogenase e Fuscoredoxina- Estudos Bioquímicos e
Espectroscópicos”
Ana Pamplona, DQ/FCT/UNL, May 2003
Supervisor(s): J.J.G.Moura
“Estudos Teóricos da Química de Interfaces”
Daniel José Viegas Antunes dos Santos, UP, Dec 2003
Supervisor: José Alberto Nunes Ferreira Gomes
“Controlo de Formulações Farmacêuticas Baseado em Sistemas de Exactidão Aferida”
Adriana Martins Pimenta, UP, Dez de 2003
Orientador(es): M.C. B. Montenegro e A N. Araújo
“Limonete, Hipericão-do-Gerês, Cardo-do-coalho, Fel-da-terra: Metodologias de Controlo de Qualidade com
Base na Fracção Fenólica e Estudos de Acção Antioxidante e Hepatoprotectora”
Patrícia Valentão, FFUP, Maio de 2003
Orientadores: Rosa Seabra, Paula Andrade e Lourdes Bastos
“Desenho de novos catalisadores: nanocompósitos preparados por imobilização de complexos de metais de
transição com bases de Schiff em carvão activado”
Ana Rosa Aires Neto da Silva, FCUP, 2003
Orientadores: Baltazar de Castro e Cristina Freire
“Desenvolvimento de m]etodos Electroanal]iticos para a determinação de biomarcadores. Diacetilo e vitalidade
de levedura em industria cervejeira e metalotioneinas em estudos ambientais”
Pedro Miguel Gonçalves Rodrigues, FCUP, 2003
Orientadores: Aquiles Barros e José António Rodrigues)
Química Verde
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M. Sc. Dissertations
“Contribuição Para o Estudo da Fermentação Maloláctica em Vinhos Portugueses”
Luz Catarino Neves Fernandes, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2003.
Supervisor: Ângela M. S. Relva, Ana M.Costa Freitas
“Comparação de Dois Métodos, SPME e SDE, Para Análise Quantitativa de Compostos do Aroma. Aplicação
do SPME Associado à GC-EM na Análise de Vinhos Monocasta”
Luís Maria Pestana de Torres Vaz-Freire, Faculdade de Ciências e Tecnologia da Universidade Nova de
Lisboa, 2003
Supervisor: Ana M. Costa Freitas, Ângela M. S. Relva
“Caracterização de filmes poliméricos de níquel(II) e cobre(II) com bases de Schiff e aplicação no
reconhecimento electroquímico de catiões de metais alacalino-terrosos”;
Luís Carlos Fernandes Neto, FCUP, 2003
Orientador: Cristina Freire
“Caracterização de filmes de poli[Ni(saltMe)] por espectrocopia de impedância electroquímica”. Elsa Maria
Carvalho Cerqueira Pereira, FCUP, 2003
“Complexos de manganês com Bases de Schiff: aplicação em catálise homogénia e heterogénia”
Mário Joaquim dos Santos Cardoso, FCUP, 2003
“Extracção de vapor para remediação de solos - Controlo dos parâmetros que limitam a descontaminação”
José Tomás Veiga Soares de Albergaria, FEUP, 2003
Orientador:
“Determinação de compostos orgânicos de grande valor comercial em extractos vegetais”
Salomé de Sousa Teixeira, FEUP, 2003
Orientador:
“Modelação da resposta de sistemas sensores ópticos fluorescentes para pH, Mg(II), Al(III) e Zn(II)”
Abel José Assunção Duarte, FCUP, 2003
Orientador:
Estudo de compostos relacionados com o envelhecimento da cerveja
José Ricardo Cunha Carneiro, FCUP, 2003
Orientador: Paulo Joaquim Almeida
Comportamento electroquímico de g-hexaclorociclo-hexano em N, N-dimetilformamida
Clélia Alexandre Claudino Milhano, FCUP, 2003
Orientadores: Aquiles Barros e Maria Irene Montenegro
Desenvolvimento de um sistema em fluxo para a análise de SO2 na cerveja acoplando um módulo de
pervaporação e detecção voltamétrica
Gabriela Maria Couto Carvalho Peres Correia, FCUP, 2003
Orientador: José António Rodrigues
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C
Patents
Química Verde
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Patents
Fases estacionárias quirais baseadas em terpenóides, C.M.M. Moiteiro, M.R.L.S. Tavares da Rosa, M.J.M.
Marcelo Curto, A.M. Lobo, Patent Aplication PT 102878, 28.11.2002.
Novos Líquidos Iónicos Baseados na Unidade Tetra-Alquil-dimetil-guanidínio, Nuno, M. M. Mateus, Luís A. A.
F. C. Branco, Carlos A. M. Afonso, Departamento de Química da FCT-UNL, Pedido de Patente Portuguesa nº
102937, 9 de Abril, 2003.
“Treatment of Aqueous Media Containing Electrically Charged Compounds”. WO 01/40118 A1. Inventores:
João Goulão Crespo e Maria Ascensão Reis. International patent, granted 19/09/2003
“Method for the obtention of aryl ethers using a silver salt as catalyst and ultra-sonic mixing”, A.C. Fernandes,
J. E. Borges, M.F. Pereira, C.J. Romão, L.M. Correia, P.B. Correia, Portuguese Patent, 102315 Z, granted
30/06/2003.
“New preparation method of the anti-ulcer compounds Omeprazole, Lansoprazole and Pantoprazole”, A.C.
Fernandes, J. E. Borges, M.F. Pereira, C.J. Romão, L.M. Correia, P.B. Correia, R, Tavares, M. Costa, F.
Teixeira, Portuguese Patent 102317 A, granted 31/10/2003.
“N-(fosfonoalquil)glycine, N-(fosfonobenzil)glycine, N-(fosfonofenetil)glycine, their production process and their
appication as herbicide”, P.B. Correia, Portuguese Patent 102716, granted 31/12/2003.
“New method for the preparation of the anti-ulcer compounds Omeprazole, Lanzoprazole and Pantoprazole”,
A.C. Fernandes, J. E. Borges, M.F. Pereira, C.C. Romão, L.M. Correia, P.B. Correia, R, Tavares, M. Costa,
F. Teixeira, International Patent WO 03/097606, granted 27/11/2003.
“Processo para a determinação voltamétrica, célula voltamétrica e dispositivo para determinação em fluxo”.
Patente de Invenção Portuguesa Nº 102608 – Patente concedida pelo Instituto Nacional da Propriedade
Industrial, em 3 de Março de 2003.
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ANNEX III
RESEARCH PROJECTS
Química Verde
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Research Projects
Funded by EU
Adsorbed Natural Gas System with Guard-Bed Device
ANGUARD: EU ENK6-CT2000-00053
José Paulo Mota
Molecular Level Devices and Machines
European network RTN1-1999-00138
Fernando Pina
The Mechanism, Specificity and Inhibition of Enzymes belonging to the Xantine Oxidase family: Medical and
Industrial Applications
European Research Network TMR: (HPRN-CT-1999-00084) (2000-2004)
Maria João Romão
Intercalation as a new approach for obtaining highly efficient pi adsorbents and supports catalysts
INTAS_00-00750/2000
Isabel Fonseca
Funded by NATO
New approach to waste recovery into selective adsorbvents of heavy metals
NATO Science for Peace Program nº 977984/2002
Isabel Fonseca
Funded by FCT
Alcaloides naturais como sintões quirais
POCTI/QUI/44501/2002
Ana M. C. Lourenço
Nova metodologia sintética de gama-amino ácidos quirais uteis na concepção de novos fármacos
Manuela A. Pereira
Synthetic Methods for N-Heterocycles based on Sigmatropic Rearrangements
POCTI/QUI/36456/99
S. Prabhakar
Development of New and Improved Processes for Palladium Catalysed Cross-Coupling Reactions for Natural
Products Synthesis
Praxis XXI, PCTI/QUI/36536
Maria Teresa Barros
Saccharose as a water soluble chiral auxiliary and as a linker for solid phase chemistry
Praxis XXI, PCTI/QUI/47973/2002
Maria Teresa Barros
Rationalising Stereoselectivity in Organic Synthesis: From Theory to Practice
António Gil de Oliveira Santos
Química Verde
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Green Processing With Ionic Liquids Coupled to Supercritical CO2 Extraction or Membrane Pervaporation
POCTI/EQU/35437/1999
Luis Paulo Rebelo, ITQB (Carlos A. M. Afonso, from REQUIMTE).
Natural Vegetal Antioxidants
Abel J.S.C. Vieira
Hopanoid composition of sediments from Lower Tagus Basin
Angela M. S. Relva.
New carbohydrate-based compounds from ethnopharmacological plants: bioactivity against pathogenic yeasts
POCTI/1999/FCB/35863
Elvira Maria Mendes Sardão Monteiro Gaspar
Removal of Cu, Cr, As and creosote from impregnated wood waste aiming its recycling
POCTI/32927/AGR/2000
Alexandra Ribeiro, DCEA, UNL, (M.D.R. Gomes da Silva, from REQUIMTE)
Desenvolvimento de um sistema de informação e gestão ambiental para o estuário do Sado
POCTI/33137/BSE/99-00
Maria Helena Costa, DCEA/IMAR, UNL, (M.D.R. Gomes da Silva, from REQUIMTE)
Compostos esterólicos como potenciais marcadores da poluição fecal e sua correlação com indicadores
biológicos
PNAT/1999/BIO/15024
José Filipe Santos Oliveira, GDEH/UBIA, UNL, (M.D.R. Gomes da Silva, from REQUIMTE)
Chemosensors based on polyammine receptors bearing fluorophoric units
Fernando Pina
Randomly ordered DNA sensors based on direct questioning of immobilised probes with wavelength shifting
pairs
POCTI/BIO/38922/2001
J.C. Lima
Molecular-Level Devices and Machines
POCTI/QUI/32442/99
Fernando Pina
Building blocks for photoactive molecular cages
António Jorge Parola
Photophysics and photochemistry of anthocyanins
POCTI/QUI/33679/99
João Carlos Lima
Química Verde
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Production and characterisation of a PDLC. New strategies for polimerisation and incorporation in supercritical
CO2.
POCTI/CTM/47363/2002
Madalena Dionísio (João Sotomayor)
Resolução de misturas racémicas de substâncias activas em contactores de membranas,
POCTI/FCB/43942/2001, 2002-2004
M. Helena Marques (FFUL), Isabel Maria Coelhoso, João G. Crespo e C.M. Afonso (REQUIMTE)
Tecnologia para a obtenção de filmes e revestimentos comestíveis para alimentos a partir de recursos
nacionais de baixo valor
POCTI/EQU/45595/2002, 2003-2006
Alberto M. C. Sereno
Mechanisms of Drug Controlled Release from Microspheres
POCTI/EQU/46715/2002, 1-Outubro-2003 -30 Setembro de 2006
Margarida Cardoso
Produção de Copolímeros PHB/PHV por Culturas Mistas
POCTI/1999/35675, 2001-2004
Bioreactor de Membrana de Permuta Iónica para Desnitrificação de Água
PRAXIS/P/BIO/14065/1998, 2002-2004
Reacção e Transporte num Reactor de Membranas de Permuta Iónica para Tratamento de Água Potável
POCTI/EQU/39482/2001, 2002-2004.
Production of copolymers phb/phv by mixed cultures
POCTI/35675/BIO/2000
Ana Maria Ramos
Phase-behavior and microscopic characterisation of micro/macro-emulsions in CO2. New strategies for
polymerisation and enzymatic catalysis
POCTI/99/QUI/35429
Ana Isabel Aguiar-Ricardo
Transesterification of vegetable oils. Aplication to the production of biodiesel
Joaquim Vital
Catalytic depolymerisation of current plastic wastes
Ana Maria Ramos
Changes in the molecular dynamics followed by dielectric spectroscopy during the formation of a Polymer
Dispersed Liquid Crystal
POCTI/CTM/37435/2001; from 5/2003 to 5/2005
Maria Madalena Alves Campos de Sousa Dionísio Andrade
Química Verde
A - 44
Production and characterization of a polymer dispersed liquid crystal. New strategies of both polymerization
and impregnation in supercritical-CO2
POCTI/CTM/47363/2002; from 11/2003 to 11/2006
Maria Madalena Alves Campos de Sousa Dionísio Andrade
Phase-behaviour and microscopic characterisation of micro/macroemulsions In CO2. New strategies for
polymerisation and enzymatic catalysis
POCTI/35429/2000; 2001/01/31 to 2004/01/30
Ana Isabel Nobre Martins Aguiar de Oliveira Ricardo.
High Pressure NMR spectroscopy of polymers and biopolymers in CO2 emulsions
Ana Isabel Nobre Martins Aguiar de Oliveira Ricardo.
Computational fluid mechanics and process dynamics of supercritical fluid extraction columns with structured
packings and static mixers
POCTI/2000/EQU/34957; 01/10/2000 a 01/10/2003
Pedro Miguel Calado Simões
Estudos Estruturais e Mecanísticos de Enzimas Chaves do Ciclo do Azoto
PRAXIS/QUI/10087/98
Isabel Moura
Enzimas chave da activação do sulfato e do metabolismo energético. Novas proteínas de Co não corrinóides
e Zn-ATP sulfurilases, cinases do adenilato
Isabel Moura
“Bacterial cytochrome c peroxidases- Activation, Enzymatic Mechanism And Structure”
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
Isabel Moura
High Pressure NMR spectroscopy of polymers and biopolymers in CO2 emulsions
Maria dos Anjos Macedo
Engenharia de Metaloproteínas: Uma Aproximação Pós-Genómica
POCTI / BME / 36152 / 99
José J.G.Moura
Estudos Electroquímicos Dinâmicos em Proteínas de Transferência Electrónica
POCTI / QUI / 42277 / 2001
José J.G.Moura
High-Pressure NMR Spectroscopy of Polymers and biopolymers in CO2 Emulsions
POCTI/QUI/42313/2001, 2002-2005
José J.G.Moura
Química Verde
A - 45
NMR structural studies of the active center of 5-aminolevulinate synthase, the first enzyme of the mammalian
heme biosynthetic pathway
POCTI/BME/39184/2001
Maria dos Anjos Macedo
Orientation of Proteins By Liquid Crystals
POCTI/QUI/42279/2001, 2002-2005
Francisco Jorge Caldeira
Vif (virion infectivity factor) structure and localization during HIV-2 infection
POCTI/ESP/36103/99
Francisco Jorge Caldeira
Structural and mechanistic studies of fatty acid desaturases. Looking for reactivity of diiron clusters
Pedro Tavares
Engenharia de Proteínas aplicada a enzimas degradadoras de nitrilos
POCTI/CEG/2508/95
Jorge Lampreia
Protease de Plasmodium chabaudi como alvo na terapia da malária
POCTI/43637/BME/2000
Jorge Lampreia
As proteases de parasitas da malária como alvo na quimioterapia
POCTI/ESP/42223/2001
Jorge Lampreia
A crystallographic contribution to the understanding of the structure and function of Mo-enzymes
POCTI/1999/BME/35078 (2000-2004)
Maria João Romão
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
M. Salette Hipólito Reis
Evaluation of membrane environment on modulation of drugs interactions with the P-glycoprotein multidrug
transporter
M. Salette Hipólito Reis
Development and chemical, structural and rheological characterisation of protein-polysaccharide mixed aqueous
systems
POCTI / QUI / 36452/2000
Maria do Pilar Gonçalves
Interaction between metal ions and buffers for the environmental and biological pH ranges
Helena M.V.M. Soares
Química Verde
A - 46
Alterações endócrinas e enzimáticas na solha (Platichthys flesus) e na tainha (Mugil cephalus) expostos a
contaminantes orgânicos e metais pesados no estuário do Douro, e seu uso como indicadores de poluição
PDCTM/P/MAR/15280/1999 (2001-2004).
Félix Dias Carvalho
Hepatotoxicity and cardiotoxicity studies of methylenedioxymetamphetamine and its metabolites in Wistar
rats
POCTI/36099/FCB/2000
Amphetamines and physical exercise. An hazardous combination?
POCTI/ACT/43562/2001
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.
Rosa Seabra
Modelling inhibitor mechanisms in radical enzymes: a QM/CM approach
POCTI/35376/QUI/2000
Maria João Ramos
Investigation of Mimetic Modifications of Bioactive Peptides by Inclusion of Novel Synthetic Amino Acids
Maria João Ramos
Influence of Procyanidin Structures on their Ability to Complex with Anthocyanins - A Molecular Interpretation
of Colour
André Melo
Experimental and Theoretical Studies on Model and Supported Catalysts: Catalytic Systems with Industrial
and Environmental Relevance
POCTI/QUI/33765/99
José Ferreira Gomes
Electrocatalytic Reactivity of Gold Chiral Surfaces
POCTI / QUI / 41704 / 2001
Maria Natália Cordeiro
Síntese, estrutura, propriedades, especiação em solução e estudos biológicos de compostos de vanádio
com potencial para o tratamento de Diabetes
Maria Rangel
Structural Factors Determinant on the Reactivity of Photolysis Products of B12 Model Compounds
Maria Rangel
Química Verde
A - 47
Estruturas poliméricas condutoras baseadas em complexos de metais de transição (Conducting/redox polymer
networks based on transition metal complexes
POCTI/QUI/ 32831/2000
Ana Cristina Freire
Aplicação de argilas com pilares funcionalizadas com complexos metálicos em catálise assimétrica
POCTI/QUI/ 42931/2001
Baltazar de Castro
Design of novel metallo-surfactants with catalytical relevance
Baltazar de Castro
Nanoestruturas de metais nobre em meios organizados:aplicações tecnológicas
Eulália Pereira
Determinação de alfa-cetoácidos e alfa-dicetonas como produtos do metabolismo celular
Aquiles Barros
Estudo da durabilidade dos geossintéticos com vista a uma melhor definição dos seus coeficientes de
segurança
POCTI/ECM/42822/2001
Aquiles de Barros
Funded by MADRP
Desenvolvimento e aplicação de metodologias expeditas de controlo e segurança no sector agro-alimentar
AGRO/273
José L. F. C. Lima
Protecção contra pragas do olival numa óptica de defesa do ambiente e do consumidor
AGRO/314
Beatriz Oliveira
Funded by ADI
Análise por voltametria em fluxo de compostos importantes para o controlo do processo de produção de
bebidas
BEERVOLT
Aquiles Barros
Funded by “Ciência Viva”
Experimentar, comunicar e transformar
PW-047 (Agência Nacional para a Cultura Científica e Tecnológica)
Gabriela Ribeiro
Química Verde
A - 48
Bilateral cooperations
Acoustic wave sensor for metal ions based on poly[M(salen)crown] recognition chemistry
Acção Integrada Luso-Britânicas B-6/03
Ana Cristina Freire
Rheological behaviour and morphology of protein/polysaccharide mixed systems
GRICES/CAPES (Portugal/Brasil)
Use of galactomannan/starch mixtures in low-oil food emulsions stabilisation
CRUP (E-19/02)
Fruit juice concentration by osmotic evaporation in membrane contactors
ICCTI/OMFB P9/01 (Portugal/Hungria)
Isabel Maria Coelhoso
Development of membrane systems for chiral separations based on molecular recognition mechanisms
Integrated Action E-39/02 Portugal- Spain
Isabel Maria Coelhoso
Etude des interactions macromoleculaires entre proteines de transfert d’electrons
ICCTI – Embaixada da França, 316 C2
José J.G.Moura
Analyse des interactions protéines-protéines par arrimage moléculaire sous contraintes RMN
Centre National de la Recherche Scientifique/ICCTI, PICS Nº 1392
José J.G.Moura
Biochemical and Spectroscopy Studies on the Enzyme Nitrate Reductase (NAP) from the sulfate-reducing
organism Desulfovibrio desulfuricans”
Projecto de Cooperação Científica e Tecnológica Portugal / Argentina
J.J.G.Moura
Desenvolvimento de mini-sondas e de sistemas automáticos visando análise de baixo impacto ambiental
FCT/GRICES/CAPES (Portugal/Brasil)
José L. F. C. Lima
Automação de análise selectoras e discriminatórias
Rui A. S. Lapa
Desenvolvimento de sistemas automatizados de fluxo com detecção voltaamperométrica
CRUP (Acção Integrada Luso-espanhola E-41/03)
Frutas tropicais de alta humidade: processamento, embalagem sob atmosfera modificada e avaliação da
qualidade
Alberto M. Sereno (Portugal), Dr. Miriam D. Hubinger (Brazil)
Química Verde
A - 49
Tecnologia de películas biodegradaveis para alimentos em ibero-américa
CYTED project XI.20
Contributo da monitorização baseada em sensores opticos e potenciométricos na eficiência do processo
produtivo e minimização do impacto ambiental da galvanoplastia industrial.
Responsável: Alberto N. Araújo
Desenvolvimento de transdutores ionicos sensiveis a fosfato na conscepção e sintese de um novo ionoforo.
CRUP (Acção Integrada Luso-Britanica B-3/03)
M. Conceição B. M. Montenegro
Química Verde

Annual Report 2003

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