Laboratório Associado
para a Química Verde
Tecnologias e Processos Limpos
REPORT
2005
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 2005 was the fourth 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 six
researchers (Investigadores Auxiliares) to carry out activities under the contract of Laboratório
Associado.
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 380
researchers, of which more than 200 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.
Laboratório Associado para a
Química Verde
iv
History
REQUIMTE stands for Rede de Química e Tecnologia and names the Chemistry and
Technolology Network that has been formed by two Research Units, the Centro de Química da
Universidade do Porto - CEQUP and the Centro de Química Fina e Biotecnologia da Universidade
Nova de Lisboa – CQFB.
Since November 2001 REQUIMTE became the “Laboratório Associado para a Química Verde
– Tecnologias e Processos Limpos” of Fundação para a Ciência e a Tecnologia of MCTES, in
which 374 researchers (208 holding a Ph.D. Degree) exploit the basic principles of Green Chemistry and aim to contribute to the practice of Sustainable Chemistry.
The need of practicing a sustainable development in order to reach the social, economic and
environmental objectives of the modern society is well accepted by governments, industrial
sector and general public. Within this scope, Chemistry which is commonly associated with
harmful products and not with materials absolutely essential for everyday life, must have a
decisive role in the maintenance and improvement of living and environmental conditions.
The awareness of contemporary society for the inevitability of the use of chemicals and chemical
processes and the understanding of the concept of sustainability lead to a new way of thinking
Chemistry. Having in mind the implementation of clean practices and clean industrial processes
in which the amount of raw materials, energy, costs and risks are reduced a scientific movement,
known as Green Chemistry, has emerged in the last quarter of the 20th century.
Green Chemistry aims to redevelop laboratory and industrial processes in order to make them
cleaner and economical viable. For that purpose scientists have to design new reactions and
processes in which principles like an economy of atoms, use of renewable materials, use of nontoxic solvents and use of clean energy sources must be followed.
The objectives of Laboratório Associado para a Química Verde – Tecnologias e Processos Limpos
are: a) to encourage the use of clean products and technologies; b) to assist industry in the
design and implementation of non-aggressive chemical processes; c) to train young researchers
in interdisciplinary areas related with the practice of sustainable chemistry; d) to make public
the principles of green chemistry and to alert society for the necessity of a sustainable practice
in everyday life.
Research is presently focused in the following thematic areas of: i) natural products, (ii) food
quality and safety, (iii) clean production technologies and processes, (iv) environmental control
and remediation and (v) catalysts, solvents and non-toxic compounds.
The sharing of multidisciplinary scientific knowledge, technology and equipment between
researchers of the two centers that formed the network has significantly contributed to the
development of new projects in green chemistry and to the enrichment of training of graduate
students by making easier the mobility of human resources.
At present the network REQUIMTE can be described as a big Laboratory that has two operating
sites, one at Universidade Nova de Lisboa and other at Universidade do Porto.
Laboratório Associado para a
Química Verde
v
REQUIMTE in 2005
2005 was the third year in which REQUIMTE operated as a —Laboratório Associado“, although
the cooperation existed since 1996. The strategic plan was to focus expertise in Analytical,
Biological, Inorganic, Organic, Physical and Theoretical Chemistry and in Chemical Engeeniring
in a contemporary unifying concept œ that of Green Chemistry.
2005 was the second year when the funding contract with FCT-MCTES was operational and
REQUIMTE hired six new researchers in order to carry out activities included in the contract with
the Ministry. 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 280, much to the dedication of its graduate students, 14 of which have completed their
doctoral thesis and 14 their master thesis in 2005.
Laboratório Associado para a
Química Verde
vi
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 Bastos (UP)
Maria Rangel (UP)
Advisory Comittee
Prof. William B. Motherwell
University College, Christopher Ingold Laboratories, 20 Gordon St., London WC 1 H OAJ, UK
Organic synthesis; Reaction mechanisms in organic chemistry
Prof. Luigi Marzilli
Department of Chemistry, Louisiana State University, Baton Rouge, LA70803-1804, USA
Spectroscopy; Inorganic synthesis; B12 chemistry; Bioinorganic chemistry; Metal based drugs
Prof. Jean L. Rivail
Université Henri Poincaré, Laboratoire de Chimie Théorique, B.P. 239, 54506 Vandoeuvre-les-Nancy,
France
Theoretical chemistry
Prof. Marek Trojanowicz
University of Warsaw, Faculty of Chemistry, Pasteura 1, PL-02-093,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
Laboratório Associado para a
Química Verde
vii
http://www.requimte.pt
LABORATÓRIO ASSOCIADO
QUÍMICA VERDE – TECNOLOGIAS E PROCESSOS LIMPOS
REQUIMTE, is the biggest network in Chemistry and Chemical Engineering established in Portugal and is recognized as the
Laboratório Associado para a Química Verde by the Portuguese Ministério de Ciência e do Ensino Superior since November 2001.
The scientific expertise and complementary knowledge available, put together by the two research centres that form the network
(Centro de Química Fina e Biotecnologia-UNL and Centro de Química-UP), allowed us to deal with the topic GREEN CHEMISTRY
- CLEAN TECHNOLOGIES AND PROCESSES with a wide range of tools and from different perspectives.
REQUIMTE has the mission of corporate in a continuous form, in a competent and efficient way in the prosecution of the specific
aims of the national scientific and technologic politics in the areas of:
1
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.
Laboratório Associado para a
Química Verde
viii
Events
The Day of Chemistry is organized every year bringing more than 600 students to the Campus
of Caparica. In 2005 it occurred on the 8th of February. In the morning there is a session in the
auditorium with live experiments and in the afternoon the students visit several laboratories in the
Chemistry Deparpment.
Every year the Faculty of Sciences and Technoly (Campus of Caparica) organizes an Open Day
with visits to the laboratories of the Chemistry Department). In 2004 a Forum of Chemistry was
organized by students and teachers of Chemistry.
The VII Jornadas Tecnológicas de Engenharia Química took place on the...
>>Igual a 2004:<<
Program Ciência Viva
In the week of the FCT Scientific Culture visits to several laboratories were organized.
In the summer period two weeks courses, for students presently attending the secondary school,
were organised at CEQUP and on the themes of Treatment of Laboratory Wastes and Toxicology.
Monographic Courses (5 days) are ministrated in the themes:
Determination of Molecular Structures by X-Ray Diffraction Methods
Nuclear Magnetic Resonance: From Theory to Practice
Liquid-liquid and gas-liquid chromatography
Organization of Conferences , Symposiums and Workshops
XIX Reunião Ibérica de Adsorção, July 2004
>>Retirei da info 2005:<<
Participation in the organization of:
- Acção de Formação de Professores “Histórias com Muita Ciência”, Lisboa, Nov 2005
Prof. P. Mata
- Acção de Formação “Gastronomia Molecular: do Laboratório para a Cozinha. Azoto Líquido e Alginatos” ,
Lisboa, Nov 2005
Prof. P. Mata
- Acção de Formação “Gastronomia Molecular: do Laboratório para a Cozinha. Gelatinas quentes e frias”,
Lisboa, Mar 2005
Prof. P. Mata
- Curso Satélite (trabalhos práticos do 12º ano) ao 4º DEDQ - Encontro Nacional da Divisão de Ensino e
Divulgação da Química, Sociedade Portuguesa de Química, Out. 2005.
Prof. M. Pereira
- 4º DEDQ – Encontro Nacional da Divisão de Educação e Divulgação da Química da Sociedade
Portuguesa de Química (http://www.spq.pt/congressos/4dedq/).
P. Tavares
- Curso Monográfico de Bioelectroquímica, 9-11 de Outubro 2005.
J.Moura / M.G. Almeida
Organização de eventos
2ª Jornada “Bebidas e Saúde do IBeSa” (Instituto de Bebidas e Saúde).
1st February 2005 in Faculdade de Farmácia including the presentation of the research work developed in
2003/2004 with grants from IBeSa and a conference intituled “Natural antioxidants, ageing and oxidative
stress” presented by Prof. Vítor Costa from IBMC. The presented work researches were compiled in a book
(ISBN 972-99931-0-6).
Laboratório Associado para a
Química Verde
ix
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 2005.
Laboratório Associado para a
Química Verde
RESEARCH REPORT
2005
Part A
2
AREA 1
NATURAL PRODUCTS: SCREENING AND SYNTHESIS
- A simple rearrangement of N-prenyl indole derivatives was uncovered and led to a simple entrancy
into cancer useful natural molecules.
- A new enantioselective inverse phase transfer reaction for the Markovinkov hydration of double
bonds was disclosed, that makes use of cyclodextrins.
- The synthesis of Ecstasy metabolites, produced by glutathione capture of the intermediate
quinones, showed that the neurotoxicity paralels its redox potential.
- Guinea-Bissau plant extracts revealed a library of cytotoxic phenolic compounds.
- Chirality codes, previously developed, were applied to the prediction of enantiomeric excess in a
combinatorial library of enantioselective reactions.
- A new method was developed to represent chemical reactions with no specification of the reac
tion center, from the difference of MOLMAP molecular descriptors of products and reactants.
- The CIP rules to specify chirality in chemistry were revised in order to improve its logic, consis
tency, scope and applicability. The work was adopted by IUPAC.
Laboratório Associado para a
Química Verde
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AREA 2
FOOD QUALITY AND SAFETY
- Participation in International Collaborative study ISO/CD 6885.
- Membership of Technical Committees: elaboration of the industry code of Hygienic Practices
and NP EN ISO documents.
- Implementation of collaborative projects with producer associations: characterization of traditional food products and safety.
- Quality Control of food products answering to industry questions and consumers concerns:
GMOs and acrylamide.
- Application of chemometric techniques (univariate, bivariate and multivariate statistics) to evaluate
the relationships between different types of parameters in a given product, and to classify new
observations in relation to predefined models.
- Modelling and optimisation of food processing.
- Measurement and prediction of physical and rheological properties.
- Studies performed in poultry for human consumption to evaluate the occurrence of vancomycin
resistant enterococci as well as in swines in Europe.
- Patented voltammetric method for the determination of diacetyl directly in beer fermentation
vessels.
- In vitro evaluation of safety/toxicity/protection of food components and beverages for human
consumption.
Laboratório Associado para a
Química Verde
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AREA 3
CLEAN PRODUCTION TECHNOLOGIES AND PROCESSES
- Membrane Processes were used for separation and/or reaction, in the recovery of aroma compounds, pre-enrichment for sample discrimination, extraction of amino acids and derivatives
using ionic liquids, vapour separation .
- Ion Exchange Membrane Bioreactos were used in the removal of polluting ions from drinking
water streams.
- Polysulfone membrane formation were prepared using a CO2-assisted phase inversion method.
The Thermodynamics of ionic liquid-containing systems was studied.
- Dynamic modelling of supercritical fractionation, nanofiltration and adsorption was carried out.
The synthesis of new polyurethanes and PDLC, as well as the preparation of polymeric systems
for drug controlled-release, were carried out in both conventional and scCO2 media.
- Studies on the optimisation of pectin extraction from orange peels and the subsequent production of model edible films were carried out.
- Process Integration of Supercritical Fluid Extraction and Membrane Separation continued to be
studied.
Laboratório Associado para a
Química Verde
5
AREA 4
ENVIRONMENTAL CONTROL AND (BIO) REMEDIATION
- Use of Ion Exchange Membrane Bioreactor (IEMB) for the removal of polluting ions (perchlorate
and nitrate) from drinking water streams.
- Biodegradation of organ-mercurial compound to metallic mercury, under aerobic conditions, by
pure bacterial cultures in CSTR, fed with a real vaccine production wastewater.
- Simultaneous removal of P and N and microbial population identification (FISH).
- Pesticide Analysis (in food and environmental samples)
- Laboratory Waste Management (waste separation/disposable strategies)
- Soil Remediation (in-pulp solvent extraction procedures to remediate hydrocarbon contamination).
- New trends on exploitation of different transducing schemes such us optical and electrochemical.
- Studies on immobilization techniques for the monitoring low levels of drugs, food and pesticides
(additionally studies for construction of flow-through voltammetry electrodes).
- Development of continuous flow systems and dedicated equipment in automatic laboratorial
determinations or in on-line control of industrial processes (multi-commutated flow methodologies and new concept of multi-pumping flow analysis).
.
Laboratório Associado para a
Química Verde
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AREA 5
CATALYSTS, SOLVENTS AND NON-TOXIC COMPOUNDS
- A new enantioselective inverse phase transfer catalysis reaction for the Markovnikov hydration
of double bonds by an oxymercuration-demercuration reaction with cyclodextrins as catalysts
was developed.
- Sugars were used as sources for new chiral materials and microwave radiation was used in
accelerated synthesis and/or in solvent free reactions.
- Hydrogenation of limonene and the synthesis of new polyurethanes were performed in supercritical
CO2.
- Quantitative structure-property relationships between the melting point and density of ionic
liquids was established, providing the design/synthesis of novel imidazolium and guanidinium
based ionic liquids. A series of ionic liquids based on imidazolium and guanidinium cations were
used as neoteric solvents in alkene asymmetric epoxidation.
- Metals (Pd and Ni) and transition metal complexes (Mn, Cu and VO) immobilised in several
supports (carbon materials, zeolites, clays and silica materials) and dispersed in PDMS matrix
were tested in reductive and oxidative catalytic reactions.
- Novel methods for the preparation of Pt and Pd nanoparticles with catalytic applications were
developed, as well as their immobilisation on graphite.
- Application of Monte Carlo methods to model the spontaneous formation of silica nanoparticles
during the early stages of zeolite synthesis yielded results that are in close qualitative agreement with the experimental observations.
- Design/optimisation of new leads for the treatment of cancer, malaria, AIDS and hypertension
have been done by molecular modelling and computer simulation, as well as determination of
inhibition mechanisms of RNR enzyme by anti cancer drugs.
- Crystallographic and functional studies on several metalloproteins, on proteins involved in cellulose (polysaccharyde) recognition and degradation and on proteins from the glyoxalase pathway.
Laboratório Associado para a
Química Verde
RESEARCH REPORT
2005
Part B
2
ANALYTICAL AND BIOORGANIC CHEMISTRY
Head of Laboratory: Elvira Maria M. Gaspar, Assistant Professor
Staff Members:
Angela M.S. Relva
Assistant Professor
Marco Diogo R.G. da Silva
Assistant Professor
José Luiz Capelo-Martinez
Assistant Researcher
Post-Doct Fellows:
Raquel Rial Otero
Ph.D. Students:
Laila H. Ribeiro
Number of articles in scientific journals : 7 (1-7)
Number of patents: 1
Number of Ph.D. Thesis: 1
Scope and description of on-going research
During 2005 the group was mainly focused in some environmental problems. New sample treatments were
applied to solve analytical problems. It was studied the hopanoid composition of sediments from lower
Tagus basin. To decode the process of host tree selection by herbivore insects (a collaborative program
with environmental scientists), namely the Mediterranean defoliator winter pine processionary moth, a
profile of pine varieties cultivated in Portugal was achieved as an indicator for insect attack in the environment. New natural oligossacharides with potential pharmacological properties were isolated from an
European Convolvulaceae plant. New chiral HPLC and enantioselective GC separation methods of an active
natural compound (also present as a fragment group of complex molecules) has been developed as new
separation techniques. Also new clean analytical methodologies for organometallic compounds speciation
in food and environmental samples were used.
Future Activities
During 2006 the group will continue to be involved in environmental / human health problems. Human health
will be focused by food analysis, monitoring the volatile profile to access the quality of important food matrixes
and also by the analysis of human volatile compounds, potential bio-markers in cancer disease. Extraction of
added value compounds from orange peel wastes and the potential of fruit stones to be used as adsorvents for
the selective removal of organic compounds will be topics of interests. Clean analytical methodologies for
organometallic compounds speciation in food and environmental samples. The creosote volatiles and other
environmental specific problems will be studied by 1D GC-TOF-MS and also by GCxGC-TOF MS in a collaborative program. New analytical methodologies to control the quality of food samples, specially antibiotics and
pesticides will be developed. Furthermore new methods for rapid protein identification based on MALDI-TOFMS will be assayed.
Laboratório Associado para a
Química Verde
3
PHOTOCHEMISTRY AND SUPRAMOLECULAR CHEMISTRY
Head of Laboratory: Fernando Pina, Full Professor
Staff Members:
A. Jorge Parola
Assistant Professor
J. Carlos Lima
Assistant Professor
M. João Melo
Assistant Professor
Carlos Lodeiro
Assistant Researcher
Alexandra Bernardo
Associate Professor
Micaela de Sousa
Invited Assistant
Ana Claro
Invited Assistant
Post-Doct Fellows:
Berta Covelo, Damián Fernández
Ph.D. Students:
Margarida Moncada, Sérgio Alves, Letícia Giestas,Carlos Pinheiro, André Vidal Pinheiro, Joana Ferreira
Project Grantee:
Bruno Pedras
Number of articles in scientific journals: 10 (8-17)
Chemosensors
In the framework of the Project POCTI/47357/QUI/2002, “Chemosensors based on polyamine receptors
containing fluorophoric units”, we have privileged our collaboration with the group of Prof. Enrique GarciaEspaña from the University of Valencia, Prof. António Bianchi e Andrea Bencini from the University of Florence,
as well as Prof. Jaume Casabó e Lluis Escriche, from the University Autónoma de Barcelona, and the group
of Prof. Rufina Bastida, from the University of Santiago de Compostela. In the case of the three first collaborations,
the chemosensor is constituted by a polyamine receptor linked to a fluorophoric unit; in the case of the last
two collaborations (papers 11, 16 and 17), the receptor is a macrocycle having oxygen or sulphur atoms in
order to modify the coordinative properties and extend these chemosensors to other target metal ions or
organic anions different from those achieved through the polyamine receptors. In the case of paper 1 the
concept of chemosensor has been extended to a catalytic system. The ATP hydrolysis takes place in a
system containing the chemosensor coordinated to Zn(II), and having non-coordinated Zn(II) in solution.
In the framework of the Project POCI/55519/QUI/2004, “Towards New Supramolecular Devices: from new
synthetic methods to multifunctional applications” we have been working in the developement of new
chemosensors provided with nitrogen-sulphur and nitrogen-oxygen atoms in their structure for the detection of
heavy metal ions, such us Hg(II), Cd(II) and Pb(II), as well as transition metal ions such as Zn(II), Ni(II), Cu(II)
and Co(II). This subject was developed mainly in collaboration with the group Prof. Jaume Casabó and Lluis
Escriche, from the University Autónoma de Barcelona, Spain (Papers 11 and 16), where a nice example of a
Laboratório Associado para a
Química Verde
4
square planar Ni(II) complex whas studied, whith colour tuning upon anion (Cl-, Br-, I-, F-, CN-) or neutral
molecules (water, methanol, acetonitrile, pyridine, bipyridine) coordination (Figure 1). This behavior allows
naked-eye detection of anions and could be used to develop new devices for detecting, for instance, iodide
ions in mixed halide solutions (Paper 9). Upon synthetic modification (addition of an anthracene unit) the
chemosensor was converted into an emissive system for Pd(II) and Cu(II) detection. (Paper 11).
4+
4+
4+
4+
2,2’-Bipy
4
4+
-
F
4+
-
Cl
Py
4+
-
H2O
Br
4+
-
CH3CN
I
S
N Ni
N
S
2+
4
Figure 1 – Color tuning upon coordination of neutral molecules of anions to a Ni(II) complex.
In collaboration with the group of Prof. Rufina Bastida, from the University of Santiago de Compostela, an
example of Energy Transfer from a macrocyclic to an Europium(IIII) or a Terbium(III) metal center was studied
(Paper 10). With the group of Prof. Manuela Raposo, from the University of Braga, Portugal, we have started
an intense study of new bisthiophene-phenanthroline systems and their Ru(II) complexes; part of this work
was presented in the 8th National Conference on Photochemistry in Coimbra.
With the aim of obtianing new “multifunctional applications”, interaction of new pyrene derived ligands with
barbituric, parabanic and cianuric acids was carried out (work in press).
The group was also involved in the study of the coordinative properties of several 1,10-phenanthroline derivatives
in the solid state, following a crystal engineering approach (paper 8). This area was developed in collaboration
with the group of Professor E. Vázquez-López from the University of Vigo, Spain. Into this collaboration one
Postdoctoral student, Berta Covelo spent one year, since September 2004 and October 2005, in our group
integrated as researcher in the project “Towards New Supramolecular Devices: from New Synthetic Methods
to Multifunctional Applications”.
Ionic liquids
This work has been carried out in the framework of POCI/QUI/57735/2004, “Photochromism of Flavylium
Compounds in Water/Ionic Liquid Biphasic Systems”.This project started on the 1st July 2005.
Two synthetic approaches are being pursued: (i) synthesis of flavylium salts with chloride counter-ions in
gram scale quantities as a source for ionic liquid synthesis; (ii) synthesis of flavylium salts with anionic
substituents (e. g., sulfonate goups).
Studies on the behaviour of flavylium salts in C8MIMPF6 and C10BMIMPF6 were also started.
Flavylium chemistry
This is a recurring subject in the research of our group. The study of the colour, and the possibility of changing
it by pH, light or electric inputs have important applications in several fields from the art pigments to the food
dyes. In particular, we are interested in the use of flavylium compounds to carry out cycles for write-readerase at the molecular level. A step further towards applications was done during the last year, through the
incorporation of flavylium compounds in water permeable matrices (papers 12 and 13). Within our attempts to
develop the concepts of multistate/multifunctional systems, we have reported an example of two connected
flavylium networks operating through different algorithms, paper 14, see Figure 2.
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Química Verde
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Figure 2 - Network of chemical reactions involving two flavylium compounds
Anthocyanin blues
NH2
4
In this project, we aim to explore the use of natural antioxidant compounds, the anthocyanins, as well as their
pH jump
pH jump
pH=1.0
7.7
synthetic analogs, the substituted flavylium salts, as sources
for blue, redCtH
and
violet colorantspH=Ctand
pigments.
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Write Unfortunately, the chemistry
In modern world, color is ubiquitous, creating a bright and colourful environment.
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used to synthesize and to process colours is not always environment friendly.
“Greener”
colorants
must hν, ∆
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hν, ∆
hopefully be developed in order to guaranty a more sustainable development.
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hν
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pH= 0
pH= 1
AH+
pH= 7.7
3
+
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B/Cc
Blue pigments based on zeolite inclusion complexes withAHanthocyanin
or flavylium salts
pH jump have been prepared
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and their photochemical and thermal stability will be studied and compared
Maya
2 with those displayed by
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hν
1
Blue, one of the most stable known blue inclusion complexes.
pH jump
pH=12.0
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This project involves a feedback approach (experimental data Û theoretical
calculations),
that is beeing used
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to gather a deep insight on the mechanisms that controlpH=0color and
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pH=1.6
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pH jump
pH= 6.0
B/Cc
It is a REQUIMTE funded project that joins the CQFB group and its knowledge of anthocyanin
and flavylium
unlock
salts chemistry and the CEQUP group with its expertise in theoretical calculations
and
predictions.
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1
The color in Art
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4
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1
+
5
pH jump
i) the study of the colour of medieval Portuguese manuscript illuminations; the Book of Birds (O livro das
Aves) and The Apocalypse of Lorvão (O Apocalipse do4'-acetamidoflavylium
Lorvão), considered a national treasure were two of the
Manuscripts that came to our Faculty for a more detailed study, namely with non invasive techniques, such
as microXRF and micro fluorescence emission.
The more relevant results obtained were submitted for publication.
Laboratório Associado para a
hν, ∆
hν
CH3
Some of the more recent projects in the area of Conservation ofO the
Cultural Heritage have acquired a nice
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pH= 6.0
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Ct
momentum, in particular:
Ct
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pH=12.0
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ii) The study of the photochemistry / photophysics of ancient and historical dyes was also a success with the
discovery of a new natural flavylium in Dragon’s blood resin from Dracaena Draco, and the full characterization
of its network of pH dependent chemical reactions. This work was also submitted for publication.
iii) Finally, the study of modern synthetic polymers used in Modern Art, in the framework of the PhD Project,
Liaisons Dangereuses, where an interdisciplinary team was created, with Polymer Scientist Ana Ramos, Art
Historian María Jesús de Àvila and Conservation Scientists Maria João Melo and Joana Ferreira for a better
understanding and preservation of Portuguese Modern Art. The artists chosen for the case studies are Joaquim
Rodrigo, Ângelo de Sousa and Lourdes Castro. The work obtained from the first analysis and characterization
of the paintings by Joaquim Rodrigo will be presented in two international Meetings, at the MoMA, New York
and at the Tate Modern, London; the work presented at the Tate will be the subject of a publication by the
Getty Conservation Institute.
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ORGANIC SYNTHESIS AND CHEMISTRY OF NATURAL PRODUCTS
Head of Laboratory: S. Prabhakar, Full Professor / Ana M. Lobo, Full Professor
Staff Members:
Pedro Abreu
Assistant Professor
Paulina Mata
Assistant Professor
Manuela Pereira
Assistant Professor
Ana M. Lourenço
Assistant Professor
Paula S. Branco
Assistant Professor
Luisa M. Ferreira
Assistant Professor
João Aires de Sousa
Assistant Professor
Maria Manuel Marques
Assistant Researcher
Post-Doct Fellows:
Yuri Binev, Sunil Gupta, Qingyou Zhang, Yong Hong Liu, Susan Matthew, Mário Gomes, Ricardo Mendonça
Ph.D. Students:
Paulo Glória, Luís Pinto, Vasco Bonifácio, Rita Noronha, Susana Gaudêncio, Goncalo Carrera, Diogo Latino
Valdemar Figueira
Project Grantee:
Marta Vilela, Artur Abreu, Carla Macedo, Catarina Soares
Number of articles in scientific journals: 15 (18-32)
Number of articles in books: 1
Number of M.Sc. Thesis: 1
ORGANIC SYNTHESIS AND CHEMISTRY OF NATURAL PRODUCTS
Debromoflustramine B is a biological active alkaloid and its synthesis was recently reported by our group,
starting with commercially available L-tryptofan. The method consisted in the stereoselective and chemoselective
introduction of the 3,3-dimethylallyl chain at C3a of the known 1,2,3,8-tetrahydropyrrolo[2,3-b]indole skeleton
with formation of two diasterioisomers with 36% ee. However, only the minor isomer could be converted into
the final alkaloid. We envisaged starting from D-tryptofan and follow the same route. After performing several
experiments, we observed the same behaviour and a mixture of two diasteriosomers were formed with 35%
ee, in which the major isomer - (2R,3aS)-methyl-1-acetyl-1,2,3,3a-tetrahydro-3a-(3-methylbut-2-enyl)pyrrolo[2,3b]indole-2-carboxylate could be used for the total synthesis of the alkaloid.
Novel rearrangements of the indole framework were studied. N-alkylation of N-acetyl L-tryptofan methyl ester
with 3,3-dimethylallyl bromide gave (S)-methyl 2-acetamido-3-(1-(3-methylbut-2-enyl)-1H-indol-3-yl)propanoate
with 78% yield. This product was treated with BF3OEt2. Several experiments were carried under diverse
conditions and migration of the allylic chain to the C2 position was observed. This study enabled the synthesis of crucial intermediates that bear the skeleton of important biologically active alkaloids, as well as furnished information about the reaction mechanism. Theoretical computer calculations of the putative transition states point out towards a considerable ionic character of this rearrangement.
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A new enantioselective Inverse Phase Transfer Catalysis (IPTC) reaction for the Markovnikov hydration of
double bounds by an oxymercuration-demercuration reaction with cyclodextrins as catalysts was disclosed.
Moderate e.e. (up to 32%) and yields (14-60%) were obtained for allylic amines and protected allylic alcohols
as starting materials. The alkenes reacted with different molar quantities of mercuric salts in a water/nhexane mixture (1:1) containing a phase transfer catalyst, resulting on the formation of a mercury species.
The correspondent alcohol was obtained after demercuration with alkaline borohydrate. The best conditions
determined for the allylic ether where further applied for the protected allylic amines.
Although there were previously reported methods for enantioselective hydration of allylic alcohols, no equivalent method was up-to-date available for the direct enantioselective hydration of allylic alcohols. The present
method was able to induce enantioselectiviy to the direct hydration of allylic amines.
Future trends:
The method will be applied to the semisyntesis of several quinolizidic alkaloids.
Retrosynthetic analyses of halipeptins structure acknowledged a oxazoline tripeptide fragment, which included unnatural isoleucine-like residue, and a polysubstituted decanoic acid fragments as major building
blocks to be synthesized. Synthetic strategies towards isoleucine-like residue took place along with its
incorporation in the tripeptide structure in large scale. Building blocks’ coupling assembled the required
carbon structure and, after some more chemical manipulations, halipeptin A was obtained along with some
epimers.
Related to benzopyran-derived bioactive molecules a new classes of compounds targeting the HIF-1 pathway
were discovered and lead optimization took place in collaboration with Prof. Van Meir at Emory University
(Atlanta, USA). Structure-activity relationship studies allowed molecules structural simplification and identified functionalities with strong impact in molecules activity.
The Brazilian plant species of Solanum cernuum V. (Solanaceae) e Cissus sulciccaulis B. (Vitaceae) are
currently used in popular medicine. Among other therapeutic activity S. cernuum exhibit potent antitumoral
activity and C. sulciccaulis is important in the treatment of arthritis and beberi diseases. The dichloromethane
and methanol extracts of these species proved to have low toxicity. The methanol extract (aerial parts) and
dichlomethane (leaves) of S. cernuum were studied for their validation. The methanol extract has 10% of
glicosyl alkaloids and peptides. The dichloromethane extract is composed of alkanes (C25-C34), steroids (were
the most abundant is cycloeucalenone) and the xanthophyll lutein. The dichloromethane extract of C.
sulciccaulis contains 1,76% of fatty acids (C14-C24), 1,71% of sitosterol, 0,14% of other terpenoid compounds
and 0,12% of long chain alcohols.
Future trends:
The work will continue with further identification of steroid and terpenoid fractions of S. cernuum and C.
sulciccaulis, together with the study of the methanol extract of C. sulciccaulis.
The research activity has been focused on the search for bioactive natural products of plant origin. The
evaluation of the anti-inflammatory and antioxidant activity of a Cuban medicinal plant extract (Pedilanthus
tithymaloides) was carried out, and the identification of the corresponding active principlkes is in progress.
From a medicinal plant (Ozoroa insignis) collected in Guinea-Bissau, a library of cytotoxic phenolic compounds has been characterized. The scientific collaboration with the University of Monastir (Tunisia) has been
developed, leading to the isolation of new ceramides, and antioxidant phenolic glycosides from Tunisian
plants. In collaboration with the Faculty of Pharmacy of Lisbon, new cytotoxic diterpenes with reversal effect
on multidrug resistance, have been isolated from Euphorbia species of Portuguese origin.
Architectural drawings on historical archives are often photoreproduced. When based in diazonium salts the
discoloration problem is frequent. An interesting aspect of drawing collections from DGEMN is the presence
of interleaving sheets of paper. This aspect gave us material to analyse the migrating coloured species
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without destruction of the museum objects. Organic extracts of the interleaving sheets were analyzed, with
aim to study the migrating species, and the only compound isolated and characterized was resorcinol. HPLC
studies of extracts of small samples of discoloured interleaving sheets and photoreproductions showed resorcinol as the only common compound. When a drawing in unable to promote the discoloration of adjacent
interleaving sheets the resorcinol is absent.
Future trends:
After the confirmation of these observations in a larger number of cases, a method for the rapid identification
of a ‘dangerous’ drawing by the use of HPLC can be applied to each new specimen before its introduction on
the archive to assess its risk to degrade the rest of the collection.
3,4-Methylenedioxymethamphetamine (MDMA or “Ecstasy”) is a widely abused, psychoactive recreational
drug. There are growing evidences that the MDMA neurotoxic profile may be highly dependent on its hepatic
metabolism, leading to the production of reactive catechols, catechol thioethers, and quinones. In this study
the electrochemical oxidation-reduction processes of MDMA main human metabolites, obtained by chemical
synthesis, were evaluated by cyclic voltammetry. Parallely the neurotoxicity of á -methyldopamine (á-MeDA),
N-methyl-a-methyldopamine (N-Me-á-MDMA) and 5-(glutathion-S-yl)- á -methyldopamine [5-(GSH)-á-MeDA]
was also evaluated in cortical neuronal cultures2 and the results correlated with their oxidation-reduction
potential. The lower oxidation potential observed for the catecholic thioethers of á -MeDA and N-Me- á -MeDA
correlated with the higher neurotoxicity of these adducts.
Future trends:
Synthesis of the monomethoxylated cysteine and glutathione adducts of 4-hydroxy-3methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) will be conducted for
neurotoxic evaluation.
Quantitative structure-property relationships (QSPR) were established for the melting point and density of
ionic liquids. These were applied to the design of new imidazolium and guanidinium ionic liquids that were
synthesized.
The chirality codes previously developed in the group were applied to the prediction of the enantiomeric
excess in a combinatorial library of enantioselective catalytic reactions. The same codes were applied to train
neural networks in order to automatically assign absolute configurations from 1D NMR data.
A new method was developed to represent chemical reactions with no specification of the reaction center,
from the difference of MOLMAP molecular descriptors of products and reactants.
Future trends:
Application of the MOLMAP representation of chemical reactions to the genome-scale classification of metabolic reactions. Development of QSAR models for the prediction of biological activity, biodegradability and
toxicity. Further development of QSPR models for ionic liquids. Application of the SPINUS system for the
simulation of 1H NMR spectra to the NMR-based classification of chemical reactions.
Research activities concerning the specification of stereogenic units in organic chemistry, and particularly in
the development of the Cahn-Ingold-Prelog System in order to improve its logic, consistency, scope and
applicability were conducted. In this context a collaboration was established with IUPAC (International Union
of Pure and Applied Chemistry) as a researcher of Requimte (Prof. Paulina Mata) belongs now to the Advisory
Subcommittee of the Chemical Nomenclature and Structure Representation Division (VIII) http://www.iupac.org/
organ/members/m/mata.html.
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In 2005 this collaboration was very intensive, because of the ongoing revision of IUPAC recommendations on
specification of molecular configuration to be included in the next IUPAC Book on Nomenclature of Organic
Compounds.
Molecular Gastronomy was also developed, through international connections with researchers in the area,
particularly Dr. Hervé This (Lab. de Chimie des Interactions Moléculaires Collége de France et Lab. de Chimie
Analytique - Institut National Agronomique Paris Grignon (INA P-G)), national collaborations (Prof. Maria da
Conceição Loureiro Dias – Dep. de Botânica e Engenharia Biológica do Instituto Superior de Agronomia) and
also the participation in the “Cours the Gastronomy Moléculaire 2004/2005 “ (INA P-G) .
Work continued in the in science teaching and divulgation front which hopes to contribute to improve science
teaching practices, to motivate students to continue their studies in science and to develop the general
scientific culture in the public at large. This involves working with other educational levels (production of
teaching aids and teacher training) and collaboration in research activities with other institutions, particularly
Instituto Superior de Psicologia Aplicada. Concerning science divulgation activities special reference should
be made to a) the collaboration with Ciência Viva and Pavilhão do Conhecimento by the participating in
demonstrations and in international projects and b) the publication of science divulgation articles in the
Portuguese press.
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SELECTIVE SYNTHESIS AND STRUCTURAL CHEMISTRY
Head of Laboratory: Maria Teresa Barros, Associate Professor
Staff Members:
Maria Teresa Avilés Perea
Assistant Professor
António Gil de Oliveira Santos
Assistant Professor
Eurico José da Silva Cabrita
Assistant Professor
Marta Morais Saraiva de Andrade
Assistant Researcher
Ana Maria Madeira M. Faísca Phillips Principal Researcher
Post-Doct Fellows:
Krasimira Petrova, Snezhana Bakalova
Ph.D. Students:
Vitor João Salgueiro Rosa, Paula Correia da Silva, Maria João Morgado, Filipe José dos Santos Duarte,
Paula Alexandra Carvalho Rodrigues
Number of articles in scientific journals: 6 (33-38)
The principles of Green Chemistry as applied to organic synthesis (economy and efficiency) needs advanced principles of organic synthesis applied to the efficient production of complex organic compounds from
renewable natural (chiral) starting materials. We have continued to investigate the reactivity of saccharose and to apply our results to the study and application of sugar as a chiral auxiliary for asymmetric
synthesis. Our final goal is to apply this chemistry to diverse problems under unusual but ecologically
favourable reaction conditions.
The selective derivatisation of carbohydrates with unsaturated moieties is one of the easiest routes for the
preparation of polymers and for the construction of asymmetric molecules using carbohydrates as chiral
auxiliaries. We have developed an efficient strategy to attach vinylic groups and dienes to the sugar nucleus
(D-glucose, D-xylose and sucrose) by two different procedures (esterification and Wittig olefination), both
under microwave irradiation. On the other way, Wittig reaction between O-formylated sugars and stabilised
phosphoranes was performed in dry medium, with the formation of the isomer E as major product. By studying the application of microwave technology, we intend to develop cleaner and more efficient methods for
synthesis, from a time and energy view point, in an attempt to contribute to a sustainable development. Water
soluble dienes are also prepared by exposure of the hydroxyl groups of the sugar auxiliary.
The chemistry of azides includes a useful synthetic applications in the preparation of 1,2,3-triazolines via 1,3
dipolar cycloaddition reactions. We have reported the preparation of several azides of low molecular weight,
which have been useful for this work.
We have been successful in the selective connection of acrylate to the sugar molecules (in some positions) and the copolymerisation with simple acrylates to form more or less dense sugar polymers. By
attaching the sugar to a polymer it is possible to alter some of its properties.
In collaboration with a research group in Biothecnology area we are preparing L-arabinose selectively
marked in C2. The increasing interest in the production of ethanol from wood pulp (cellulose) has generated
studies related to the metabolism of L- Arabinose (the second most abundant pentose in cellulose) by
yeasts, which are excellent fermentative microorganisms. In order to elucidate the metabolic pathway of
arabinose in selected yeasts. The technique to be used is in vivo NMR using C2-13C labelled L-arabinose.
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Asymmetric synthesis and homogeneous catalysis The syntheses of chiral bis(oxazolines) derived from
(R,R)-(+)-tartaric acid and their application as ligands in the enantioselective Michael addition of diethylzinc to
enones, already underway during 2004, was completed. The results were published.
During this period, methodology for the syntheses of novel chiral diamines and sulfur-containing compounds
derived from tartaric acid was also developed and continued. The possibility of using these compounds either
as chiral organocatalysts or as ligands for metal-catalyzed processes, was investigated. A bis-Nmethylmethanamine derivative was found to be an organocatalyst for the diastereoselective and asymmetric
Michael addition of cyclohexanone to b-nitrostyrene. The results were published in 2006.
Stereoselective behavior of chiral a-haloacyl compounds in nucleophilic substitution reactions,
under dynamic kinetic resolution (DKR) conditions. We studied, theoretically, the stereoselective behavior of nucleophilic substitution reactions of ?-haloacyl compounds, using a chiral auxiliary approach, based
on ephedrine derived imidazolidinones, under DKR conditions. New chiral auxiliaries were proposed and
some derivatives were synthesised and tested, confirming the theoretical previsions of expected high final
diastereoisomeric excesses.
Catalysts in asymmetric synthesis: A complexation study.
The use of Lewis acids (LA) to enhance the reactivity and stereoselectivity of a number of reactions involving
N-acyloxazolidinones and N-acylimidazolidinones is well known. For some transformations the stereoselectivity
induced by the LA complexation can’t be entirely explained by the accepted models. We have undertaken a
NMR and molecular modeling study of this type of complexation with different LA. Since the molecules in
question have two key Lewis base sites, several solution complexes are possible, including chelated ones,
which render the study very complex. In an attempt to simplify it, we have prepared several model compounds
and studied their complexation with non-chelating (BF3.OEt2) and chelating (AlEtCl2, TiCl4 and Mg(ClO4)2)
LAs. Only for Mg(ClO4)2 evidence for chelated forms in solution was found. This information can be of major
importance in the revision of the accepted mechanisms for all reactions where N-acyloxazolidinones and Nacylimidazolidinones are used, prompting us to the proposal of new mechanistic pathways.
Asymmetric additions to alkenes. Our interest in this subject has been mainly related with Diels-Alder
reactions and the addition of electrophilic species (e.g amines) to unsaturated N-acyloxazolididinones and Nacylimidazolidinones. The accepted mechanism, proposed by Evans, more than 20 years ago is unable to
explain many unexpected experimental observations made during these years. Based on our previous work
on DKR reactions, we started the development of a full theoretical and experimental study (based on NMR
techniques), with the aim of clarifying possible mechanisms and to propose structural variations, able of
increasing the final diastereoisomeric excesses. Our studies brought us to a new proposal, which can explain
the observed stereochemistry, both in Diels-Alder and in electrophilic additions. We also studied similar
additions to alkenes connected to different chiral auxiliaries (as, for instance, Ethyl-S-lactyl acrylate) with
more flexible structures, showing that even in these cases no chelated transition states are theoretically
expected (results published in 2006).
Intramolecular asymmetric aldol reactions. Based on our previous experimental results, we are currently
studying the mechanistic aspects of asymmetric aldol reactions, using theoretical and experimental tools.
Our aim is the understanding of the large amount of literature information on chiral and achiral systems. Until
the moment, in spite of many reports describing the use of chiral catalysts and auxiliaries, there is no real
understanding of the factors governing the mechanistic pathways of even the achiral processes. Our first
studies indicate a strong dependence on electronic effects which, if confirmed, can reduce substantially the
variety of structures able to work as starting materials.
Application of NMR techniques to the study of intermolecular interactions. The complementary between the information obtained from NOE and relaxation studies with that from diffusion NMR was explored in
the study of 1-buthyl-3-methylimidazolium (BMIM) ionic liquids. The determination of internuclear dis-
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tances, relaxation rates and relative strength of ion pair association have been interpreted in terms of rotational motion, molecular structure and molecular properties and allowed the proposal of a solvation model for
[BMIM]PF6 dimethylsulfoxide solutions for a wide range of concentrations.
The study of the molecular determinants of ligand specifity in family 11 Carbohydrate Binding Module (CBM11) is being conducted by NMR Saturation transfer in combination with ligand titrations of the
protein. The protein was labelled with 13C and 15N and several experiments are being conducted in order to
study the enzyme complexed with its carbohydrate ligands;
In Organometallic chemistry, we have continued with the synthesis of cobalt (II) complexes of the general
formula CoX2( a-diimine) where X= Cl, or I and the a-diimines are bis(aryl)acenaphthenequinonediimine) (ArBIAN) and 1,4-diaryl-2,3-dimethyl-1,4-diaza-1,3-butadiene (Ar-DAB) in order to study their structural and
magnetic properties. The synthesised complexes are, [Co(Ph-DAB)Cl2], 1a; [Co(o,o’,p-Me3C6H2-DAB)Cl2], 1b;
[Co(o,o’ –iPr2C6H3-DAB)Cl2], 1c; [Co(o,o’,p-Me3C6H2-DAB)I2], 1’b; [Co(o,o’-iPr2C6H3-DAB)I2], 1’c; [Co(o,o’,pMe3C6H2-BIAN)Cl2], 2b ; [Co(o,o’-iPr2C6H3-BIAN)Cl2], 2c; [Co(o,o’,p-Me3C6H2-BIAN)I2], 2’b; [Co(o,o’-iPr2C6H3BIAN)I2], 2’c. They were characterised by elemental analyses, mass spectrometry, IR spectrometry, and
EPR, in some cases we were able to grow crystals suitable for X-ray structural determination. X-band EPR
measurements in polycrystalline samples performed on all compounds indicate high-spin Co(II) ion (S=3/2) in
an axially distorted environment. Single crystal experiments on compounds 1b and 1c allowed us to evaluate
the orientation of the g-tensor in the molecular frame. This work is almost completed and it will be submitted
soon for publication.
2006 activities to be developed
Several polymers which we have recently synthesised and are based upon saccharose are being studied for
their biodegradability. This work is being carried out in collaboration with another University.
We are interested to use alternative energy source (microwaves) and alternative reaction conditions (use of
ecologically friendly solvents such as water or no solvent).
In a related project we pretend to study the use of concentrated saccharose solutions in accelerating
stereoselective reactions. It is hoped that the structurally ordered sugar solution will both direct the reagents
and accelerate the reaction in such a way that the product is obtained with high stereoselectivity and affords
optically active products. Pericyclic reactions which can be carried out under aqueous conditions should be
influenced by a chiral environment and organization of the reagents should occur.
In another part of this project we hope to study the glycosylation of steroids. A number of important groups
of drugs are glycosides. The sugars seem to play a key role in the interaction of the drugs with their receptors
All the synthetic methodologies will be designed in order to develop cleaner technologies as a major
emphasis in green chemistry. Among the several aspects, when is possible, the synthetic methodologies will
be performed in solvent free conditions, or water-based medium, and/or under microwave radiation as
alternative energy source.
In another project, we intend to produce environment-friendly, ready-biodegradable complexones at a
low cost, which may compete at industrial scale production with the well established and cheap synthetic
NTA and EDTA compounds. For this purpose, the synthesis pathway will be based mainly in the life cycle;
block units of stereo specific amino acids will be used for asymmetric synthesis of natural
aminopolycarboxilates ligands (or derivates) potentially better biodegradability (i.e. they will be decomposed into non-toxic compounds, environmentally benign substances). In addition, the synthesis will be
designed in order to be performed in a water-based medium, for reducing the use of organic solvents.
Collaboration in a European project related to the synthesis and physical properties of azides will continue.
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Research aimed at the development of novel organocatalysts or ligands for metal-catalyzed asymmetric reactions, particularly using amino acids as chiral synthons, is currently underway. Concerning the Lewis
acids - carbonyl compounds complexation studies, we plan to publish the results obtained in the last years
and to continue to use the information obtained with these studies in the clarification of the mechanisms
involved in asymmetric Diels-Alder reactions and other additions to double bonds.
We expect also to continue our work in order to clarify several aspects dealing with intramolecular aldol
reactions based on molecular modelling, NMR and synthetic work. Our aim is to understand the achiral
systems and to concentrate efforts in understanding part of the literature information and, in special, the
experimental results obtained in our research group during the last couple of years.
We will continue to apply NMR techniques to the study of intermolecular interactions, especially ionpair strength in the BMIM family of ionic-liquids and also solute-solvent interactions. The study of the molecular determinants of ligand specifity in CBM11 will continue. The 2D 15N-HSQC will be assigned using double
and triple resonance NMR experiments in order to identify the residues involved in ligand recognition. As
before this work continues in strong collaboration with groups integrated in other research themes: Protein
Crystallography and Biomimetic Systems and Simulation.
We will continue with the synthesis of a-diimine cobalt complexes, trying to introduce ligands such as
indenyl, cyclopentadienyl, alkyl, and hydride. New a-diimine ligands will be synthesised as well as new
Schiff base ligands, The complexing properties of the newly synthesised ligands towards metal cations of
environmental, analytical or catalytic interest, such as Co(II), Ni(II), Pd(II), Ag(I), Cu(I), Zn(II), Cd(II) and
Hg(II), will be tested.
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PHYSICAL ORGANIC CHEMISTRY / RADICAL CHEMISTRY
Head of Laboratory: Abel J. Vieira, Associate Professor
Staff Members:
João Paulo Noronha
Assistant Professor
Ph.D. Students:
Pedro Manuel C. C. P. dos Santos, Mónica Pombinho de Matos, João Adalberto A. Lourenço
Project Grantee:
Patrique Nunes
UndergraduateStudent:
Marisa de Barros Sousa e Silva
Number of articles in scientific journals: 3 (37, 39,40)
Radical reactions of DNA bases and other biologically relevant compounds. Effect of antioxidants.
Radical oxidation of caffeic acid, cinnamic acid and tetrahydrocannabinol. Identification of final products.
Relative antioxidant properties of the above referred compounds.
Synthesis of new precursors of alkyl radicals. Radical alkylation of xanthines.
Evaluation of the in vitro antioxidant activity of non-steroidal anti-inflammatory drugs (NSAIDs)
Chiral Quantitative Structure-Property Relationship / Counterpropagation Neural Networks study of enantiomeric separation on two HPLC columns
Natural Products Research – Portuguese Autochthon Plants – Bioactivity Studies
Arson analysis
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CHEMICAL ENGINEERING SCIENCE
Head of Laboratory: Manuel Nunes da Ponte, Full Professor
Staff Members:
Pedro Brito Correia
Invited Full Professor
João Goulão 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
Assistant Researcher
Post-Doct Fellows:
Adrian Oehmen, Ewa Bogel-Lukasik, Filomena Feitas, Gilda Carvalho, Isabel Esteves, José Castanheiro,
Luisa Serafim, Marta Eiroa, Paulo Lemos, Raquel Fortunato, Rui Ruivo, Svetlana Lyubchik , Teresa Casimiro,
Vesna Najdanovic-Visak, Vitor Alves
Ph.D. students:
Ana Teixeira, Carla Brazinha, Carla Portugal, Carlota Veiga de Macedo, Cláudia Galinha, Cristina Matos,
Graça Albuquerque, João Dias, João Fernandes, José Luís Santos, Liliana Guerreiro, Márcio Temtem, Maria
Teresa Viciosa, Mariana Sousa e Costa, Mário Eusébio, Patrícia Oliveira, Pedro Vidinha, Rafal Bogel-Lukasik,
Sofia Nunes Barata
Project grantees:
Ana Rita Brás, Angela Pereira Machado, Ricardo Couto
Number of articles in scientific journals: 48 (5, 6, 38, 41-85)
Number of patents: 5
Number of articles in books: 9 (2-10)
Number of Ph.D thesis: 5
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Scientific interests
The scientific interests of the Chemical Engineering Science Area in 2005 spanned a wide range of subjects:
- Adsorption processes
- Biocatalysis in nonaqueous solvents
- Bioprocess modelling, monitoring, optimisation and control
- Bioremediation
- Chemical reaction and supercritical carbon dioxide
- Depolymerisation studies and characterisation of biopolymers
- Dynamic modelling of supercritical fractionation processes
- Drug controlled-release systems
- Dynamical changes upon crystallization in a biopolymer
- Extraction of biopolymers from industrial wastes for edible films applications
- High-pressure NMR
- Membrane Processing and Monitoring
- Modelling of nanofiltration processes
- Polymer processing and membrane formation in clean solvents
- Polymeric Catalytic Membranes & Heterogeneous Catalysts
- Polymer dispersed Liquid Crystal formation in both conventional and scCO2 media
- Supercritical fluid extraction of liquid mixtures
- Thermodynamics of Ionic Liquids
- Viscous fluid mixing. Experiments and CFD
The interplay of these multiple research interests resulted in a research effort leading, as an ultimate, strategic goal, to the study of Process Integration. The main areas of research can be grouped under three headings:
- Clean Solvents and Clean Processes,
- Process Simulation and
- Polymeric Materials
The above-described research activities led to collaborative efforts that joined different groups, in order to
develop
- Integration of Processes
RESEARCH ACTIVITIES IN 2005
Clean Solvents and Clean Processes
Adsorption processes
1- Activated carbon metal supported catalysts were tested on chlorobenzene hydrodechlorination. The types
of materials used in this study were the following: synthetic activated carbon (SCN) and carbonaceous porous
supports obtained from polystyrene-based organic salts pyrolysis.
The introduction of the metal (palladium and nickel) on this support was performed using the method of
reductive adsorption, discarding the need of subsequent reduction (in the case of palladium). These catalysts
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were successfully tested on the hydrodechlorination of chlorobenzene at room temperature and under H2
atmospheric pressure.
2- A novel porous material obtained from carbon-containing liquid and solid wastes has been used as adsorbents
for 3d–transition metals removal from multi-components solutions. The activated carbon was prepared from
the co-mingled natural organic wastes: sunflower husks of 25 %, petroleum waste of 50 % and low–grade
bituminous coal of 25 %. The adsorption of the 3d-transition metals: copper (II), cobalt (III), nickel (II), iron (II)
& (III), manganese (II) and chromium (III), on novel activated carbons were investigated from multi-components
model and technological solutions. Emphasis has been done to the kinetics and thermodynamic studies of
the simultaneous adsorption of the 3d–transition metals in a static mode.
Polymeric Catalytic Membranes
1 - The limonene oxidation was performed in liquid phase, over cobalt acetylacetonate encapsulated in the
supercages of zeolite Y and dispersed in a polydimethylsiloxane (PDMS) matrix. t-Butyl hydroperoxide was
used as oxygen supplier. When the oxidation reaction was carried out over the PDMS embedded catalyst,
the main product obtained was limonene oxide. On the other hand, when the Y-zeolite encapsulated cobalt
Schiff base was directly used as catalyst, limonene oxide was also formed but the main product obtained was
a polymer. The PDMS matrix seems to play an important role in tuning the reactants concentration near the
catalyst active sites in such a way that polymerisation is inhibited.
2 - Poly(vinyl alcohol) (PVA) membranes were prepared using sulfosuccinic acid (SSA) by varying the amount
of SSA (5-40 mol%) and were used for the esterification of acetic acid by isoamilic alcohol. The conversion of
isoamilic alcohol increases when the amount of sulfosuccinic acid used in the polymer crosslinking is increased
from 5% to 20%.
3 - The transesterification of soybean oil with methanol was studied using as catalysts two Nafion commercial
membranes with different thickness and a poly(vinyl alcohol) membrane crosslinked with sulfossucinic acid
(PVA-SO3H). The reactions were carried out at 60 ºC, in a batch reactor using the membrane cut in small
pieces. The initial activities of the membranes are compared being observed an increase of initial activity with
the amount of sulfonic acid groups.
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. New approaches for selective recovery
of solutes from the permeating stream are under development.
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 and for the reuse of
catalysts in integrated reaction systems.
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5 - Extraction of amino acids and derivatives using ionic liquids was studied using different membrane
configurations. The transport mechanism that regulates solute transport, diffusion through water clusters
inside the ionic liquid, was compared for both configurations.
6 - Stability of supported ionic liquid membranes for vapour separation
The use of a room-temperature ionic liquid (RTIL) immobilized in the pores of a supporting membrane is
particularly interesting owing to the non-volatile character of RTIL´s which opens the possibility to obtain very
stable liquid membranes. This new concept will allow the developpment of “taylor-made” membranes using an
IL specifically designed to separate target solutes from vapour streams.
7 - Development of non-invasive, on-line monitoring techniques. used on-line mass spectrometry, Steadystate 2D-fluorescence, Fluorescence anisotropy, Time-decay fluorescence, Confocal Laser Scanning Microscopy and Raman Confocal Microscopy. 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.
Bioremediation
1- Membrane Bioreactors
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. The process efficiency was compared using different types of membranes with a special emphasis on
such having a comparatively low commercial price in order to promote the practical implementation of this
technology.
A study on the removal of other emerging water micropollutants (arsenate, ionic mercury etc.) was initiated.
The arsenate separation potential of a number of anion-exchange membranes was evaluated. Batch tests
under controlled conditions were performed in order to evaluate the ionic mercury reduction to metallic mercury
by pure and mixed cultures. The AAS method for mercury determination was adapted and optimised for both
liquid and gas phase measurements, which allowed for closing the corresponding mass balances.
2-Conventional biological processes.
The simultaneous phosphorus and nitrogen removal was studied in two systems fed with propionate and
acetate. The system adapted to propionate was more stable that the acetate one, that collapsed and lost the
capacity of phosphorus removal. In the propionate reactor it was shown that the organisms able to remove
both nutrients (DPAO, denitrifying polyphosphate accumulating organisms) are different from the classical
organisms for phosphorus removal (PAO, polyphosphate accumulating organisms). Using Fluorescence in
situ hybridisation (FISH) for the identification of phosphorus accumulating organisms, two different morphotypes
were present in the propionate reactor, a dominant rod-shaped organism and a bacilli-shape one. Further
research is being carried out for the identification of these two morphotypes
The biodegradation organomercurial compound to metallic mercury, under aerobic conditions, by a pure
culture of Pseudomonas putida spi3 strain was studied in a continuous stirred tank reactor (CSTR) fed with
a real vaccine production wastewater.
Chemical reaction in supercritical carbon dioxide
In mixtures of high pressure carbon dioxide with liquids just below the critical pressure of the mixture, liquidvapour equilibrium shows a peculiar behaviour. While the gas phase remains essentially pure CO2, the liquid
incorporates large amounts of carbon dioxide, which increase with pressure, dramatically changing its physical properties. Hydrogenation of limonene was performed in this pressure range, either in one phase or
biphasic conditions, with surprising results inn terms of selectivity of products. The interpretation of the
reactivity needed detailed measurements and correlation of phase equilibria. The hydrogen distribution ratios
between the phases wee revealed to be extremely sensitive to pressure.
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Thermodynamics of Ionic Liquid-containing systems
An extensive programme of measurement of phase equilibrium properties of ionic liquid mixtures with water,
alcohols and high-pressure carbon dioxide continued.
Biocatalysis in nonaqueous media
Research efforts were directed towards the:
(i) Generation of experimental data to test/upgrade the existing model for cutinase enantioselectivity (E),
through studies on the impact of temperature and solvent on E of cutinase and of a lipase at temperatures
down to -20 ºC (ii) Elucidation of the interactions between cutinase and sol-gel matrices prepared with different sol-gel precursors, by combining information obtained from experiments with sol-gel matrices containing
additives, from fluorescence spectroscopy studies based on the single tryptophan residue of the enzyme,
and from a calculation of the charge distribution at the surface of cutinase based on the existing model for the
enzyme (iii) Implementation of a reaction/separation scheme, combining the chiral resolution of an alcohol in
an ionic liquid catalysed by a lipase with extraction with supercritical CO2 (iv) Synthesis of geranyl acetate in
a continuous packed-bed reactor via the esterification of geraniol with acetic acid catalyzed by a lipase in
supercritical CO2 and in supercritical ethane, with an emphasis on water activity effects/control.
Process Simulation and Modelling
Dynamic modelling of supercritical fractionation processes
A dynamic model of a supercritical fluid extraction plant was developed. The model incorporates a series of
simulation models for the main pieces of equipment of the SFE plant, namely, the countercurrent structured
packed column, the regeneration column, the heat exchanger for solvent warm-up and the make-up unit. The
dynamic model of the heat exchanger takes into account the resistances to heat transfer in the gas as well
as in the heating fluid (liquid water at ambient pressure) and across the stainless steel wall of the inner tube.
Validation of the dynamic model was done by comparing predicted results with experimental data obtained for
the fractionation of the squalene + methyl oleate with SC CO2. A good agreement was obtained between
measured and predicted temperature and composition profile of the gas and liquid phases through the packed
bed and extraction time.
Modelling of biological processes
1-Metabolic model of Polyhydroxybutyrate production by mixed microbial cultures. The model is based on 7
metabolic reactions. Theoretical yields were evaluated. Experiments were performed with and without ammonia
supply enabling the analysis of PHB formation independently of the cell growth process. The experimental
yields partially confirmed the theoretical values. A full kinetic model was synthesized. The final model exhibited
high accuracy in describing the process state of most experiments performed, thus opening good perspectives
for model-based optimisation studies.
2- Modelling and optimisation of a recombinant BHK-21 cultures. A model-based optimisation study of fedbatch BHK-21 cultures expressing the human fusion glycoprotein IgG1-IL2 was performed. A hybrid mechanistic/
chemometric model identified the BHK metabolic fluxes. The optimisation studies showed that the glutamine
and glucose concentrations should be maintained at low levels during the growth phase and then glutamine
feeding should be increased.
3- Modelling and Control of Rotavirus Virus-Like Particle Production. Rotavirus-like particles were produced
using the baculovirus-insect cells expression system. A mathematical model was developed to describe the
intracellular dynamics of DNA, corresponding to the three structural genes of rotavirus, the respective mRNA
concentration, single protein concentration (VP2, VP6 and VP7), and assembled VLPs concentration.
Modelling of nanofiltration processes
1 - Modeling Rejection in Aqueous Nanofiltration – Solute Geometry and Orientation Effects
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A new approach to model the rejection of uncharged solutes by nanofiltration membranes was developed. A
mathematical description of the solute size and geometry was developed, using the concept of geometric
radius, in which the molecular geometry was represented by a prolate revolution ellipsoid. Additionally, the
preferential orientation angle of the solute towards the membrane, and during permeation, was taken into
consideration and, consequently, its effect on rejection was also introduced.
2 -Modelling the Transport in Solvent-resistant Nanofiltration
The transport of solvents through a solvent resistant NF membrane was analysed by a hybrid modelling
approach, integrating mechanistic and chemometric models that allowed for a better description of the
experimental results. The permeation of water, n-butanol, iso-butanol and tert-butanol as single solvents as
well as in their binary mixtures was studied and successfully modelled.
3- Application of CDF for Simulation and Optimization of the Flow Patterns
CFD (computational fluid dynamics) was applied to characterize the flow in membrane modules, which
allowed for studying velocity, pressure, concentration and pressure drop profiles inside the channels. The
introduction of ladder-type spacers was found to disrupt the boundary layer near the membrane surface, thus
decreasing its thickness, and therefore enhancing the mass transfer.
Modelling of adsorption and adsorption processes
Cyclic adsorption processes, including Pressure Swing Adsorption, Simulated Moving Bed and hybrid systems integrating adsorption and membrane permeation were modelled and optimized using state-of-the-art
computational tools. A novel single-column mimetic alternative to multicolumn continuous counter-current
chromatography has been developed. A novel approach for structural characterization of carbon nanotubes
using molecular simulation and adsorption of probe molecules was developed and experimentally validated.
Polymeric Materials
Polymer processing in clean solvents
New polyurethanes were synthesized in supercritical CO2, using as monomers the CO2 and aziridines.
Aziridines have been reported to react with CO2 to give cyclic urethanes and polymers consisting of urethane
and amine units. Substituted aziridines copolymerize with carbon dioxide to give copolymers of polyurethane
in the presence or absence of catalysts. The use of supercritical CO2 simultaneously as reaction medium
and reactant offers the opportunity to manipulate the outcome of these reactions, including the polymer
structure. A new experimental apparatus was developed for carrying out reactions up to 120ºC and 34 MPa.
The polymers obtained were analyzed by MALDI-TOF, NMR and DSC.
Membrane formation in scCO2
Polysulfone (PS) membranes were prepared using a CO2-assisted phase inversion method. The effect of the
solvent affinity and depressurization rate in the morphology of the membranes was investigated. These parameters had a significant effect upon the pore size of the membranes. Different casting solutions were
prepared using the following solvents: methylpyrrolidone, dimethylformamide, dimethylacetamide, chloroform, dimethylsulfoxide and dimethylpropionamide. The morphology of the membranes obtained by fast depressurization (less than one minute) and slow (approximately one hour) were compared. Higher depressurization rates produce larger pores. Solvents with higher affinity to CO2 produce membranes with smaller
pores and with narrow pore size distributions. Molecular size of the solvent and depressurization rate could be
additional parameters used in CO2 – assisted phase inversion method to control membrane morphology. This
technique was able to produce homogeneous membranes with a good interconnectivity between pores.
Dynamical changes upon crystallization in a biopolymer
Dielectric relaxation spectroscopy was used to monitor the development of crystallinity in poly(L-lactic acid)
(PLLA) at 80ºC. Loss data was modeled considering the evolution of three relaxation processes: the aprocess of the bulk-like (non-restricted) amorphous phase, the a-process of the amorphous fraction influ-
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enced by the crystalline structure and the b-relaxation. It was found that the shape parameters and the
position of the loss peaks are essentially constants during crystallisation, just with their dielectric strengths
varying. This indicates that (i) the two segmental dynamics process develops independently and (ii) the
dynamics features of the (slower) confined amorphous phase do not change during crystallization
PDLC formation in both conventional and scCO2 media
Polymer dispersed liquid crystals were produced by conventional thermally initiated polymerization of
TrEGDMA+AIBN/E7 mixtures in different ratios, the 30/70 composition ratio revealing suitable electro-optical
response. The formation process of the PDLC having the 40/60 composition as precursor was followed by
dielectric relaxation spectroscopy in a pioneer study in literature.
The degree of conversion of the monomers was evaluated by FTIR and its dependence on polymerization
temperature, initiator concentration, E7 concentration, radiation power and radiation time were analysed.
SEM was used to examine the morphology of the polymer dispersed liquid crystal (PDLC) produced. Differential Scanning Calorimetry (DSC) studies were made for not polymerized and partial polymerized samples to
test the degree of conversion calculations. Dielectric Relaxation Spectroscopy (DRS) were used to compare
the results obtained by this technique and FTIR in the same experimental conditions.
In order to control the secondary thermal reactions in the photo-polymerization mechanism, a photoiniferter
was used, p-xylylene bis(N,N-diethyl dithiocarbamate) (XDT), as a initiator in a living radical polymerization.
Electro-optical studies of the PDLC film were made in order to evaluate the voltage needed to change the
transmission characteristics of the film. These electro-optical characteristics are dependent on the polymer
structure and thus the nature and the rate of polymerization mechanism, that means, size and number of the
E7 microdroplets in the polymer matrix.
High-pressure NMR
High-pressure nuclear magnetic resonance, HP-NMR was applied to investigate the CO2-philicity of Krytox
and the molecular interactions between different stabilizers, monomers and carbon dioxide. A new polyether
ketone (PEEK) cell was built, with 10 mm o.d., suitable to record NMR spectra on a Brucker system.
Pressure-dependent NMR spectra of CO2, CO2 + stabilizer and CO2 + stabilizer + monomer were obtained at
334 K and pressures from 15 MPa up to 22 MPa.
Depolymerisation and monomer recovering
The catalytic depolymerisation of polymethylmethacrylate (PMMA) was studied in order to obtain the pure
monomer for further polymerisation, aiming at to maximize conversion at lower temperatures than those
industrially used in simple pyrolysis. The ongoing studies involved the preparation and characterisation of
catalysts based on La, Ce, Cs, Cu and Ni oxides inserted in the structure of mesoporous carbon xerogels,
during the sol-gel synthesis of the polymer carbon precursor (a resorcinol-formaldehyde resin). These novel
catalysts were tested in the depolymerisation of pure and waste PMMA, under previously optimised reaction
conditions. The additives of the waste PPMA showed to play a synergetic catalytic effect on the reaction.
Modelling of kinetic data was performed and a reaction scheme was proposed.
Extraction of biopolymers from industrial wastes for edible films applications
Studies on the optimisation of pectin extraction from orange peels (residue from juice manufacturing) by the
flash pressure method (pressure from 2-5 bars) were carried out under a financed REQUIMTE project. Pectin
extracts were chemically characterised (monosaccharide content, degree of esterification, galacturonic acid
content, average molecular weights and polydispersity. The viscoelastic properties of pectin solutions were
determined by the CEQUP group. The final purpose of this study is the preparation of edible films pectincarrageenan, being the carrageenan extracted from natural resources (seaweeds from the Portuguese coast
–CEQUP group). The film preparation and the transport studies are carried out by another CQFB group. The
thermophysical and mechanical characteristics of the films are performed by the CEQUP group. Three CQFB
groups are involved in this project.
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Drug controlled-release systems
Studies on polymeric formulations for drug controlled release have been performed. Microspheres containing
anti-tumour drugs from the taxoid group and the antracicline group have been prepared using conventional
methods, namely solvent evaporation method, and by an emulsion-membrane technique. Drug release studies were performed and mass transfer models were developed.
Polymeric systems containing anti-inflammatory drugs were produced using supercritical fluid technology in
different conditions: impregnation studies and SAS technology. The process efficiency and drug release
behaviours were compared with the solvent evaporation method.
Drug release formulations for target delivery of anti cancer drugs in the colon were developed using polymers
pH dependent.
Production of Biopolymers
Strategies to obtain high cell concentrations of mixed microbial cultures without loosing the PHA storage
capacity have been implemented. Experiments were carried out in Sequencing Batch Reactors (SBR). The
effect of operating parameters such as substrate and ammonia concentrations, sludge age and reactor feeding strategies on the growth yield and specific growth rate were evaluated. Furthermore, microbial population
was characterized by molecular biological techniques such Fluorescence in situ hybridisation (FISH) and the
impact of the operating parameters on the microbial community structure was evaluated.
A two-stage process including a fermentation step, in which the sugars of the molasses are converted into
organic acids, CO2 and H2, and a PHA production step, in which the organic acids serve as the precursors to
the formation of PHAs was implemented. The effect of different operating conditions on sugar to organic acids
conversion yields and organic acids concentration profiles has been evaluated. The quality of the fermented
molasses as a substrate for the production of PHAs was determined.
Formulation and characterization of edible films for foods
Model edible films based on commercial k-carrageenan and pectin from citrus fruit, were prepared, and their
hygroscopic and mechanical properties, as well as their water vapour permeance, were determined. The
model carrageenan-pectin films studied showed increased mechanical properties, lower glass transition
temperatures, increased water permeability and hydrophilic properties with increased carrageenan content. A
levelling off of the water permeance was observed at high carrageenan content and a maximum was seen in
the mechanical properties. Film properties do not comply with simple mixing rules of carrageenan and pectin
properties, suggesting a phase separation between pectin rich and carrageenan rich domains.
Thermal-polymerization induced by 2,2’-azobisisobutyronitrile (AIBN) and photo-polymerization induced by
2,2-dimethoxy-2-phenylacetophenone (DMPA) of triethylene glycol dimethacrylate and their composites with
and without the low molecular weight nematic liquid crystal E7 were studied.
The degree of conversion of the monomers was evaluated by FTIR and its dependence on polymerization
temperature, initiator concentration, E7 concentration, radiation power and radiation time were analysed.
SEM was used to examine the morphology of the polymer dispersed liquid crystal (PDLC) produced. Differential Scanning Calorimetry (DSC) studies were made for not polymerized and partial polymerized samples to
test the degree of conversion calculations. Dielectric Relaxation Spectroscopy (DRS) were used to compare
the results obtained by this technique and FTIR in the same experimental conditions.
In order to control the secondary thermal reactions in the photo-polymerization mechanism, a photoiniferter
was used, p-xylylene bis(N,N-diethyl dithiocarbamate) (XDT), as a initiator in a living radical polymerization.
Electro-optical studies of the PDLC film were made in order to evaluate the voltage needed to change the
transmission characteristics of the film. These electro-optical characteristics are dependent on the polymer
structure and thus the nature and the rate of polymerization mechanism, that means, size and number of the
E7 microdroplets in the polymer matrix.
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Integration of Processes
Process Integration of Supercritical Fluid Extraction and Membrane Separation
The objective of this project is to explore the feasibility of combining two “green” separation technologies,
based on different separation principles, for the separation of squalene from free fatty acids presented in the
olive oil deodorized distillates (OODD): supercritical fluid extraction (SFE) and membrane technology. The
first part of the project dealt with the determination of the vapor-liquid phase equilibria of the system squalene,
oleic acid and carbon dioxide, to determine the best conditions for the subsequent step, the SFE/membrane
extraction. A static mixer was used for this purpose. Different sets of pressure and temperature conditions
were evaluated. Subsequently, a new apparatus for supercritical fluid extraction has been built where it
incorporates the membrane module needed to the following project tasks.
RESEARCH ACTIVITIES FOR 2006
Clean Solvents and Clean Processes
Membrane Processing and Monitoring
1 - Membrane Design of New Selective Nafion Membranes Using Room Temperature Ionic Liquids
This work aims the designing of new membranes with a high chemically, mechanically and thermal stability,
exhibiting unique characteristics which are conferred by the incorporation of Room Temperature Ionic Liquids
(RTILs) with distinct properties. These membranes may be used for different applications exhibiting a high
selectivity, namely for gas and vapour permeation as well as for low resistance devices. Different characterization
techniques will be used to evaluate if the cation is incorporated inside the membrane, namely Confocal
Raman Spectroscopy, X-ray Photoelectron Spectroscopy and impedance measurements.
2 - Development of processes based on “smart” membranes
This project will be focused on the development of membranes able to respond to environmental and/or
external stimulus. This project will be directed to the conception of new membranes with the following (aimed)
properties: self-cleaning, self-adjusted flux and rejection. This new materials will be applied for barrier packaging
films, fractionation of biomolecules, catalytic degradation of pollutants.
Bioremediation
1- Membrane Bioreactors
Future work will be focused on integration of this biotransformation with the simultaneous transport of ionic
mercury and arsenate through the pre-selected cation exchange membranes. Process optimisation will be
performed with a real water and a different membrane configuration.
2-Conventional biological processes.
The study on simultaneous phosphorus and nitrogen removal will proceed namely on the identification of the
microbial species involved in the process.
A new process concerning the biodegradation of xenobiotic compounds will be initiated
Process Simulation and Modelling
Modelling of biological processes
PHA metabolic model. A metabolic model for PHA production from mixtures of volatile fatty acids by mixed
microbial cultures will be developed.
On-line monitoring of BHK-21 metabolic fluxes. Measurement techniques based on fluorescence spectra and
automated sampling and analysis of glucose, glutamine, glutamate, lactate and ammonia (delivered by a
bioprofiler device) will be integrated for on-line monitoring of BHK-21 cells’ metabolic fluxes.
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On-line optimising control of BHK-21 fed-batch cultures. Based on the previous information acquired on-line,
the glutamine and glucose feeding strategies will be optimised on-line according to a model predictive control
scheme.
Genetic control in the baculovirus-isnsect cells expression system (BEVS). A mathematical model will be
developed for gene expression in the BEVS taking into account the length of genes, strength of promoter and
interaction with the host.
Stochastic infection kinetics in the BEVS system. A model for the infection by three different recombinant
baculovirus at low multiplicity of infection will be developed
Culture screening based on hybrid modelling. A method for culture screening supported by hybrid models will
be developed and applied to PHA producing bacteria.
Membrane modelling. A predictive hybrid model for the flux of mixtures of solvents in nanofiltration membranes
will be developed based on basic descriptors of solvents and membranes. Modelling of solute fractionation in
vapour permeation / pervaporation will developed.
Process Simulation and Modelling / Nanofiltration
The planned activities will be focused on improving the application of the hybrid approach, and coupling the
mechanistic with chemometric models for describing the solvent transport in nanofiltration in order to improve
the predictive power and usefulness of this type of modelling. Furthermore, the CDF approach will be used to
simulate and model the flow patterns. Spacers, of a different geometry and for a varying distance between two
consecutive filaments, will be tested in a specially designed transparent cell with a rectangular channel under
different flow conditions in order to identify the most efficient spacer and to validate the CDF simulations
results.
Polymeric Materials
Drug controlled-release systems
Polymers with functional groups will be produced using supercritical polymerization. Drug release formulations
will be developed for target delivery of anti cancer drugs using polymers pH dependent as well as polymers
with functional groups.
Polymeric systems containing peptides for oral administration will produced.
Design of biodegradable composite films for food packaging
The objective of this work is to develop model composite films based on commercial pectin and carrageenan,
and characterize them in terms of their hygroscopic and mechanical properties, and permeability to gases
(oxygen and carbon dioxide) and water vapour. In order to design films with specific permeability properties,
reactive compounds (ascorbic acid and/or calcium hydroxide to interact with oxygen and carbon dioxide,
respectively) are added to the polymer matrix, while mica flakes are used as impermeable barriers.
Ongoing work is focused on the characterization of the films in terms of permeability to gases (oxygen and
carbon dioxide). The preliminary results obtained suggest that, these films are more permeable to water than
to oxygen and carbon dioxide, and it is anticipated a decrease of the gas permeability with the inclusion of
mica flakes. Moreover, the inclusion of ascorbic acid, which reacts with oxygen, leads to an improvement of
the films selectivity (CO2/O2).
Production of Biopolymers
Research on the Production of PHA by mixed cultures from renewable sources will be continued. The analysis
of the microbial population with a high PHA accumulation capacity will be followed by molecular probes. The
process optimization based on on-line monitoring and control will be followed.
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Polymeric catalytic membranes
The studies on limonene oxidation will be continued with the development of bifunctional catalysts able to not
only epoxidise limonene to limonene oxide, but also to open the epoxide ring to the correspondent glycol and
dehydrate this last to carveol. The optimisation of polymeric catalytic membranes and membrane reactors
capable of minimise polymerisation and working under continuous feed of the oxidant, will be performed.
The studies aiming the development of novel catalysts and polymeric catalytic membranes effective in the
environmental friendly production of biodiesel, will be continued.
Preparation of novel carbons from wastes and removal of organic compounds and heavy metals from wastewaters will be continued. The studies on the use of activated carbon as catalyst support for dioxins model
compounds conversion will be continued.
The studies on recovering of methylmethacrylate from waste polymethylmetacrylate will be continued, using
as catalysts perovskites , pillared clays (argentine bentonites), exchanged with rare earth metals, and the
same metals supported on mesoporous carbons.
The characterisation of polyhydroxyalkanoates obtained by biosynthesis, in co-operation with other group of
the same scientific area will go on.
The characterisation of natural polysaccharides (concerning average molecular weights and polydispersity,
by SEC) carrageenan extracted from seaweeds of the Portuguese coast, and pectins extracted from orange
peels (waste of juice industries) will be performed, under cooperation projects involving three groups of CQFB,
two of the same area, and a group from CEQUP/REQUIMTE. A novel method of pectin extraction, under
pressure, will be studied and the operation conditions, pressure, temperature and time will be optimized in
order to obtain the maximum pectin yield, keeping the desired pectin characteristics.
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BIOCHEMISTRY AND BIOPHYSICS OF PROTEINS
and
BIOINORGANIC CHEMISTRY AND PROTEIN ENGINEERING
Head of Laboratories: Isabel Moura, Full Professor / José J. G. Moura, Full Professor
Staff Members:
Jorge Lampreia
Assistant Professor
Cristina Costa
Assistant Professor
Anjos Macedo
Assistant Professor
Jorge Caldeira
Associate Professor
Pedro Tavares
Assistant Professor
Alice Pereira
Assistant Professor
Carlos Salgueiro
Assistant Professor
Ricardo Franco
Assistant Professor
Stephane Besson
Assistant Professor
Gabriela Almeida
Assistant Professor
Sergey Bursakov
Assistant Researcher
Post-Doct Fellows:
Rui Duarte, Patricia Sousa, Cristina Timóteo, Francoise Auchere, Ludwig Krippahl, Sofia Pauleta, Krisztina
István
Ph.D. Students:
Patricia Raleiras, Cristina Cordas, Gabriela Rivas, Pablo Gonzales, Jorge Dias, Ana Martins, Filipe Folgosa,
Carlos Martins, Joana Raimundo, Márcia Guilherme, António Nunes, Inês Isabel Fernandes Gomes.
Project Grantee:
Celina Santos , Américo Duarte, Andreia Barateiro, Miriam Sousa, Vanessa Cabral, Célia Silveira, Vanessa
Cabral, Ricardo Alves, Sara Berguete, Ricardo Pais, Cristiano Mota, Duarte Alves , Ana Rita Fins , Andrea
Mestre, Mª João Baleizão, Simmone dell´Acqua, Ana Teresa Lopes.
Number of articles in scientific journals: 15 (86-100)
Number of Ph.D Thesis: 1
DENITRIFICATION AND THE DINITROGEN BIOCYCLE
Denitrification is a stepwise sequential 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 obtained, as well as on ET
proteins involved and Nitrate reductases (see section B.)
Nitrous oxide 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,
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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 m4-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. The activity of the Pseudomonas nautical N2OR was measured after periods of incubation of the enzyme in excess methyl viologen and dithionite solution. It was
observed that the enzyme activity is dependent on the incubation time in the presence of reductant. It initially
increases rapidly and then saturates slowly after 40 min, when highest activity is obtained.
EPR measurements of the sample in the same reducing conditions were performed at periods of similar
incubation times at which activities were measured to correlate with the activity assays. The characteristic
EPR signal (gII=2.16) of the resting enzyme decreases with increasing incubation time.
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 m-1,3-bridging mode. We demonstrate now that
the redox 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.
Nitric Oxide Reductase
A Nitric Oxide Reductase (NOR) was purified from Pseudomonas nautica. It is a membrane bound enzyme,
so the detergent dodecyl maltoside was used for solubilization. The purified enzyme consists of two subunits:
NOR B which has a molecular weight of about 56 KDa and contains two b type hemes and one non-heme
iron; and NOR C which has a molecular weight of about 17 KDa and contains one c type heme.
In the native form of the enzyme, the main features of the EPR spectrum consist of a signal at gz=3.6 which
belongs to a highly anisotropic heme c and a set of signals with a gz=2.99, which belong to a low spin heme
b. Quantification of these signals gave a ratio of about 1:1. Since no other EPR signals are detected (apart
from a small amount of a high-spin heme signal) it is thought that the non-heme iron is spin cupled to the other
heme b originating a S=0. A typical signal of heme bound to NO with a spin quantification of only 0.2, appears
after reduction and incubation with NO. Simulation of this signal revealed that it is a mixture of hexacoordinated
and pentacoordinated heme.
Copper containing Nitrite reductase
The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type
1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct
determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper
(T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be
inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to
the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum
of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu
is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of
T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO(2) (-). Electrochemical experiments show that azurin from
the same organism can donate electrons to this enzyme.
Pseudoazurin
Pseudoazurin isolated from Paracoccus pantotrophus periplasm has a type I copper centre, coordinated by
the N side chains of two His, S methione and S? cysteine residue, in a distorted tetrahedral geometry. This
protein exhibits a spectrum with absorption bands around 450 nm, 590 nm and 760 nm in the oxidised form.
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The bands at 450 nm and 590 nm have a L-metal charge transfer character (Scys – Cu ion; 590 nm: CysS p
® Cu x2 – y2 and 450 nm: CysS s ® Cu x2 – y2). Pseudoazurin also presents a characteristic rhombic EPR
spectrum with EPR lines split by a I=3/2 nucleus with a hyperfine constant of 70 gauss.
We are carrying out studies in order to characterize two pH transitions. One was observed by visible spectroscopy, at low pH values; and the other by EPR at high pH values. Direct electrochemistry revealed two pKa
values corresponding to these two transitions.
In order to determine the mean g-values of the g-tensor of Cu site of pseudoazurin protein samples were
crystallized as single and multiple crystal forms. EPR spectra of several crystals were recorded in the three
mean axis. The protein was crystallized in the oxidized spectrum as a single crystal and the structure was
determined at a resolution of 1.3 Å. The structure of pseudoazurin was determined in collaboration with the
Prof. Maria João Romão group.
STRUCTURE AND MECHANISMS IN MOLYBDENUM AND TUNGSTEN ENZYMES
Membrane-bound nitrate reductase P. chlororaphis DSM 50135– initial step of Denitrification
A nitrate reductase was solubilized with Triton X-100 from the membranes of Pseudomonas chlororaphis DSM
50135 grown microaerobically in the presence of nitrate. Like other membrane-bound nitrate reductases, it
contains three subunits, of 129, 66 (64) and 24 kDa, referred to in the literature as alpha, beta and gamma,
respectively. Electrocatalytic studies revealed that only the membrane-bound, not the solubilized form of the
enzyme, can accept electrons from a menaquinone analog, menadione, whereas both forms can accept
electrons from methylviologen. The isolated enzyme possesses several iron-sulfur clusters and a molybdopterin
guanine dinucleotide active center. The iron-sulfur clusters can be grouped in two classes according to their
redox properties, the high-potential and low-potential clusters. In the as-isolated enzyme, two forms of the
molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy.
The low-pH form shows a hyperfine splitting due to a proton, suggesting the presence of an -OHx ligand.
Dithionite reduces the Mo(V) center to Mo(IV) and subsequent reoxidization with nitrate originates a new
Mo(V) signal, identical to the oxidized low-pH form but lacking its characteristic hyperfine splitting. The
isolated preparation also contains heme c (in a sub-stoichiometric amount) with the ability to relay electrons
to the molybdenum center, suggesting that this nitrate reductase may contain heme c instead of the heme b
usually found in this class of enzymes.
EPR and kinetic studies on perilplasmic Nitrate reductase from D. desulfuricans ATCC 27774 Ammonification
Periplasmic Nitrate Reductase from the sulphate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774
(Dd NapA) is an 80 KDa monomeric periplasmic enzyme having the bis-molybdopterin guanine dinucleotide
cofactor (bis-MGD) and a [4Fe-4S] cluster. EPR and enzymatic studies were performed to identify catalytic
and non-catalytic relevant species. The sample in the as-prepared form presents a Mo(V) EPR signal that
was assigned as non-catalytic relevant. The Dithionite reduced sample lacks Mo(V) ion EPR signal and
presents a rhombic signal that is associated with the [4Fe-4S] cluster. The addition of nitrate to this sample
yielded a Mo(V) rhombic signal with resonance lines split by a weakly interacting proton which is not solvent
exchangeable. Addition of cyanide to the fully reduced sample yielded a new Mo(V) signal and decrease the
g3 component of the Fe-S EPR signal. Kinetic studies show that Dd Nap has a complex enzymatic mechanism
with more than one binding site for nitrate. Inhibition studies demonstrated that cyanide and azide inhibits
nitrate depletion.
Study of the spin-spin interactions between the metal centers of aldehyde oxidoreductase
The aldehyde oxidoreductase from Desulfovibrio gigas belongs to the family of molybdenum hydroxylases.
Besides a molybdenum cofactor which constitutes their active site, these enzymes contain two [2Fe-2S](2+,1+)
clusters which are believed to transfer the electrons provided by the substrate to an acceptor which is either
a FAD group or an electron-transferring protein. When the three metal centers of D. gigas AOR are simultaneously paramagnetic, splittings due to intercenter spin-spin interactions are visible when the EPR spectra
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are recorded at low temperatures. By studying quantitatively these interactions with a model based on the Xray crystal structure, which takes into consideration the interactions between the magnetic moments carried
by all the metal sites of the system, it is possible to determine the location of the reducible sites of the [2Fe2S] clusters. When combined with the electron-transfer pathways proposed on the basis of the X-ray crystal
structure, the results provide a detailed description of the electron-transfer system of D. gigas AOR.
ANTAGONISTS Mo AND Cu
Heterometallic clusters present on novel proteins
Orange protein
This protein contains a Mo centre containing other metals and had high sequence homology to proteins from
iron-molybdenum cofactors biosynthesis family. Determination of the 3D structure of the apo-protein using
NMR spectroscopy is being carried out.
The coding sequence of the Orange protein (ORP) was obtain using Polymerase Chain Reaction (PCR) in
which degenerated primers, based on the N-terminus and C-terminus, where applied. The DNA fragment of
350 base pairs (bp) obtained has revealed an open reading frame (ORF) that matched the known sequence of
the ORP. In order to clarify the sequence of the protein, genome walker technique was applied, using a DNA
library of Desulfovibrio gigas. The correct coding sequence was attained, revealing a length of 351 bp corresponding to a polypeptide chain of 117 amino acids.
The ORP coding sequence was amplified with a new pair of non-degenerated primers. These primers contained in the 5’ end sequence for different restriction enzymes. The gene was inserted in the pET-21-d vector
using NheI and HindIII, and the resultant vector was cloned in Escherichea coli DH5a ultra competent cells
(Invitrogen). The resultant protein (rORP) differs from the native one in the N-terminus site, by the addition of
4 aminoacids. The new plasmid containing the ORP gene was transformed using Epicurian Coli BL21 (DE3)
competent cells (Stratagene).
The overexpression of the rORP was performed in different growth media and the induction of the target
protein was made by the addiction of isopropyl â-D-thiogalactopyranoside (IPTG) when the bacterial culture
was in the middle of the exponential phase. Sodium dodecyl sulphate-polycrylamide gel electrophoresis
(SDS-PAGE) analysis of the total protein amount was performed to ensure the presence of the rORP. Optimal
growth conditions were obtained (growth media and temperature, IPTG concentration) and large scale volumes (5 litres) of bacterial culture produced.
The purification of rORP from the crude extract was accomplished in two chromatographic steps (1) gel
filtration, using superdex 75 equilibrated with 300 mM Tris-HCl pH 7.6 and (2) an ion exchange diethylaminoethyl
(DEAE) sepharose equilibrated with 10 mM Tris-HCl pH 7.6, using a linear gradient to a final concentration of
500 mM Tris-HCl pH 7.6. The purity of the protein after each step was followed by SDS-PAGE only as no
colour was possible to observe in any of the fractions obtained.
Toxicity of copper and molybdenum on a free living and in biofilm growing cells of D. gigas were studied to
understand the chemistry and nature of copper, molybdenum and sulphur interactions. The toxic effect of
copper was more emphasized in free than in biofilm cultures and biofilm cells were more competent to remove
metals from solution.
Blue Protein
Blue Protein was purified from Desulfovibrio aminophilus. Cells were suspended and disruptured in a French
press. After centrifugation and ultracentrifugation the supernatant was dialyzed. The soluble extract was
subjected to a three step purification protocol that included: anion exchange chromatography (DEAE-52
cellulose), a Hydroxyapatite column and gel filtration chromatography (Superdex 200). The purity grade was
determined by SDS-PAGE electrophoresis. Molecular mass determination. The molecular mass of the native
protein was determined using gel filtration and a single symmetrical elution peak was detected. Molecular
mass of the subunits was determined by mass spectrometryand SDS PAGE. Determination of the Free
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Sulfhydryl and Disulfide Bond Content. In order to determine free sulphydryl groups and disulphide bonds
Desulfovibrio aminophilus, Blue protein was incubated with either iodoacetamide or with DTT and iodoacetamide
and then the samples were then analysed by matrix-assisted laser-desorption ionisation time-of-flight mass
spectroscopy.
Aminoacid residues sequencing. Peptides were obtained by proteolytic digestion and separated by reverse
phase chromatography. Aminoacid sequencing was performed on an automatic amino acid sequenator. The
peptide sequences were aligned against Blue protein sequence from other sulphate reducer bacteria in order
to find out conserved regions.
DNA sequence determination. In a first attempt multiplex PCR was used in order to amplify a DNA fragment
corresponding to Blue protein. Degenerated primers were designed based on reverse translation of aminoacid
sequences from peptides generated by proteolytic digestion. PCR gaves a single which was isolated, cloned
and sequenced. In a next step, inverse PCR protocol was used to amplify flanking sequences using genomic
DNA as template. Specific primers pointing away from the known sequences were designed. PCR products
were isolated and sequenced. Currently 169 amino acids of the protein sequence have been determined.
CYTOCHROME c PEROXIDASE – CCP
A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase
Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d)
of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change.
Sedimentation velocity experiments confirmed the presence of a single site, although results at higher
pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c
peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking
simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were
situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with
the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electrontransferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of
electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the
electron-transferring heme.
P. pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase
The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a
heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were
shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was reevaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in
a weak monomer/dimer equilibrium that is ionic-strength-dependent. The pseudoazurin was shown to be a
highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence
consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of
which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a
wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of
NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These
lysines are associated with acidic residues just back from the rim, the resonances of which are also affected
by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of
the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.
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Crystallization and preliminary X-ray diffraction analysis of the di-haem cytochrome c peroxidase
from Pseudomonas stutzeri
Crystals of cytochrome c peroxidase from Pseudomonas stutzeri were obtained using sodium citrate and
PEG 8000 as precipitants. A complete data set was collected to a resolution of 1.6 A under cryogenic
conditions using synchrotron radiation at the ESRF. The crystals belong to space group P2(1), with unit-cell
parameters a = 69.29, b = 143.31, c = 76.83 A, beta = 100.78 degrees. Four CCP molecules were found in the
asymmetric unit, corresponding to a pair of dimers related by local dyads. The crystal packing in the structure
shows that the functional dimers can dimerize, as suggested by previous biochemical studies.
Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from P. stutzeri
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.Two crystal forms of cytochrome c peroxidase from
Pseudomonas nautica were obtained and reported last year.
Study of the cytochrome c peroxidase (CCP) electron transfer complexes, specially the one between CCP and the cupredoxin – pseudoazurin.«
Determination of the parameters of binding using different techniques; and structural characterization of the
electron transfer complexes between these two proteins. Kinetic characterization of cytochrome c peroxidase using pseudoazurin as the electron donor.
Identification of the important residues for the formation of the complex and/or for the electron transfer rate.
In this study we have mutated some residues that are proposed to be important to drive the complex formation
(residues belonging to the ring of lysines) or involved in the electron transfer pathway (residues belonging to
the hydrophobic patch of cupredoxins). In the first stage, the mutant pseudoazurins are being biochemical
characterized and then different parameters will be determined, such as the binding affinity, electron transfer
rates and a structural characterization of the complexes will also be attempted.
Structural and mechanistic studies of the effect of the calcium ion in the activation of the enzyme, using
resonance Raman spectroscopy and also UV-visible spectroscopy.
The electrochemical characterization of this enzyme is being done in collaboration with Prof. M.M. Correia
dos Santos.
Structural basis for the mechanism of Ca2+ activation of the di-heme CCP from P.nautica 617
Cloning and overexpression of P.nautica CCP is under way.
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STRUCTURAL STABILITY OF ADENYLATE KINASE
Adenylate kinase was cloned and expressed in E. coli. Expression in E. coli was studied to understand the
mechanisms of metal incorporation into adenylate kinase in vivo conditions. Bacteria were grown on the
controlled media and essentiality and toxicity of metals were determined. Optimal conditions for the bacterial
growth and recombinant protein production with different metals present were obtained. Cobalt, manganese
and nickel used are the metals the most close by its characteristics to the zinc. Impacts of metals concentration on cell growth and on expressed enzyme activity were studied. Kinetic parameters and maximum
rates of cell growth were determined.
A cobalt-porphyrin(CoPf) containing protein was isolated from D. gigas and characterized. Molecular mass of
the protein and porphyrin were established by application of MALDI-TOFF and ESI-MS spectrometry. The
presence of one dissulfide bridge in the CoPf was demonstrated by molecule modification and following MS
mass determination. Amino acid and nucleotide sequences of CoPf were obtained and used to identify this
protein as a g-subunit of sulfite reductase of D. gigas.
NANOSURFACES AND NANOPARTICLES
The study of the interaction of biological molecules, namely proteins and nucleic acids, with noble metal
surfaces including silver electrodes, nanostructured surfaces, and silver and gold nanoparticles. Our strategy
is two-fold: (1) direct interaction of proteins with the metal surfaces and (2) coating the nanoparticles and
nanostructured surfaces with self-assembled monolayers (SAM) of alkanethiols. This coating leads to stabilization and prevents uncontrolled growth and aggregation of the nanostructures. Colloidal gold is also being
used to develop a new class of nano-biosensors that is able to recognize and detect specific DNA sequences
and single-base mutations in a homogeneous format. The aggregation of the nanoprobes is accompanied by
a change of color from red to blue, providing the means for detection. Understanding the physical and chemical conditions that control the specific hybridization of gold nanoprobes to DNA and/or RNA targets will enable
the design and preparation of probes to be used in the clinical environment. Transmission Electron Microscopy (TEM) is used to characterize the morphology and dimensions of the nanoprobes. Atomic Force Microscopy (AFM) imaging allows us to measure high resolution topographic images for characterization of the
steps involved in the formation of the gold nanoprobe-DNA aggregates.
New nanostructured surfaces functionalized with new ligands are being used to study heme-containing membrane proteins with high selectivity and sensitivity using Surface Enhanced Resonance Raman Spectroscopy
(SERRS). New surfaces for SERRS are being generated including silver electrodes, nanostructured surfaces,
and silver and gold nanoparticles with alkanethiols as capping agents. New modified alkanethiols for the
formation of adequate Self-Assembled Monolayers (SAMs) for the immobilization of membrane proteins are
being synthesized and used to cover the former surfaces. Two examples of the work in progress are the
selective binding of an histidine-tagged protein to a SAM made of a Ni(II)-NTA-terminated ligand, and the
utilization of a thiol-derivatised neoglycoconjugate of maltose to form a SAM at a electroactive silver surface.
The former ligand is being synthesized by two different methods using the S-protected-mercaptocarboxylic
acids: a) “Hell-Volhard-Zelinsky” type synthesis with N,N-bis(carboxymethyl)amine; b) coupling with Ná,Nábis(carboxymethyl)-L-lysine. The latter neoglycoconjugate mimics n-dodecyl-b-D-maltoside, a detergent widely
used for membrane protein solubilization, and was utilized for adsorption and spectroelectrochemical studies
of a tetraheme-containing membrane protein solubilized in that detergent.
DEVELOPMENT OF ELECTROCHEMICAL BIOSENSORS
In recent years, the recurrent anthropologic uses of nitrites have raised several public concerns. As a consequence, there is a growing demand to quantify these oxyanions in food and environmental samples. The
development of analytical probes based on stable enzymes with high selectivity and specific activity to nitrite,
such as the multihemic nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774, seems to be
a good alternative to conventional protocols. However, previous attempts to design a ccNiR-based electro-
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chemical sensor were not fully successful from the stability and/or sensitivity viewpoints, due to an inefficient
enzyme-electrode link. In the pursuit of better enzyme immobilization matrices, we constructed and characterized different bioelectrode configurations, using the following strategies:
- entrappment of ccNiR in a ionomeric polymer (Nafion) loaded with methyl viologen as redox mediator;
- attachment of biotinylated ccNiR molecules via avidin bridges to polypyrrole films bearing biotin groups; to
establish the electronic communication between the enzyme redox centers and the solid electrode, the
polypyrrole polymers were also functionalized with viologen groups.
The kinetic behaviour of ccNiR was also investigated by homogeneous enzyme assays and direct electrochemistry.
SIMPLE METAL SITES
Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-ray
The structure of Treponema pallidum (Tp) SOR was solved in collaboration with the X-Ray group at REQUIMTE
(resp. Profª M.J.Romão).
The structure determination of D.gidas SOR and Tp Rubredoxin are underway using NMR methodologies.
VANADIUM(V)-COMPLEXES AND CALCIUM PUMP
Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR).
To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely
(NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O,
potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR
spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate
(VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A
1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM
oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM
tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the
same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM
dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a
1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM
tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/
ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the
interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1
and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of
calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity
into the nature of key species forming upon dissolution of the title complexes in the reaction media.
OXYGEN ACTIVATION
Di-iron sites
It is our research objectives the study of metal cofactors in biology, in particular the ones involved in molecular
oxygen activation and detoxification mechanisms using fast kinetics coupled to spectroscopies such as
Mössbauer spectroscopy. Is an integral part of these objectives the development of software tools that enable
the necessary spectral data analysis. Currently, our interests are centered in two main projects.
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Although reactions of molecular oxygen with organic compounds are energetically favorable, they are kinetically stable (making aerobic life possible). To control dioxygen reactivity nature developed two different ironcontaining cofactors: heme and diiron clusters. While heme-containing enzymes are well-studied systems,
only recently diiron cluster-containing proteins started to be studied in detail. Thus, understanding diiron
cluster reactivity is understanding how nature overcomes the kinetic stability and controls oxygen reactivity,
which in turn is of fundamental importance. It is our understanding that this type of study can only be successful if based in different techniques, such as spectroscopic, kinetic and electrochemical tools.
Also, Mössbauer data analysis is becoming an emerging problem since the software used is incompatible
with recent operating systems. This software is often developed by the user (becoming highly specific) or
limited to basic functionalities, not open to third party plug-ins and usually expensive. We are currently
developing a Mössbauer data analysis program that makes possible the analysis of spectra of biological
samples (including kinetic data analysis).
Mechanistic and Structural Studies of Iron Oxidation and Storage by Fast Ferritins
Our research interests are centered in metalloenzymes involved in iron metabolism and cellular detoxification. Ferritin serves the dual functions of iron detoxification and storage; it catalyzes the oxidation of the toxic
Fe2+ ions in the cells to the less toxic Fe3+ ions (ferroxidation) and stores the oxidized Fe3+ ions within its
protein shell in a mineral form similar to ferrihydrite (mineralization). The rapid freeze-rapid quench technique
in conjunction with Mössbauer and EPR spectroscopies has been used to study the mechanism of ferritin
ferroxidation and mineralization. It is our purpose to further understand the kinetic and spectroscopic observations on the iron uptake mechanism of ferritins. Our short term objectives are to understand how ferritin
binds Fe2+ ions in solution, catalyzes their oxidation, and directs the oxidized Fe3+ ions into the inner protein
cavity to form the ferrihydrite mineral core. Since the structure of the putative ferroxidase site is highly homologous to those of the binuclear iron centers found in the R2 subunit of E. coli RNR and in MMOH, this
study will also provide more general information concerning the chemistry of carboxylate-bridged diiron centers in proteins.
MULTIHEME ENZYMES
The Geobacteraceae, a family of dissimilatory Fe(III)- has been found to dominate the microbial communities
present in a diversity of subsurface environments in which Fe(III) reduction is the terminal electron-accepting
process. In the environment, Fe(III) is found mainly in the form of insoluble iron oxide particles and coatings,
and many species of the family Geobacteraceae have evolved the ability to reduce a variety of electron
acceptors that are either insoluble or too large to be transported into the cytoplasm. These include Fe(III) and
Mn(IV) oxides. The members of the family Geobacteraceae are also capable of reducing many soluble metals, including the radionuclide U(VI). From the analysis of completed genome of G. sulfurreducens over 100
putative c-type cytochrome genes have been identified. Many of these cytochrome genes are more highly
expressed during growth on Fe (III) than with fumarate as the electron acceptor and deletion of some of these
cytochrome genes greatly reduces the capacity for Fe (III) reduction.
The main goal of our research is to characterize thermodynamically Geobacter sulfurreducens multiheme
cytochromes involved in such pathways using visible spectroscopy and NMR techniques to understand the
functional mechanism of these electron transfer proteins.
The present target proteins are:
- Cytochorme c7 (PpcA) – trihaem cytochrome with 71 residues and with heme bis-His axial coordination
which is involved in Fe(III) and UV(VI) reduction pathways. This protein shares high structural homology with
a cytochrome c7 from D. acetoxidans and cytochromes c3 from Desulfovibrionacea family.
- PpcA homologues in a total of four that were revealed from the analysis of the G. sulfurreducens genome.
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- Polymers of cytochrome c7-type domains. The polymer under study consists of four c7-type domains with a
total of 12. Interestingly, the individual domains of this polymer contain a heme which is coordinated by a
methionine and a histidine residue (having the other two hemes bis-His axial coordination), and thus representative of a new type of cytochromes.
PROTEIN-PROTEIN INTERACTIONS
We developed a new algorithm for (BIGGER/CHEMERA) for macromolecular interactions.
Synechocystis ferredoxin/ferredoxin-NADP(+)-reductase/NADP+ complex: Structural model obtained
by NMR-restrained docking
Ferredoxin (Fd) and ferredoxin-NADP(+)-reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)-restrained-docking approach, two alternative structural models of the Fd-FNR complex in the presence of NADP+ are proposed.
The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1
stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The
NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes.
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PROTEIN CRYSTALLOGRAPHY
Head of Laboratory: Maria João Romão, Associate Professor
Staff Members:
Ana Luisa Carvalho
Assistant Researcher
Cecília Bonifácio
Laboratory Technician
Pedro Assunção
Computer System Administrator
Post-Doct Fellows:
José Trincão, Shabir Najmudin, Roeland Boer
Ph.D. Students:
Teresa Santos Silva
Research Students:
Catarina Coelho, Filipe Freire, Aldino Viegas, Lídia Barata
Number of articles in scientific journals: 5 (89, 101-104)
We have focused our work on structural and functional studies of different systems grouped into (A)
Metalloproteins; (B) Proteins involved in cellulose (polysaccharyde) recognition and degradation; (C) Proteins
from the glyoxalase pathway.
An important focus of our research have been metalloenzymes, in particular those dependent on the
molybdopterin cofactor. In addition, we have pursued, together with the Faculty of Veterinary Medicine of
Lisbon, structural and functional studies on several new components of the Cellulosome assembly, a complex
machinery responsible for plant cell wall degradation. More recently we started, in collaboration with the
Faculty of Sciences of Lisbon, a new project on enzymes from the pathogenic organism Leishmania infantum.
METALLOPROTEINS
Isoquinoline 1-oxidoreductase from Brevundimonas diminuta
Isoquinoline 1-oxidoreductase from B. diminuta catalyzes the first step of isoquinoline degradation to
isoquinoline-1-one. It consists of two subunits, one of 80 kDa, containing the Mo active center, and another of
16 kDa, containing two [2Fe-2S] clusters. Its primary sequence and behavior, specifically in its substrate
specificity and lipophilicity, differ from other members in the family. A
crystal structure of the enzyme is expected to provide us with an
explanation for these differences. We have optimized a previously
described purification procedure and crystallized the enzyme. A structure
Mo site
[2Fe-2S]
solution was obtained and model building and refinement to 2.5 Å
resolution are under way.
[2Fe-2S]
Periplasmic nitrate reductases of Ralstonia eutropha and
Desulfovibrio desulfuricans Aiming at studying the amino acid residues
important for its enzymatic specificity, we used nitrate reductase from
R. eutropha (R.e. NAP) as a model system for mutational studies of the
active site. As a first step we have been trying to develop an efficient
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expression system in order to produce adequate amounts of pure protein. Aiming at clarifying the reaction
mechanism for this class of enzymes we have performed several crystallographic studies on reduced, inhibited
and substrate-bound forms of the enzyme from D.desulfuricans. With this purpose, most of the experiments
were carried out in an anaerobic chamber. Crystals were obtained in anaerobic conditions using protein
reduced with either methyl viologen or dithionite. This protein was also co-crystallized in the presence of
substrate, substrate analogues and inhibitors.
Comparing the crystal structures of oxidized and reduced Cytochrome c Peroxidase from P. stutzeri
We have solved the P. stutzeri Cytochrome c Peroxidase crystal structure in the oxidized as well as in the
reduced form. The latter crystals were grown in an anaerobic chamber, under an argon atmosphere. Crystals
were cryo-cooled in liquid nitrogen inside the glove box. A complete dataset was collected in-house and was
used to solve the structure. The structural differences between the two redox states of the enzyme contribute
to clarify mechanistic aspects for CCPs.
The non-heme Superoxide Reductase from Treponema pallidum
Superoxide reductase is responsible for the scavenging of
superoxide radicals in the cell. The crystal structure of Treponema
pallidum (Tp) SOR was determined to 1.55 Å resolution. Unlike
class I SOR, in TpSOR the desulforedoxin-type structural Fe centre
is absent but the N-terminal domain is maintained and exhibits a
desulforedoxin-like fold. The C-terminal domain harbors the
Fe(His)4(Cys) active site. The participation of a glutamate as the
sixth ligand of the iron centre in P. furiosus SOR was not observed
in TpSOR. As suggested for PfSOR, X-ray photoreduction could
be affecting the oxidized form of the TpSOR centre, preventing
hexacoordination of the iron atom.
The CELLULOSOME
Structural basis for the specificity of Carbohydrate Binding Modules (CBM 30 and CBM 44)
The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a
fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has
considerable industrial potential. Enzyme systems that attack the plant cell wall contain non-catalytic
carbohydrate-binding modules (CBMs), that mediate attachment to this composite structure and play a pivotal
role in maximizing the hydrolytic process.
Although xyloglucan is a key component of the plant cell wall, CBMs
that bind to this polymer have not been identified. The C-terminal domain
of the modular Clostridium thermocellum enzyme
ctCel9D-Cel44A comprises a novel CBM44 which
binds to cellulose and xyloglucan with equal
affinity. The crystal structures of CBM44 and
another CBM (CBM30) show how
celluloligosaccharides can be accomodated.
CBM30
Mutagenesis studies confirm that the cleft
comprises the ligand binding site and that a
hydrophobic platform plays a pivotal role in ligand recognition.
CBM 44
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Insights into the Structural Determinants of Cohesin—Dockerin Specificity
Cellulosomes assemble via the Type I dockerins on the catalytic subunits binding to the reiterated Type I
cohesins in the molecular scaffold, while Type II dockerin-cohesin interactions anchor the complex onto the
bacterial cell surface. Type I and Type II cohesin-dockerin pairs show no cross-specificity. The structure of
CohII is very similar to Type I cohesins, and the dockerin binding site is likely to be conserved in the two
proteins. Subtle differences in the topology of the binding sites and a lack of sequence identity in the betastrands that comprise the core of the dockerin binding site explain why Type I and Type II cohesins display
such distinct specificities for their target dockerins.
PROTEINS FROM THE GLYOXALASE PATHWAY
Crystallographic studies on the glyoxalase II from Leishmania infantum
One of the most remarkable features of trypanosomatids is the functional replacement of glutathione by
N1N8bisglutathionilspermidine (tripanothione). Cloned glyoxalase II from Leishmania infantum was
overexpressed in E. coli, purified and crystallized. The structure was solved using the human glyoxalase II
structure as a model. These results, together with future detailed characterization using other substrate
analogues will contribute to the understanding of why trypanosomatids evolved to make use of trypanothione
instead of glutathione.
Planned research activities for 2006
Metalloproteins
Multi-heme nitrite reductase from D. desulfuricans: Crystallization of the membrane hetero-oligomeric complex
of NIR; Crystal structures of inhibitor and substrate-analogue bound complexes as well as structures of
different redox states of the enzyme.
Nitrate reductase from Ralstonia eutropha – Optimization of a suitable expression system. Mutagenesis
studies and purification of wild-type and mutants. Crystallographic studies of the purified proteins.
Co/Zn containing adenylate kinase – Crystallization of Co-containing enzyme and structure solution.
Superoxide reductase from T. pallidum – Mutagenesis studies and crystal structures of the mutants.
Plant cell wall degrading enzymes
Novel studies on the structure and mechanisms that determine the molecular recognition of carbohydrates
towards different CBM modules. It will involve mutagenesis and structural studies. The latter will include a
combined approach of crystallographic, NMR and computational chemistry methods (in collaboration with
other researchers from Requimte).
Proteins from the Glyoxalase Pathway
Structural and mutagenesis studies of L. infantum glyoxalase II in order to clarify the reaction mechanism;
Crystallographic studies on other enzymes from the glyoxalase pathway
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BIOLOGICAL TRANSPORT
Head of Laboratory: Teresa Maria Fonseca de Moura, Associate Professor
Staff Members:
Hugo Gil Ferreira
Invited Full Professor
Karin Tonnies Gil Ferreira
Associate Professor
Isabel Borges Coutinho de Medeiro
Assistant Professor
Maria da Graça Soveral Rodrigues
Assistant Professor
Ana Isabel Dias Bicho dos Santos
Assistant Researcher
Project Grantee:
João Filipe da Custódia Dias, Rui Manuel Lucas Gameiro Domingues Tostões
Membrane Transport
1. Detection and role of aquaporins in different Strains of S. cerevisiae (in collaboration with Prof. Conceição
Dias from Instituto superior de Agronomia)
The functional expression of aquaporins was investigated S. cerevisiae strains differing in the expression level
of AQY1 and AQY2: double mutant, parental and overexpressing AQY1 or AQY2. The functionality of these
proteins was accessed in vivo by measuring the activation energy of water fluxes in protoplasts. The behavior
of the different strains when submitted to osmotic challenges revealed to be different and dependent on the
type of aquaporin expressed, giving values of the osmotic permeability coefficient and activation energy that
reflect the preferred pathway for water transport in each strain.
2. Inhibition of the mitochondrial pyruvate carrier by valproate and its metabolites
(in collaboration with Prof. Margarida Silva from Faculdade de Farmácia de Lisboa)
Pyruvate driven oxygen consumption and ATP synthesis is severely inhibited by the antiepileptic Valproic
acid (VPA). To test whether this effect results from an inhibition of the mitochondrial pyruvate carrier by VPA
or by its â-oxidation metabolites 3-keto-VPA and 4-Delta-VPA we used inverted submitochondrial vesicles
(ISMV) prepared from rat liver cells. We found a marked inhibition on pyruvate transport with the metabolite 4Delta-VPA at physiological concentrations (0.5 mM and 1 mM). The inhibition mechanism and the kinetic
parameters are under study.
3. Functional characterization of a P-ATPase from Nicotiana tabacum: Patch Clamp studies
(in collaboration with the Plant Development group of Prof. José Feijó from Institute Gulbenkian for Science)
The proton P-ATPase from Nicotiana tabacum is responsible for proton fluxes observed during pollen tube
growth (4µm/sec, one of the fastest polarized cellular growth in nature). The electrophysiological patch-clamp
technique can be applied to access the proton currents through this ATPase and to access the mechanisms
by which pump activity is regulated.
cDNAs for the wilt-type of the protein and a construct with GFP (green fluorescence protein) on the C-terminal
were subcloned into pcDNA with the CMV promoter for expression in mammalian cells. The conctruct cDNA
has been used to transiently transfect mammalian HEK293 cells for patch-clamp characterization. Expression of the protein in transfected cells has been accessed by means of a laser light (with 473 nm wavelength).
So far, the detected fluorescence levels (24-48hrs after transfection) are low. A control of the transfection
protocol was made with GFP alone with almost 60% of the cells showing fluorescence after 48hrs. Preliminary results obtained in the whole-cell configuration show an increase of the cell current upon application of
40µM fusicoccin, a fungal toxin known to enhance proton pumping by this pump.
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4. Electrophysiological studies on membrane transporters
(in collaboration with Prof. Constanta Ganea of the Biophysics Department, “Carol Davila” University of Medicine and Pharmaceutics, Romania; our lab. received the visit of Anca Popescu, Ph.D. student, Out-Dez 2005,
for this joint project)
This line or research consists on studying the effects of various physico-chemical factors of general interest,
as heavy metals (Ni, Cd, etc.), food supplements, pollutants and/or drugs (quercetin, hypericin, galantamine,
etc) on the electrical properties of different membrane transporters and ion channels, by means of the sensitive patch-clamp (CQFB/FCT/UNL) and BLM, black lipid membranes (“Carol Davila U.M.P.). The results will
provide better understanding of molecular mechanisms underlying membrane transport and of the influence of
external factors on these mechanisms.
To start this project a culture of TE671LH cells which express the nicotinic acetylcholine receptor was established and maintained. A fast perfusion system that allows accurate temporal control of application of solutions and drugs to the cells (on the millisecond range) was built. The whole-cell configuration of the patchclamp technique was used to examine the effect of quercetin (an antioxidant of plant origin used as food
supplement) on the acetylcholine induced transient currents by the neuronal receptor. Patch-clamp results
show a possible decrease of the currents after exposure to quercetine and alteration of the receptor kinetics.
5. Patch clamp characterization of ionic currents of HEK293 cells
HEK293 cells are commonly used for transfection of heterologous membrane proteins (channels and transporters). It is therefore important to know which endogenous membrane transporters these cells have. Several
types of potassium and anionic channels were characterized. However, an integrative study of HEK293 cells
is lacking to understand the magnitude of these endogenous currents under experimental conditions, and the
relative contribution of sodium, chloride and potassium to the total current of a cell.
Ion substitution patch-clamp experiments have shown that the total ionic current by a cell does not account
for the summation of the individual currents of these three ions.
Mathematical models of biological systems
Mathematical models for cells and epithelial systems are being developed. Numerical simulations are implemented in the Berkeley Madonna package (http://www.kagi.com/authors/madonna).
1. Tubular organ - thick ascending limb of the Henle´s loop - TAL
The flat sheet cTAL model, developed for the behaviour of the thick ascending limb of the Henle´s loop of
mammalian kidney, was extended to a tubular geometry in order to simulate the behavior of cTAL in in vitro
and in vivo conditions as referred in the last report. The model is now able to fit experimental reported results
and predict behaviors where data is not yet available. In particular the model: i) simulates reported output
values for the ion concentrations and predicts their steady state profiles along the lumen; ii) simulates the
increase of the trans-epithelial electrical potential difference, in contrast with the potential difference across
the basolateral barrier that remains unchanged, when perfusing at high rates; iii) predicts the same value for
the static head independently of the NaCl load at the entrance of the tubule.
2. Calcium homeostasis - A minimal model
(col. with Dr. J.F. Raposo and Prof. L. Sobrinho from Instituto Português de Oncologia Lisboa, and Dr. A.
Pires from the Hospital Amadora-Sintra)
In mammals the calcium concentration in the extracellular compartment (ECC) is finely regulated (Calcium
Homeostasis) and controls a large number of physiological processes: blood coagulation, the excitability of
nerve and muscle cells, the epithelial secretion, the secretion of parathormone (PTH), the functional state of
many ionic channels, etc. As a result of the development of reliable and easy techniques of measurement of
the main variables of this control system (Calcium, PTH and Calcitriol) it is now possible to characterize with
some detail many of the system’s disturbances which are frequently seen by the endocrinologists. Although
there is now published information on the behaviour of a number of the components of the calcium control
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system we lack tools that may help us to analyze the behaviour of the system as a whole. This is an attempt
to develop such a tool. A first version of the model covering acute situations was published (J. Clin. End. Met.,
2002, 87, 4930-40). Most of 2005 was dedicated to data gathering from several hospitals (Instituto Portugues
de Oncologia, Hospital de Santa Cruz, Hospital de Setúbal) and to its statistical analysis.
Studies on Food Components
1. Analysis of the mineral and trace elements composition of human milk (in collaboration with Prof. Matilde
Castro and Dr. Lino Mendes from Faculdade de Farmácia de Lisboa)
As stated in the report of 2004, the mineral and trace elements composition of human milk was characterized.
The screening of potentially toxic mineral elements in breastfeeding mothers was performed and correlated
with socio-demografic data and dietary patterns through a food frequency questionnaire. The great variability
of the values determined in the different samples of maternal milk (n= 140) has to be emphasized. It was
possible to detect the presence of major minerals and trace minerals in the human milk, especially Mn, Si, B,
Ba, Sr among others. Hg was detected in 15 samples, showing a correlation between fish consumption and
its presence.
All these studies are being performed using the ICP (Induced Coupled Plasma) technique.
2. Behaviour of natural pigments in solution and in organized media
NMR 1D and 2D data was obtained for aqueous solutions of anthocyanin isolated from the peel of Passiflora
edulis (Sims) fruit. Preliminary results showed it to be a monoglucoside derivative of a non-substituted
aglycone.
UV-Vis spectroscopy absorption and emission data were obtained for several fluorescein derivatives of the
protein bovine serum albumine (BSA), namely Erythrosine (food color E 127), Eosin and Rose Bengal in
water and in aqueous solutions. For all systems, phosphorescence was readily observed at room temperature in well deareated solutions both with and without protein. Complementary studies using the triplet-emitter
bromonaphtalene in deareated water-BSA systems also showed phosphorescence at room temperature
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DEVELOPMENT OF TRANSDUCERS
Head of Laboratory: José Luís Costa Lima, Full Professor
Staff Members:
Alberto Nova Araújo
Associate Professor
Rui Alexandre Santos Lapa
Associate Professor
Mª Conceição B. S. M. Montenegro
Full Professor
Maria Beatriz V. N. Q. G. Junqueiro
Associate Professor
Agostinho Almiro Almeida
Assistant Professor
João Luís Machado Santos
Assistant Professor
Maria Lúcia M. F. Sousa Saraiva
Assistant Professor
João Pedro Almeida Lopes
Assistant Reseracher
Rita Isabel Lemos Catarino
Assistant Professor
Paula Cristina Gerónimo
Assistant Professor
Ivone Valente Oliveira
Assistant Professor
Cristina Maria C. Morais Couto
Assistant Professor
Adriana Martins Pimenta
Assistant Professor
Maria Isabel Almeida Cardoso
Technical Staff
João Rodrigo da Silva Santos
Laboratory Technician
Cristina Maria Delerue-Matos
Professor Coordenador
Maria do Carmo V. F.Vaz
Professor Coordenador
Abel José Assunção Duarte
Assistant
Ph.D. Students:
Maria Luísa Soares Silva, Sofia Alexandra Lopes da Piedade Gomes, Emília Maria Gonçalves Santos, Célia
Maria Pinto Gomes Amorim
M.Sc. Students:
Branca Sofia Teixeira, Ricardo Nuno Mendes Jorge Pascoa
Number of articles in scientific journals: 7 (105-111)
Number of Ph.D thesis: 1
Number of M.Sc thesis: 1
New trends were investigated based on exploitation of different transducing schemes such us optical and
electrochemical. Studies on immobilization techniques were implemented for the monitoring low levels of
drugs, food and pesticides. Additionally studies were developed concerning the construction of tubular and
wall-jet electrodes dedicated to the implementation of manifolds with FIA multi-side detection or multicommutated
flow systems.
In more detail it can be referred the following results:
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- A new nitrate-selective electrode was been constructed based on the analytical potentialities of the oxaazamacrocycles as potentiometric ionophores with improved selectivity characteristics when compared with
commercial available unities. The working characteristics of this new electrode constructed without inner
reference solution, made possible its application to the determination of nitrate in different types of vegetables
and bottled mineral waters without the use of a support electrolyte for elimination of possible interferences.
- An inexpensive and easy to construct miniaturized biosensor for the determination of uric acid in biological
fluids was been developed. The amperometric biosensor was prepared by using a carbon paste electrode
prepared with uricase from Arthrobacter globiforms and tetracyanoquinodimethane as electron transfer mediator.
- A tubular amperometric detector coupled to a multicommutated flow system was been constructed and
applied in the determination of uric acid in urine. The exploitation of the analytical potentialities of the
multicommutated flow manifold allowed the implementation of the on-line sample dilution based on the zone
sampling approach. The dilution capability exhibited by the developed methodology allowed a direct insertion
of the samples in the flow system without any pre-treatment.
- Tin (IV) porphyrins were used as ionophores for phthalate selective electrodes preparation. The influence on
the electrode response of the ionophore structure and the membrane composition, namely the amount of the
incorporated ionic sites was been studied. The analytical usefulness of the electrodes was been assessed by
potentiometric determination of phthalate in water and industrial products.
- The reagent generation by ultrasonic irradiation to produce chloride and hypochloride for analytical purposes
derived from the residues of organochlorines of laboratory effluents was been studied. The dependence between
the production of chloride obtained from the sonication of the effluents containing organochlorines and the
irradiation time was evaluated for CCl4, CHCl3, CH2Cl2. It was observed that under saturation conditions the
production of chloride was highest for CHCl3. The extent of sonochemical generation of hypochlorite was also
evaluated in the presence of several NaOH concentrations.
- An optical biosensor based on immobilised carbonic anhydrase and its application to the determination of
the anti-glaucoma agent acetazolamide by enzyme inhibition measurement, was been evaluated. The enzyme
and a pH indicator dye, cresol red, were physically immobilised in overlapped sol-gel thin films, in a dual-layer
format. The sensor was integrated in a flow cell and coupled to a continuous flow system operating on a
multicommutation and binary sampling approach.
- Two types of ion selective electrodes (with and without inner reference solution) have been developed using
selective membranes based on iron oxyhydroxide embedded on silica gel mixed with ultra pure graphite at a
2/29 (w/w) ratio. The working characteristics of the electrodes and the effect of interfering ions was investigated.
The accuracy and the precision of the determinations with the constructed ESI’s have been tested on arsenicfree drinking water spiked with known amounts of arsenic and groundwater samples containing high levels of
arsenic.
Fiber-optic Fluorescence optodes
Sensors regarding an optical transduction are also known as optodes and constitute an increasing area in
green chemistry. They are suitable for chemical recognition and quantification, allowing selective readings at
low levels and with low sample volumes.
Within this context, fiber-optic fluorescent optodes are currently being developed for the determination of ionic
species in aqueous solutions. Optimization of the integration of the different components among optodes is
carried out. Chemometric techniques to process sensor signals under steady state or lifetime fluorescence
are also under development.
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AUTOMATION AND INSTRUMENTATION
Head of Laboratory: José Luís Costa Lima, Full Professor
Staff Members:
Alberto Nova Araújo
Associate Professor
Rui Alexandre Santos Lapa
Associate Professor
Mª Conceição B. S. M. Montenegro
Full Professor
Maria Beatriz V. N. Q. G. Junqueiro
Associate Professor
Agostinho Almiro Almeida
Assistant Professor
João Luís Machado Santos
Assistant Professor
Maria Lúcia M. F. Sousa Saraiva
Assistant Professor
Marcela Alves Segundo
Assistant Researcher
Adriano Fachini
Assistant Researcher
IIvone Valente Oliveira
Assistant Professor
Cristina Maria C. Morais Couto
Assistant Professor
Adriana Martins Pimenta
Assistant Professor
João Alexandre Velho Prior
Assistant
João Rodrigo da Silva Santos
Laboratory Technician
Ph.D. Students:
Paula Cristina into, Cristina Vieira, Pedro Maia, Marta Filipa Ribeiro, Karine Marques, Luís Miguel Magalhães,
Eunice Rodrigues, Hugo Oliveira, Marieta Passos, André Araújo
M.Sc. Students:
Daniel Ribeiro, Ana Isabel de Castro, Cristina Silvestre
Number of articles in scientific journals: 22 (112-133)
Number of Ph.D thesis: 1
Number of M.Sc. Thesis: 1
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 sequential injection analysis. Additionally new flow
analysis concepts and methodologies was been present and evaluated, namely multi-pumping flow analysis
and single reaction interface flow analysis.
In this theme the following activities can be stressed:
- The development of a new separation method based on a novel reversed-phase sequential injection
chromatography (SIC) technique was used for simultaneous determination of ambroxol hydrochloride and
doxycycline in pharmaceutical preparations. The chromatographic resolution between peak compounds was
lower than 5.0 and analysis time was less than 9 minutes under the optimal conditions. The method was
found to be applicable for rotine analysis of the active compounds in several pharmaceutical preparations.
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- The development of a sequential injection analysis system based on the sensitized effect of cationic surfactants
on the reaction between metal ions and chelating dyes. The quality of the results obtained with the developed
systems for 18 water samples were evaluated by comparation with conventional procedures, with no statistically
significant differences for a 95% confidence level.
- A critical review on flow-injection systems with multi-side detection was been published. The paper stress
the improvements in system simplicity, ruggedness and performance of the multi-side detection and emphasize
situations where relocating the detector results in significant advantages over traditional FIA systems.
- Two review on sequential injection analysis (SIA) was also published. One of the papers stressed that SIA
with electrochemical detection is an important tool for the automation of chemical procedures and presents a
overview of the principles of operation and applications and the other referred the application of this methodology
in food analysis.
- The new concept of single interface in flow analysis was been present and discussed. The dual or multiple
interface concept, commonly associated to the distinct flow techniques, was replaced by a single interface
concept, which do not rely on the utilisation of a well-defined and compelling sample volume but only on
mutual penetration of sample and reagent zones at a single reaction interface where both sample and reagent
met together prior to detection. The detector is positioned at the core of the manifold (not in the conventional
terminal position) and repetitive flow reversal enable interface manipulations, including multi-detection of the
entire reaction interface or the monitoring of the evolution of a pre-selected interface zone by using reversal
cycle times.
- A fast and sensitive flow-based procedure for chemiluminometric determination of carvedilol was been proposed.
The developed methodology takes advantage of the antioxidant capacity of carvedilol to inhibit the
chemiluminescence response resulting from the oxidation of luminol by hypochlorite, by acting as a hypochlorite
scavenger.
- A sensitive sequential injection analysis methodology for the fluorimetric determination of naproxen is proposed.
The developed automatic analytical procedure is based on the complexation of naproxen with cyclodextrin
yielding an enhanced fluorimetric signal and applied to the determination of naproxen in pharmaceutical
preparations. The results obtained with the flow system were compared with those furnished by the reference
procedure and the relative deviations were lower than 3.6%.
- A flow injection multisite detection system was developed for correction of the sample blank in a colorimetric
determination. By using detector relocation, a single spectrophotometer is used for the sequential reading of
the sample blank and the coloured product; the sample crosses the flow cell to measure intrinsic absorption,
the colour reagent is subsequently added, and the the flow cell is relocated to provide the reading of the
resulting plug.
- A fully automated analytical methodology for chemilluminometric determination of propranolol hydrochloride
in pharmaceutical preparations was been proposed. The developed procedure was based on the oxidation of
propranolol by potassium permanganate in acidic medium and was implemented in a multicommutated flow
system.
- Restricted acess material (RAM) column containing 25µm C18 alkyl-diol support was integrated into the
sequential injection analysis (SIA) manifold and the SIA-RAM system was tested for direct determination of
furosemide in serum. The integration of RAM material into SIA enabled creation of a comprehensive on-line
sample clean-up technique combined with fluorescence determination of the analyte.
- A multi-pumping flow system for the chemiluminometric determination of the hypoglycaemic drug metformin
was implemented. The developed methodology was based on the metfotmin-induced inhibition (metformin
acts as Cu(II) scavenger) of the catalytic effect of Cu(II) ions on the chemiluminescent reaction between
luminal and hydrogen peroxide.
- A computer-controlled multi-syringe flow injection system was proposed to perform the spectrophotometric
determination of avaible iron and boron in soil extracts. The methodologies were based on the formation of
ferroin complex (determination of iron) and azomethine-H reaction (determination of boron). Both determinations
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were performed in manifolds with similar configurations by changing the reagents present in the different
syringes.
- A multi-syringe flow injection system for the determination of total phenolics based on the 4-amino-antipyrine
reaction was been proposed. In line preconcentration using solid-phase extraction was implemented, offering
an environmentally friend alternative to organic solvent extraction performed in batch procedure and in previous
described flow methodologies.
- A new flow methodology exploiting the multi-pumping approach was developed for the spectrophotometric
determination of ambroxol hydrochloride in pharmaceutical preparations. The flow manifold was implemented
by using exclusively multiple solenoid actuated micro-pumps which acted simultaneously as sample insertion,
solutions propelling and reagents commutation units.
- The development of a pulsed flow sequential injection analysis (SIA) system based on the utilisation of pulse
generating solenoid micro-pumps, wich replace the conventional solutions propeplling units commonly used
in SIA systems. The influence on sample/reagent zones interpenetration and reaction zone formation of the
reproducible pulsed flow produced by micro-pumps operation, which is characterised by a chaotic solutions
movement, was comparatively evaluated with the typical laminar flow conditions of conventional SIA systems.
- Four configurations for sample introduction (volume or time based) were tested in order to establish if the
different strategies affect the analytical signal in multi-syringe (MSFIA) systems. The influence of the best
sampling approaches were also evaluated in MSFIA systems for the spectrophotometric determination of
phenolic compounds and the potentiometric determination of chloride.
- A potentiometric flow system has been developed for the direct determination of arsenic (V) in water samples
after on-line preconcentration in a minicolumn packed with a new adsorbent material selective to As(V). The
flow system has been successfully applied to the determination of inorganic arsenic species in groundwater
samples collected in areas affected by arsenic contamination.
- An enzymatic flow analysis approach, based on the Griess-Ilosvay reaction, was developed for the evaluation
of nitrite and nitrate aiming an environmental benign alternative reduction method using nitrate reductase and
the development of on-line dilution procedure for the analysis of water samples, with a wide range of nitrate
concentrations.
- An automatic system that performs two analytical procedures allowing the evaluation of the alternative
antioxidant capacity of wine samples, was evaluated. Automation was carried out using sequential analysis
system that allowed the development of the two methodologies that were evaluated using trolox as standard
and subsequently using other antioxidant substances which are abundant in wine and whose antioxidant
activities were compared to that shown by trolox.
- An automatic methodology was been proposed based on multi-syringe flow injection analysis for the
determination of avaible phosphorous in soil extracts. This fully computerized flow technique allowed the
development of a flow network where sample and reagents were intercalated and sent further towards detection
system.
- A novel sampling approach in flow analysis was proposed and applied to the spectrophotometric determination
of glucose and fructose in invert sugar syrups. The samples are collected in gelatine capsules and brought to
the laboratory. The capsules are placed directly into a dissolution chamber of a flow-batch system, therefore,
the manual sample dissolution step is not required.
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QUALITY CONTROL AND AUTHENTICITY OF FOOD PRODUCTS
Head of laboratory: Rosa Seabra, Full Professor / Beatriz Oliveira, Assistant Professor
Staff Members:
Isabel Maria P. L. V. O. Ferreira
Assistant Professor
Paula Andrade
Assistant Professor
José Fernandes
Assistant Professor
Patrícia Valentão
Assistant Professor
Susana Casal
Assistant Professor
Rui Alves
Professor Coordenador
Olívia Pinho
Associate Professor
Isabel Mafra
Assistant Researcher
Luísa Maria S. Vieira Peixe
Assistant Professor
Helena Maria Neto Ferreira
Assistant Professor
Miguel F. de Albuquerque Cabral
Assistant Professor
Cristina Réu
Technician
Cristina Maria Delerue-Matos
Professor Coordenador
Maria do Carmo V. F. Vaz
Professor Coordenador
Maria Goreti Ferreira Sales
Assistant
Maria de Fátima S. Barroso
Assistant
Post-Doct Fellows:
Sónia Dopico
Ph.D. Students:
Sara Cunha, Miguel Faria, Joana Amaral, Bárbara Ribeiro, Sandra Maria Basílio Quinteira, Carla Alexandra
Novais Oliveira e Silva, Patrícia Sofia Carneiro Antunes, Elisabete Maria Pereira Machado, Sandra Quinteira,
Ana Raquel Freitas
M.Sc. Students:
Ana Filipa Gomes, Áurea Roxo, Carla Barbosa, Lúcia Maia, Sílvia Marisa da Cruz Barros, Sónia Mendes,
Alexandra Soares, Cristina Soares, Aida Reis, Filipa Grosso.
Number of articles in scientific journals: 27 (134-160)
Number of articles in books: 2
Number of Ph.D. Thesis: 2
Number of M.Sc. Thesis: 4
The leading research lines of our group are quality control, authenticity and safety of foodstuffs, with emphasis
on macronutrients (fatty acids, proteins, triglycerides), micronutrients (vitamin E, phytosterols, carotenoids,
organic acids) residues and contaminants (biogenic amines, pesticides, acrylamide, heterocyclic amines,
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polycyclic aromatic hydrocarbons) of conventional and traditional food products, with “Protected Designation
of Origin”, PDO. In this context, the development of analytical procedures is a constant challenge and the
goal of our research team.
The principal activities under development in 2005 were:
Molecular biology techniques for the evaluation of food safety and food authenticity
The research includes the use of DNA based techniques (PCR Polymerase Chain Reaction) applied to food
products. The identification of species of milk producers in dairy products, namely cheeses PDO (Protected
Designation of Origin), has been carried out for the detection of fraudulent procedures. Particularly, the “Queijo
de Cabra Transmontano” is a product with PDO, whose manufacture should include only milk of one caprine
breed, the “Serrana Transmontana”. A duplex PCR technique for the simultaneous detection of bovine and
caprine milks in cheeses has been developed. By means of a linear normalized curve between the log of the
ratio of the bovine band intensity and the sum of bovine and caprine intensities vs. the log of cow’s milk
percentage, it was possible to quantify adulterations with bovine milk in the range of 1-50%. The method was
successfully applied to “blind” and commercial caprine cheeses. This work has resulted in an M.Sc, thesis to
be presented next year.
Vitis vinifera clonal identification is of great importance as the choice of the best clones can contribute for
wine quality and authenticity. By means of an optimized detection method of clones identification, it was
possible to obtain 14% discrimination within Touriga Nacional (TN) clones, identifying two different groups.
The polyclonal origin of TN clones was previously verified with eight microsatellite markers. This research line
resulted in a PhD thesis presented in December 2005.
Next year we intend to develop Molecular PCR-based techniques for Olive Oil authenticity evaluation. In the
international scene there are particular signs that olive oil has been adulterated with chemically similar oils,
like hazelnut oil (Corylus avellana L.), and in some cases with other crops. To develop highly specific PCR
methods one needs to obtain (1) highly pure DNA extracts using alternate extraction techniques and (2) PCR
primers throughout the analysis of DNA sequences publicly available like the ones of NCBI (GenBank) and
EMBL. This last goal can be achieved only by knowing how to analyse and retrieve data from these huge
databases using tools frequently used by Bioinformatics. This work programme will be a basis of the
Postdoctoral Grant concerning the safety and authenticity of foodstuffs by biological molecular procedures.
The detection of genetically modified (GM) soybean RR (Roundup Ready) with herbicide tolerance (glifosato)
has been carried out by PCR techniques. The method under development included the design of specific
primers for the lectin gene and the 3’ NOS/plant junction specific of RR soybean. Those primers were
successfully used to detect the RR soybean in foodstuffs by conventional PCR and to quantify it by real-time
PCR with SYBR Green as fluorescence dye. The planned research for the future includes the comparison of
several DNA extraction methods from foodstuffs, the detection of Bt maize in raw materials and foodstuffs and
the development of more specific real-time PCR techniques with hydrolysis probes (such as TaqMan).
Chemical Markers of Food Authenticity
Characterization of conventional and traditional foodstuffs is a custom, being examples of the developed work:
Physico-chemical characterization of PDO cheeses, that includes lipolytic and proteolytic products
characterization, and correlation between volatile components (determined by SPME/ GC/MS) and sensory
characteristics. Proteolysis and authenticity criteria based in lactoglobulins HPLC analyses for identification
of homologous proteins in milk mixtures from different species were also evaluated. This work resulted in a M.
Sc. Thesis.
(Isabel Ferreira, Beatriz Veiros, Olívia Pinho)
Some coffee constituents were determined in both green and roasted coffee and their behaviour with roasting
evaluated and discussed. Actually was under evaluation the presence of heterocyclic amines correlated with
beneficial effects in neurological system. The evaluation of Harman and norharman, two beta-carbolines that
might be involved in the protection for Parkinson disease, was accomplished with 17 samples analysed in the
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raw state and after three different roasting degrees. It was verified that the amounts are proportional to the
roasting intensity and that robusta coffee amounts are always higher. These conclusions are important for the
consumption of “expresso” coffee, where high degrees of roast and blends with robusta are a must for a good
final quality. This research has resulted in an M.Sc, thesis to be presented next year as well as a Grant from
IBeSa.
Several quality parameters are determined in varietal olive oils in order to characterize some cultivars of Olea
europaea (fatty acids, sterols, tocopherols, triglycerides) and were applied to PDO olive oils “Azeite de Trásos-Montes”. This research line concerns to the PhD Grant about safety and authenticity of olives and olive oils
in preparation.
An HPLC methodology to characterize the carotenoid profile (lutein and ??carotene) of olive oils was
implemented. This methodology was developed, validated and applied to several PDO Portuguese olive oils
from different regions (Trás-Os-Montes and Alentejo). This subject will be described in a M.Sc. thesis in
preparation.
The chemical composition (including fatty acid, sterols, tocopherols and tocotrienols and triacylglycerol profiles)
of different cultivars of hazelnuts (Corylus avellana L.) and walnuts (Juglans regia L.), grown in different
geographical areas was evaluated as well as the importance of these parameters in the discrimination of the
cultivars in study by chemometric methods. The experimental work is concluded and a PhD thesis is finished
and presented to evaluation.
Sheep milks of two autochthonous Portuguese breeds were evaluated for their conjugated linoleic acid (CLA)
levels. The results suggest that this milk can be considered an interesting source of CLA. In prosecution of
this line a similar study with Spanish breeds was performed in collaboration with a Spanish Faculty aiming the
detection of interesting differences for genetic improvement of the breeds. The conjugated linoleic acid levels
(CLA), total fatty acid profile and total fat were determined in the adipocite depots of suckling lambs from 4
different breeds in a total of 122 animals. The same parameters were also evaluated on the fat from their
ingested milk. The variability of the results seems to be correlated with the “ganaderia”, lamb age, breed and
the mother’s milk production per day. This research line continues with more milk samples from another
breeding as well as with traditional Portuguese sausages aiming protected geographical indication (PGI) as a
form to obtain added value from the regional products of underprivileged regions.
Measurement of flavour characters in food by sensory and instrumental techniques is other goal in development.
Optimization of SPME/GC/MS methods for separation and identification of volatile compounds in foodstuffs
and beverages like, cheese, honey, beer and coffee for extraction of a wide variety of compounds belonging to
very heterogeneous groups, such as, esters, alcohols, acids, aldehydes, ketones, hydrocarbons, eters,
sulphur compounds, alicyclic compounds, aromatic compounds, heterocyclic compounds, etc. will be performed.
Train of sensory panels to establish Quantitative Descriptive Analysis of those products is also part of this
research line.
Within the scope of a research project regarding the chemical characterization and the evaluation of the
antioxidant potential of tronchuda cabbage (Brassica oleracea L. var. costata DC). seventeen phenolic
compounds from the internal leaves of this cabbage were characterized and quantified by reversed-phase
HPLC-DAD-ESI-MSn and HPLC/DAD, respectively: quercetin 3-O-sophoroside-7-O-glucoside, 3-pcoumaroylquinic acid, kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-(caffeoyl)-sophoroside-7O-glucoside, sinapoyl glucoside acid, kaempferol 3-O-(sinapoyl)-sophoroside-7-O-glucoside, kaempferol 3O-(feruloyl)-sophoroside-7-O-glucoside, kaempferol 3-O-(p-coumaroyl)-sophoroside-7-O-glucoside, 4-pcoumaroylquinic acid, sinapic acid, kaempferol 3-O-sophoroside, 3 isomeric forms of 1,2-disinapoylgentiobiose,
1-sinapoyl-2-feruloylgentiobiose, 1,2,2-trisinapoylgentiobiose and 1,2’-disinapoyl-2-feruloylgentiobiose.
A phytochemical study was also undertaken on in vivo material cultivated under conventional and organic
practices and collected at different times. Six organic acids (aconitic, citric, ascorbic, malic, shikimic and
fumaric acids) were identified and quantified by HPLC-UV. Qualitative and quantitative differences were noticed
between internal and external leaves. Analysis of the internal leaves phenolics was achieved by HPLC-DAD,
and the phenolic profile obtained revealed to be distinct from that of the external leaves previously determined.
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By this means it were identified and quantified eleven compounds: 3-p-coumaroylquinic acid, kaempferol 3-Osophoroside-7-O-glucoside, kaempferol 3-O-(caffeoyl)-sophoroside-7-O-glucoside, kaempferol 3-O-(sinapoyl)sophoroside-7-O-glucoside, kaempferol 3-O-(feruloyl)-sophoroside-7-O-glucoside, kaempferol 3-O-sophoroside,
two isomeric forms of 1,2-disinapoylgentiobiose, 1-sinapoyl-2-feruloylgentiobiose, 1,2,2’-trisinapoylgentiobiose
and 1,2’-disinapoyl-2-feruloylgentiobiose. In general, internal leaves exhibited more constant chemical profiles.
Tronchuda cabbage seeds were also studied for their chemical composition. Thirteen phenolic compounds
were characterized and quantified by reversed-phase HPLC-DAD-MS/MS-ESI and HPLC-DAD, respectively:
two sinapoylgentiobiose isomers, three sinapoylglucose isomers, kaempferol-3-(sinapoyl)sophorotrioside-7glucoside, sinapoylcholine, kaempferol-3,7-diglucoside-4’-(sinapoyl)glucoside, three disinapoylgentiobiose
isomers, 1,2,2’-trisinapoylgentiobiose and 1,2-disinapoylglucose. 1,2-Disinapoylgentiobiose was the compound
present in highest amounts, followed by kaempferol-3-(sinapoyl)sophorotrioside-7-glucoside and 1sinapoylglucose, being 1,2-disinapoylgentiobiose isomer the minor compound. Seven organic acids (aconitic,
citric, ascorbic, malic, quinic, shikimic and fumaric acids) were also identified and quantified by HPLC-UV.
Ascorbic acid was the main compound, followed by citric acid.
Studies regarding the influence of the white cabbage butterfly Pieris brassicae on tronchuda cabbage chemical
composition are in course.
Within the scope of a Ph.D. project the phenolic compounds and the organic acids composition of Edible
mushroom species, the edible beefsteak fungus Fistulina hepatica was determined by HPLC/DAD and HPLC/
UV, respectively. The results showed a profile composed by five phenolic compounds (caffeic, p-coumaric and
ellagic acids, hyperoside and quercetin) and six organic acids (oxalic, aconitic, citric, malic, ascorbic and
fumaric acids). The quantification of the identified compounds revealed that ellagic acid and malic acid are the
main compounds in this species. In a general way the phenolic profile revealed to be more constant than the
organic acids one and could be more useful for the quality control of the species. Studies with other edible
mushroom species are in course.
Within the scope of a post-Doctoral project the analytical methodology for the chemical characterization of
white “Vinho Verde” grapes, by their most important phenolic compounds and organic acids, was achieved by
the sequential application of the experimental design that allows a large number of factors to be screened
simultaneously, to determine which of them has a significant effect on the analytical signal instead of a
traditional strategy of optimizing each variable separately. Plackett-Burman design was used as a screening
method in order to select the variables that have influence on the system and subsequently a statistical
model of central composite design was used to calculate the optimum value for each variable. The developed
method combines solid-liquid extraction of both phenolic compounds and organic acids from grapes and a
subsequent clean-up step using solid-phase extraction (SPE). Phenolic compounds and organic acids were
determined by HPLC/DAD and HPLC/UV, respectively. The identified and quantified phenolic compounds
were: quercetin-3-o-glucose, kaempferol-3-o-rutinoside, isohamnetin 3 glucose, quercetin, kaempferol and
epicatequin. The determined organic acids were oxalic, citric, tartaric, malic, shykimic and fumaric acids.
The methodology will be applied to the determination of these compounds in white “Vinho Verde” grapes from
several varieties.
In another work the anthocyanins, organic acids and voltatile phenols composition of red wine obtained from
Touriga Nacional grapes growing in Dão region (Portugal) was determined by HPLC/DAD, HPLC/UV and GC/
FID, respectively. By these means nine anthocianic compounds (malvidin-3,5-O-diglucoside, cyanidin-3-Ogalactoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin, cyanidin,
pelargonidin and malvidin), six organic acids (ketoglutaric, tartaric, malic, quinic, lactic and shikimic acids)
and two volatile phenols (4-ethylguaiacol and 4-ethylphenol) were identified and quantified. Malvidin-3-Oglucoside, the pair lactic plus shikimic acids and 4-ethylguaiacol were the main anthocyanin, organic acids
and volatile phenol, respectively. The effect of nine different Dekkera bruxellensis strains on these chemical
parameters was also evaluated. The results obtained indicate that some strains of D. bruxellensis yeast are
able to deteriorate red wine from Dão region during its maturation by the production of volatile phenols, namely
4-ethylphenol.
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Ballota nigra (Lamiaceae), known as black horehound, is a perennial herb which is commonly distributed in
most areas of the world that have mild climate. It is a small herb, which flowered aerial parts are gathered,
dried and used for medicinal purposes. A phytochemical study was undertaken in this species, and seven
phenolic compounds (chlorogenic, caffeic and caffeoylmalic acids, ballotetroside, forsythoside B, verbascoside
and allysonoside) and eight organic acids (oxalic, aconitic, citric, ascorbic, malic, quinic, shikimic and fumaric
acids) were identified and quantified by HPLC/DAD and HPLC/UV, respectively. Forsythoside B and quinic
acid revealed to be the main compounds in its infusion.
The phytochemical characterization of Linaria vulgaris (Scrophulariaceae), known as yellow toadflax, is in
course.
Food Safety
In the field of food contaminants, the project (approved and financed by FCT) aiming the assessment of the
dietary exposure of Portuguese consumers to acrylamide proceeds. With this goal an isotope labelled GCMS analytical methodology was developed and allowed the accurate quantification of the compound in distinct
kind of food matrixes. The referred method was applied to the determination of the levels of the compound in
some starchy food products that include chips, potato crisps, breakfast cereals, biscuits and crisp bread.
Furthermore, we started to study the role of some raw material properties in acrylamide formation during food
processing, namely in cheap frying.
A special attention has been devoted to study the presence of acrylamide in coffee and coffee derivatives,
probably one of the most important sources of acrylamide for Portuguese consumers. A specific SPE/GC-MS
analytical methodology was optimised and used for screening the presence of the compound in this kind of
products. Furthermore, studies to explain the formation of the compound during the processing of coffee rawmaterials are in execution. An MSc thesis will be presented next year concerning this subject.
Also pesticides residues are a real concern in food chain and, used without knowledge, can contribute to the
expansion of serious environmental and public health effects. Either in their original chemical structure or in
metabolic or degradation products, it is therefore common to find trace amounts in agricultural and agroalimentary products.
Pesticides remain the first line of defence in pest control when crop injuries and losses become economic.
They are the only answer to a severe pest outbreak or emergency and are typically dosed close to or post
harvest. Even though some times unexpected, another problem arising at pesticide control is the presence of
metabolites or conversion products of the analyte. Depending of its pathway at the environment, different
chemical structures can be found within the several matrices. Additionally, the concentration levels expected
here are, in most cases, much lower than those of the original pesticide but much more toxic. This is the case
of Fenthion (I) an organosphophorus insecticide, widely used in the control of olive fruit fly (Bactrocera oleae
Gml.) pest. It is rapidly converted to fenthion sulfoxide (II) and subsequently by oxidation gives origin to
fenthion sulfone (III). Another oxidative process due to the plant enzymatic action can involve the other sulphur
atom in the fenthion moiety, thus leading to the formation of another three metabolites (fenoxon (IV), fenoxon
sulfoxide (V) and fenoxon sulfone (VI). All the metabolites show higher toxicity than the parent compound: the
letal dose (LD50) is 220 mg/kg for I, 125 mg/kg for II-IV, 50 mg/kg for V and 30mg/kg for VI.
Due to the possibility of the presence of these compounds olives and olive oils a matrix solid-phase dispersion
(MSPD) methodology has been developed to extract the fenthion and its 5 metabolites in olives being the
method compared with a conventional liquid-liquid extraction.
The extracted compounds were analyzed by gas chromatography using a nitrogen phosphorus detector for
quantification and mass spectrometry detection for identification. The developed methodology MSPD provide
alternative method to liquid–liquid extraction and offers additional benefits due to its high sensitivity, simplicity
and small size of sample required. Two papers in this field are in preparation. In the same context and similar
approach Fhosmete were also determined in olives after the development of specific techniques for it extraction
as well as its metabolites. The results obtained are in evaluation and a paper will be submitted to publication.
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Dimethoate are another pesticide in study but it determination and metabolites will be performed in the
Microbial Biophysics and Residue Chemistry Research Unit - Eastern Regional Research Center (USA) with
supervision of Prof. Steven J. Lehotay.
Olive trees are attacked by a variety of insects and other pests, which cause a reduction in the quality and
quantity of the olives and oil produced. Many of these are controlled by organophosphorus pesticides, endosulfan
and several synthetic pyrethroid pesticides, which are registered for use in olive groves in some UE members
(Greece). The compounds are lipophilic, with high n-octanol-water partition coefficients ranging from 4.7 to 7.0
(log values), which suggests that residues will concentrate in the oil during extraction from olive oils. Portugal
is a olive oil producer and it will be important to control the presence or not of these products or their
metabolites in order to answer to health concerns and simultaneously to prevent unethical operating and
marketing practices and implement the value of regional or traditional references. To answer these questions
a GC/NPD methodology was implemented for the multiresidual determination and quantification of
organophosphorus pesticides being applied to olives and virgin olive oils from biological and conventional
agriculture. Samples harvested in different regions of Portugal and submitted to phytossanitary treatments
with the referred product are in evaluation.
The presence of biogenic amines in foodstuffs constitutes an important food safety problem, due to its implication
in phenomena of food intolerance and intoxication. Traditional foods have been an important part of the
Portuguese diet and it was observed that a high intake of harmful amounts of biogenic amines from traditional
Portuguese fermented foods is possible, since the few data available for Portuguese traditional cured sausages
and cheeses indicate the presence of high contents of putrescine, cadaverine, tyramine and in some cases
histamine. Methodologies previously validated for characterization of biogenic amines of Protected Denomination
of Origin (DOP) Portuguese meat products and cheeses were applied to extend the current limited database
on this topic, and contribute to evaluation of exposure of the Portuguese consumer to biogenic amines and in
some cases propose modifications in traditional manufacture to reduce biogenic amines.
Oils and fats are rich sources of essential fatty acids and other nutrients but, when used over prolonged
periods, their biological value decreases and several hazard substances are formed by oxidation, hydrolysis,
and polymerization. The quality of the frying oil is a critical factor to preserve high quality and produce safe
fried products. The used oil rejection should be easily determined. The polar components (TPC) comprise a
multitude of different artefacts and their content is a suited indicator of fat quality, being the basis of legislation
in many countries for the control of used frying fats. Nevertheless, the official methodology is time consuming,
and unsuitable for on-line assessment. Several quick tests measuring colour and dielectric constant have
been developed and are commercially available. An MSc. student developed this subject including the study:
1. test the efficiency of several quick tests while comparing with official methodologies; and 2. evaluate
several frying operations in a restoration unit. Different foods and frying times were evaluated until the usual
rejection point in that unit, based in visual inspection. The quick tests evaluated were simple to use, relatively
inexpensive, “green”, and mostly able to stand up to the rigors of frying and handling by workers with reduced
analytical knowledge. The samples collected in the restoration unit clearly demonstrate that the oils are
precociously rejected comparatively with the legislation limits, being therefore on no health concern. With the
diffusion of these quick tests the oils will probably be used for longer periods, still within legislation, with
advantages for both industry and environment. The higher concerns are indeed the small restaurants were the
oils are intermittently used, for uncontrollable periods, and were no quality control is implemented. A MSc.
thesis is practically concluded.
In this context another MSc. student will develop an HPLC methodology to determine polymerized triacylglycerol
contents in the previously referred oils correlating these contents with TPC and safety of the frying products.
Also in this subject some studies to evaluate the performance of several oils and fats to be used in industrial
frying (catering and industry) are being performed by request of an oil producer. The catering frying procedures
are simulated in laboratory and the quality of the frying oils evaluated along the frying time.
Trans fatty acids are naturally present in animal fats (ruminants) and formed in vegetable oils and fats by
hydrogenation. Some health concerns (e.g. increase blood cholesterol levels) about its presence in foodstuffs
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conducted to the government’s decisions to limit its contents of certain fatty foodstuffs, the agreement between
manufacturers in Europe to this effect and USA’s ruling that this content must be declared. As a result of the
decisions mentioned above, the edible oils and fats industry is required to lower and control the trans content
of its products while maintaining a fatty acid composition acceptable in terms of health nutrition. To know the
scenery in Portuguese products, namely cookies / biscuits, a project accepted and supported by UP rectorate
are in execution with the collaboration of GRAC. The objective is the implementation of environmental friendly
procedures to fat extraction (microwave). The project includes the evaluation of 100 brands of cookies marketed
and available to the consumers.
Polycyclic aromatic hydrocarbons (PAHs) are a diversified family of more than 100 lipophilic organic
contaminants composed by two or more fused aromatic rings. These compounds are mostly formed by
incomplete combustion of organic matter as a consequence of a series of natural and anthropogenic processes.
Due to their multiple potential sources of contamination, PAHs are ubiquitously distributed in nature. Therefore,
human exposition is virtually unavoidable, which raises an important public health concern due to their recognized
carcinogenic activity. Exposition to PAHs was epidemiologicly associated with an increased risk of skin and
lung cancer. Sixteen PAHs are actually classified as priority pollutants by Environmental Protection Agency
(EPA) on the basis of their occurrence and carcinogenicity being six with 4-6 rings classified as heavy PHAs
the potential toxic in food chain.
Diet is the major non-occupational source of PAHs for non-smokers. Due to their lipophilic nature, PAHs may
contaminate fats and oils, which represent, along with cereal products, the principal food sources but fish and
smoked foodstuffs are also subject of concern. In order to determine the dietary exposure of Portuguese
consumers to PHAs a work program was drew up and will be developed along the next year.
Beneficial compounds of foodstuffs
It is important to emphasize that the major chemical markers of authenticity referred above are also beneficial
compounds of foodstuffs but will not referred in this item.
Separation and characterization of bioactive peptides from whey hydrolysis to be applied in food and
pharmaceutical industries: Whey is a by-product in the manufacture of cheese. Although whey protein content
is relatively low (0.6%), finding alternative uses for whey solutions is a concern for the dairy industry. Hydrolysis
of whey proteins by enzymes immobilized on an inert support can either change or evidence functional
properties of the produced peptides, thereby increasing their applications as food ingredients. A process for
separation of bioactive peptides based on affinity chromatography and using continuous systems will be
developed for ALPMHIR, the most potent antihypertensive peptide obtained from trypsin hydrolysis of blactoglobulin. Similar process will be developed for other bioactive peptides with antimicrobiane action. This
process will enable recovery of bioactive peptides with minimal destruction thus enabling utilization by returning
these active peptides to functional cheese. Other peptides that can be used as ingradientes in infant formula
industry will also be recovered.Bioactive peptide profile of cheeses rich in probiotic microorganisms with low
fat content and appreciated sensory characteristics will be compared with functional cheeses enriched with
selected peptides from whey.
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 were evaluated, indicating that our feeding style is
already far from the original and health claimed “Mediterranean diet”. The nutritional composition of the traditional
Portuguese sausage “alheira” was determined and evaluated the modifications that occur with different cooking
processes.
In this field were also evaluated the nutritional compositions of confectionery cakes.
(Susana Casal, Eulália Mendes, Rita Carneiro)
“Expresso” coffee is being studied for its chemical and sensorial attributes in order to provide a safer product
by reducing the levels of unhealthy substances and increasing those with known health benefits. Different
blends and degrees of roast will be studied. This subject will be developed by a PhD student that obtained a
Grant from FCT.
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Garlic (Allium sativum) has the reported benefits of lowering cholesterol levels, reduction hypertension, and
acting as an antimicrobial agent in living systems. The active ingredients in garlic are thought to be the
sulphur containing compounds, or thiosulfinates, present in the volatile oil. Allin, which converts to allicin (Sallyl-2-propenthiosulfinate) in the presence of the enzyme allinase, is present at about 1% in garlic cloves.
About 60 accessions belonging to the Portuguese garlic germplasm collection held at Banco Português de
Germoplasma Vegetal, Braga, are currently analyzed for the allicin content using an RP-HPLC-UV method.
Preliminary results indicate a high degree of variation in the allicin content which can be very valuable for garlic
breeding and improvement of Portuguese varieties. This line research will be included in a MSc. thesis in
execution in collaboration with CIBIO-CEQUP.
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.
Beer composition and beer foam stability
Comparison between composition of two types of beer: with alcohol and without alcohol. Study of beer foam
stability, by characterization of protein profiles using reversed phase and size exclusion –HPLC. Contribution
of iso-alpha acids, organic acids, sugars, amino acids and others to foam stability.
Development of an HPLC method using a “light scattering” detector was developed for separation and
quantification of sugars in food matrices (beer, honey, dairy products).
Chemometric techniques
Given the great number of analyses and techniques applied to the same foodstuffs, chemometric techniques
were used in order to evaluate the relationships between different types of parameters, to determine the best
parameters to characterize a given product, and to classify new observations in relation to predefined models.
Univariate, bivariate and multivariate statistics were applied, mainly principal components, canonical variate
and discriminant analysis, canonical correlation, procrusts and generalized procrusts analysis, as well as 3way Tucker analysis. Complete algorithms were written in order to developed three main lines of work: (i)
producing detailed analysis for pedagogic purposes, (ii) enhancing the use of statistics in the department
through the application of predictive biplots for data description and analysis, and through interpolative biplots
for routine laboratory classification purposes, (iii) generalizing and evaluating the advantages and disadvantages
of biplots in current industrial and investigational practices. This work resulted in a PhD thesis that will be
presented next year. Simultaneously a book in this subject is in preparation by orders from the editor.
Microbiology
The rapid emergence and spread of several antibiotic resistant bacteria in both clinical and community settings
during the last years have been facilitated by the use of antimicrobial agents in hospitals and in human
husbandry. In an attempt to control the problem of antibiotic resistance the European Union banned the use
of antibiotics that are analogues to those used in human medicine for purposes of growth enhancement in
food-producing animals. Portugal is one of the EU countries with the highest antibiotic use in veterinary
medicine and with the highest rates of vancomycin resistance in enterococci. Thus, studies on diversity of
both resistant bacterial strains and their genetic elements involved in antimicrobial resistance are essential
for the control of antimicrobial resistance dissemination. Following, the achievements obtained in this field
were:
High occurrence of antibiotic resistant enterococci suggests maintenance of selective pressure by the use of
antibiotics/other substances in the Portuguese poultry production. Persistence of a number of widespread
PFGE types containing different resistance genes might reflect environmental/host adapted enterococcal
strains that might contribute to the maintenance of antibiotic resistance, thus constituting a resistance reservoir
that is non-sensitive to banning interventions.
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The simultaneous occurrence of a vancomycin-resistant E. faecium (VREF) strain among swine in four distant
European countries for at least a 4 year period and the presence of a common Tn1546 type D indicate
stability of strains and vancomycin genetic elements in swine production. The finding of a group of genetically
closely related strains, including both vancomycin-susceptible E. faecium and VREF isolates, harbouring a
particular purK allele previously associated to E. faecium swine strains, might mirror wide dissemination of a
host specific clone more prone than others to acquire and spread different antibiotic resistance as reported for
human clinical E. faecium isolates.
The study with Portuguese Salmonella isolates indicates that antibiotic resistance mediated by integrons is
common among diverse serotypes. The presence of several categories of these genetic elements shared
among human and food isolates suggest that resistance determinants among animals may contribute to the
problem of multi-drug resistant salmonella in humans.
The newly described sul3 gene which confers resistance to sulphonamides has now been identified in 14
Salmonella isolates from three serotypes collected from human and nonhuman sources in Portugal, being
mainly observed in isolates from swine food products. The consumption of sulphonamides for veterinary use is
generally widespread in Portugal, particularly for swine production. So, the appearance of a newly described
gene and the simultaneous presence of several sul genes may reflect its high usage in food-producing animals.
The persistence of several sulphonamide resistance genes may be the result of the successive pressure
exerted by sulphonamides and other antimicrobial agents that are also commonly used and may be mitigated
by the fact that not all sulphonamide-resistant determinants exert a fitness cost, as described for the sul2encoding plasmid.
Quantification of food aditives
Never before has the range and choice of foods been so wide either in supermarkets, specialist food shops or
when eating out. Food additives play a key role in maintaining the food qualities and characteristics that
consumers demand, keeping food safe and protected from many environmental adverse conditions, such as
temperature changes, oxidation and exposure to microbes, which can change their original composition.
All food additives must have a demonstrated useful purpose and undergo a rigorous scientific safety evaluation
before they can be approved for use. Each commercialized product must also meet all legal requirements,
which includes controlling most levels of food additives. This creates the necessity of performing food analytical
controls in a routine fashion. Thus, simple, quick and selective methods producing low levels of effluents of
small toxicity and with reduced reagent consumption are required to address main environmental and economical
aspects of today’s society. Electrochemical techniques are explored for this purpose, and applied to the
determination of acidity regulators and flavour enhancers in foods of different sources.
Determinations of glutamate and maltol, two flavour enhancers, are being developed by means of voltammetric
procedures with gold and mercury electrodes. Both analytes are electrochemically reduced. The inherent
mechanism was studied after cyclic voltammetric experiments. Main analytical features such as detection
limit, quantification limit, slope, reproducibility, and selectivity were evaluated. Developed methods were meant
for analysis of soups, sauces and cakes. Microwave assisted procedures were also carried out to assist
preparation of complex food samples
Regarding acidity regulators, tartaric acid was determined by means of spectrophotometric procedures, carried
out in flow injection analysis set-ups. Solid-phase reactors are also in preparation for this purpose. Main flow
parameters are optimized and the optimum conditions are selected for analytical application of the method.
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ENVIRONMENTAL CONTROL AND REMEDIATION
Head of laboratory: Cristina Delerue Matos, Professor Coordenador / Helena Soares, Assistant Professor
Staff Members:
Maria do Carmo Vaz
Professor Coordenador
Simone Barreira Morais
Assistant
Maria Goreti Ferreira Sales
Assistant
Sónia Adriana Figueiredo
Assistant
Maria Isabel Branco Alves Martins
Professor Adjunto
Maria Teresa de Oliva Teles Moreira
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
Salomé Sousa Teixeira
Assistant
Maria Isabel Limpo de Serra
Assistant
José Tomás Soares de Albergaria
Technician
Maria Aurora Soares da Silva
Technician
Sérgio Alberto Monteiro de Morais
Technician
Paula Celeste Baptista Paíga
Technician
Ph.D. Students:
Carina Machado, Cristina Maria Rodrigues Ferreira Alves, Oriza Paula Guedes Tavares
Number of articles in scientific journals: 10 (161-170)
Number of Ph.D Thesis: 2
Pesticide Analysis
Determination of pesticide residues in food and environmental samples has received much attention in the
last few decades. Following the need to develop faster, cleaner and more reliable analytical methodologies
new chromatographic and electroanalytical methods have been developed.
Concerning chromatographic procedures such as LC-DAD, LC-MS, GC-ECD or MS, they have been applied
for the analysis of tomato samples, wine, must, grape and water. Microwave-assisted solvent extraction,
liquid-liquid extraction (LLE), and solid-phase extraction (SPE) and microextraction (SPME) have been used
to extract the analytes under study for chromatographic analysis.
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Voltammetric procedures for the determination of azinphos-methyl, parathion-methyl and diuron in soil samples
using gold ultramicroelectrodes were developed. Regarding soil pretreatment, microwave-assisted solvent
extraction was performed. This strategy is effective when compared to traditional extraction techniques,
providing reduced extraction times, reduced solvent consumption and increased sample throughput.
Other electroanalytical techniques were used to analyse natural waters and commercial phytopharmaceuticals.
Voltammetric, amperometric and potentiometric based procedures were used for this purpose. These were
applied to the determination of metam and molinate. Experiments were adapted to flow conditions, allowing
reduced time, cost and operator intervention. These electroanalytical approaches generated also effluents of
small toxicity.
Laboratory waste management
“Greening” of chemistry has clearly brought many advantages. Still, wastes from chemical analytical laboratories
are typically hazardous and present a high diversity. The fact that only small quantities are generated has
justified negligence of their existence or has induced laboratories to hire specific companies to perform this
task.
Thus, a waste management program was created and currently implemented for supporting all laboratory
activities of students in a Chemical Engineering degree. This program is responsible for waste separation/
disposal strategy, collection, and characterisation of all inorganic liquid toxic wastes produced. Once entering
the research laboratory, a proper chemical treatment is investigated and implemented.
Resulting solids are isolated, purified, and evaluated in terms of purity and contaminants. When possible,
solids are redirected as reagents towards another laboratory experiment. This feature provides a sustainable
perspective to the waste management program. Before discard in the public sewage system, all liquid effluents
emanating from suitable treatment are chemically controlled. Still, fulfilling chemical regulations does not
ensure lack of adverse effects in living organisms when aquatic environment is chosen as final destiny for
liquid treated wastes. Thus, chlorella vulgaris has been exposed to these wastes in order estimate inherent
toxicity effects. As comparison, untreated wastes have also been subject of ecotoxicity trials.
Soils remediation
Soil contamination is closely related with inadequate practices of some industrial activities, disposal of industrial
and municipal wastes or environmental accidents. Minute amounts of an organic compound are able to
contaminate soil and groundwater, which grants serious risks for environment if toxic or carcinogenic compounds
are concerned. Several technologies, like soil vapour extraction (SVE), in-pulp solvent extraction and
bioremediation, are suitable to enable remediation at low cost.
SVE is simple, relatively cheap and very efficient to remove from soils volatile organic compounds, usually
correlated to underground fuel reservoirs. This strategy has been applied to remediation of soils of sand,
organic matter and clay contaminated with benzene, toluene, ethylbenzene, xylene, trichloroethylene and
tetrachloroethylene. SVE experiments are performed in a pilot installation with vacuum pumps to produce
airflow. This is meant to enable estimation of remediation time, a feature that a conventional SVE procedure
does not preview.
In-pulp solvent extraction procedures were used to remediate soils with petroleum hydrocarbons. Ternary
systems composed by solvents of small environmental concern and able to establish a single phase mixture
were used for this purpose. Contaminants used in this study were 2,4-trimethylpenthene, xylene, naphthalene
and hexadecane. Main analytical features for an improved efficiency were established. Effect of soil parameters
and possibility of solvent regeneration were also appraised.
Bioremediation by means of dual bio-augmentation is a novel approach that enhances degradation of organic
pollutants regarding the use of microorganisms resistant to toxic metals. Four robust strains (Methylobacterium
strains PM1, Mi1 and F5.4 and Methylophilus str. EHg7) are currently used to improve degradation of
trichloroethylene (TCE) and methyl tert-butyl ether (MTBE) by soil microbial communities. Ability of natural
microflora to degrade organic compounds in soils is under study prior to its enrichment with Methylosinus
trichosporium str. OB3b as active degrader. It is proposed to set up microcosm experiments in the presence
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or absence of inocula of the 4 bacterial cultures in the presence and absence of heavy metals (Cd, Cr or Pb)
and to study the effect of the addition of the selected organisms.
Gas chromatographic methodologies are developed for the quantification of pollutants and process monitoring.
Removal of toxic compounds by means of adsorption strategies
Adsorption plays a key role in modern industries, especially in the field of environmental protection engineering,
with the increasing environmental awareness of people all over the world. Many companies are looking at
tertiary treatment processes to remove contaminants from their effluents.
Several studies are in development with (inexpensive) natural adsorbents: cork, algae and wastes from corn
production. One of them explores the adsorption capacity of cork (Quercus suber L.) to remove pyrethroids
from waters, in order to attenuate the impact of this compound in aquatic life. An ecotoxicological evaluation
showed that synthetic pyrethroids are amongst the most toxic pesticides for aquatic organisms.
Other study aims to explore the ability of Portuguese macro algae species (Ascophyllum nodosum, Sargassum
muticum, Fucus Spiralis and Pelvetia canaliculata) for the removal of toxic metals (Cd(II), Zn(II), Ni(II), Cr(III),
Cu(II) and Pb(II)) from aqueous solutions is currently under development. This work is divided in the following
parts: (i) the effect of temperature, pH and initial concentration of Pb (II) and Cu(II) in sorption process onto
marine macro algae Ascophyllum nodosum was studied by using a Box-Behnken factorial design method,
which gives a mathematical model that shows the influence of each variable and their interactions; (ii) various
types of reagents, mineral acids (HCl and H2SO4) and aqueous solutions of ethylenediaminetetracetic acid
(EDTA) disodium salt, calcium chloride and sodium chloride were investigated for their efficiency to recover
Cu adsorbed by Ascophyllum nodosum; (iii) and finally equilibrium studies were conducted with mono and bicomponent metal solutions using batch adsorption experiments.
Finally, wastes (Zea mais) from corn production have been tested successfully as adsorbents for textile
dyestuffs. Batch equilibrium and Kinetic studies were performed. Kinetic studies were conducted using batch
adsorption experiments and fixed-bed columns and the experimental results were fitted to models.
Interaction between pH buffers and metal ions
In this project (POCTI/39950/QUI/2001), the complexation properties of several M-(buffer)x-OHy systems involving
six metal ions (Cd2+, Co2+, Cu2+, Pb2+, Ni2+, and Zn2+) and seven pH buffers (CAPS, CAPSO, CHES, TAPSO,
TAPS and AMPSO) were characterized. No complexation was detected between three buffers (CAPS, CAPSO
and CHES) and four metal ions (Cd2+, Cu2+, Pb2+, and Zn2+) tested. For the other systems, results indicated
complexation and the final models of thirteen systems were fully defined.
Biorremediation of wastewaters using microorganisms
The main aim of this project, which started in 2005, is to develop a clean technique, using yeast brewer’s
cells, for the treatment of wastewaters containing heavy metals
The evaluation of yeasts flocculation capacity in the presence of heavy metals, as well as the optimization of
metal ions accumulation conditions by the biomass was studied (financial support: POCTI/CTA/47875/2002
and UP/CGD /2005).
New environment-friendly chelating agents for industrial and domestic applications
This is a multidisciplinary project, financed by REQUIMTE and FCT (POCI/QUI/57891/2004), which involves
researchers from the two centers of REQUIMTE.
The project started in 2005 with the synthesis of the new ligands. Then, biodegradation and metal complexation
studies will be performed to check the environmental impact of these ligands and their strength as metal
chelators, respectively.
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ANALYTICAL METHODOLOGIES
Head of laboratory: Aquiles de Barros, Associate Professor
Research Team:
José António Maia Rodrigues
Assistant Professor
Paulo Joaquim Ferreira de Almeida
Assistant Professor
Luís Guilherme de Lima Ferreira Guido
Assistant
Maria Isabel Afonso Rocha
Assessor
Maria Fernanda Rocha M. L. O. Cabral
Associate Researcher
Cristina Maria F. Delerue Alvim de Matos
Professor Coordenador
Simone Barreira Morais
Assistant
Maria do Carmo Vaz
Professor Coordenador
Maria Goreti Ferreira Sales
Assistant
Simone Barreira Morais
Assistant
Maria de Fátima Sá Barroso
Assistant
Hendricus Petrus Antonius Nouws
Assistant
Paula Celeste Baptista Paíga
Assistant
Post-Doct Students:
Pedro Miguel Gonçalves Rodrigues
Ph.D. Students:
Andreia Filipa da Silva Curto
M.Sc. Students:
Marta Sofia Roma Pires, Maria Fernanda Andrade Resende, Sérgio José Pinto Teixeira
Number of articles in scientific journals: 7 (171-177)
Number of M.Sc. Thesis: 1
Activity in 2005
The strong lines of the project announced for the triennium 2003-2005 are the consolidation of the growing
investigation related with beer and the diversification of the analytical techniques used. In this context a
special reference is made to the research that is being devoted to the studies related with the problem of beer
ageing, no doubt the main particular topic considered in the development of the project. Anyway, it is important
to note that voltammetric analysis still represents an important field of investigation in the context of the
project, as a continuation of the research produced before 2003.
The main activities under development during 2005 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 the three years joint project Beervolt, approved by “Agência
de Inovação” in 2004, was continued. This project, initiated in April 2004, is based on a patent meanwhile
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submitted (already approved in Portugal and in phase of EC approval). It consists in the development of a
equipment for the voltammetric determination of diacetyl directly in the fermentation vessels and involves two
other companies: Carlsberg S/A and CAI.
The cooperation with Carlsberg S/A, Denmark - The joint project previously referred involving this important
beer company will allow the intensification of a collaboration that exists since the year 2000.
The cooperation with Institut Français de la Brasserie et de la Malterie, I. F. B. M., Nancy, France In the
context of a collaboration involving Unicer and I. F. B. M., the Ph. D. student of this group Andreia Curto was
involved in a post-graduate training of 6 months at I. F. B. M., in the context of a Ph. D. “mix grant” that she
obtained from FCT. The main objective of this joint research was the study of the impact of several technological
factors of the malting and brewing processes on the organoleptic stability of beer.
The cooperation with the Instituto Superior de Engenharia do Porto (ISEP) – This collaboration consists
essentially in the co-supervision of the Ph. D. work of Henri Nouws, lecturer at the ISEP. The work is cosupervised by Aquiles Barros and Cristina Delerue-Matos. A HMDE flow cell developed in our group is being
used in the investigation, and some results were already presented in scientific meetings and published in
scientific journals.
Another branch of the research worthy of mention is:
The cooperation with the Department of Civil Engineering of the Faculty of Engineering of Porto – It is a
collaboration essentially devoted to studies on the accelerated degradation of geosynthetics; the work involves
the investigation of the behaviour of these materials after being submitted to the effect of some chemical
agents, under especially intense degradation conditions.
Plan for 2006
For 2006, the objective of the group is to continue the activity of 2005, with special focus on:
— 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;
— 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);
— 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.
— opening of a new line of investigation focused on analytical, industrial and biochemical studies of some
phenolic compounds present in beer and in hop.
— opening of a new line of investigation focused on the use of membrane based extraction techniques with
application in the development of analytical methodologies in flow for food analysis.
Electroanalysis
Electrochemistry is often required in the analytical chemistry field because of its selectivity, sensitivity, and
generation of effluents of low toxicity. It may also provide information concerning oxidative/reductive mechanisms.
Different voltammetry-based methods were developed, exploiting oxidation/reduction of the analyte as well as
its adsorption, using conventional electrodes like glassy carbon and hanging mercury drop electrodes or
ultramicroelectrodes. Development of flow methods was done by coupling amperometric and stripping
voltammetric detectors to flow-injection analysis set-ups. Different working electrodes were used here, including
glassy carbon, carbon paste, chemically modified and hanging mercury drop electrodes. This strategy was
applied to the analysis of different compounds within pharmaceutical and environmental fields.
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Ion-selective electrodes are also constructed for presenting the attractive feature of selectivity. Selective
membrane composition and configuration were regarded. Preparation of polymeric sensors was selected
after evaluating the effect of ionophore and plasticizer natures. Devices of cylindrical configuration were prepared
for flow set-ups. This arrangement ensured a laminar flow, decreasing dead volumes and the response time of
the detector.
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PHYSICOCHEMICAL CHARACTERIZATION OF FOOD PRODUCTS
Head of laboratory: Maria do Pilar Gonçalves, Associate Professor / Alberto Sereno, Associate Professor
Staff Members:
Loïc Hilliou
Assistant Researcher
Post-Doct Fellows:
Marta Isabel de Glória V. M. Silva
Ph.D. Students:
Luis Mayor Lopez, Wancheng Sittikijyothin, Fabio Donato Soares Larrotonda
Number of articles in scientific journals: 3 (178-180)
Number of M.Sc Thesis: 1
Rheological behaviour and morphology of protein-polysaccharide mixed systems
Studies were conducted on the effect of shear on b-lactoglobulin gels, pure or mixed with galactomannans
using dynamic oscillatory measurements. Different shear rates, time of shear and temperature conditions
were tested. The results showed that the rheological behaviour of the pure and mixed gels was very sensitive
to shear treatment. In all cases studied, the time needed to reach the gel point decreased and the gel
strength increased with increasing shear rate. The strain at fracture for mixed gels subjected to shear was
higher than for unsheared gels; whereas the contrary was observed for pure gels. The mechanical spectra
were analyzed quantitatively by fitting a Cole-Cole model to the storage and loss compliance versus frequency
data. The fits matched reasonably well the data in the high frequency region for the mixed gels.
The work is in its final stage. In 2006, some articles will be submitted to international journals and a PhD
dissertation will be also submitted by Wancheng Sittikijyothin in the Faculty of Engineering of the University
of Porto (FEUP).
Cold gelation of globular proteins
a) The denaturation conditions, namely temperature and holding time, and the protein concentration were
systematically varied in order to screen their impact on the resulting heat denatured whey protein isolate (HDWPI) solution viscosity and gel elasticity. Results show that the former variables can be reconciled in a single
parameter - the aggregates concentration - which controls the rheological properties of the HD-WPI solutions
and the texture characteristics of the corresponding cold-set gels.
b) In the frame of a scientific collaboration with a Brazilian working group, a study about the rheological
behavior of ‘Polpa de Açaí (Euterpe oleaceae)’ was initiated.
c) In the frame of a CAPES/GRICES project an investigation about biodegradable superabsorbent polymers
(SAP) has been developed. The swelling ratio, the degree of swelling, the water sorption isotherms and the
texture profile of the new materials were determined. The rheological behavior of the SAPs is also under
investigation.
Work plan for 2006
The studies mentioned in points b) and c) of the above paragraphs will be concluded at the beginnings of
2006. In the frame of our study about cold-set gelation of WPI, it will be developed the necessary hardware (a
transparent measurement cell and an optical train) to make structural studies during the gelation process.
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Technology for edible biodegradable films and coatings for foods
In this FCT project (POCTI/EQU/45595/2002), phycocolloids were extracted from Portuguese seaweeds. The
research activity was continued by mixing the isolated polysaccharides (k-i hybrid carrageenans) with starch.
Films were obtained from these mixtures by casting. These films were characterized about their physical and
chemical properties. The results of this characterization together with a comparison with model films prepared
from commercial k-carrageenan and starch were reported at the ENPROMER 2005 and the CIBIA 2005
meetings.
Workplan for 2006
Efforts in film formulation will primarily aim at lowering film hydrophilic properties by incorporating hydrophobic
chemicals. Finally, film formulation will be extended to pectins as planned in the work plan of a separated
project (see REQUIMTE project below).
COMFOOD: Controlling the microstructure of food products by rheo-optical methods.
This FCT funded project started in October.
Workplan for 2006
Building of an optical train and preliminary rheo-optical characterization of model food systems.
Physical characterization of PHA biopolymers
This FCT funded project started in September.
Workplan for 2006
PHA samples, produced by our partners at CQFB, will be characterized by Differential Scanning Calorimetry
(DSC) and rheology.
Characterization of biopolymer mixtures for food applications.
This GRICES/DAAD cooperation aims at studying commercial k- and i-carrageenan gels as well as k-i hybrid
carrageenans extracted from Mastocarpus stellatus seaweeds (see project POCTI/EQU/45595/2002) by means
of Fourier transform rheology (FTR) and new Magnetic Resonance Imaging (MRI) techniques. In a preliminary
study, a steady shear applied on cooling carrageenans solutions was shown to impede the gelation mechanism.
After shear cessation, gels can set with an elasticity that depends on the applied shear. The FTR signatures
of i- and k-i hybrid carrageenans gels are virtually similar, thus indicating resembling structures. The FTR
behavior of -carrageenans gels is more complex as it appears to be a combination of hard gel-like behavior at
low strains and of soft gel-like behavior at larger strains.
Workplan for 2006
Results reported above will be presented at the ISFRS 2006 and AERC 2006 meetings and 2 articles will be
prepared right after additional structural characterization (optical microscopy and/or cryoSEM imaging). Then,
polysaccharide mixtures such as gelatin-carrageenan blends should be studied with the same set of techniques.
Extraction of added value compounds from orange peel wastes: application to mixed biodegradable
films.
This project is funded by REQUIMTE. Films containing commercial pectins have been first characterized. The
rheological characterization of pectins produced by our CQFB partners has been initiated.
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Work plan for 2006
Characterization of newly extracted pectins by rheology and DSC. These new products will then be used in
films whose properties will be compared to commercial pectin-based films.
Studies in Food Structure
The last year studies about changes in food structure during processing were performed and some mathematical
models were applied in order to establish relations quality-macrostructure-microstructure of processed food
materials. This work focused on the study of structural changes during osmotic dehydration of pumpkin fruits.
The work is in its final stage, and in the next year some articles will be submitted to international journals in
the area of Food Science and Technology.
A Ph Degree dissertation, with the title “Characterization and modelling of structural changes in fruits and
vegetable tissue submitted to dehydration processes”, will be also submitted by Luis Mayor López in the
Faculty of Engineering of the University of Porto (FEUP).
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TOXICITY AND PROTECTION STUDIES
Head of Laboratory: Maria de Lourdes Bastos, Full Professor
Staff Members:
Rosa Seabra
Full Professor
Paula Andrade
Assistant Professor
Felix Carvalho
Assistant Professor
Fernando Remião
Assistant Professor
Helena Carmo
Assistant
Maria Elisa Soares
Assessor Principal
Eulália Mendes
Assessor Principal
Carla Rodrigues
Technician
Maria João Marques
Researcher
Ph.D. Students:
João Paulo Soares Capela, Ricardo Jorge Dinis Oliveira , Helena Pontes, Vera Costa, Miguel Lopes, Ema
Alves, Márcia Carvalho
M.Sc. Students:
Luis Correia, Renata Silva
Number of articles in scientific journals: 14 (181-194)
Biological activity of plant extracts and beverages
The hot water extracts of tronchuda cabbage internal and external leaves and of the seeds were investigated
for their capacity to act as scavengers of DPPH· radical and reactive oxygen species (superoxide radical,
hydroxyl radical and hypochlorous acid), exhibiting antioxidant capacity in a concentration dependent manner
against all radicals. Despite the antioxidant capacity exhibited by tronchuda cabbage internal leaves, in
general terms they exhibited lower antioxidant potential than external leaves. This can be ascribed to the
higher content of both phenolics and organic acids in the external leaves.
According to the results obtained in all assays, and in comparison with data from both tronchuda cabbage
internal and external leaves, it could be observed that, in general terms, tronchuda cabbage seeds exhibit
higher antioxidant potential than its leaves. This is not surprising once seeds often contain the highest
concentration of lipids of any plant tissue, with high levels of polyunsaturated fatty acids. The occurrence of
high amounts of phenolic compounds, particularly of hydroxycinnamic derivatives, and organic acids, namely
ascorbic acid, in tronchuda cabbage seeds suggest that these compounds protect storage lipids from oxidation.
Studies regarding the antioxidant capacity of tronchuda cabbage against reactive nitrogen species (nitric
oxide and peroxynitrite) are in course.
Beefsteak fungus Fistulina hepatica was investigated for its capacity to act as a scavenger of DPPH· radical
and reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid). Good results were
obtained against DPPH and superoxide radicals and hypochlorous acid but a prooxidant effect was observed
for hydroxyl radical. The study of the antioxidant potential of other edible mushroom species is in course.
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The antioxidant ability of the medicinal species Ballota nigra (Lamiaceae) infusion against DPPH radical,
reactive oxygen species (superoxide radical, hydroxyl radical, hypochlorous acid) and nitric oxide was
investigated. The tested infusion mainly exhibited a potent scavenging effect on DPPH, nitric oxide and
superoxide radicals. In hydroxyl radical assay a potent pro-oxidant activity was noticed. No effect was found
against hypochlorous acid.
Studies regarding the antioxidative capacity of Linaria vulgaris (Scrophulariaceae) are in course.
Due to the high content of antioxidant compounds, some foods could be able to delay or even revert the
neurodegenerative events related to the increased oxidative stress. The central nervous system is
particularly vulnerable to this phenomenon, a manifold process that plays a pivotal role during aging, after
chronic ethanol consumption and in some neurodegenerative diseases. This vulnerability is related to the
generation of harmful reactive oxygen species that lead easily to a depletion of antioxidant defences.
Polyphenols present powerful in vivo antioxidant properties and are particularly abundant in red wine and
green tea. Thus, we thought of interest to test the antioxidant efficacy of red wine and green tea after
prolonged consumption in the hippocampus and in the cerebellum of the adult rat. Using biochemical
methods we intended to quantify the activity of antioxidant enzymes, lipid and protein oxidation,
glutathione levels and transcriptional activity changes in these brain areas. These studies are in progress.
Studies of mechanisms of toxicity using in vitro and in vivo models
An increasing body of data has being demonstrating that mammalian cells have elaborate networks of molecular
signalling in counteracting heat shock and in developing adaptation to oxidative stress to avoid cell death.
However, the precise mechanisms linking heat shock, oxidative stress and cell survival/cell death mechanisms
are not yet clearly understood. The purpose of this study was thus to determine the usefulness of the freshly
isolated mice hepatocytes model for the study of the time course of hyperthermia-induced oxidative stress
and cellular signalling. Freshly isolated mouse hepatocytes obtained from Charles River CD1 male adult
mice were incubated under normothermia and hyperthermia. The toxic effects were evaluated by measuring
cellular viability and oxidative stress. Hyperthermia-induced cell signalling was evaluated through the
measurement of HSF1 activation and HSP70 expression. The results accomplished in this work demonstrated
that mild continuous hyperthermia leads to oxidative stress and loss of cellular viability in a time-dependent
manner. Additionally, it was also found that hyperthermia was capable of inducing a heat shock response
reflected the activation of HSF1, which emerged before the formation of HSP70 levels.
Continuing our mechanistic toxicity studies of amphetamine derivatives, a new in vitro model was
implemented in our lab in order to extend our research to the brain, an important target organ of the toxicity
of these compounds.
The widely abused psychoactive recreational drug 3,4-Methylenedioxymethamphetamine (MDMA or
“Ecstasy”) and MDMA-metabolites-induced were evaluated for their neurotoxicity in neuronal cultures from
rat cortex under normal (36.5ºC) and hyperthermic conditions (40ºC). Our studies showed that MDMA and
MDMA-metabolites produce a neurotoxicity that is potentiated under Hyperthermia. Also that MDMAinduced neurotoxicity is dependent on its 5-HT2A-receptor agonism and on the generation of reactive
oxygen species (ROS)/reactive nitrogen species (RNS). These studies bring new insights on the
mechanisms by which MDMA and its metabolites elicit neurotoxicity.
Amphetamine and it’s alike are substances with the ability of inducing pleasant sensations in those who use
them, being classified as psychostimulant drugs of abuse. The consumption of amphetamine analogous
compounds, in particular MDMA, increased in an expressive way in the last years. The clinical and experimental
evidences of the amphetamine and it’s alike toxicity, allied to the consumption in the younger population,
specially in the reproductive period, imposes the analysis of the situation. It’s our purpose to contribute to the
characterization of the behaviour, and in a long term, of the exposure to MDMA, using adolescent Wistar rats.
It’s being used a dosage of MDMA known to induce neurotoxicity. With the purpose of identifying the biochemical
and cellular modifications responsible for the behaviour changes, are being executed a set of biochemical
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quantifications. These studies are being accomplished in brain areas known to be specially damaged by
MDMA, namely hippocampus, striatum, accumbens, rafe and pre-frontal cortex. All these studies will be
equally executed to determine the simultaneous consumption of MDMA and ethanol gives the high frequency
with which they are consumed in the younger population.
Since ethanol and MDMA are usually consumed simultaneously, it is very relevant to evaluate the effect of
the association of these two substances on the hepatic cells as they are involved in the metabolism of
both drugs. Additionally, hyperthermia has been recognized as a life-threatening effect associated with
MDMA exposure. Therefore, a comparative in vitro study is being performed in isolated mouse hepatocytes
to evaluate the hepatotoxicity of MDMA, ethanol, and their association under normothermic and
hyperthermic conditions. These studies are in course.
The pesticide paraquat is responsible for several acute fatal intoxications being the lung the main target
organ of toxicity. However, no antidote or effective treatment was developed until now to decrease the
paraquat accumulation in the lung or to disrupt its toxicity. In vivo studies, particularly those directed to
decrease paraquat lung accumulation and those to interfere with the subjacent signaling pathways, which
lead to edema and subsequent fibrosis, have been performed.
All these toxicity studies are being developed by the PhD students and consistent results are expected in the
next year.
Some validated in vitro models, namely the microcrustaceous Daphnia magna and Artemia parthenogenica,
as well as the microalgae Tetraselmis chuii and the mosquitofish Gambusia holbrooki were used for the
evaluation of environmental contamination by acethylcholinesterase inhibitors and pharmaceutical drugs.
Biotransformation of compounds: in vivo and in vitro studies
In this research line important biomarkers of exposure (metabolites) and biomarkers of effect are being
identified and validated and we hope they will be of great value to establish toxicity mechanisms involving
the biotransformation of xenobiotics.
Sustained high levels of circulating catecholamines can lead to cardiotoxicity and neurotoxicity. We have
previously shown that the catecholamines oxidation products can further conjugate with GSH in isolated
rat cardiomyocytes and hepatocytes leading to the formation of potentially more toxic species. The next
points were studied during 2005:
1) An isocratic reverse-phase HPLC with photodiode array and/or coulochem detection methodology was
developed in order to analyse biogenic catecholamines-GSH adducts.
2) A sample treatment procedure for catecholamines-GSH adducts analysis in biological samples was also
developed and applied to human serum. The obtained recoveries were between 60 and 70% and the
chromatograms were clean.
3) (Glutathion-S-yl)-catecholamines were characterized by their UV and mass spectrometry (MS).
4) The development of this methodology allows a direct analysis of catecholamines-GSH adducts in blood,
enabling the study of the catecholamines oxidation process.
5) To establish the possible toxicity of cathecolamines in the presence or absence of a system capable of
generating oxygen free radicals (Xanthine and Xanthine Oxidase) on calcium tolerant cardiomyocytes
isolated from adults rats.
Results are being analysed and it is expected to be published in current year.
The influence of genetic polymorphism of the main metabolizing enzymes of the designer drugs of abuse 4MTA, 2C-B, and MDMA in their toxic effects was evaluated. By using cell lines that express the genetic
variants of human metabolizing enzymes (CYP2D6, CYP3A4) we are able to identify the enzymes that are
mostly involved in the detoxification of these drugs and also the relationship between polymorphic
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metabolism and toxicity. Subsequent use of human liver microsomal fractions profiled for the enzymes of
interest and incubation with susceptible cell lines will be performed in order to determine if genetic
polymorphism is in fact related with the overexpression of the toxic effects of these designer drugs of
abuse. With this aim we have developed the following tasks:
1. Evaluation of the influence of polymorphic metabolism in the cytotoxic effects of MDMA and 4-MTA.
Studies with V79 cell lines genetically engineered for the expression of polymorphic variants of human
CYP2D6 enzyme.
2. Development and validation of an analytical technique for the quantification of the oxidative metabolite of
MDMA, N-methyl-á-methyldopamine.
3. Characterization of the oxidative metabolism of MDMA catalyzed by CYP2D6 in V79 cell lines
genetically engineered for the expression of polymorphic variants of human CYP2D6 enzyme. Identification
and quantification of metabolites.
4. Evaluation of the cytotoxicity of N-methyl-á-methyldopamine towards the addopted in vitro experimental
model. Comparison with the parent compound MDMA
Important results were obtained and were already submitted for publication and other studies under this
subject are in course
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BIOMIMETIC SYSTEMS AND SIMULATION
Head of Laboratory: Maria João Ramos, Associate Professor
Staff Members:
Alexandre Magalhães
Assistant Professor
André Melo
Assistant Professor
José Augusto Pereira
Assistant Professor
Pedro Fernandes
Invited Assistant Professor
Agostinho Antunes Pereira
Assistant Researcher
Nelson Brito
Technician
Ph.D. Students:
Akapong Suwattanamala, Rute Fonseca, Susana Pereira, Nuno Cerqueira, Fátima Lucas, Vineet Pande,
Sérgio Sousa, Ricardo Branco, Alexandra Carvalho, Irina Moreira, Zenaida Mourão, Alexandre Carvalho, Marco
Preto, Daniel Dourado, José Rui Marques, Alexandra Marques, Bruno Tamames, Juan Tamames.
M.Sc. Students:
Eva Cunha, Daniel Osório
Project Grantee:
Natércia Brás, Sónia Patrício
Number of articles in scientific journals: 23 (195-217)
Number of articles in books: 1
Number of M.Sc Thesis: 2
Just as in the past few years, we have been generally interested in the molecular modelling of biological and
chemical systems. To further these studies, we have been using both molecular simulations and quantum
mechanics techniques, as well as quantum mechanics/molecular mechanics (QM/MM) or quantum mechanics/
quantum mechanics (QM/QM) hybrid methods. Last year we have added to our present, general interest in
proteomics, a new line of computational genomics. Our idea is to eventually establish a close link between
both areas, which we have already started doing. A more detailed description concerning our main present
interests, which have derived from year 2004 and will continue in 2006, follows:
I. Disease Related Research
Cancer
(i)The study of the catalytic and inhibition mechanisms of enzymes P450 1A2 1,2 (an enzyme involved in
the metabolic pathway of carcinogenesis) RNR 3-5 (ribonuclease reductase, an enzyme thought to be involved
in cancer), and other enzymes 6-11, resorting to both quantum mechanics, QM/QM and QM/MM methods, has
advanced very much. These studies have been financed by the National Foundation for Cancer Research,
U.S.A. (P450 1A2) and by the Fundação para a Ciência e Tecnologia, Portugal (RNR) via project POCTI/
35376/QUI/2000.
(ii)We are further involved in the study of the catalytic and inhibition mechanisms of other enzymatic reactions,
some of them also thought to be involved in cancer, such as superoxide dismutase, glutathione transferase
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and farnesyl transferase12-16. These studies have been financed by the Fundação para a Ciência e Tecnologia,
Portugal (RNR) via project POCI/QUI/61563/2004.
(iii)Continuation of the screening of 3,5 billion small molecules as potential inhibitors of 16 proteins,
directly involved in cancer, which are current targets of the pharmaceutical industry. This project is part of the
work carried out by the NFCR Centre for Computational Drug Design, based at Oxford, and directed by Prof.
Graham Richard of which Maria João Ramos is an Associate Director, being financed by the National Foundation
for Cancer Research, U.S.A 17.
A.I.D.S.
(iv) The study of the problem of new potential therapeutic agents for the treatment of acquired
immunodeficiency syndrome (AIDS) by investigating the binding modes of different anti-AIDS chelators like
ATA, HPH and other analogs to achieve a better design of anti-HIV metal chelators, continued. Several
research works were published on this theme, as well as some reviews18-22. This project is being carried out in
close collaboration with two experimental organic chemists, Dr. R. Sharma, in India, and Prof. M. Otsuka, in
Japan.
Hypertension
(v) To help on the modelling of mimetic modifications of bioactive peptides by inclusion of novel synthetic
amino acids, we have run extensive molecular dynamics on angiotensin II in both environments, water and
DMSO23. 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 projects POCTI/35380/QUI/2000 and POCI/QUI/
55673/2004.
II. Drug Design Complementary Studies
(vii)Alanine scanning computational mutagenesis has been studied and a new methodology proposed. For
this reason, extensive molecular dynamics simulations of protein complexes has been carried out and
computational mutagenesis performed on the interface of these complexes, which has allowed the establishment
of the respective hot spots 24-27.
(viii) Continuation of the development of new formalisms to analyse the molecular interactions in association
processes 28. This involves the partitioning of the physical observables of a molecular system into spatial and
physical meaningful components. These decomposition methodologies can be used as powerful tools for
molecular modelling and design of biological systems.
(ix)Calix[4]arenes are molecules that possess 4 aromatic moieties which can form p-electron-rich cavities
and behave as host compounds for ions and neutral species. Therefore, the adsorption of calix[4]arenes in
electrode/electrolyte interfaces is an interesting field to be explored to get more insight about the role and
properties of adsorbed molecules, as well as the nature of those interfaces and its applications to ionselective electrodes or potentiometric sensors. The work developed in this period has been concerned with
the study of thialcalix[4]arenes and their complexation with transition metal ions. We are further involved in
the design of new calixarene derivatives to help the experimental synthesis of potential host compounds of
this type 29,30.
(x)Efforts have also been made with imparting education on drug design 31
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III. Computational Genomics
We have started a line of research on Computational Genomics with the goal of expanding the team’s research
experience on Bioinformatics, allowing the simultaneous integration of genomic 32,33 and proteomic data. The
study of insertions of fragments of mtDNA into nuclear chromosomes is an important mechanism for the
evolution of genomes, and when targeted to gene loci, such insertions can be mutagenic, causing disease.
We have been studying this subject in more detail and additionally, we have been supervising research on the
evolution and comparative genomics of genes with important biological functions, namely the gene coding the
human enzyme ABAD that is linked to the mitochondrial toxicity in Alzheimer’s disease.
III. Requimte’s collaboration projects
Requimte itself has provided grants for collaborative projects between different groups in Porto and Lisbon and
we have won two of those projects, with other different groups also belonging to Requimte, and have been
investigating on:
CBM11
Molecular determinants of ligand specificity in the family 11 Carbohydrate Binding Modules.
Anthocyanin Blues
A green approach towards food colorants and new pigments based on anthocyanins and synthetic flavylium
salts.
References:
[1] R. Fonseca, M. Marini, A. Melo, M. C. Menziani; M. J. Ramos Medicinal Chemistry, 2005, 1, 355-360
[2] F. Iori; R. Fonseca; M. J. Ramos, Bioorganic Medicinal Chemistry, 2005, 13; 4366-4374
[3] N. M. F. S. A. Cerqueira; S. Pereira; P. A. Fernandes; M. J. Ramos, Current Medicinal Chemistry, 2005,
12, 1283-1294
[4] S. Pereira; P.A. Fernandes; M. J. Ramos, Journal of the Americal Chemical Society, 2005, 127; 51745179
[5] N. M. F. S. A. Cerqueira; P. A. Fernandes; L. A. Eriksson; M. J. Ramos, Biophysical Journal, 2005- In
press
[6] M. F. Lucas; M. J. Ramos, Journal of the Americal Chemical Society, 2005, 127; 6902-6909
[7] M. J. Ramos; P. J. Silva, Journal of Physical Chemistry B, 2005, Vol.109; 18195-18200
[8] A. P. Carvalho; P. Fernandes; M. J. Ramos, Progress in biophysics and molecular biology, 2005- In press
[9] A. P. Carvalho; P. Fernandes; M. J. Ramos, Journal of Computational Chemistry, 2005- In press
[10] A. P. Carvalho; P. Fernandes; M. J. Ramos, Journal of Physical Chemistry B, 2005- In press
[11] A. P. Carvalho; P. Fernandes; M. J. Ramos, Mini-Reviews in Medicinal Chemistry, 2005- In press
[12] R. J. F. Branco; P. A. Fernandes; M. J. Ramos, Journal of Molecular Structure: Theochem, 2005, 729,
141–146
[13] R. J. F. Branco; P. A. Fernandes; M. J. Ramos, Theoretical Chemistry Accounts, 2005- In press
[14] S. F. Sousa; P. A. Fernandes; M. J. Ramos, J. Biol. Inorg. Chem., 2005, 10, 3-10
[15] S. F. Sousa; P. A. Fernandes; M. J. Ramos, Journal of Molecular Structure: Theochem, 2005,729, 125–
129
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[16] S. F. Sousa; P. A. Fernandes; M. J. Ramos, Biophysical Journal, 2005, 88, 483-494
[17] http://www.chem.ox.ac.uk/cancer/ccdd.html
[18] P. A. Fernandes et al, Theoretical Chemistry Accounts, 2005, 113; 197-204
[19] V. Pande; M. J. Ramos, Bioorganic & Medicinal Chemistry Letters, 2005, 15; 4057-4063
[20] V. Pande; M. J. Ramos, Current Medicinal Chemistry, 2005, 12; 357-374
[21] V. Pande; M. J. Ramos, Bioorganic & Medicinal Chemistry Letters, 2005, 15; 5129-5135
[22] R. K. Sharma; V. Pande; M. J. Ramos; H. K. Rajor; S. Chopra; K. Meguro; J. Inoue; M. Otsuka,
Bioorganic Chemistry, 2005, 33; 67-81
[23] M. A. C. Preto; A. Melo; H. L. S. Maia; T. Mavromoustakos; M. J. Ramos, Journal of Physical Chemistry
B, 2005, 109, 17743-17751
[24] I.S. Moreira; P. A. Fernandes; M. J. Ramos, Proteins, Structure, Function and Bioinformatics, 2005- In
press
[25] I.S. Moreira; P. A. Fernandes; M. J. Ramos, Int. J. Quantum Chem., 2005- In press
[26] I.S. Moreira; P. A. Fernandes; M. J. Ramos, J. Phys. Chem. B, 2005- In press
[27] I.S. Moreira; P. A. Fernandes; M. J. Ramos, Theor. Chem. Acc., 2005- In press
[28] A. R. F. Carvalho; A. Melo, International Journal of Quantum Chemistry, 2005, 104, 240-248
[29] A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes, Journal of Molecular Structure: Theochem,
2005, 729, 83-90
[30] A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes, Chemical Physics, 2005, 310, 109-122
[31] M. J. Ramos; P. A. Fernandes, Journal of Chemical Education, 2005, 82; 1021-1025
[32] A. Antunes; M. J. Ramos, Genomics, 2005, 86; 708-717
[33] P. Gaubert; A. Antunes, Journal of Mammalogy, 2005, 86, 1068-1074.
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APPLIED BIOPHYSICS AND BIOCHEMISTRY
Staff Members:
Baltazar de Castro
Full Professor
José Costa Lima
Full Professor
Paula Gameiro
Assistant Professor
Maria Salette Reis
Assistant Professor
Eduarda Graça R. Fernandes
Assistant Professor
Mª José Feio
Invited Assistant Professor
Carla Manuela S. Matos
Assistant Professor
Catarina Mansilha
Assistant Professor
Ph.D. Students:
Patrícia Neves, Sofia Costa Lima, Sónia garcia, Marlene Susana Dionísio Lucio, Helena Machado Ferreira,
David Costa, Christophe Siquet, Ana Maria Gomes
M.Sc. Students:
Cláudia Daniela Oliveira de Lacerda Nunes, Célia Tavares de Sousa, Marisa de Freitas
Project Grantee:
Adriana Key
Number of articles in scientific journals: 12 (218-229)
Number of M.Sc. Thesis: 2
Quenching studies were performed in OmpF/DMPC proteoliposomes and a markedly difference in protein
conformation (compared to that on mixed micelles) was observed. OmpF fluorescence, in the absence or
presence of four different quinolones, was quenched by acrylamide or iodide and the differentiate accessibility
of these quenchers to the OmpF tryptophans provide information on the possible entry of the quinolones into
the cell showing that this entry is related to physico-chemical characteristics of the drugs. Proteoliposomes
with other lipids are beeng performed and the interaction of fluoroquinolone analyzed by fluorescence
spectroscopy.
Ion current though single trimeric OmpF porins reconstituted into planar lipid bilayers were performed to study
translocation of some quinolones of different size. Favorably interacting quinolones occlude the pore during
their translocation and cause random transient blockages of the ion current through the channel. Analyzing
these fluctuations, we observe relatively high affinity of moxifloxacin for OmpF. These studies will be performed
for other fluroquinolones.
The minimum inhibitory concentration (MIC) of copper(II)/norfloxacin, and copper(II)/ofloxacin and of the ternary
complexes copper(II)/norfloxacin/phenanthroline copper(II)/ofloxacin/phenanthroline have been studied in several
bacterial stains with series of porin mutants which lack one or several major outer membrane proteins and the
results show that porins in general are very important for the uptake of these possible metalloantibiotics, and
that their activity is much similar to that of the fluoroquinolones allow. Furthermore it seems that for ofloxacin
and norfloxacin OmpC can also be important. Synthesis of other metal (Cu, Zn, Co, Ni) complexes with
fluoroquinolonas and ternary metal/fluoroquinolone/phenanthroline complexes are beginning to be performed
and also some studies to determine a possible activity of these new compounds in the bacteria stains. If these
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compounds show to be active some toxicological studies will be performed.
Intrinsic Trp fluorescence of P-glycoprotein (Pgp) was used to perform fluorescence quenching studies with
tuberculostatics in presence and absence of ATP and a resonance energy transfer was observed between
protein and these drugs. Methodologies and formalisms of resonance energy transfer are being applied to the
experimental results to extract information of the possible transport mechanism of Pgp. In vitro flow cytometry
methods are being implemented to quantitatively assess inhibitors and/or modulators of P-glycoprotein.
In cyanobacterial and some eukaryotic algal photosynthetic systems, electron transfer between the cytochrome
b6-f complex and photosystem I is mediated by either plastocyanin or cytochrome c6 . The structure-function
relationship that allows these two soluble proteins to carry out the same cellular role despite their structural
differences has been the focus of considerable research efforts in recent years.
The comparative study of the effect of temperature upon the stability of the plastocyanins from the thermophilic
cyanobacterium Phormidium laminosum and a mesophilic counterpart Synechoccystis sp. revealed considerable
differences in the thermodynamic and kinetic properties of their unfolding process. Results previously obtained
with cytochrome c6 from Nostoc sp. PCC 7119, a thermo tolerant cyanobacterium seem to suggest that a
similar evolutionary pattern was followed.
The work under way aims at studying the unfolding process of this protein functionally homologous to plastocyanin
and check whether a similar stability pattern can be observed. We will therefore use cytochrome c6 from
mesophilic and thermophilic cyanobacterial species overexpressed in E. coli and purified from periplasmic
extracts to study their thermal unfolding process. We will use a combination of UV/vis, fluorescence and EPR
spectroscopies to obtain thermodynamic and kinetic data. If the process proves to be reversible, we will also
resort to DSC to obtain some of the process’ thermodynamic parameters.
Biophysics and Biochemistry of organized media
The main objective of this work is to study the role of the NSAIDs induced perturbation on the bilayer structure
and dynamic properties, trying to clarify the mechanism of action of these drugs and to elucidate the role of
these effects on their anti-inflammatory mechanisms of action.
Liposomes were used as membrane mimetic models to study the effect of several non steroidal anti-inflammatory
drugs (NSAIDs) on some biophysical parameters of biological membranes. It is well known that the principal
mechanism of action for NSAIDs is the inhibition of the conversion of arachidonic acid through the cyclooxygenase
or lipoxygenase pathways resulting in the formation of the prostaglandins or leukotrienes. Moreover, the
development of inhibitors that selectively binds to COX-2 for which it is been expected anti-inflammatory action
in vivo with minimal gastric side effects, shows that the sequence of events resulting from cyclooxygenase
inhibition does not totally explain the overall gastric toxicity of NSAIDs. Such mechanisms are complex and
the cascade of events leading to mucosal damage has yet to be characterized and can also be related to
NSAIDs’ topical irritancy.
We investigated the ability of NSAIDs to bind to surface-active phospholipids, as well as their effect on the lipid
dynamic properties of membrane models, once the results may elucidate about the effects of these drugs
compromising the integrity of gastric mucosal barrier. For this purpose, three different NSAIDs (indomethacin,
acemetacin and nimesulide) were selected and their influence on the biophysical properties of 1, 2dipalmitoylphosphatidylcholine (DPPC) liposomes was studied using different and complementary techniques.
Structural changes involved in the DPPC thermal transitions upon the interaction of the bilayers with NSAIDs
were evaluated by small angle X-ray scattering (SAXS). The short-range organisation of the phases was
characterised by using wide angle scattering (WAXS). These x-ray scattering techniques were also compared
to the results yielded by differential scanning calorimetry studies (DSC) which have proven to be very useful for
the study of the interactions between drugs and lipid membranes.
Along with the studies in bilayers systems, models consisting of phospholipids spread at the air– water
interface as a Langmuir monolayer were used. The interest in phospholipid monolayers is prompted by the fact
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that it allows varying such physical-chemical parameters of the interface as molecular density, lateral pressure,
surface potential, or ionic conditions and thus, it provides a simple model for studying drugs penetration.
Structural changes within the DPPC monolayer upon adsorption of indomethacin were investigated both by
synchrotron X-ray experiments at grazing incidence (GIXD) and pressure–area isotherms. These experiments
were carried out to obtain information on the structure of the penetrated monolayer and to gain additional
knowledge to the interpretation of the penetration mechanism of indomethacin over the lipid phase, once this
NSAID revealed a high perturbing effect both in the monolayer and bilayer systems.
In these studies we have also evaluated the partition coefficient of NSAIDs, the location of that drugs on the
membrane, their effect on membrane fluidity and also on the surface potential.
The determinations of partition coefficient 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 charged drug in media.
Location and membrane fluidity were evaluated by fluorescence using fluorescent probe molecules. Some of
these probes are capable of sensing “fluidity” gradient through the bilayer leaflet. The results obtained shows
that all the NSAIDs studied increase the membrane fluidity. Only the diclofenac decrease the fluidity of the lipid
membrane.
It has also been suggested a relation between the ability of drugs to change membrane fluidity and their
membrane antioxidant action namely their role on lipid peroxidation process. Lipid peroxidation 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. We have studied the effects of NAIDs on the lipid peroxidation
using a hydrophilic initiator (AAPH) and fluorescence probes with different hidrophobicity properties. Fluorescein
was used as a hydrophilic probe, DADF as a fluorescein derivative probe that have a lipophilic moisture and
consequently the fluorescein part is located near the membrane surface, and DPH-PA as a hydrophobic probe
that is located inside the lipid bilayer. The studies were performed in aqueous buffers and in liposomal systems
trying to get information concerning to the NSAIDs scavenging activity to peroxyl radical and to the effect of
their partition in lipid membrane. Results show that, to understand the effect of NSAIDs on lipid peroxidation it
must be considered not only their scavenging activity but also the location of the drugs in the membrane and
their interactions with the membrane surface. The 3-(4-(6-phenyl)-1,3,5-hexatrienyl) phenylpropionic acid (DPHPA) was also used as fluorescent probe to monitoring the antiperoxidation activity of the NSAIDs and the
changes in membrane fluidity during the peroxidation. With the experimental assays developed it was possible
to evaluate that 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 against peroxidative injury.
We have also performed the study of the effect of NSAIDs on lipid peroxidation using a hydrophobic initiator
(AMVN). The results obtained show that are a relation between the effect of the NSAIDs on the membrane
fluidity and their protective effect on the lipid peroxidation.
The studies of the effect of NSAIDs on their interaction with membrane enzymes that are usually involved in the
inflammation processes were initiated. Phospholipase A2 (PLA2) plays an important role in the regulation of
the phospholipid composition of biological membranes and in the release of physiologically and
pathophysiologically important inflammation mediators as prostaglandines, leukotrienes and thromboxanes15. Although the structure of several PLA2s and their catalytic sites are known with atomic resolution, their
mode of action on the larger scale of the membrane is not understood and one of the consequences is that
their exact metabolic role also remains unresolved. Therefore, the investigation of the mechanism of the PLA2
enzymatic reaction and the search for opportunities to influence this process is of great interest for the therapy
of inflammation and allergic diseases. In addition, drugs which are able of inhibiting PLA2 activity remain a
potential target for the development of new drugs in the treatment of diseases like Alzheimer.
Some NSAIDs like indomethacin, although regarded as cyclooxygenase inhibitors, also inhibit PLA2 but the
precise mechanism by which indomethacin blocks the activity of PLA2 remains an unresolved issue.
We have implemented to different methodologies (a spectrophotometric and a fluorimetric) to evaluate the
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PLA2 activity. We are also studding the effect of nature and composition of lipid matrix on the PLA2 activity
using liposomes prepared with different kinds of phospholipids with or without cholesterol.
Scavenging activity studies for reactive oxygen and nitrogen species by non-steroidal antiinflammatory drugs (NSAIDs), â-adrenergic antagonists and plant extracts
Endogenously produced pro-oxidant reactive species are essential to life, being involved in several biological
functions. However, when overproduced (e.g. due to exogenous stimulation), or when the levels of antioxidants
become severely depleted, these reactive species become highly harmful, causing oxidative stress through
the oxidation of biomolecules, leading to cellular damage that may become irreversible and cause cell death.
The scientific research in the field of reactive oxygen species (ROS) and reactive nitrogen species (RNS)
associated biological functions and/or deleterious effects is continuously requiring new sensitive and specific
tools in order to enable a deeper insight on its action mechanisms. However, reactive species present some
characteristics that make them difficult to detect, namely their very short lifetime and the variety of antioxidants
existing in vivo, capable of capturing these reactive species. It is, therefore, essential to develop methodologies
capable of overcoming this type of obstacles. Our group has been involved in the application of chemiluminiscent
and fluorescent probes as sensors for ROS and RNS, with the objective of their use in scavenging assays.
Once implemented, the methodologies have been subsequently applied in scavenging activity studies for ROS
and RNS by non-steroidal anti-inflammatory drugs (NSAIDs), â-adrenergic antagonists and plant extracts, as
briefly explained below.
We have recently proposed that several NSAIDs may react with reactive oxygen species (ROS), namely
peroxyl radical (ROO.), hydroxyl radical (HO.), superoxide radical (O2.-), hydrogen peroxide (H2O2), and
hypochlorous acid (HOCl), and reactive nitrogen species (RNS), namely nitric oxide (.NO) and peroxynitrite
(ONOO-) produced at sites of inflammation. This type of effect has potential therapeutic relevance since the
antioxidant activity may strongly contribute for the final anti-inflammatory outcome to be attained by these
compounds. The scavenging activity is being evaluated using non-cellular in vitro methodologies, as well as
cellular systems, using Human neutrophils.
The therapeutic effects of â-blockers are normally explained by their capacity to block the â-adrenoceptors,
however, some of the beneficial cardiovascular effects shown by this group of compounds have already been
associated with the antioxidant properties that some of them seem to possess. The â-blockers atenolol,
labetalol, metoprolol, pindolol, propranolol, sotalol, timolol, and carvedilol were tested for their putative scavenging
activity for ROS (O2.-, H2O2, HO., HOCl and ROO.) and RNS (.NO and ONOO-). Some of the studied compounds
are effective ROS and/or RNS scavengers, these effects being possibly useful in preventing oxidative damage
verified in hypertension as well as in other cardiovascular diseases that frequently emerge in association with
oxidative stress.
Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae) is a low tropical American shrub with a reported wide
range of healing properties. In Cuba, this species is traditionally used as a tincture in the treatment of
stomatological infections, as stomatitis and gingivitis. A tincture from P. tithymaloides collected in Cuba has
been evaluated for its in vitro scavenging effects on reactive oxygen species (ROS) (HOÿ, O2ÿ•, HOCl, ROOÿ
and H2O2), reactive nitrogen species (RNS) (ONOO• and ÿNO).
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BIOINORGANIC AND MEDICINAL INORGANIC CHEMISTRY
Head of Laboratory: Maria Rangel, Associate Professor
Staff Members:
Baltazar de Castro
Full Professor
Paula Gameiro
Assistant Professor
João Gomes
Assistant Professor
Ph.D. Students:
Ana Claúdia Nunes
Project Grantee:
Andreia Daniela Leite, André Neto da Silva
Number of articles in scientific journals: 1 (230)
Design of orally active insulin-mimetic drugs. In order to compare the properties of complexes formed by
multidentate and bidentate ligands we have initiated the synthesis of multidentate ligands derived from 3hydroxy-4-pyridinone type and their corresponding metal complexes with VO(IV) and Zn(II). The complexes
formed are being characterized in the solid state and in solution. Evaluation of the insulin-like action of the
synthesized complexes will be performed in collaboration with Prof. Hiromu Sakurai (Kyoto University). This
work is being developed under contract POCI/QUI/56949/2004 (FCT, Portugal).
Design of functionalized siderophores to target infection processes. The design of new chelators is
crucial for treatment of diseases associated with iron overload and to deprieve bacteria and virus associated
to infectious processes such as TUBERCULOSIS and AIDS, from iron which is one of its essential nutrients.
The work that has been done in the current year and that will be pursued in the next one is focused on the
design of novel molecules to target infection and act as intracellular fluorescent probes. The work can be
summarized as the synthesis of functionalized units in order to produce macromolecules that can act as iron
chelators and also as fluorescence probes. This work is being developed under contract POCI/QUI/56214/
2004 (FCT, Portugal).
Reactivity of B12 model compounds. The work in this project aims the establishment of structural factors
that may be determinant on the reactivity of photolysis products of B12 model compounds. We have identified
and characterized the photolysis products of cobaloximes and imino/oxime compounds with variable organic
radicals and nitrogen and phosphorus bases. The results obtained evidence that the primary products are
strongly dependent on the nature of the organic radical and are identical for the two different equatorial
moieties. This work is being developed under contract POCTI/QUI/46231/2002 (FCT, Portugal).
During the year of 2006 we will pursue the objectives of the above projects in which new compounds will be
synthesized and characterized from the chemical and biological point of view.
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COMPUTATIONAL STUDY OF METAL SURFACES
Head of Laboratory: José Ferreira Gomes, Full Professor
Staff Members:
Natália Cordeiro
Associate Professor
Post-Doct Fellows:
Shuwen Yao
Ph.D. Students:
Hugo Santos, Akapong Suwattanamala
M.Sc. Students:
Ana Sofia Pinto
Number of articles in scientific journals: 6 (231-236)
We are particularly interested in understanding the physical and chemical behaviour of heterogeneous systems,
looking in particular to the modelling of interfacial phenomena. The theoretical study of such systems and
phenomena is interesting on its own and is of such difficulty that experimentalists find it very useful to
complement their work with computational results; many of our studies are thus being carried out in close
collaboration with experimentalists. We have been using quantum chemical methods, such as DFT, as well
as molecular simulations techniques (MC and MD simulations). Representative of the studies performed in
2005 and to be pursued in 2006, follows:
Modelling and reaction of species adsorbed on metal surfaces.
We have been studying the interaction of methoxy radical with copper and ruthenium surfaces using a cluster
model approach. The DFT results show that this approach gives a reasonable description of the surfaces
allowing a good prediction of the structural and energetics features of the adsorbate. Furthermore, it shows
that a reliable description of the infrared vibration spectra of adsorbed methoxy on the clean and low oxygen
precovered Ru(0001) surfaces may be obtained. In addition, the preferential sites for the adsorption of OH
(radical and anionic species) on the elemental gold surfaces, (111), (110) and (100), has been studied by the
same approach. This work is now being extended to new reactions and different metals, namely to the study
of the reactions pertaining to the methanol synthesis on single-crystal Cu and Ru surfaces as well as
enantiomerically pure metal catalysts, which can transmit chirality information and thereby be applied in
enantioselective synthesis/detection. In those studies, more realistic methods like periodic DFT methods will
be applied.
Modelling nanoporous materials.
We have been applying molecular simulation methods (MC and MD), both at atomistic and at mesoscale
levels, to study the structure and the synthesis of nanoporous materials. This research area is at its infancy
in our laboratory. So far, we have applied Monte Carlo methods on a simple cubic lattice to model the
spontaneous formation of silica nanoparticles during the early stages of zeolite synthesis. In spite of the
simplicity of the model, it is able to describe the self-assembly of nanoparticles from clear solution, yielding
results that are in close qualitative agreement with experimental observations.
We have also begun to apply the above methods to study the synthesis of periodic mesoporous silica materials
(e.g., MCM-41). The lattice model is being extended to cope with micelle formation from chain surfactants,
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which are precursors in the mesoporous silica synthesis. Molecular dynamics and Kinetic Monte Carlo will
be employed to model micelle self-assembly in the presence of silica condensation.
Recently, we have also started a collaboration with an experimental group, with the aim of developing a
theoretical model for enantioselective catalytically active metal complexes grafted onto nanoporous supports.
Information about the structure of the material, as well as the coverage and conformations of the complexes
will be obtained from MC simulations. DFT methods will be used to model reactions catalysed by the
immobilized complexes.
Modelling and ion-transfer at liquid-liquid interfaces.
Following our previous work, we have been using MD simulations to study liquid-liquid interfaces like the
strongly associating liquid interfaces formed between water and a linear alcohol. Emphasis has been given to
the effects of increasing the length of the alcohol chain on the structure and dynamics of such interfaces.
Present work focuses on the study of other liquid interfaces currently being used in ion transfer experiments,
such as those formed between water and 2-octanol, nitrobenzene (NB) or 2-nitrophenyloctyl ether (NPOE).
Notice that 2-octanol is a chiral organic liquid which may specially favour the transfer of chiral ionic species.
On the other hand, NPOE is quite interesting due to its low toxicity, which enables its use on large-scale
industrial processes. We have recently completed a detailed comparative study of the pure and water-saturated
NB and NPOE liquids, including thermodynamic and dynamic properties, as well as ion solvation properties.
The simulation of the interfaces between water and these liquids is underway.
Selected References:
R Gulaboski, CM Pereira, MNDS. Cordeiro, I Bogeski, AF Silva, J. Solid State Electrochem., 2005, 9(6), 469474
R Gulaboski, CM Pereira, MNDS. Cordeiro, I Bogeski, E Ferreira, D Ribeiro, M Chirea, AF Silva, J. Phys.
Chem. B, 2005, 109(25), 12549-12559
JE Rodrígez-Borges, X García-Mera, F Fernández, VHC Lopes, AL Magalhães, MNDS Cordeiro, Tetrahedron,
2005, 61(46), 10951-10957
M Jorge, SM Auerbach, PA Monson, J. Am. Chem. Soc., 2005, 127(41), 14388-14400
R Gulaboski, V Mirceski, CM Pereira, MNDS. Cordeiro, AF Silva, F Quentel, M L´Her, e M. Lovric, Langmuir,
2005, in press.
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NOVEL HETEROGENEOUS CATALYSTS
Head of Laboratory: Ana Cristina Freire, Associate Professor / Baltazar de Castro, Full Professor
Research Team:
Eulália Pereira
Assistant Professor
Uwe Pischel
Assistant Researcher
Rita Ferreira
Assistant
Ana Rosa Silva
Assistant Researcher
Post-Doc Students:
Pankaj Das, Iwona K-Biernacka, Iwona Kiersztyn, Peter Eaton, Sunita Arun Salunke, Elzbieta Skiba, Issam
Oueslati
Ph.D. Students:
Magda Martins, Andrea Carneiro, João Tedim, Joana Fonseca, Adelaide Miranda, Marek Kluciar
M.Sc. Students:
Rosa Bessada, Miguel de Sousa, Bruno Jarrais, Jorge Teixeira, Tiago Borrego, Ana Isabel Cruz
Project Grantee:
Sónia Patrício, Frederico Calheiros
Number of articles in scientific journals: 18 (237-254)
Number of Ph.D thesis: 1
Number of Ms.C. thesis: 1
Novel catalysts by heterogenisation of metal complexes
We pursued the preparation and characterisation of chiral manganese(III) salen complexes for immobilisation
on new room temperature ionic liquids, activated carbons, carbon xerogels, new mesoporous aluminium
pillared clays, clays and mesoporous silica-based materials. The catalytic activity of the new complexes in
the asymmetric epoxidation of several alkenes (styrene, á-methylstyrene and 6-ciano-2,2-dimethylchromene)
was evaluated in CH2Cl2 using NaOCl, m-chloroperoxybenzoic acid (m-CPBA) and PhIO (in CH3CN) as oxygen
sources. The experiments were performed at low to room temperature and reaction time, the alkene conversion,
chemical selectivity of products, respective yields and enantiomeric excesses (ee%) were determined by
GC-FID. All the complexes showed catalytic activity in the experimental conditions used.
The asymmetric epoxidation of 6-ciano-2,2-dimethylchromene by chiral manganese(III) salen complexes was
performed in a series of new room temperature ionic liquids based on imidazolium and guanidinium cation
using NaOCl, PhIO, H2O2 and H2O2.urea as oxygen sources. The new room temperature ionic liquids could be
used as neoteric solvents for the 6-ciano-2,2-dimethylchromene asymmetric epoxidation, at room temperature;
nevertheless, compared with the reaction performed on CH2Cl2 its shows a decrease in ee%, but could be
reused up to 3 times, although with some decrease in ee% values.
To understand and to model the membrane ûux and retention behaviour as a function of the solvent/catalyst/
membrane system and to optimise the integrated case-studies, preliminary studies on the nanofiltration of a
manganese(III) salen complex were done. The SRNF membrane STARMEM 120 (Grace-Davison, USA) was
chosen for this system. It has a MWCO of about 200 Da, and is stable for ethanol – water mixture. It was
observed that catalyst rejection was higher than 95% for a 1 mM manganese complex solution, after processed
by NF at different pressure values, which indicates that the catalyst is almost totally retained by the chosen
membrane.
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Chiral Mn(III) salen complexes were anchored using several strategies developed in our lab onto several
supports: activated carbons, carbon xerogels, mesoporous pillared clays (PILCs), clays (Laponite) and
mesoporous silica materials (HMS, MCM-41 and FSM-16). All the materials were characterised by elemental
analyses, XPS, XRD (inorganic materials), nitrogen adsorption at 77 K, temperature programmed desorption
(carbon materials), FTIR (inorganic materials) and Uv-vis spectroscopies (inorganic materials). The catalytic
properties in the asymmetric epoxidation of several alkenes of the new catalysts were determined using
equivalent experimental conditions of those used in homogeneous phase. For these catalysts the leaching of
the active phase and their reusabibility was evaluated. All the catalysts showed catalytic activity: the reaction
times were larger than those observed in homogeneous phase, in some cases the alkene conversion decreased,
as well as the ee%; in some cases ee% did not decrease with reuse.
Copper(II) and vanadyl(IV) acetylacetones have also been heterogenised through Schiff condensation onto
amine functionalised activated carbons, carbon xerogels, new mesoporous aluminium pillared clays, clays
(Laponite and K10 montmorillonite) and hexagonal mesoporous silica (HMS). All the materials were characterised
by elemental analyses, XPS, XRD, nitrogen adsorption at 77 K, termogravimetry, FTIR, Uv-vis spectroscopies
and EPR. Their catalytic properties in the aziridination of styrene and epoxidation of allylic alcohols using tertbutylhydroperoxide as oxygen source, respectively, have been evaluated. All the new heterogeneous catalysts
were active and could be reused in more catalytic cycles.
Finally, 4-triethoxysilylaniline (TESA) was used as an organo-silane coupling reagent for surface functionalization
of several inorganic materials with different textural properties: Laponite, K10 montmorillonite, silica gel,
MCM-41 and FSM-16. All the materials were characterised by 13C and 29Si MAS NMR, FTIR and nitrogen
elemental analysis.
FUTURE WORK
(1) Design and preparation of new chiral complexes based on the Schiff base ligands with other transition
metals with homogeneous catalytic properties.
(2) Determine the Reichardt’s e Kamlet-Taft polarity parameters of the new room temperature ionic liquids
used as neoteric solvents for the asymmetric epoxidation of alkenes. Characterise the room temperature
ionic liquids by XPS.
(3) Explore the use of nanofiltration as a new method for the separation of homogeneous catalysts.
(4) Anchoring/encapsulation of chiral Mn complexes onto several supports: mesoporous silica materials,
mesoporous carbons, nanotubes, mesoporous PILCs and clays.
(5) Homogeneous/heterogeneous catalytic reactions (epoxidation and aziridination of alkenes and epoxidation
of allylic alcohols). For the heterogeneous catalysts, reutilisation of the catalysts will be also evaluated.
(6) Use other organo-silane coupling reagents for surface functionalisation of several inorganic mesoporous
matrixes.
Chemosensors based on transition metal complexes
We have pursed the preparation of nickel and copper complexes with Schiff base ligands functionalised with
groups that can act as coordination sites for representative and lanthanide cations and for anions (halides,
H2PO4-, HSO4-, NO3-) by methodologies developed in our laboratory. Several functionalities were introduced in
the aldehyde moiety and in the diimine bridge: crown ether and pseudo crown ethers groups. These complexes
were characterised by the usual techniques: elemental analyses, mass spectra, EPR, FTIR and UV-Vis
spectroscopies, cyclic voltammetry.
The sensing properties of the complexes were performed in several solvents, using cyclic voltammetry, UVvis; for the complexes that can polymerise and give stable films recognition studies were also done by cyclic
voltammetry, in-situ FTIR and UV-Vis, electroacoustic impedance and electrochemical impedance.
In solution, it was possible to determine equilibrium constants for the complexation of lanthanides and alkalineearth cations for the metal complexes functionalised with pseudo-crown groups by Uv-vis spectroscopy.
Cyclic voltammetry allowed the semi-quantiative evalutation of the interaction of all the cations tested (+1, +2
and +3). For the complexes functionalised with crown ethers in the imine bridge, no interaction was detected
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by UV-Vis, since the complexation site does not influence the CT bands used to monitor the interaction
between the complex and the cations.
All the polymeric films adsorbed the metal cations tested (+1, +2), although they exhibited different behaviour:
those with higher change induced higher changes in the electrochemical, UV-Vis and IR properties of the
films. The coordination site was unambiguously identified as the crown ether or pseudo-crown ether by IR
spectroscopy and XAS of Ni and Ba K-edge. The adsorption of cations by the polymeric films was also
monitored by electrochemical quartz crystal microbalance: there was an increase in mass during the adsorption
process and during reduction the cations were expelled into the solutions; this property was confirmed by the
cyclic voltammetric studies of the films with adsorbed cations in free supporting electrolyte and confirm the
films could be reuse in successive sensing experiments without loosing their electrochemical and chemical
stability.
FUTURE WORK
(1) Design, preparation and characterisation of new complexes with sensing properties for cations and anions.
(2) Preparation and characterisation of their corresponding films.
(3) Evaluation of the association constants for the interaction of cations and anions with the complexes by
new techniques: fluorescence, calorimetric titration and conductimetry
(4) Study of the interactions of cations and anions with the polymeric films by new techniques: electrochemical
quartz crystal microbalance, XAS and neutron reflectivity
(5) Morphological characterisation of the films by SEM and AFM.
(6) Cation and anion selectivity studies and for polymeric films reutilisation studies.
Molecular materials with non-linear optical properties
The synthesis of materials with non linear optical properties (NLO) is a current research area due to the
potential application of these materials for the fabrication of high performance electro-optical switching elements
for telecommunication and optical information processing. In this context, transition metal complexes, which
show in some cases intrinsic NLO properties, are important building blocks for the preparation of material with
this type of properties.
During 2005 new nickel and copper complexes with salen type ligands functionalised with donor-acceptor
groups, designated generically as [M(DA-salen)] were prepared. The new complexes were electropolymerised
by methods developed in our group and the linear (UV-Vis) and 3d NLO properties of the compounds and films
have been determined. We have started to prepare films with the complexes by using the self-assembly layerby-layer technique.
The 3d NLO properties were evaluated using the z-scan technique, which is based on the self-focusing or
defocusing of a converging beam of known direction (Nd:YAG laser, l=1064 nm). The technique permits the
evaluation of non linear refractive index (n2) and the non linear absorption coefficient (b).
The 3d NLO properties of the complexes, polymeric films and layer-by-layer films were measured. The complexes
showed a great variation on their NLO properties; the differences were attributed to the donor/acceptor properties
of the substituents within the ligand. The polymeric films show 3d NLO properties which have been strongly
enhanced when compared to the monomers in solution, and are a result of the charge carriers generated
during the oxidation of the polymeric films. The films prepared by layer-by-layer technique did not exhibit NLO
properties due to their very thin thickness.
FUTURE WORK
(1) Design, preparation and characterisation of Pd and Pt complexes
(2) Preparation and characterisation of their corresponding films.
(3) Evaluation of linear (UV-Vis) and 3d NLO properties for Pd and Pt complexes and corresponding films.
(4) Preparation and characterisation of new films by self-assembly layer-by-layer method.
(5) Evaluation of 3d NLO properties for films prepared by self-assembly layer-by-layer method.
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Metallosurfactants. Deposition of monolayer and mutilayer films of the amphiphilic complexes
[Fe(RR’bpy)2(CN)2] on ITO and mica was performed using the Langmuir-Blodgett technique, and the resulting
modified surfaces were studied by UV/vis spectroscopy, cyclic voltammetry and AFM. In addition, the
aggregation behavior of these compounds in methanol/water was studied by AFM in order to clarify the
influence of the drying conditions on the morphology of the nanocapsules formed.
Several novel ruthenium(II) and ruthenium(III) complexes with alkyl substituted bipyridine ligands were
synthesized and characterized by the usual techniques (UV/vis, FTIR, NMR, FAB-MS, CV). Their aggregation
behavior was studied in several different solvents, and solvent mixtures by SEM, AFM and DLS. The preliminary
results obtained show that the morphology of the nanoparticles obtained is strongly dependent on solvent
polarity.
Ongoing and future work include further characterization of Langmuir-Blodgett films, including determination
of Langmuir isotherms, and UV/vis, IR, CV and AFM studies.
Metal Nanoassemblies. Application of a photocatalytic method to gold reduction enabled us to prepare gold
sols with different nanoparticle morphology, namely triangular prisms, spheres and disks. TEM and AFM were
used to study the growing process of the nanocrystals. The results obtained show that the morphology of the
nanoparticles is strongly dependent on pH and amount of photocatalyst used.
In addition, novel methods for the preparation of Pt and Pd nanoparticles with catalytic applications were
developed using a similar approach. Homogeneous suspensions of 2-5 nm nanoparticles were prepared. The
method is being modified in order to obtain larger nanoparticles. Immobilization of this nanoparticles in solid
supports (graphite) was also used, but in this case the nanoparticles obtained showed a very broad size
distribution. 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 nanoparticles.
Application of nanoparticles to biological systems: A novel method for the detection of gene expression
was developed in collaboration with Prof. Pedro Baptista and Prof. Ricardo Franco (FCT-UNL). The method is
based on the different aggregation properties of gold nanoparticles derivatized with a specific oligonucleotide
as the probe, in the presence or absence of the complementary sequence. We have used AFM to study the
nanoparticle-probe-DNA interactions. Interestingly, hybridizing the DNA plasmids in the presence of gold
nanoparticle-probe leads to extremely large concatemeric structures forming from individual DNA molecules.
The synthesis of other nanoparticles with appropriate physical properties for detection and/or separation of
DNA plasmids, namely FexOy/Au is in progress. The preliminary results obtained show that the method used
is successful for the preparation of mixed FexOy/Au. Nevertheless the particles show a broad size distribution
and irregular distribution of gold at the surface of the iron oxide core. The synthetic method is being optimized
in order to obtain homogeneous size distribution and capping of the iron oxide core. Future developments
include optimization of the synthetic procedures, microscopy characterization of the nanopartcicles (TEM
and AFM), determination of the magnetic properties (Mossbauer, SQUID), and development of a new method
for detection and removal of specific DNA plasmids from biological samples.
Photoactive Compounds and Solid-State Materials
The research activities in the year 2005 can be divided in four main projects: (a) development and photophysical
characterization of novel lanthanide-calixarene complexes (published in 2006); (b) development of novel
molecular logic gate functions by using phthalimide-terbium conjugates (published in 2006); (c) synthesis
and characterization of bistable molecular fluorescent switches with aromatic dicarboximides (published in
2005); (d) investigation of energy transfer phenomena in organic-inorganic hybrid materials doped with europium
(collaboration with Prof. Carlos, CICECO University of Aveiro). Furthermore, two more works related to the
investigation of stereoselective phenomena in excited states were published in collaboration with Prof. Miranda
in Valencia.
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ANNEXES
2005
A - 2
ANNEX I
RESEARCH TEAM
Laboratório Associado para a
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A - 3
A
MEMBERS OF STAFF
Laboratório Associado para a
Química Verde
A - 4
Alberto Sundaresan Prabhakar
Ana Maria F. T. Lobo
Baltazar Manuel Romão de Castro
Fernando J. S. Pina
Hugo Gil Ferreira
Isabel Maria A. M. G. de Moura
Jose Alberto Nunes Ferreira Gomes
José J. G. de Moura
Jose Luis Fontes Costa Lima
Luis F. G. Sousa Lobo
Manuel M. Nunes da Ponte
Maria Lurdes Souteiro Bastos
Maria Conceição B. S. M Montenegro
Pedro Brito Correia
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 S. S. Rangel
Maria João Ribeiro Nunes Ramos
Maria João L. R. M. C. Romão
Maria Natália Dias Soeiro Cordeiro
Maria Pilar Figueroa Gonçalves
Maria Teresa Barros Silva
Rosa Maria Moreira Seabra Pinto
Rui Alexandre Santos Lapa
Susana Filipe Barreiros
Teresa Maria Fonseca de Moura
Cristina Maria Delerue Alvim Matos
Maria Leonor Oliveira Madureira Pinto
Maria Carmo Veiga Fernandes Vaz
Rui Alves
Agostinho Almiro Almeida
Alberta Paula Gameiro Santos
Alexandre Lopes Magalhães
Ana I. N. M. Aguiar Ricardo
Ana M. F. C. Lourenço
Ana Maria Martelo Ramos
Andre Alberto Sousa Melo
Ângela M. S. Relva
António Gil de Oliveira Santos
António Jorge Parola
Carlos S. S. Pereira Lima
Eduarda Graças Rodrigues Fernandes
Elvira Maria M. Gaspar
Eulália Fernanda Carvalho Pereira
Eurico José da Silva Cabrita
Felix Dias Carvalho
Fernando Manuel Gomes Remião
Helena Maria Neto Ferreira
Helena Maria Vieira Monteiro Soares
Henrique J. R. Guedes
Isabel Borges Coutinho de Medeiro
Isabel Maria Ligeiro da Fonseca
Isabel Maria Viegas Oliveira Ferreira
Isabel Maria Rola Coelhoso
João Carlos S. B. Sotomayor
João Carlos Lima
João Luis Machado Santos
João M. Aires de Sousa
João Paulo C. Noronha
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Professora Associada
Professora Associada
Professora Associada
Professora Associada
Professor Associado
Professora Associada
Professora Associada
Professora Coordenadora
Professora Coordenadora
Professora Coordenadora
Professor Coordenador
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Laboratório Associado para a
UNL
UNL
FCUP
UNL
UNL
UNL
FCUP
UNL
FFUP
UNL
UNL
FFUP
FFUP
UNL
UNL
FEUP
FFUP
FCUP
FCUP
FCUP
UNL
ICBAS-UP
UNL
ICBAS-UP
FCUP
UNL
FCUP
FEUP
UNL
FFUP
FFUP
UNL
UNL
ISEP
ISEP
ISEP
IPVC
FFUP
FCUP
FCUP
UNL
UNL
UNL
FCUP
UNL
UNL
UNL
UNL
FFUP
UNL
FCUP
UNL
FFUP
FEUP
FFUP
FEUP
UNL
UNL
UNL
FFUP
UNL
UNL
UNL
FFUP
UNL
UNL
Química Verde
A - 5
Joaquim Silvério Marques Vital
Jorge M. P. Lampreia Pereira
José António Maia Rodrigues
Jose Augusto Caldeira Pereira
Jose Oliveira Fernandes
José Paulo Barbosa Mota
Luís Guilherme L. Ferreira Guido
Luisa M. S. P. Ferreira
Luísa Maria S. Vieira Peixe
Marco Diogo R.Gomes da Silva
Maria Alice S. Pereira
Maria Ascenção Reis
Maria Beatriz Prior Pinto Oliveira
Maria Beatriz Guerra Junqueiro
Maria Cristina O. Costa
Maria D’Anjos L. Macedo
Maria Gabriela Teles Cepeda Ribeiro
Maria João Melo
Maria José Feio
Maria Lúcia Sousa Saraiva
M. Madalena A.C.S.Dionísio de Andrade
Maria Manuela M. A.Pereira
Maria Margarida C.M. A. Cardoso
Maria Salete Reis Dias Rodrigues
Maria Teresa Avilés Perea
Marco Diogo Silva
Miguel Freire de A. Cabral
Olivia Maria de Castro Pinho
Patrícia Carla Ribeiro Valentão
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
Ricardo Franco
Rui Manuel Freitas Oliveira
Susana Isabel Pereira Casal
Ermelinda Manuela Jesus Garrido
Jorge Manuel Pinto Jesus Garrido
Alexandra Bernardo
Adriana Martins Pimenta
Carla Manuela Soares Matos
Catarina Isabel Guerra Rodrigues
Cristina Maria C. Morais Couto
Ivone Valente Oliveira
João Luís Tavares de Matos Gomes
Francisco Jorge Fernandes Caldeira
Lígia Maria da Silva Rebelo Gomes
Luísa Lima Gonçalves
Maria Alexandra Bernardo
Maria Gabriela Almeida
Maria da Graça Soveral Rodrigues
Maria Helena Reis Prado de Castro
Paula Cristina Gerónimo
Rita Isabel Lemos Catarino
Sara Isabel Xavier Candeias
Stephane Besson
Ana Maria Philips
Maria Fernanda Cabral
Adriano Fachini
Agostinho Pereira
Ana Bicho dos Santos
Ana Luísa Carvalho
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar Convidada
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar Convidado
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Assistente
Professora Adjunta
Professor Adjunto
Professora Associado
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Investigadora Principal
Investigadora Principal
Investigador Auxiliar
Investigador Auxiliar
Investigadora Auxiliar
Investigadora Auxiliar
Laboratório Associado para a
UNL
UNL
FCUP
ICBAS-UP
FFUP
UNL
FCUP
UNL
FFUP
UNL
UNL
UNL
FFUP
FFUP
UNL
UNL
FCUP
UNL
FCUP
FFUP
UNL
UNL
UNL
FFUP
UNL
UNL
FFUP
FCNAUP
FFUP
FFUP
UNL
UNL
FCUP
UNL
UNL
FCUP
UNL
UNL
UNL
FFUP
ISEP
ISEP
ISCS
UFP
UFP
ISCSN
ISCSN
ISCSN
UFP
ISCSS
ISCSN
ISCSS
ISCSS
ISCSS
FFUL
ISCSN
UFP
UFP
U. Lusófona
U. Lusófona
UNL
FCUP
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
Química Verde
A - 6
Ana Rosa Silva
Carlos Lodeiro Espino
Isabel Mafra
João Pedro Lopes
Marcela Alves Segundo
Maria Manuel Marques
Marta Andrade
Peter Eaton
Loïc Hilliou
Owen Catchpole
Serguey Bursakov
Svetlozar Velizarov
Uwe Pischel
Manuel Rui Azevedo Alves
Maria Isabel Branco Alves Martins
Maria Teresa de Oliva Teles Moreira
Ana Claro
Helena Carmo
Abel José Assunção Duarte
Florinda F. Martins
Hendrikus Petrus Antonius Nouws
João Velho Prior
Maria de Fátima de Sá Barroso
Maria Goreti Ferreira Sales
Maria Isabel Brito Limpo de Serra
Maria João D. Ramalhosa Ferreira
Maria Manuela Barbosa Correia
Micaela de Sousa
Olga Manuela Matos de Freitas
Rita Isabel Simões Ferreira
Simone Barreira Morais
Salomé de Sousa Teixeira
Sónia Adriana R. C. Figueiredo
Valentina Maria FernandesDomingues
Maria Elisa A. M. Fernandes Soares
Eulalia Maria Bernardino Mendes
Maria Isabel Afonso da Rocha
Maria Isabel Almeida Cardoso
José Tomás Soares de Albergaria
Paula Paíga
Carla Rodrigues
Cecília Bonifácio
Cristina Réu
João Fraga
João Rodrigo da Silva Santos
Nelson Brito
Pedro Assunção
Rui Viegas
Maria Aurora Soares da Silva
Sérgio Cruz Monteiro de Morais
Investigadora Auxiliar
Investigador Auxiliar
Investigadora Auxiliar
Investigador Auxiliar
Investigadora Auxiliar
Investigadora Auxiliar
Investigadora Auxiliar
Investigador Auxiliar
Investigador Auxiliar
Investigador Auxiliar
Investigador Auxiliar
Investigador Auxiliar
Investigador Auxiliar
Professor Coordenador
Professora Adjunta
Professora Adjunta
Assistente Convidada
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente Convidada
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assessora Principal
Assessora Principal
Assessora
Assessora
Técnico
Técnica
Téc. Sup. 2.ª Classe
Téc. Sup. 2.ª Classe
Téc. Sup. 2.ª Classe
Téc. Informática
Téc. Sup. 2.ª Classe
Téc. Informática
Téc. Informática
Téc. Sup. 2.ª Classe
Encarregada de trabalhos
Encarregado de trabalhos
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
IPVC
ISEP
ISEP
UNL
FFUP
ISEP
ISEP
ISEP
FFUP
ISEP
ISEP
ISEP
ISEP
ISEP
UNL
ISEP
IPVC
ISEP
ISEP
ISEP
ISEP
FFUP
FFUP
FCUP
FFUP
ISEP
ISEP
REQUIMTE
REQUIMTE
FFUP
REQUIMTE
REQUIMTE
REQUIMTE
UNL
REQUIMTE
ISEP
ISEP
KEY
UNL
FCUP
FFUP
FCNAUP
ISEP
ISCSN
UFP
Universidade Nova de Lisboa
Universidade do Porto - Faculdade de Ciências
Universidade do Porto - Faculdade de Farmácia
Universidade do Porto - Fac.Ciências da Nutrição
Instituto Superior de Engenharia do Porto
Instituto Superior de Ciências de Saúde (Norte)
Universidade Fernando Pessoa
FFUL
FEUP
ICBAS-UP
UAl
IPVC
ISCSS
U. Lusófona
Universidade de Lisboa - Faculdade de Farmácia
Universidade do Porto - Faculdade de Engenharia
Universidade do Porto - Inst. Ciências Biomédicas
Universidade do Algarve
Inst. Politécnico de Viana do Castelo
Instituto Superior de Ciências de Saúde (Sul)
Universidade Lusófona
Laboratório Associado para a
Química Verde
A - 7
B
POST-DOCTORAL FELLOWS
Laboratório Associado para a
Química Verde
A - 8
Adrian Oehmen
Berta Covelo
Cristina Timóteo
Damián Fernández
Elzbieta Skiba
Ewa Bogel Lukasik
Françoise Auchère
Filomena Freitas
Gilda Carvalho
Isabel Esteves
Issam Oueslati
Iwona Biernacka
Iwona Kiersztyn
José Castanheiro
José Trincão
Krasimira Petrova
Krisztina Istvan
Luísa Serafim
Ludwig Kripahal
Mário Gomes
Marta Eiroa
Marta Isabel Silva
Patrícia Sousa
PauloLemos
Pankas Das
Pedro Rodrigues
Sofia Pauleta
Quingyou Zhang
Raquel Fortunato
Raquel Otero
Ricardo Mendonça
Roeland Boer
Rui Duarte
Rui Ruivo
Shabir Najmudin
Shuwen Yao
Snezhana Bakalova
Sonia Dopico
Sunil Gupta
Sunita Salunke
Susan Matthews
Svetlana Lyubchik
Svetlozar Velizarov
Teresa Casimiro
Vesna Nasdanovic-Visak
Vitor Alves
Yonh Hong Liu
Yuri Binev
Laboratório Associado para a
Química Verde
A - 9
C
Ph. D. STUDENTS
Laboratório Associado para a
Química Verde
A - 10
Adelaide Miranda
Akapong Suwattanamala
Alexandra Carvalho
Alexandra Marques
Alexandre Carvalho
Ana Freitas
Ana Gomes
Ana Nunes
Ana Martins
Ana Teixeira
André Araújo
André Pinheiro
Andrea Carneiro
Andreia Filipa Curto
António Nunes
Bárbara Ribeiro
Bruno Tamames
Juan Tamames
Carina Machado
Carla Brazinha
Carla Portugal
Carla Silva
Carlos Martins
Carlos Pinheiro
Carlota Macedo
Célia Amorim
Christophe Siquet
Claudia Galinha
Cristina Alves
Cristina Cordas
Cristina Matos
Cristina Vieira
Daniel Dourado
David Costa
Diogo Latino
Emília Santos
Ema Alves
Elizabete Machado
Eunice Rodrigues
Fábio Larotonda
Fátima Lucas
Filipe Duarte
Filipe Folgosa
Francisco SIlva
Gabriela Rivas
Gonçalo Carrera
Graça Albuquerque
Helena Ferreira
Helena Pontes
Hugo Oliveira
Hugo Santos
Inês Gomes
Isabel Esteves
Irina Moreira
Joana Amaral
Joana Ferreira
Joana Fonseca
Joana Raimundo
João Lourenço
João Dias
João Fernandes
João Capela
João Tedim
Jorge Dias
José dos Santos
José Marques
Karine Marques
Laila Ribeiro
Leticia Giestas
Liliana Guerreiro
Luís Magalhães
Luis Lopez
Luís Pinto
Magda Martins
Márcia Guilherme
Márcio Temtem
Marco Preto
Marek Kluciar
Margarida Moncada
Maria João Morgado
Maria Luisa Soares Silva
Maria Teresa Viciosa
Mariana Costa
Marieta Passos
Mário Eusébio
Marlene Lucio
Marta Filipa Ribeiro
Miguel Faria
Miguel Lopes
Mónica Matos
Nuno Cerqueira
Oriza Tavares
Pablo Gonzalez
Patrícia Antunes
Patrícia Neves
Patricia Oliveira
Patrícia Raleiras
Paula Rodrigues
Paula Gomes Pinto
Paula Silva
Paulo Glória
Pedro Manuel Santos
Pedro Maia
Pedro Vidinha
Rafal Lukasik
Ricardo Branco
Ricardo Oliveira
Rita Noronha
Rute Fonseca
Sara Cristina Cunha
Sandra Quinteira
Sérgio Alves
Sérgio Sousa
Sónia Garcia
Sofia Barata
Sofia Costa Lima
Sofia Gomes
Susana Pereira
Susana Gaudêncio
Teresa Santos Silva
Valdemar Figueira
Vasco Bonifácio
Vera Costa
Victor Rosa
Vineet Pande
Wancheng Sittikijyothin
Zenaida Mourão
Laboratório Associado para a
Química Verde
A - 11
D
M. Sc. STUDENTS
and
other research students
Laboratório Associado para a
Química Verde
A - 12
M. Sc. Students
Aida Reis
Alexandra Soares
Ana Castro
Ana Filipa Gomes
Ana Isabel Cruz
Ana Maria Gomes
Ana Sofia Pinto
Aúrea Roxo
Branca Teixeira
Bruno Jarrais
Carla Barbosa
Célia Sousa
Claúdia Nunes
Cristina Silvestre
Cristina Soares
Daniel Osório
Daniel Ribeiro
Eva Cunha
Filipa Grosso
Jorge Páscoa
Jorge Teixeira
Lúcia Maia
Luís Correia
Maria Resende
Marisa de Freitas
Marta Sofia Pires
Miguel Sousa
Renata Silva
Ricardo Mendes
Rosa Bessada
Sergio Teixeira
Sílvia Barros
Sónia Mendes
Tiago Borrego
Other Research Students
Adriana Key
Aldino Viegas
Américo Duarte
Ana Brás
Ana Fins
Ana Lopes
André Neto da Silva
Andrea Mestre
Andreia Barateiro
Andreia Leite
Ângela Machado
Artur Abreu
Bruno Pedras
Carlota Macedo
Catarina Coelho
Catarina Soares
Célia Silveira
Celina Santos
Cristiano Mota
Duarte Alves
Filipe Freire
Frederico Calheiros
João Dias
Lídia Barata
Maria Baleizão
Marisa Silva
Marta Vilela
Miriam Sousa
Natércia Brás
Patrick Nunes
Sara Berguet
Sónia Patrício
Ricardo Alves
Ricardo Couto
Ricardo Pais
Rui Tostões
Simonne dell’Acqua
Vanessa Cabral
Laboratório Associado para a
Química Verde
A - 13
E
Profiles of the new researchers
contracted under the Associate
Laboratory Program
Laboratório Associado para a
Química Verde
A - 14
Dr. Peter Eaton studied chemistry at the University of York, UK and obtained his BSc in 1994. He
then obtained an MSc in Polymer Technology from Loughborough University, before beginning his PhD at the
Materials Research Institute, Sheffield Hallam University. His doctorate title was “ATR-FTIR Spectroscopy
and Raman Microscopy Studies of Organosilane Diffusion and Hydrolysis on PVC Films”.
Since 1998 Peter has been using Atomic Force Microscopy(AFM). Firstly, at the University of Portsmouth,
UK, he developed novel surface analysis techniques with the AFM; including adhesion force and nanoindentation
mapping.These novel techniques were applied to a variety of samples, including ultralow surface energy
polymers. Then he moved to the IIQ, Seville, Spain, where he used AFM to study glyconanoparticle aggregation,
and began to study the interactions between nanoparticles and DNA. This work has been continued since
2004 in the REQUIMTE at the department of chemistry, Porto, Portugal, where he is working as an associate
researcher and also using the AFM to study a wide variety of chemical and biological systems including
electroactive polymer films, organometallic vesicle structure, protein binding by biosensors and model
proteoliposome surfaces.
Dr. Ana Rosa Silva started her research work in her late undergraduate Chemistry studies (1995) and
Master (1996/1998) at LAQUIPAI (FCUP) synthesising transition metal complexes with biologically important
ligands and characterising their spectroscopic and magnetic properties. Then, in her Ph.D. (1998/2002) at
CEQUP (FCUP) besides of synthesising and characterising transition metal complexes with Schiff base
ligands, she anchored these transition metal complexes onto activated carbons, using several methodologies,
and applied them as successful heterogeneous catalysts in the epoxidation of alkenes.
The Post-Doctoral research work (2003/2005) at CEQUP (FCUP) was focused on the heterogenisation of
chiral homogeneous catalysts using activated carbon, mesoporous hexagonal silica and room temperature
ionic liquids for the heterogeneous asymmetric epoxidation of alkenes. During this period she visited for 9
months the Green Chemistry Group/Clean Technology Centre of the University of York (2004/2005) where
she also became interested in the use of renewable resources as supports and the use of microwave technology
in Chemistry. In September of 2005 joined REQUIMTE and her current research interests are the synthesis
of new chiral homogeneous catalysts, mainly transition metal complexes with Schiff base ligands, and the
development of sustainable chemical catalytic processes by heterogenisation of homogeneous catalysts in
several solid supports (activated carbons, mesoporous carbons, carbon nanotubes, mesoporous silicas,
clays, pillared clays), by using non-conventional solvents and by using nanofiltration membranes for separation
and recycling of homogeneous catalysts.
Dr. Marta Morais Saraiva de Andrade obtained her degree in Organic Chemistry in 1999 at the
Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa. In 1997 carried out a research period in
the Institut für Organische Chemie der Universität Wien (Viena, Austria). Since 2003/2004 she is an Invited
Assistant in the Structural Organic Chemistry Group. In 2005 she obtained a doctoral degree in Chemistry,
specialization in Organic Chemistry, from the Universidade Nova de Lisboa. Since December 2005 she is an
Assistant Researcher at REQUIMTE - Laboratório Associado - Centro de Química Fina e Biotecnologia. Her
main research interests are selective organic synthesis, with special attention to the development of new
strategies in green organic chemistry. She has published, as main author/co-author, 2 papers in international
scientific periodicals with referees and presented 2 oral communications by invitation and 9 posters in scientific
meetings. She has co-supervised undergraduate students.
At the moment, she is involved, as a team research member, in two different research projects, namely the
development of carbohydrates-based chiral auxiliaries as green raw materials for new methodologies in Organic
Synthesis and in the synthesis, complexation and biodegradation studies of chiral chelators for industrial and
domestic application. These studies are directed toward the development of cleaner (greener) organic chemistry.
Laboratório Associado para a
Química Verde
A - 15
Dr. Rui O. Duarte, born in Lisbon, 1968, graduated in 1991 on Applied Chemistry (Biotechnology) from
the Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa (FCT/UNL). He completed his PhD
thesis in 2002 in Inorganic Chemistry, titled “Proteínas contendo Molibdénio e Tungsténio”. From 1998 to
2002, he was Invited as Assistant in the Departamento de Química e Bioquímica of Universidade do Algarve.
From 2002 to 2005 he had a post-doctoral position in the BioVanadium research group, in collaboration with
the Bioquímica Física de Proteínas group headed by Prof. Isabel Moura (FCT/UNL). He is currently Invited
Assistant Professor in Departamento de Química (FCT/UNL) and has recently (2005) joined the Associated
Lab Requimte as an Assistant Researcher. Within the present research interests are the isolation and
characterization of metalloproteins, the use of molecular biology techniques for cloning and overexpression
and characterization of novel Iron-Sulfur centers by Electron Paramagnetic Resonance (EPR) and Mössbauer
spectroscopies. He also has a special interest in spectroscopic studies applied to medical and biological
problems. He is the responsible person for the Bruker X-band EPR spectrometer located at FCT/UNL.
Dr. Ulrich Scheven obtained a bachelors degree in Physics at the University of California at Berkeley
in 1989, a masters degree in Physics from Princeton University in 1991, and a Ph.D. degree in Physics from
Princeton University in 1995. His experimental Ph.D. research focussed on the electronic properties of low
dimensional organic metals at cryogenic temperatures, in high magnetic fields. After a two year post-doctoral
appointment at Princeton, during which he continued work with organic metals, he joined Schlumberger Doll
Research in Ridgefield, Connecticut, to work in Schlumberger's NMR group doing research relevant to the
oilfield services industry. This led to in the development of novel measurement techniques. Later on he pursued
NMR studies of fluid flow and dispersion in porous media, first in in collaboration with Professor Cory's group at
MIT in Boston and, after moving to Schlumberger's laboratory in Cambridge (UK), in collaboration with members
of the Magnetic Resonance Research Centre at Cambridge University. At MIT and Cambridge he has cosupervised Ph.D. and postdoctoral researchers. Dr. Scheven has (co-)authored 14 publications, presented at
numerous conferences, and is co-inventor on two patents.
In December 2005 he joined REQUIMTE, where he intends to use NMR techniques to study non-linear
spatiotemporal phenomena associated with flow (non-Newtonian / multiphase) and reactions in porous media,
such as rocks or packed bed rectors employed in heterogeneous catalysis.
Dr. João Almeida Lopes joined the REQUIMTE (Laboratório Associado) in December 2005 as assistant
researcher in chemometrics and process spectroscopy. He graduated in Chemical Engineering (biotechnology
branch) in 1997 (Instituto Superior Técnico). He completed his PhD in Biotechnology in the area of chemometrics
in the pharmaceutical industry in 2001 working in chemometrics modelling of industrial pharmaceutical
production processes (Instituto Superior Técnico). Since then, he consolidated the development and application
of chemometrics and spectroscopy for systems identification in the chemical, biochemical and pharmaceutical
fields working as post-doc researcher at Instituto Superior Técnico. In 2004, he and three co-workers at the
time in the Center for Biological and Chemical Engineering (CEBQ) of IST were awarded with the first prize in
the entrepreneurship contest Bioempreendedor (http://www.bioempreendedor.com) where a FTIR spectroscopy/
chemometrics based methodology was proposed for rapid and cost effective identification of human bacteria
pathogens. Vibrational spectroscopy (Near infrared, Mid infrared and Raman spectroscopy) in biotechnology
has been there since one of his major research interests.
Current research activity is focused on the development and application of chemometrics and vibrational
spectroscopy systems in the context of green-chemistry (non-destructive, non-invasive, reagent-free): 1)
pharmaceutical processes and pharmaceutical quality control, 2) analytical methods development and
optimization, 3) biochemical, biological and environmental systems monitoring and 4) improved microrganisms
identification for infection control.
Laboratório Associado para a
Química Verde
A - 16
ANNEX II
PUBLICATIONS
Laboratório Associado para a
Química Verde
A - 17
A
Scientific Articles
——
Book Chapters
——
Articles in Procedings
Laboratório Associado para a
Química Verde
A - 18
Articles in Scientific Journals
1.
“Ultra fast trypsin digestion of proteins by high intensity focused ultrasound”
D. Lopez-Ferrer; JL Capelo; J. Vazquez
Journal of Proteome Research, 2005, Vol. 4, 1569-1574
2.
“Micro-focused ultrasonic solid-liquid extraction (mu FUSLE) combined with HPLC and fluorescence
detection for PAHs determination in sediments: optimization and linking with the analytical minimalism
concept”
J.L. Capelo, M.M. Galesio, G.M. Felisberto, C. Vaz and J. Costa Pessoa
Talanta, 2005, Vol.66, 1272-1280
3.
“Focused ultrasound versus microwave digestion for the determination of lead in must by electrothermalatomic absorption spectrometry”
J.L.Capelo; S. Catarino; A.S. Curvelo-Garcia and M. Vaiao
Journal of AOAC International, 2005, Vol. 88, 585-591
4.
“Discussion of parameters associated with the ultrasonic solid-liquid extraction for elemental analysis
(total content) by electrothermal atomic absorption spectrometry. An overview”
J.L. Capelo; C. Maduro; C. Vilhena
Ultrasonics Sonochemistry, 2005, Vol. 12, 225-232
5.
“Effect of zeolites on lipase catalyzed esterification in nonaqueous media”
C. Peres; N. Harper; M. D. R. Gomes da Silva; S. Barreiros
Enzyme and Microbial Technology, 2005, Vol. 37 145-149
6.
“Cutinase activity in supercritical and organic media: water activity, solvatation and acid-base effects”
S. Garcia; P. Vidinha; H. Arvana; M. D. R. Gomes da Silva; M.O. Ferreira; J.M.S. Cabral; E.A. Macedo;
N. Harper; S. Barreiros
The Journal of Supercritical Fluids, 2005, Vol. 35, 62-69
7.
“Ultrasonication for Analytical Chemistry”
J.L. Capelo and A. Mota
Current Analytical Chemistry, 2005, Vol. 1, 193-20
8.
“A Zinc(II)-based receptor for ATP binding and hydrolysis”
C. Bazzicalupi; A. Bencini; A. Bianchi; A. Danesi; C. Giorni; C. Lodeiro; F. Pina; S. Santarelli; B.
Valtancoli
Chem. Commun. 2005, 2630-2632
9.
“A New Zn(II) Chemosensor Based on a Tweezer Pyridine-Naphthalene System. An Off-On-Off System
Working in a Biological pH window”
R. Aucejo; J. Alarcón; E. García-España; J. M. Llinares; K. L. Marchin; C. Soriano; C. Lodeiro; M. A.
Bernardo; F. Pina; J. Pina; J. S. Melo
Eur. J. Inorg. Chem. 2005, 4301-4308
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“Spectroscopy and Coordination Chemistry of a New Tetranucleating Tetra(fluorophoric) Ligand Displaying
Sensing Ability for Metal Cations”
J. Pina; J. S. de Melo; F. Pina; C. Lodeiro; J. C. Lima; A. J. Parola; M. P. Clares; Mª T. Albelda; C.
Soriano; R. Aucejo; E. García-España
Inorg. Chem., 2005, 44, 7449-7458
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“New Fluorescence PET System Based on N2S2 Pyridine-Anthracene-Containing Macrocyclic Ligands.
Spectrophotometric, Spectrofluorimetric, and Metal Ion Binding Studies”
A. Tamayo; C. Lodeiro; L. Escriche; J. Casabó; B. Covelo; P. González
Inorg. Chem., 2005, 44, 8105-8115
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“Write-Read-Erase Molecular Switching System Trapped in a Polymer Hydrogel Matrix”
F. Galindo; J. C. Lima; S. V. Luis; A. J. Parola; F. Pina
Adv. Funct. Mat., 2005, 15, 541-545
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“Water/humidity, and ammonia sensor, based on a polymer hydrogel matrix containing a fluorescent
flavylium compound”
F. Galindo; J. C. Lima; S. V. Luis; M. J. Melo; A. J. Parola; F. Pina
J. Mater. Chem., 2005, 15, 2840 – 2847
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“Bio-Inspired Multistate Networks Responsive to Light, pH and Thermal Inputs. An Example of a Multistate
System Operating Through Different Algorithms”
L. Giestas; F. Folgosa; J. C. Lima; A. J. Parola; F. Pina
Eur. J. Org. Chem., 2005, 4187-4200
15.
“One-dimensional fluorescent stacking structure based on zinc mixed-complex salt encapsulated within
an ‘ice-like’ three-dimensional hydrogen-bonded water network”
R. Carballo; B. Covelo; C. Lodeiro; E. M. Vazquez-López Cryst
Eng. Commun., 2005, 7(48) 294-296
16.
“Self-Assembly of a Hydrophobically Modified Naphthalene-Labeled Poly(acrylic acid) Polyelectrolyte
in Water:Organic Solvent Mixtures Followed by Steady-State and Time-Resolved Fluorescence”
T. Costa; M. G. Miguel; B. Lindman; K. Schillen; J. C. Lima; J. Seixas de Melo
J. Phys. Chem. B., 2005, 109, 3243-3251
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“Charge-Transfer Complexation as a General Phenomenon in the Copigmentation of Anthocyanins”P.
F. da Silva; J. C. Lima; A. A. Freitas; K. Shimizu; A. L. Maçanita; F. H. Quina
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“Identification of a Novel Small-Molecule Inhibitor of the Hypoxia-Inducible Factor 1 Pathway”
C. Tan; R. G. de Noronha; A. J. Roecker; B. Pyrzynska; F. Khwaja; Z. Zhang; H. Zhang; Q. Teng; A. C.
Nicholson; P. Giannakakou; W. Zhou; J. J. Olson; M. M. Pereira; K.C. Nicolaou; E. G. V. Meir
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“Total Synthesis of Halipeptins: Isolation of Halipeptin D and Synthesis of Oxazoline Halipeptin
Analogues”
K. C. Nicolaou; D. Schlawe; D. W. Kim; D. A. Longbottom; R. G. de Noronha; D. E. Lizos; R. Rao
Manam; D. John Faulkner
Chemistry European Journal, 2005, Vol. 11, 6197–6211
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“Total Synthesis of Halipeptins A and D and Analogues”
K. C. Nicolaou; David W. Kim; D. Schlawe; D. E. Lizos; R. G. de Noronha; D: A. Longbottom
Angewandte Chem. Int. Ed., 2005, Vol. 44, 2–7
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“New Enantioselective Method for Hydration of Alkenes Using Cyclodextrins as Phase Transfer Catalyst”
A. R. Abreu; I. Costa; C. Rosa; L. M. Ferreira; A. Lourenço; P. P. Santos
Tetrahedron, 2005, Vol. 61, 11986-11990
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“Estimation of melting points of pyridinium bromides ionic liquids with decision trees and neural networks”
G. Carrera; J. Aires-de-Sousa
Green Chemistry, 2005, Vol. 7, 20-27
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“Prediction of Enantiomeric Excess in a Combinatorial Library of Catalytic Enantioselective Reactions”
J. Aires-de-Sousa; J. Gasteiger
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Q.-Y. Zhang; G. Carrera; M. J. S. Gomes; J. Aires-de-Sousa
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S. Caetano; J. Aires-de-Sousa; M. Daszykowski; Y. Vander Heyden
Anal. Chim. Acta, 2005, Vol. 544, 315-326
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Q.-Y. Zhang; J. Aires-de-Sousa
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“Antioxidant Phenolic Glycosides from Moricandia arvensis”
H. Braham, Z. Mighri, H. Ben Jannet, S. Matthew, P. M. Abreu
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“New Ceramides from Rantherium suaveolens”
M. H. Oueslati, H. Ben Jannet, Z. Mighri, P. M. Abreu
Lipids, 2005, Vol 40, 1075-1079
29.
“High-Performance Liquid Chromatographic Determination of Glycoalkaloids in Potatoes from
Conventional, Integrated, and Organic Crop Systems”
P. Abreu, A. Relva, S. Matthew, Z. Gomes, Z. Morais
Food Control, 2005, (in press, doi:10.1016/j.foodcont.2005.08.005)
30.
“The Cahn, Ingold and Prelog System: eliminating ambiguity in the comparison of diastereomorphic
and enantiomorphic ligands”
P. Mata; A. M. Lobo
Tetrahedron: Asymmetry, 2005, 16, 2215-2223
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Paulo M. C. Glória
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M. T. Barros; M. Adilia Charmier, Christopher D. Maycock and Thierry Michaud,
Tetrahedron, 2005, 61, 7960-7966
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“Novel chiral bis(oxazolines): synthesis and application as ligands in the copper-catalyzed
enantioselective conjugate addition of diethylzinc to enones”
M. T. Barros, C. D. Maycock, and A. M. Faísca Phillips
Tetrahedron: Asymmetry, 2005, vol. 16, 2946-2953
35.
“Contrasting behavior in azide pyrolyses: an investigation of the thermal decomposition of methyl
azidoformate, ethyl azidoformate and 2-azido-N, N-dimethylacetamide by ultraviolet photoelectron
spectroscopy and matrix isolation infrared spectroscopy”
J.M. Dyke, G. Levita, A. Morris, J.S. Ogden, A.A. Dias, M. Algarra, J.P. Santos, M.L. Costa, P. Rodrigues,
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T. Brand; E. J. Cabrita; S. Berger
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37.
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Z. Tostão, J. P. Noronha; E. J. Cabrita; J. Medeiros; J. Justino; J. Bermejo; A. P.
Fitoterapia, 2005, 76, 173-180
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“New Chiral Auxiliaries for Dynamic Kinetic Resolutions. From Theory to Practice”
A. G. Santos; J. Pereira; C.A.M. Afonso; G. Frenking
Chem. Eur. J., 2005, 11, 330-343
39.
“ESR and ENDOR Investigations on Various Wurster’s Radical Cations in Solution. Experimental Results,
Theoretical ab initio, and DFT Calculations”
Grampp, G.; Kelterer, A-M.; Landgraf, S.; Sacher, M.; Niethammer, D.; Telo, J.P., Dias, R.M.B.; Vieira
A.J.S.C., Monathshefte für Chemie, 2005, 136, 519-536
40.
“A new dihydroxysterol from the marine phytoplankton Diacronema sp. (Haptophyta)”
Tostão, Z.; Noronha, J.P.; Cabrita, E.J.; Medeiros, J.; Justino, J; Bermejo, J., Rauter, A.P.
Fitoterapia, 2005, 76, 433-438
41.
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ARE Bras; T Casimiro; J Caldeira; A Aguiar-Ricardo
Journal of Chemical and Engineering Data, 2005, 50, 1857-1860
42.
“Heterogeneous Polymerisation of Diethylene Glycol Dimethacrylate in Supercritical Carbon Dioxide”
T Casimiro; AM Banet-Osuna; AMRamos; M Nunes da Ponte; A Aguiar-Ricardo
European Polymer Journal, 2005, 41, 1947-1953
43.
“Phase behavior studies of a perfluoropolyether in high-pressure carbon dioxide”
T Casimiro; A Shariati; CJ Peters; M Nunes da Ponte; A Aguiar-Ricardo
Fluid Phase Equilibria, 2005, 228, 367-371
44.
“Hydration of alpha-pinene over molybdophosphoric acid immobilized in hydrophobically modified PVA
membranes”
JE Castanheiro; IM Fonseca; AM Ramos; R Oliveira; J Vital
Catalysis Today, 2005, 104, 296-304
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“Thermophysical and thermodynamic properties of ionic liquids over an extended pressure range:
[bmim][NTf2] and [hmim][NTf2]”
RG Azevedo; JMSS Esperanca; J Szydlowski; JP Visak; PF Pires; HJR Guedes; LPN Rebelo
Journal of Chemical Thermodynamics, 2005, 37, 888-899
46.
“Thermophysical and thermodynamic properties of 1-butyl-3-methylimidazolium tetrafluoroborate and
1-butyl-3-methylimidazolium hexafluorophosphate over an extended pressure range”
RG Azevedo; JMSS Esperanca; V Najdanovic-Visak; ZP Visak; HJR Guedes; M Nunes da Ponte; LPR
Rebelo
Journal of Chemical and Engineering Data, 2005, 50, 997-1008
47.
“Mathematical modelling of a mixed culture cultivation process for the production of polyhydroxybutyrate”
JML Dias; LS Serafim; PC Lemos; MAM Reis; R. Oliveira
Biotechnology and Bioengineering, 2005, 92, 209-222
48.
“Glass transition dynamics of poly(L-lactic acid) during isothermal crystallisation monitored by realtime dielectric relaxation spectroscopy measurements”
M Dionisio; MT Viciosa;YM Wang; et al
Macromolecular Rapid Communicationd, 2005, 26, 1423-1427
49.
“Sonocatalysis and Alkaline Doped Carbons. An Efficient Method for the Synthesis of Chalcones in
Heterogeneous Media”
CJ Durán-Valle; IM Fonseca; V Calvino-Casilda; M Picallo; AJ López-Peinado; RM Martín-Aranda
Catalysis Today, 2005, 107-108, 500-5006
50.
“Automatic filtering and reodorization of adsorbed natural gas storage systems”
IAAC Esteves; MS Lopes; MP Nunes, PM; et al
Adsorption-Journal of the International Adsorption Society, 2005, 11: 905-910 Suppl
51.
“Biodegradation of thiomersal containing effluents by a mercury resistant Pseudomonas putida strain”
R Fortunato; JG Crespo; MAMReis
Water Research, 2005, 39, 3511-3522
52.
“Stability of supported ionic liquid membranes as studied by X-ray photoelectron spectroscopy”
R Fortunato;CAM Afonso;J Benavente; JG Crespo
Journal of Membrane Science,2005, 256, 216-223
53.
“Preparation and catalytic testing of sulfonic acid functionalized activated carbons”
l. Guerreiro; I Fonseca; RM Martin-Aranda; AM Ramos;AM Botelho do Rego; JVital
Phosphorus Sulfur and Silicon and the Related Elements, 2005, 180 1485-1486
54.
“Enantioselective separation of propranolol by chiral activated membranes”
T Gumi; C Palet; Q Ferreira; JG Crespo; IM Coelhoso; C Palet
Separation Science and Technology, 2005, 40 773-789
55.
“Enhanced esterification conversion in a room temperature ionic liquid by integrated water removal with
pervaporation”
P Izak; NMM Mateus; CAM Afonso; JG Crespo
Separation and Purification Technology, 2005, 41, 141-145
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56.
“Ternary-phase equilibria for CO2+3-methyl-1-butanol+2-phenylethanol”
JA Lopes; MN da Ponte
Journal of Supercritical Fluids, 2005, 34, 189-194
57.
“Deviations from ideality in mixtures of two ionic liquids containing a common ion”
JNC Lopes; TC Cordeiro; JMSS Esperanca; HJR Guedes; S Huk;LPN Rebelo; K Seddon
Journal of Physical Chemistry B, 2005, 109 : 3519-3525
58.
“Biphasic hydrogenation of alpha-pinene in high-pressure carbon dioxide”
A Milewska; AMB Osuna; IM Fonseca; M. Nunes da Ponte
Green Chemistry, 2005, 7, 726-732
59.
“Single-column simulated-moving-bed process with recycle lag”
JPB Mota; JMM Araujo
Aiche Journal, 2005, 51, 1641-1653
60.
“Molecular simulation of gas separation by equilibrium-based adsorption processes”
JPB Mota; IAAC Esteves; RCR Rodrigues; et al.
Adsorption-Journal of the International Adsorption Society, 2005, 11 319-324 .
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“Oxidation of limonene over carbon anchored transition metal Schiff base complexes: Effect of the
linking agent”
P Oliveira; AM Ramos;I Fonseca; A Botelho do Rego; J Vital
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“Direct integration of pervaporation as a sample preparation method for a dedicated “electronic nose”
C Pinheiro; T Schafer; JG Crespo
Analytical Chemistry, 2005, 77, 4927-4935
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“Sorption of aroma compounds in poly (octylmethylsiloxane) (POMS)”
T Schafer; A Heintz; JG Crespo
Journal of Membrane Science, 2005, 254, 259-265
64.
“Elucidating interactions of ionic liquids with polymer films using confocal Raman spectroscopy”
T Schafer; RE Di Paolo; R Franco; JG Crespo
Chemical Communications, 2005, 2594-2596
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“Dynamic model of a supercritical carbon dioxide heat exchanger”
PC Simoes;J Fernandes; JP Mota
Journal of Supercritical Fluids, 2005, 35, 167-173
66.
“Modelling the enantioselective hydrolysis of a meso-diester using pig liver esterase in a two-phase
hollow fibre reactor”
HA Sousa; CAM Afonso;JPB Mota; JG Crespo
Chemical Engineering Research & Design, 2005, 83, 285-294
67.
“Removal of inorganic charged micropollutants in an ion-exchange membrane bioreactor”
S Velizarov;C Matos; MAM Reis; JG Crespo
Desalination, 2005, 178, 203-210
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“Changes in molecular dynamics of the tri-ethylene glycol dimethacrylate monomer upon polymerization”
MT Viciosa; CM Rodrigues; M Dionisio
Journal of Non-Crystalline Solids, 2005, 351, 14-22
69.
“Non-mechanistic modelling of complex biofilm reactors and the role of process operation history”
G Wolf;JS Almeida; MAM Reis; JG Crespo
Journal of Biotechnology, 2005, 117, 367-383
70.
“Structural Characterization of Single-Walled Carbon Nanotube Bundles by
Experiment and Molecular Simulation”
S Agnihotri; JPB Mota; M Rostam-Abadi; MJ Rood
Langmuir 2005, 21, 896-904
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“Accessing Gelling Ability of Vegetable Proteins using Rheological and Fluorescence Techniques”
AP Baptista; CAM Portugal; I Sousa;JG Crespo; A Raymundo
Int. Journal of Biological Macromolecules, 2005, 36, 134-143
72.
“Characterization of a Nematic Mixture and its Components by Reversed-Phase HPLC and UV
Spectroscopy Analysis: an Application to Phase Behavior Studies in Liquid Crystal-CO2 Systems”
Brás, A.R. E.; Henriques, S; Casimiro, T; Aguiar-Ricardo A.; Sotomayor J., Caldeira J., Santos C.,
Dionísio M
Electronic Liquid Crystals Communications, Online 2005
73.
“N2O Conversion Using Manganese Binary Mixtures Supported on Activated Carbon”
SA Carabineiro; f Brás Fernandes; JS Vital;AM Ramos;. IM Fonseca
Applied Catalysis B, 2005, 59, 185-190
74.
“Microbial Population Response to Changes of the Operating Conditions in a Dynamic Nutrient Removal
Sequencing Batch Reactor”
F Freitas; M Temudo; MAM Reis
Bioprocess And Biosystems Engineering 2005, 28, 3-8
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“Evidence For Lower Critical Solution Behavior In Ionic Liquid Solutions”
J Lachwa; J Szydlowski; J Najdanovic-Visak; LPN Rebelo; KR Seddon; M Nunes da Ponte; JMSS
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“Optimization of the Conditions of Cr (III) Adsorption on the Activated Carbon”
SB LyubchiK; I Perepichka.; OL Galushko; AI Lyubchik; ES Lygina; IM Fonseca
Adsorption, 2005, 11, 581-593
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CT Matos; S Velizarov; JG Crespo; MAM Reis
Water Science And Technology :Water Supply, 2005, 5, 9-14
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“Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation
in conditions of oxygen transfer limitation”
R Oliveira; JJ Clemente;AE Cunha; MJT Carrondo
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“Deuterium Isotope Differences In 2-propanone, (CH3)2CO/(CD3)2CO: A High-Pressure Sound-Speed,
Density, and Heat Capacities Study”
J Szydlowski; R Gomes de Azevedo; LPN Rebelo;JMSS Esperança; HJR Guedes
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A Teixeira; H Cruz; A Cunha; J Clemente; P Alves; MJT Carrondo; R Oliveira
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and Process Operation History”
G Wolf;JS Almeida; MAM Reis; JG Crespo
Water Science And Technology, 2005, 51, 51-58
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“Rotational Mobility in a Crystal studied by Dielectric Relaxation Spectrocopy. An Experiment for the
Physical Chemistry Laboratory”
MSC Dionísio; HP Diogo; JPS Farinha; J Moura-Ramos,
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“Clean osmium-catalyzed asymmetric dihydroxylation of olefins in ionic liquids and supercritical CO2
product recovery”
Luís C. Branco; Ana Serbanovic; Manuel Nunes da Ponte; Carlos A. M. Afonso
Chem. Comm., 2005, 107-109
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“Liquid membranes using ionic liquids: the influence of water on solute transport”
Raquel Fortunato; Maria Jesus González-Muñoz; Monika Kubasiewicz; Susana Luque; J. R. Alvarez;
Carlos A. M. Afonso; Isabel M. Coelhoso; João G. Crespo
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85.
“Osmium catalyzed asymmetric dihydroxylation of methyl trans-cinnamate in ionic liquids, followed by
supercritical CO2 product recovery”
Ana Serbanovic; Luis C. Branco; Manuel Nunes da Ponte and Carlos A.M. Afonso,
J. Organometallic Chemistry, 2005, 690, 3600-3608
86.
“Purification and characterisation of tetraheme cytochrome c3 and adenylylsulfate reductase from the
peptidolytic sulfate_reducing bacterium desulfovibrio aminophilus DSM 12254”
A.L. Cortés, S. Bursakov, A. Figueiredo, A.E. Thapper, S. Todorovic, J.J.G. Moura, B. Ollivier, I. Moura
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Bioinorg Chem Applic, 2005, 3, 81-91
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“Synechocystis ferredoxin/ferredoxin – nadp+ reductase/nadp+ complex: structural model obtained by
nmr-restrained docking”
P.N. Palma, B. Lagoutte, L. Krippahl, J.J.G. Moura, F. Guerlesquin
FEBS, 2005,579, 4585-4590
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“Study of the spin – spin interactions between the metal centers of desulfovibrio gigas aldehyde
oxidoreductase: identification of the reducible sites of the [2fe-2s]1+,2,+ clusters”
C. More, M. Asso, G. Roger, B. Guigliarelli, J. Caldeira, J.J.G. Moura and P Bertrand
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“Superoxide reductase from the syphilis spirochete treponema pallidum: crystallization and structure
determination using soft x-rays”
T. S. Santos, J. Trincão, A. Carvalho, C. Bonifácio, F. Auchere, I. Moura, J.J.G. Moura and M.J. Romão
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“Interactions of v(V)-citrate complexes with the sarcoplasmic reticulum calcium pump”
M. Aureliano, T. Tiago, R.M. Gandara, A. Sousa, A. Moderno, M. Kaliva, A. Salifoglou, R.O. Duarte,
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C. E. Martins, C. M. Cordas, C. G. Timóteo, P. Tavares, A. S. Pereira, J. J. G. Moura and I. Moura
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“EPR and Kinetic Studies on NapA from Desulfovibrio desulfuricans”
P.J. González, M.G. Rivas, C.D. Brondino, I. Moura and J.J.G. Moura.
Eur Biophys J, 2005, 34, 661
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“Best guess catalytic cycle for marinibacterhydrocarbonoclasticus 617 menbrane nitrate reductase”
C.Correia, S.Besson, C.D.Brondino, G.Fauque, J.Lampreia, I.Moura and J.J.G.Moura
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M. Guilherme, P. Tavares and A.S. Pereira
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“Isolation and spectroscopic characterization of the membrane-bound nitrate reductase from
Pseudomonas chlororaphis DSM 50135"
D. Pinho ,S. Besson, P.J. Silva, B.Castro , I. Moura
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“Prediction of Signal Peptides and Signal Anchors of Cytocrome c Nitrite Reductase from Desulfovibrio
desulfuricans ATCC 27774 Using Bioinformatic Tools”
L.L. Gonçalves, M.G. Almeida, J. Lampreia, J.J.G. Moura and I. Moura
Essays in Bioinformatics, ed. D.S. Moss, S. Jelaska, S. Pongor, IOS Press, 2005,vol. 368, 203-208
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“1H, 15N and 13C Resonance assignments of the heme-binding protein murine p22hbp”
Dias JS, Macedo AL, Ferreira GC, Jeanty N, Taketani S, Goodfellow BJ, Peterson FC, Volkman BF.
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Multicentre Redox Protein”
C. A. Salgueiro; L. Morgado; B. Fonseca; P. Lamosa;T. Catarino; D. L. Turner; R. O. Louro
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101. “On the purification and preliminary crystallographic analysis of isoquinoline 1-oxidoreductase from
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D. Roeland Boer, Axel Müller, Susanne Fetzner, David J. Lowe and Maria João Romão.
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102. “Insights into the Structural Determinants of Cohesin-Dockerin Specificity Revealed by the Crystal
Structure of the Type II Cohesin from Clostridium thermocellum SdbA”
A.L. Carvalho, V.M.R. Pires, T.M. Gloster, J.P. Turkenburg, J.A.M. Prates, L.M.A. Ferreira, M.J. Romão,
G.J. Davies, C.M.G.A. Fontes, H.J. Gilbert
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103. “Overexpression, purification and crystallization of the two C-terminal domains of the bi-functional
cellulase CtCel9D-Cel44A from Clostridium thermocellum”
S. Najmudin C.P. D. Guerreiro, L.M. A. Ferreira, M. J. Romão, C. M. G. A. Fontes and J. A. M. Prates
Acta Cryst. F, Structure Biology & Cryst. Comm F61, 1043-1045. (2005)
104. “Xyloglucan is recognised by carbohydrate-binding modules that bind to beta -glucan chains.”
Najmudin S, Guerreiro CI, Carvalho AL, Prates JA, Correia MA, Alves VD, Ferreira LM, Romão MJ,
Gilbert HJ, Bolam DN, Fontes CM.
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105. “New PVC nitrate-selective electrode: application to vegetables and mineral waters”
P. Kong Thoo Lin; A. N. Araújo; M. C. B. S. Montenegro; R. Pérez-Olmos
J. Agric. Food Chem., 2005, 53, 211-215
106. “An Inexpensive biosensor for uric acid determination in human serum by flow-injection analysis”
R. F. Dutra; K. A. Moreira; M. I. P. Oliveira; A. N. Araújo; M.C. B. S. Montenegro; J. L. L. Filho; V .L.
Silva
Electroanalysis, 2005, Vol. 17, 701-705
107. “Multicommutated flow system with amperometric detection. Determination of uric acid in urine”
M. L. S. Silva; M. B. Q. Garcia; L.F.C. Lima; J. L. M. Santos; E. Barrado
Electroanalysis, 2005, Vol. 17, 2156-2161
108. “Use of tin (IV) porhyrins as ionophores for the construction of phthalate-selective electrodes: Influence
of the structure and membrane composition on their response properties”
E. M.G. Santos; A. N. Araújo; C. M.C.M. Couto; M. C. B.S.M. Montenegro
Electroanalysis, 2005, Vol. 17, 1945-1951
109. “Production of chloride and hypochlorite for analytical purposes by sonochemical degradation of
organochlorines”
M. Korn; M. S. M. S. F. Acevedo; S. S. Borges; J. L.F.C. Lima
J. Braz. Chem. Soc., 2005, Vol. 16, 988-994
110.
“Flow-through sol-gel optical biosensor for the colorimetric determination of acetazolamide”
P. C. A. Jerónimo; A. N. Araújo; M. C. B.S.M. Montenegro; D. Šatínský; P. Solich;
Analyst , 2005, Vol. 130, 1190-1197
111.
“Construction and evalution of As (V) selective electrodes based on iron oxyhydroxide embedded in
silica gel membrane”
J.A. Rodriguez; E. Barrado; M. Vega; F. Prieto; J.L.F.C. Lima
Anal. Chim. Acta, 2005, Vol. 539, 229-236
Laboratório Associado para a
Química Verde
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112.
“Sequential injection chromatographic determination of ambroxol hydrochloride and doxycycline in
pharmaceutical preparations”
D. Šatínský; L. M. L. Dos Santos; H. Sklenárová; P. Solich; M. C. B.S.M. Montenegro; A. N. Araújo
Talanta, 2005, Vol. 68, 214-218
113.
“Application of sequential injection analysis to the determination of cationic surfactants based on the
sensitised molybdenum- bromopyrogallol red reaction”
M. L.C. Passos; M. L. M. F. S. Saraiva; L.F.C. Lima
Anal. Sci., 2005, Vol. 21, 1509-1514
114.
“Flow injection systems with multi-site detection”
V. Grassi; E. A.G. Zagatto, J. L.F.C. Lima
Trends Anal. Chem., 2005, Vol. 24, 880-886
115.
“Sequential injection analysis using electrochemical detection: A review”
R. Pérez-Olmos; J.C. Soto; N. Zárate; A. N. Araújo; M. C. B. S. M. Montenegro
Anal. Chim. Acta., 2005, Vol. 554, 1-16
116.
“Single reaction interface in flow analysis”
M. F. T. Ribeiro; J. L. M. Santos; J. L. F. C. Lima; A. C. B. Dias; E. A. G. Zagatto
Talanta, 2005, Vol. 68, 351-358
117.
“Chemiluminometric determination of carvedilol in a multi-pumping flow system”
C. K. Pires; K. L. Marques ; J. L. M. Santos; R. A. S. Lapa; J. L. F. C. Lima; E. A. G. Zagatto
Talanta, 2005, Vol. 68, 239-244
118.
“Use of flow injection multisite detection as a novel approach for blank signal correction in a
spectrophotometric determination”
T. A. C. Oliveira; R. B. R. Mesquita; J. L. F. C. Lima; A. O. S. S. Ramgel
J.AOAC Int., 2005, Vol. 88, 1511-1515
119.
“Chemiluminometric determination of propanolol in an automated multicommutated flow system”
K. L. Marques; J. L. M. Santos; J. L. F. C. Lima
J. Pharm. Biom. Anal., 2005, Vol. 39, 886-891
120. “Sequential injection extration based on restricted access material for determination of furosemide in
serum”
J. Huclová; D. Šatínský; T. Maia; R. Karlícek; P. Solich; A. N. Araújo
J. Chromatogr. A , 2005, Vol. 1087, 245-251
121. “A catalytic multi-pumping flow system for the chemiluminometric determination of metformin”
K. L. Marques; J. L.M. Santos; J.L.F.C. Lima
Anal. Bioanal. Chem., 2005, Vol. 382, 452-457
122. “Spectrophotometric determination of iron and boron in soil extracts using a multi-syringe flow injection
system”
D.M.C. Gomes; M.A. Segundo; J.L.F.C. Lima; A.O.S.S. Rangel
Talanta, 2005, Vol. 66, 703-711
Laboratório Associado para a
Química Verde
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123. “Potentiometric studies on the complexation of copper (II) by phenolic acids as discrete ligand models
of humic substances”
F. Borges; C. Guimarães; J. L.F.C. Lima; I. Pinto; S. Reis
Talanta, 2005, Vol. 66, 670-673
124. “Multi-syringe flow injection system with in-line pre-concentration for the determination of total phenolic
compounds”
H. M. Oliveira; M. A. Segundo; S. Reis; J.L.F.C. Lima
Microchim. Acta, 2005, Vol. 150, 187-196
125. “Determination of ambroxol in an automated multi-pumping pulsed flow system”
J. L. M. Santos; A. Clausse; J.L.F.C. Lima; M.L.M.F.S. Saraiva; A. O.S. Rangel
Anal. Sci., 2005, Vol. 21, 461-464
126. “A pulsed sequential injection analysis flow system for the fluorimetric determination of indomethacin
in pharmaceutical preparations”
P. C.A.G. Pinto; M. L.M.F.S. Saraiva; J. L.M. Santos; J.L.F.C. Lima
Anal. Chim. Acta, 2005, Vol. 539, 173-179
127. “Sample introduction in multi-syringe flow injection systems: comparison between time-based and
volume based strategies”
M. A. Segundo; H. M. Oliveira; J.L.F.C. Lima; M. I. G.S. Almeida; A. O.S. Rangel
Anal. Chim. Acta, 2005, Vol. 537, 207-214
128. “Speciation of inorganic arsenic in waters by potentiometric flow analysis with on-line preconcentration”
J.A. Rodríguez; E. Barrado; M. Veja; J.L.F.C. Lima
Electroanalysis, 2005, Vol. 17, 504-511
129. “An enzimatic flow analysis methodology for the determination of nitrates and nitrites in waters”
P. C. A. G. Pinto; J. L. F. C. Lima; M. L. M. F. S. Saraiva
Intern. J. Environ. Anal. Chem., 2005, Vol. 85, 29-40
130. “Automatic sequential determination of the hydrogen peroxide scavenging activity and evaluation of the
antioxidant potencial by the 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assay
in wines by sequential injection analysis”
P. C. A. G. Pinto; M. L. M. F. S. Saraiva; S. Reis; J. L. F. C. Lima
Anal. Chim. Acta, 2005, Vol. 531, 25-32
131. “Multi-syringe flow injection system for the determination of available phosphorus in soil samples “
M. I. G. S. Almeida; M. A. Segundo; J. L. F.C. Lima; A. O. S. S. Rangel
Intern. J. Environ. Anal. Chem., 2005, Vol. 85, 51-62
132. “ An improved sampling multi-pumping flow system applied to the spectrophotometric determination of
glucose and fructose in syrups”
J. M. Toloti Carneiro; A. C. B. Dias; E. A.G. Zagatto; J. L. M. Santos; J. L. F. C. Lima
Anal. Chim. Acta, 2005, Vol. 531, 279-284
133. “Application of sequencial injection analysis (SIA)to food analysis “
R. Pérez-Olmos; J.C. Soto; N. Zárate; A.N. Araújo; J. L.F.C. Lima; M. L. M. F. S.Saraiva
Food Chem., 2005, Vol. 90, 471-490
Laboratório Associado para a
Química Verde
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134. “Roast Effects on Coffee Amino Acid Enantiomers”
S. Casal; E. Mendes; M. B. P.P. Oliveira; M.A. Ferreira
Food Chemistry, 2005, vol. 89, 333-340
135. “Triacylglycerol Profile by HPLC/ELSD as a Discriminant Parameter of Varietal Olive Oils from Portugal”
S.C. Cunha; S. Casal; M. B. P.P. Oliveira
Ital. J. of Food Sci., 2005, vol. 4, 17, 447-454
136. “Composition Of Quince (Cydonia oblonga Miller) Seeds: Phenolic Compounds, Organic Acids And
Free Amino Acids”
B.M. Silva; P.B. Andrade; F. Ferreres; R.M. Seabra; M. B. P.P. Oliveira; M.A. Ferreira
Natural Product Research, 2005, vol. 19, 3, 275-281
137. “Phenolic Profiles of Portuguese Olive Fruits (Olea europaea L.): Influences of Cultivar and Geographical
Origin”
A.F. Vinha; F. Ferreres; B.M. Silva; P. Valentão; A. Gonçalves; J.A. Pereira; M. B. P.P. Oliveira; R.M.
Seabra; P.B. Andrade
Food Chemistry, 2005, vol. 89, 561-568
138. “Development and Evaluation of a Normal Phase Liquid Chromatographic Method for the Determination
of Tocopherols and Tocotrienols in Walnuts”
J.S. Amaral; S. Casal; R.M. Seabra; M. B. P.P. Oliveira
J. Liq. Chromatogr. & Related Technol., 2005, vol. 28, 785-795
139. “Simultaneous Determination of Tocopherols and Tocotrienols in Hazelnuts by a Normal Phase Liquid
Chromatographic Method”
J. S. Amaral; S. Casal; D. Torres; R.M. Seabra; M. B. P.P. Oliveira
Analytical Sciences, 2005, vol. 21, 1545-1548
140. “Vitamin E Composition of Walnuts (Juglans regia L.): a 3-Year Comparative Study of Different Cultivars”
J. S. Amaral; M.R. Alves; R. Seabra; MBPP. Oliveira
J. Agric. Food Chem., 2005, vol. 53, 26, 5467-5472
141. “Classification of PDO Olive Oils on the Basis of their Sterol Composition by Multivariate Analysis”
M. Rui Alves; SC. Cunha; JS. Amaral; JA Pereira; M. B. P.P. Oliveira
Analytica Chimica Acta, 2005, vol. 549, 166-178
142. “Determination of Ethyl Carbamate in Alcoholic Beverages: An Interlaboratory Study to Compare HPLCFLD with GC-MS Method”
S. Melo Abreu; A. Alves; M. B. P.P. Oliveira; P. Herbert
Anal. Bioanal. Chem,, 2005, 382, 498-503
143. “Effects of the Combination of Hydrophobic Polypeptides, Iso-?-Acids Composition, and Malto
Oligosaccharides on Beer Foam Stability”
I.M.P.L.V.O. Ferreira; K. Jorge; L.C. Nogueira; F. Silva; L.C. Trugo
J. Agric. Food Chem., 2005, vol. 53, 4976-4981
144. “Separation and Quantification of Beer Carbohydrates by High-Performance Liquid Chromatography
with Evaporative Light Scattering Detection”
L. C. Nogueira; F Silva; I. M. P. L. V. O. Ferreira
Journal of Chromatography A, 2005, vol. 1065, 207-210
Laboratório Associado para a
Química Verde
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145. “Roast Effects on Coffee Free and Conjugated Polyamines”
S Casal, E Mendes, MBPP Oliveira, MA Ferreira
Electronic Journal of Environmental, Agricultural and Food Chemistry, 2005, vol. 4, 1063-1068
146. “Analysis of Non-coloured Phenolics in Red Wine: Effect of Dekkera bruxellensis Yeast”
L. R. Silva; P. Andrade; P. Valentão; R. Seabra; M. E. Trujillo; E. Velázquez
Food Chemistry, 2005, Vol. 89, 185-189
147. “Analysis and Quantification of Flavonoidic Compounds from Portuguese Olive (Olea europaea L.) Leaf
Cultivars”
J. Meirinhos; B. Silva; P. Valentão; R. Seabra; J. Pereira; A. Dias; P. Andrade; F. Ferreres
Nat Prod Res, 2005, vol 19, 189-195
148. “Quince (Cydonia oblonga Miller) Fruit Characterization Using Principal Component Analysis”
B. Silva; P. Andrade; R. C. Martins; P. Valentão; F. Ferreres; R. Seabra; M. A Ferreira
J. Agric Food Chem, 2005, vol 53, 111-122
149. “Phenolic Compounds in External Leaves of Tronchuda Cabbage (Brassica oleraceae L. var costata)”
F. Ferreres, P. Valentão, R. Llorach, C. Pinheiro, L. Cardoso, J. A. Pereira, C. Sousa, R. Seabra, P.
Andrade
J. Agric Food Chem, 2005, vol 53, 2901-2907
150. “Quantitation of Nine Organic Acids in Wild Mushrooms”
P. Valentão; G. Lopes; M. Valente; P. Barbosa; P. Andrade; B Silva; P. Baptista; R Seabra
J. Agric Food Chem, 2005, vol 53, 3626-3630
151. “Effect of the Conservation Procedure on the Contents of the Phenolic Compounds and Organic Acids
in Chanterell (Cantharellus cibarius) Mushroom”
P. Valentão; P. Andrade; J. Rangel; B. Ribeiro; B. Silva; P. Baptista; R. Seabra
J. Agric Food Chem, 2005, vol 53, 4925-4931
152. “Influence of Two Fertilization Regimens on the Amounts of Organic Acids and Phenolic Compounds of
Tronchuda Cabage (Brassica oleracea L. var costata DC)”
C. Sousa; P. Valentão; J. Rangel; G. Lopes; J. A. Pereira; F. Ferreres; R. Seabra; P. Andrade
J. Agric Food Chem, 2005, vol 53, 9128-9132
153. “Characterisation of the Phenolic Profile of Boerhaavia diffusa L. by HPLC-PAD-MS/MS as a Tool for
Quality Control”
F. Ferreres; C. Sousa; M. Justin; P. Valentão; P. Andrade; R. Llorach; A. Rodrigues; R. Seabra; A.
Leitão
Phytochemical Analysis, 2005, vol 16, 451-458
154. “Phenolic Profile of Hazelnut (Corylus avellana L.) Leaves Cultivars Grown in Portugal”
J. S. Amaral; F. Ferreres; P. Andrade; P. Valentão; C. Pinheiro; A. Santos; R. Seabra
Nat. Prod. Res, 2005, vol19, 157-163
155. “Dissemination Of Sulfonamide Resistance Genes (sul1, sul2 and sul3) in Portuguese Salmonella
enterica Strains And Relation With Integrons”
P. Antunes; J. Machado; JC Sousa; L Peixe
Antimicrobial Agents and Chemotherapy, 2005, Vol. 49, 836-839
Laboratório Associado para a
Química Verde
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156. “The Integron Content of E. coli Strains with Extended-Spectrum Beta-Lactamases Along Twelve Years
in a Single Hospital in Madrid, Spain”
E. Machado; R. Cantón; J.C. Galán; A. Rollán; L. Peixe; F. Baquero; T.M. Coque
Antimicrobial Agents and Chemotherapy, 2005, Vol. 49(5), 1823-1829
157. “First isolation of blaVIM-2 in an environmental isolate of Pseudomonas pseudoalcaligenes”
S. Quinteira; H. Ferreira; L. Peixe
Antimicrobial Agents and Chemotherapy, 2005, Vol. 49, 2140-2141
158. “Characterization of In100, a new integron carrying a metallo-{beta}-lactamase and a carbenicillinase,
from Pseudomonas aeruginosa”
S. Quinteira; H. Ferreira; L. Peixe
Antimicrobial Agents Chemotherapy. 2005, Vol. 49, 451-453
159. “High Occurrence and Persistence of Antibiotic Resistant Enterococci in Poultry Food Samples in
Portugal”
C.Novais; T.M. Coque; M. J. Costa; J.C. Sousa; F. Baquero, L.V. Peixe
J. Antimicrob. Chemother , 2005, Vol. 56, 1139-1143
160. “Vancomycin Resistant Enterococcus Faecium Clone Among in Swine, Europe”
C.Novais; T.M. Coque; P. Boerlin; I. Herrero; M. Moreno; L. Dominguez; L. Peixe
Emerg. Infect. Dis., 2005, Vol. 11, 1985-1987
161. “Molecular Characterization of Glycopeptide-resistant Enterococcus faecium Isolates from Portuguese
Hospitals”
C.Novais; J.C. Sousa; T.M. Coque; L. Peixe; The Portuguese Resistance Study Group
Antimicrobial Agents Chemotherapy, 2005, Vol. 49, 3073-3079
162. “Environmental Contamination with Vancomycin-Resistant Enterococci from Hospital Sewage in Portugal”
C. Novais; T. M. Coque; H. Ferreira; J. C. Sousa; L. Peixe
Appl. Environ. Microbiol. 2005, Vol. 71, 3364-3368
163. “Study of the voltammetric behaviour of metam and its application to an amperometric flow system”
M. F. Barroso; P. Paíga; M. C. V. F. Vaz; C. Delerue-Matos
Anal. Bioanal. Chem., 2005, Vol. 383, 880-885
164. “Screening of grapes and wine for azoxystrobin, kresoxim-methyl and trifloxystrobin fungicides by
HPLC with diode array detection”
S. M. Abreu; M. Correia; P. Herbert; L. Santos; A. Alves
Food Addit. Contam., 2005, Vol. 22, 549-556
165. “Determination of Ametryn in Soils via Microwave-Assisted Solvent Extraction Coupled to Anodic Stripping
Voltammetry With a Gold Ultramicroelectrode”
O. Tavares; S. Morais; P. B. Paíga; C. Delerue-Matos
Anal. Bioanal. Chem., 2005, Vol. 382, 477-484
166. “Use of solvent extraction to remediate soils contaminated with hydrocarbons”
A. Silva; C. Delerue-Matos; A. Fiúza
J. Haz. Mat. B, 2005, Vol. 124, 224–229
Laboratório Associado para a
Química Verde
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167. “Determination of free furfuryl alcohol in foundry resins by chromatographic techniques”
M. T. Oliva-Teles; C. Delerue-Matos; M. C. M. Alvim-Ferraz
Anal. Chim. Acta, 2005, Vol. 537, 47-5
168. “Natural waste materials containing chitin as adsorbents for textile dyestuffs: batch and continuous
studies”
S. A. Figueiredo, J. M. Loureiro, R. A. Boaventura
Water Res., 2005, vol. 39, 4142-4152
169. “Sorption behaviour of bifenthrinon cork”
V. Domingues, A. Alves, M. Cabral, C. Delerue-Matos
J. Chromatogr. A, 2005, Vol. 1069, 127-132
170. “Graphic data analysis and complex formation curves as modelling and optimisation tools in
potentiometric and voltammetric speciation studies of a Pb – (TAPSO)x – (OH)y system”
C.M.M. Machado; I. Cukrowski; H. M. V. M. Soares
Electroanalysis, 2005, Vol. 518, 1291-1301
171. “An Early Development of the Nonenal Potential in the Malting Process”
L. F. Guido, P. Boivin, N. Benismail, C. R. Gonçalves, A. A. Barros
European Food Research Technology, 2005, Vol. 220, 200-206
172. “The Impact of Sulphur Dioxide and Oxygen on the Behaviour of 2-Furaldehyde in Beer: an Industrial
Approach”
J. R. Carneiro, L. F. Guido, P. J. Almeida, J. A. Rodrigues, A. A. Barros
International Journal of Food Science and Thechnology, 2005, Vol. 40, 1-8
173. “Electroanalytical Study of the Antidepressant Sertraline”
H. P. A. Nouws, C. Delerue-Matos, A. A. Barros, J. A. Rodrigues
Journal of Pharmaceutical and Biomedical Analysis, 2005, Vol. 39, 290-293
174
“Electroanalytical Study of Fluvoxamine”
H. P. A. Nouws, C. Delerue-Matos, A. A. Barros J. A. Rodrigues, A. Santos-Silva
Analytical and Bionalytical Chemistry, 2005, Vol. 382, 1662-1668
175. “How Do Sulfites Help to Control Beer Ageing”
L. F. Guido
Cerevisia, Belgian Journal of Brewing and Technology, 2005, Vol. 30, 132-138
176. “Square-wave voltametric method for determination of molinate concentration in a biological process
using a hanging mercury drop electrode”
M. F. Barroso; O. C. Nunes; M. C. Vaz; C.Delerue-Matos
Anal. Bioanal. Chem., 2005, Vol. 381, 879-883
177. “Voltammetric Determination of Dialifos in Soils With a Mercury Film Ultramicroelectrode”
S. Morais; O. Tavares; C. Delerue-Matos
Anal. Lett., 2005, Vol. 38, 1275-1288
178. “Modelling the rheological behaviour of galactomannan aqueous solutions”
W. Sittikijyothin; D.Torres; M.P. Gonçalves
Carbohydrate Polymers, 2005, Vol. 59, 339-350
Laboratório Associado para a
Química Verde
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179. “Microstructural changes during drying of apple slices”
L. Mayor; M.A. Silva; A.M. Sereno
Drying Technology, 2005, Vol. 23 (9-11), 2261-2276
180. “Water sorption isotherms of fresh and partially osmotic dehydrated pumpkin parenchyma and seeds
at several temperatures”
L. Mayor; L., R. Moreira; F. Chenlo; A.M. Sereno
European Food Research and Technology, 2005, Vol. 220, 163-167
181. “Metabolic pathways of 4-bromo-2,5-dimethoxyphenethylamine (2C-B): analysis of phase I metabolism
with hepatocytes of six species including human”
H. Carmo; J. G. Hengstler; D. de Boer; M.l Ringel; F. Remião; F. Carvalho; E. Fernandes; L. dos Reys;
F. Oesch; M. L. Bastos
Toxicology, 2005, Vol. 206, 75-89
182. “Atropine Prevents Amphetamine-induced Hyperthermia in Mice”.
F. Carvalho; J. A. Duarte; C. Sousa; F. Remião; H. Carmo; M. L. Bastos
Toxicology Letters, 2005, 158S: S76
183. “Are reactive oxygen species (ROS) involved in transcriptional repression induced by sodium arsenate
in bladder cancer (ECV 304) cell line?”
R. Pires-Das-Neves; M. L. Pereira; F. Carvalho; M. L. Bastos; F. J. Iborra
Toxicology Letter, 2005,158S: S173
184. “Detection of GSH adducts of catecholamines in biological samples”
R. Silva; V. Costa; S. Boldt; H. Carmo; F. Carvalho; M. Carvalho; R. Carvalho; M. L. Bastos; F. Remião
Toxicology Letters, 2005, 158S: S189
185. “Influence of sodium depletion on the kinetics of paraquat in the isolated rat lung”
R. J. D. Oliveira; M. J. Valle; M. L. Bastos; F. Carvalho; A. S. Navarro
Toxicology Letters, 2005, 158S: S213
186. “Acute paraquat poisoning: report of a survival case following intake of a potential lethal dose”
R. J. D. Oliveira; A. M. Sarmento; P. Reis; A. Amaro; F. Remião; M. L. Bastos; F. Carvalho
Toxicology Letters, 2005, 158S: S241
187. “E-learningand risk communication. Report of an experience at the Faculty of Pharmacy of the University
of Porto”
F. Remião; H. Carmo; F. Carvalho; M. L. Bastos
Toxicology Letters, 2005, 158S: S76
188. “Effect of the NADPH oxidase inhibitior, apocynin, on systolic blood pressure and on vascular antioxidant
defenses in hypertensive rats with activation of the renin-angiotensin system”
T. Sousa; M. Morato; D. Pinho; E. Fernandes; F. Carvalho; A. Albino-Teixeira
Journal of Hypertension, 2005, 23, S361: P3.305
189. “Voluntary fatal intoxication with inorganic mercury. Evaluation of the distribution of the metal in several
autopsy samples”
P. Triunfante; M.E. Soares; M.L. Bastos
Toxicology Letters, 2005, 158S: S169
Laboratório Associado para a
Química Verde
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190. “The Drinking of a Salvia officinalis Infusion Improves Liver Antioxidant Status in Mice and Rats”
C. F. Lima; P. Andrade; R. Seabra; M. Fernandes-Ferreira; C. Pereira-Wilson
J of Ethnopharmacology, 2005, vol 97, 383-389
191. “Acute Toxicity of Widely Used Pharmaceuticals in Aquatic Species: Gambusia holbrooki, Artemia
parthenogenetica and Tetraselmis chuii”
B. Nunes; F. Carvalho; L. Guilhermino
Ecotoxicology and Environmental Safety , 2005, vol 61(3), 413-419
192. “Scavenging of Nitric Oxide by an Antagonist of Adenosine receptors”
T. Sousa; E. Fernandes; C. Nunes; J. Laranjinha; F. Carvalho; D. Pinho; M. Morato, A. Albino-Teixeira
Journal of Pharmacy and Pharmacology, 2005, vol 57, 399–404
193. “Characterization and Use of the Total Head Soluble Cholinesterases from Mosquitofish (Gambusia
holbrooki) for the Assessment of SDS Anticholinesterasic Activity“
B. Nunes; F. Carvalho; L. Guilhermino
Journal of Enzyme Inhibition and Medicinal Chemistry ,2005, vol 20(4), 369–376
194. “Do Metals Inhibit Acetylcholinesterase (AChE)? Implementation of Assay Conditions for the Use of
AChE Activity as a Biomarker of Metal Toxicity”
M. F. Frasco; D. Fournier; F. Carvalho; L. Guilhermino
Biomarkers, 2005, vol 10(5), 360-375
195. “A Collection of Papers Presented at the 30th International Congress of Theoretical Chemists of Latin
Expression, Porto, Portugal, 8-12 September 2004- Foreword”
M. J. Ramos; A. L. Magalhães; A. S, Melo; P. A. Fernandes
Journal of Molcular Structure – Teochem, 2005, Vol.729, 1-2
196. “Computational Insight into Anti-mutagenic Properties of CYP1A Flavonoid Ligands”
R. Fonseca, M. Marini, A. Melo, M. C. Menziani e M. J. Ramos
Medicinal Chemistry, 2005, Vol.1, 355-360
197. “Molecular Dynamics Simulations of Angiotensin II in Aqueous and Dimethyl Sulfoxide Environments”
M. A. C. Preto; A. Melo; H. L. S. Maia; T. Mavromoustakos; M. J. Ramos
Journal of Physical Chemistry B, 2005, Vol. 109, 17743-17751
198. “Computer Modeling and Research in the Classroom”
M. J. Ramos; P. A. Fernandes
Journal of Chemical Education, 2005, Vol.82 (7); 1021-1025
199. “Density-Functional Calculations of the Cu, Zn Superoxide Dismutase Redox Potential: The Influence
of Active Site Distortion”
R. J. F. Branco; P. A. Fernandes; M. J. Ramos
Journal of Molecular Structure: Theochem, 2005, Vol. 729, 141–146
200. “Cu, Zn Superoxide Dismutase: Distorted Active Site Binds Substrate without Significant Energetic
Cost”
R. J. F. Branco; P. A. Fernandes; M. J. Ramos
Theoretical Chemistry Accounts, 2005, DOI: 10.1007/s00214-005-0672-x
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201. “Overview of Ribonucleotide Reductase Inhibitors: An Appealing Target in Anti-1Tumor Therapy”
N. M. F. S. A. Cerqueira; S. Pereira; P. A. Fernandes; M. J. Ramos
Current Medicinal Chemistry, 2005, Vol.12, 1283-1294
202. “Accuracy of the Numerical Solution of the Poisson–Boltzmann Equation”
I. S. Moreira; P. A. Fernandes; M. J. Ramos
Journal of Molecular Structure: Theochem, 2005, Vol. 729, 11–18
203. “Theoretical Study on the Inhibition of Ribonucleotide Reductase by 2 -Mercapto-2 -Deoxyribonucleoside5 -Diphosphates”
S. Pereira; P.A. Fernandes; M. J. Ramos
Journal of the Americal Chemical Society, 2005, Vol.127; 5174-5179
204. “Unraveling the Mechanism of the Farnesyltransferase Enzyme”
S. F. Sousa; P. A. Fernandes; M. J. Ramos
J. Biol. Inorg. Chem., 2005, Vol. 10, 3-10
205. “Farnesyltransferase: Theoretical Studies on Peptide Substrate Entrance Thiol or Thiolate Coordination?”
S. F. Sousa; P. A. Fernandes; M. J. Ramos
Journal of Molecular Structure: Theochem, 2005, Vol. 729, 125–129
206. “Farnesyltransferase – New Insights into the Zin-Coordination Sphere Paradigm: Evidence for a
Carboxylate-Shift Mechanism”
S. F. Sousa; P. A. Fernandes; M. J. Ramos
Biophysical Journal, 2005, Vol. 88, 483-494
207. “New Designs for Inhibitors of the NF-kappa B: DNA Binding”
P. A. Fernandes; A. R. R. Maia; A. A. S. Almeida; B.F.B.Silva; C.M.S.Ribeiro; C.F.B.Ribeiro; D.S.M.
Ribeiro; D.A.P.Fonseca; E.S.Henriques; E.M.S.Cunha; F.R.N.C.Maia; J.A.A.Pereira; J.P.G.Pacheco;
J.A.A.D.Ferreira; L.R.C.Matos; M.A.B.P.Pinto; M.C.S.Borges; P.J.C.R.Magalhães; P.F.R.D.Teixeira;
P.N.B.C.Veloso; R.J.F.Ferreira; S.S.Gomes; T.F.Barros; T.S.J.T.Selão; V.Pande; V.M.M.C.Fernandes;
M. J. Ramos
Theoretical Chemistry Accounts, 2005, Vol.113; 197-204
208. “Discovery of a Large Number of Previously Unrecognized Mitochondrial Pseudogenes in Fish Genomes”
A. Antunes; M. J. Ramos
Genomics, 2005, Vol.86; 708-717
209. “Theoretical Quantitative Structure-Activity Relationships of Flavone Ligands Interacting with Cytochrome
P450 1A1 and 1A2 Isozymes”
F. Iori; R. Fonseca; M. J. Ramos
Bioorganic Medicinal Chemistry, 2005, Vol.13; 4366-4374
210. “Theoretical Study of the Suicide Inhibition Mechanism of the Enzyme Pyruvate Formate Lyase by
Methacrylate”
M. F. Lucas; M. J. Ramos
Journal of the Americal Chemical Society, 2005, Vol.127; 6902-6909
211.
“Molecular Recognition of 15-deoxy-Delta(12,14)-Prostaglandin J(2) by Nuclear Factor-kappa B and
other Cellular Proteins”
V. Pande; M. J. Ramos
Bioorganic & Medicinal Chemistry Letters, 2005, Vol. 15; 4057-4063
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212. “NF-kB in Human Disease: Current Inibitors and Prospects for De Novo Structured Based Design of
Inhibitors”
V. Pande; M. J. Ramos
Current Medicinal Chemistry, 2005, Vol.12; 357-374
213. “Structural Basis for the GSK-3b Binding Affinity and Selectivity Against CDK-2 of 1-(4Aminofurazan3yl)-5-Dialkylaminomethyl- 1-H-[1,2,3] Triazole-4-Carboxylic Acid Derivatives”
V. Pande; M. J. Ramos
Bioorganic & Medicinal Chemistry Letters, 2005, Vol. 15; 5129-5135
214. “Inhibitory Activity of Polyhydroxycarboxylate Chelators Against Reconbinant NF-kappa B p50 ProteinDNA Binding”
R. K. Sharma; V. Pande; M. J. Ramos; H. K. Rajor; S. Chopra; K. Meguro; J. Inoue; M. Otsuka
Bioorganic Chemistry, 2005, Vol.33; 67-81
215. “Density-Functional Study of Mechanisms for the Cofactor-Free Decarboxylation Performed by
Uroporphyrinogen III Decarboxylase”
M. J. Ramos; P. J. Silva
Journal of Physical Chemistry B, 2005, Vol.109; 18195-18200
216. “Energy Partitioning in Association Processes”
A. R. F. Carvalho; A. Melo
International Journal of Quantum Chemistry, 2005, Vol.104, 240-248
217. “Assessing the Taxonomic Status of the Palawan Pangolin Manis Culionensis (Pholidota) Using Discrete
Morphological Characters”
P. Gaubert; A. Antunes
Journal of Mammalogy, 2005, Vol.86, 1068-1074
218. “Interaction between quinolones antibiotics and bacterial outer membrane porin OmpF”
P. Neves, E. Berkane, P. Gameiro, M. Winterhalter, B. de Castro
Biophys. Chem., 2005, Vol. 113, 123-128
219. “Surfactant removal for reconstitution of ATPase activity prtoteoliposomes of mouse MDR3 P-glycoprotein”
Sofia A.C. Lima, Anabela Cordeiro-da-Silva, Paula Gameiro, Baltazar de Castro
Anal. Biochem., 2005, Vol. 338, 350-353
220. “Antioxidant properties of prepared blueberry (Vaccicium myrtillus) extracts”
Faria A. Oliveira J., Neves P., P. Gameiro, Santos-Buelga C., Freitas V. Mateus N
J. Agricul. Food Chem., 2005, Vol. 53, 6896-6902
221. “Fluorescence probes used for detection of reactive oxygen species”
A. Gomes; E. Fernandes; J. L.F.C. Lima
J. Biochem. Biophys. Meth., 2005, Vol. 65, 45-80
222. “Sensitive sequential injection determination of naproxen based on interaction with b-cyclodextrin”
E. P. Zisiou; P. C.A.G. Pinto; M. L. M. F. S. Saraiva; C. Siquet; J. L.F.C. Lima
Talanta, 2005, Vol. 68, 226-230
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223. “Evalution of the total antioxidant capacity by using a multipumping flow system with chemiluminescent
detection”
S. R.P. Meneses; K. L. Marques; C. K. Pires; J. L.M. Santos; E. Fernandes; J. L.F.C. Lima; E. A.G.
Zagatto
Anal. Biochem., 2005, Vol. 345, 90-95
224. “Structure-property studies on the antioxidant activity of flavonoids present in diet”
S. Teixeira; C.e Siquet; C. Alves; I. Boal; M. P. Marques; F. Borges; J. L.F.C. Lima; S. Reis
Free Rad. Biol. Med., 2005, Vol. 39, 1099-1108
225. “Pindolol is a potent scavenger of reactive nitrogen species”
E. Fernandes; A. Gomes; D. Costa; J. L.F.C. Lima
Life Sci., 2005, Vol. 77, 1983-1992
226. “Effects of diclofenac on EPC liposome membrane properties”
H. Ferreira; M. Lúcio; J. L.F.C. Lima; C. Matos; S. Reis
Anal. Bioanal. Chem., 2005, Vol. 382, 1256-1264
227. “Interaction of clonixin with EPC liposomes used as membrane models”
H. Ferreira; M. Lúcio; J.L.F.C. Lima; C. Matos; S. Reis
J. Pharm. Sci., 2005, Vol. 94, 1277-1287
228. “Hydrogen peroxide scavenging activity by non-steroidal anti-inflammatory drugs”
D. Costa; A. Gomes; S. Reis; J.L.F.C. Lima; E. Fernandes
Life Sciences, 2005, Vol. 76, 2841-2848
229. “Effect of anti-inflammatory drugs on splenocyte membrane fluidity”
H. Ferreira; M. Lúcio; J. L.F.C. Lima; A. Cordeiro-da-Silva; J. Tavares; S. Reis
Anal. Biochem., 2005, Vol. 339, 144-149
230. “Photolysis secondary products of cobaloximes and imino/oxime compounds controlled by steric
hindrance imposed by the Lewis base”
M. Rangel; A. Leite; J. Gomes; B. Castro
Organometallics, 2005, 24, 3500-3507
231. “A New Strategy to Model the Si(100) Surface”
H. R. R. Santos; M. J. Ramos; J. A.N. F. Gomes
Comptes Rendus Chimie, 2005, Vol. 8, 1461–1468
232. “Adsorption of Protected and Unprotected Amino-Cyclopentene at the Si(100) Surface Modeled with a
Hybrid Quantum Mechanical Cluster Technique”
H. R. R. Santos; M. J. Ramos; J. A. N. F. Gomes
Physical Review B, 2005, Vol. 72; 075445
233. “Comparative Study of Novel Sulfonylcalix [4] Arene Derivative Compounds”
A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes
Journal of Molecular Structure: Theochem, 2005, Vol.729, 83-90
234. “Computational Study of calix [4] Arene Derivatives and Complexation with Zn2+”
A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes
Chemical Physics, 2005, Vol.310, 109-122.
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235. “Enzymatic Formation of Ions and their Detection at a Three-Phase Electrode”
R. Gulaboski; C. M. Pereira; M. N. D. S. Cordeiro; et al.
Journal of Solid State Electrochemistry, 2005, Vol.9; 469-474
236. “Electrochemical Study of Ion Transfer of Acetylcholine Across the Interface of Water and a LipidModified 1,2-Dichloroethane”
R. Gulaboski; C. M. Pereira; M. N. D. S. Cordeiro; et al.
Journal of Physical Chemistry B, 2005, Vol. 109; 12549-12559
237. “Catalytic Properties of a Mn(III) Salen Complex Immobilised in a Pillared Clay by Simultaneous Pillaring/
Encapsulation Procedures”
B. Cardoso; J. Pires; A. P. Carvalho; M. B. Carvalho; I. Kuzniarska-Biernacka; A. R. Silva; C. Freire; B.
de Castro
Eur. J. Inorg. Chem., 2005, 837-844
238. “Synthesis of Ferrocenyldiimine Metal Carbonyl Complexes and an Investigation of the Mo Adduct
Encapsulated in Cyclodextrin”
Z. Petrovski; S. S. Braga; S. S. Rodrigues; C. C. L. Pereira; I. S. Gonçalves; M. Pillinger; C. Freire; C.
C. Romão
New J. Chem., 2005, Vol. 29, 347-354
239. “Copper(II) Acetylacetonate Anchored onto an Activated Carbon as a Heterogeneous Catalyst for the
Aziridination of Styrene”
A. R. Silva; J. L. Figueiredo; B. de Castro; C. Freire
Cat. Today, 2005, Vol. 102-103C, 154-159
240. “Styrene Epoxidation Catalysed by Manganese(III) Salen Complex Supported onto Activated Carbons”
M. Cardoso; A. R. Silva; B. de Castro; C. Freire
Apply Cat. A, General, 2005, Vol. 285, 110-118
241. “Synthesis and Characterisation of Salen-Type Ligands Functionalised with Pyrrole Derivative Pendant
Arms”
M. Andrade; C. Sousa; J. E. Borges; C. Freire
J. Phys. Org. Chem., 2005, Vol.18, 935-940
242. “Chiral Manganese(III) Schiff Base Complexes Anchored onto Activated Carbon as Enantioselective
Heterogeneous Catalysts for Alkene Epoxidation”
A. R. Silva; V.y Budarin; J. H. Clark; B. de Castro; C. Freire
Carbon, 2005, Vol. 43, 2096-2105
243. “Spectroelectrochemical Characterisation of Copper(II) Salen-Based Modified Electrodes”
M. Martins; M. Vilas Boas; C. Freire; A. R. Hillman
Electrochimica Acta, 2005, Vol. 51, 304-314
244. “Asymmetric Epoxidation of Alkenes by a Chiral Manganese(III) Salen Complex Anchored onto a
Functionalised Hexagonal Mesoporous Silica”
A. R. Silva; K. Wilson; J.H. Clark; C. Freire
Stud. Surf. Sci. Catal., 2005, Vol. 158, 1525-1532
245. “Anchoring of Vanadyl Acetylacetonate onto Amine Functionalised Activated Carbons and Catalytic
Activity in the Epoxidation of an Allylic Alcohol”
B. Jarrais; A. R. Silva; C. Freire
Eur. J. Inorg. Chem., 2005, 4582-4589
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246. “Mn(III) Salen Complex Immobilised into Pillared Clays by in Situ and Simultaneous Pillaring/
Encapsulation Procedures. Application in the Heterogeneous Epoxidation of Styrene”
B. Cardoso; J. Pires; A. P. Carvalho; I. Kuzniarska-Biernacka; A. R. Silva; B. de Castro; C. Freire
Microp. Mesoporous Mat., 2005, Vol.86, 295-302
247. “(Salen)Nickel-Catalysed Epoxidations in the Homogeneous and Heterogeneous
Phase: The Implication of Oxygen on the Efficiency and Product Selectivity”
R. Ferreira; H. Garcia; B. de Castro; C. Freire
Eur. J. Inorg. Chem., 2005, 4272-4270
248. “Organo-Laponites as Novel Mesoporous Supports for Manganese (III) Salen Catalysts”
I. Kuzniarska-Biernacka; A. R. Silva; A. P. Carvalho; J. Pires; C. Freire
Langmuir, 2005, Vol. 21, 10825-10834
249. “Thermodynamic Evidence for Ca2+-Mediated Self-Aggregation of Lewis X Gold Glyconanoparticles. A
Model for Cell Adhesion via Carbohydrate- Carbohydrate Interaction”
J. M. de la Fuente, P. Eaton, A. G. Barrientos, M. Menéndez, S. Penadés
J. Am. Chem. Soc., 2005, Vol 127, 6192-6197
250. “Tribological and Structural Studies of Nanocrystalline Ti-C-N Composites Coatings Prepared by Reactive
Sputtering”
D. Martínez-Martínez, J.C. Sánchez-López, T.C. Rojas, A. Fernández, P. Eaton, M. Belin
Thin Solid Films, 2005, Vol 472 (1-2), 64-70
251. “Structural Characterization of Self-Assembled Monolayers of Neoglycoconjugates using Atomic Force
Microscopy”
C. Tromas, P. Eaton, J. Mimault, J.Rojo, S. Penadés
Langmuir, 2005, Vol 21, 6142 - 6144
252. “Intramolecular electron transfer in diastereomeric naphthalene-amine dyads: a fluorescence and laser
flash photolysis study”
S. Abad; U. Pischel; M. A. Miranda
Photochem. Photobiol. Sci., 2005, Vol. 4, 69-74
253. “Wavelength-dependent stereodifferentiation in the fluorescence quenching of asymmetric naphthalenebased dyads by amines”
S. Abad; U. Pischel; M. A. Miranda
J. Phys. Chem. A, 2005, Vol. 109, 2711-2717
254. “Proton-induced fluorescence switching in novel naphthalimide-dansylamide dyads”
S. Abad; M. Kluciar; M. A. Miranda; U. Pischel
J. Org. Chem., 2005, Vol. 70, 10565-10568
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Books and book chapters
1.
P.M. Abreu; P. Branco; S. Matthew
“Natural product-like combinatorial libraries towards the discovery of lead compounds”
in Combinatorial Synthesis of Natural Product Based Libraries, Ed. A.M Boldi
in Combinatorial Chemistry Series, CRC Press, 257-352, 2005
2.
Afonso, Carlos A. M.; Crespo, João P. S. G.
“Green Separation Processes: Fundamentals and Applications”
1nd ed. Wiley-VCH, Weinheim, 2005
3.
T Schafer; LC Branco; R Fortunato; P Izak; CM Rodrigues; CAM Afonso; JG Crespo
“Opportunities for membrane separation processes using ionic liquids”
in ACS Symposium Series, 902: 97-110 (2005)
4.
V Najdanovic-Visak; A Serbanovic; JMSS Esperanca; HJR Guedes; LPN Rebelo; M Nunes da Ponte
“Multiphase equilibrium in mixtures of [C(4)mim][PF6] with supercritical carbon dioxide, water, and
ethanol: Applications in catalysis”
in ACS Symposium Series, 901: 301-310 (2005)
5.
A Banet Osuna; A Serbanovic; M Nunes da Ponte
“Supercritical Fluids”
in Green Separation Processes, eds. C.A.M. Afonso and J. G. Crespo Eds., Wiley-VCH, Weinheim
(2005)
6.
T Schäfer, JG Crespo
“Vapour Permeation and Pervaporation”
in: Green Separation Processes, Carlos M. Afonso and João G. Crespo (Eds), Wiley-VCH
Publishers (2005)
7.
LPN Rebelo; V Najdanovic-Visak; R Gomes de Azevedo; JMSS Esperança; M Nunes da Ponte; HJR
Guedes; ZP Visak;. HC de Sousa; J Szydlowski; JN Canongia Lopes; TC Cordeiro
“Phase behavior and thermodynamic properties of ionic liquids, ionic liquid mixtures, and ionic liquid
solutions”
ACS Symposium Series, 901: 270-291 (2005)
8.
CA Cerdeiriña; J Troncoso; C Paz Ramos; L Romani; V Najdanovic-Visak; JMSS Esperança; M
Nunes da Ponte; HJR Guedes; ZP Visak; LPN Rebelo
“Criticality Of The [C4mim][Bf4] + Water System”
ACS Symposium Series, 901: 175-186, (2005)
9.
M. Sousa, M.J. Melo, A. Aguiar-Ricardo, P. Cruz,
“A Green Approach to Antique Textile Cleaning”
in The 14th Triennal Meeting The Hague Preprints, Vol II, Paterakis, A.B. (Ed.), ICOM Committee for
Conservation, 944-953 (2005)
10.
JPB Mota; IAAC Esteves
“Fundamentos dos Processos de Adsorção”
in Descontaminación Ambiental Mediante Adsorbentes, ed. F. Rodriguez-Reinoso, Cap. 1, 1-23,
Ediciones CYTED, Madrid, Spain (2005)
Laboratório Associado para a
Química Verde
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11.
P. Valentão; P. Andrade; R. Seabra
“Análise Macroscópica e Microscópica em Farmacognosia”
in Farmacognosia e Fitoquímica, ed. A. Proença da Cunha, Fundação Calouste Gulbenkian, 17-35
(2005)
12.
M.P.L.V.O. Ferreira
“RP-HPLC methods for separation of milk proteins: application on quality control and detection of
species adulteration in dairy products”
in Encyclopedia of Chromatography, eds. J. Cazes, Taylor & Francis, 1457 (2005).
13.
I.S. Moreira; P. A. Fernandes; M. J. Ramos
“Computational Determination of the Relative Free Energy of Binding- Application to Alanine
Scanning Mutagenesis”
in Molecular Materials with Specific Interactions: Modeling and Design, ed W.Andrzej Sokalski,
Springer, (2005)
Laboratório Associado para a
Química Verde
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Articles in Proceedings
“Synthesis of Polyurethanes in Supercritical CO2”
C. Veiga de Macedo, T. Casimiro, A. C. Fernandes, C. Romão, A. Aguiar-Ricardo
in Proceedings of the 10th Meeting on Supercritical Fluids, ed. M. Perrut, ISASF: Strasbourg / Colmar,
France (2005).
“Membrane Formation with CO2-Assisted Phase Inversion Method. Solvent Power and Depressurization
Effect”
M. Temtem, T. Casimiro, J. F. Mano, A. Aguiar-Ricardo
in Proceedings of the 10th Meeting on Supercritical Fluids, ed. M. Perrut, ISASF: Strasbourg / Colmar,
France (2005).
“Nonisothermal Dynamic Model of a SFE Structured Packing Column”
J. B. Fernandes, R. Ruivo, J. P. Mota, P. C. Simões
in Proceedings of the 10th Meeting on Supercritical Fluids, ed. M. Perrut, ISASF: Strasbourg / Colmar,
France (2005).
“Solubility of Liquid Crystal, E7, in Supercritical Carbon Dioxide”
A.R.E. Brás, T. Casimiro, J. Caldeira, M. Dionísio, A. Aguiar-Ricardo
in Proceedings of the 9th International Chemical Engineering Conference-Chempor, Coimbra, (2005).
”Evaluation of CO2-philicity of Perfluoropolyethers using High-pressure NMR Spectroscopy”
M. Temtem, T. Casimiro, E. J. Cabrita, A. L. Macedo, A. Aguiar-Ricardo
in Proceedings of the 9th International Chemical Engineering Conference - Chempor, Coimbra (2005).
“Study Of Cold-Set Whey Protein Gels Prepared With Mg2+”
M. Vázquez da Silva, L. Hilliou, M.P. Gonçalves
in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento
de Engenharia Química da Universidade de Coimbra, paper QL056-6 pages (2005) (ISBN 972-8055-13-7).
“Extraction of Biopolymers From Natural Resources And Industrial Wastes For Edible Films Applications”
A.M. Ramos, H. Coelho, L. Guerreiro, A.M. Relva, L. Hilliou, F.D.S. Larotonda and A.M. Sereno
in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento
de Engenharia Química da Universidade de Coimbra, paper QL078-6 pages (2005) (ISBN 972-8055-13-7).
“Characterization Of Model Edible Films Using Hydrocolloids And Pectins”
V.D. Alves, L. Hilliou, F.D.S. Larotonda, I.M. Coelhoso, M.P. Gonçalves, A.M. Sereno
in Proceedings of the 9th International Chemical Engineering
Conference CHEMPOR 2005, Departamento de Engenharia Química da Universidade de Coimbra, paper
QL096-6 pages (2005) (ISBN 972-8055-13-7).
“Effect Of Seasonal Variation On The Chemical And Physical Properties Of Carrageenans Extracted From
Mastocarpus Stellatus And Gigartina Pistillata”
L. Hilliou, F.D.S. Larotonda, P. Abreu, A.M. Ramos, M.P. Gonçalves, A.M. Sereno,
in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento
de Engenharia Química da Universidade de Coimbra, paper QL086-6 pages (2005) (ISBN 972-8055-13-7).
“Trypsin immobilisation in zeolites”
C. Rocha, M.P. Gonçalves, J.A.Teixeira
in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento
de Engenharia Química da Universidade de Coimbra, paper QL0102- 6 pages (2005) (ISBN 972-8055-13-7).
Laboratório Associado para a
Química Verde
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”Simultaneous Determination of Tocopherols and Tocotrienols in Virgin Olive Oils by Normal Phase HPLC
Coupled with Diode-Array / Fluorescence detector”
S.C. Cunha; J.S. Amaral; JO. Fernandes; MBPP. Oliveira
in Proceedings of INTRADFOOD 2005 Innovations in Traditional Foods, Valência, 2005, 353-356.
”Tocopherols and Tocotrienols Composition of Hazelnuts”
SC. Cunha; JS. Amaral; JO. Fernandes, MBPP. Oliveira
in Proceedings of INTRADFOOD 2005 Innovations in Traditional Foods, Valência, 2005, 353-356.
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B
Dissertations
Laboratório Associado para a
Química Verde
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Ph. D. Disertations
“Caracterização Química do Aroma de Azeites Virgens com Atributos Positivos e Negativos”
Laila H. Ribeiro, Organic Chemistry PhD; Dec 2005
Supervisors: M. D. R. Gomes da Silva.
“Monitoring of the structural alterations of proteins induced during ultrafiltration processes using
fluorescence techniques”
Carla Portugal, UNL, Dec 2005
Supervisors: J.G.Crespo, J.C. Lima
“Synthesis of Terpene Esters In Compressed Gases”
Célia Maria Simões Peres, UNL, 2005
Supervisor: S. F. Barreiros
“Medidas Acústicas Aplicadas à Determinação de Pontos Críticos de Misturas”
Nuno Rui Alves Ribeiro, UNL, Jun 2005
Supervisor: Ana Aguiar Ricardo
“Gas separation processes by integrated adsorption and Permeation technologies”
Isabel Esteves, UNL, Jul 2005
Supervisor: José Paulo Mota
“Mistura de Fluidos e Transferência de Calor em escoamentos Laminares Caóticos”
António Rodrigo, UNL, Jul 2005
Supervisors: José Paulo Mota, Esteban Saatdjian
“Superoxide Reductases: Structural anf Functional Aspects”
Patricia Raleiras, UNL, 2005
Supervisor: J. J. G. Moura
“Sensores Ópticos de Matriz Sol-Gel com Interesse na Análise Farmacêutica”
Paula Cristina de Almeida Jerónimo, Dez de 2005
Supervisors: Alberto Araújo and Conceição Montenegro
“Sistemas Automáticos em Fluxo Continuo para o Controlo de Formulações Farmacêuticas”
João Alexandre Velho Prior, Dez de 2005
Sistemas Automáticos em Fluxo Continuo para o Controlo de Formulações Farmaceutricas
Supervisors: João Luís Santos and Costa Lima
“Marmelo (Cydonia oblonga Miller) e Marmelada: Perfil em Compostos Fenólicos, Ácidos Orgânicos e
Aminoácidos Livres e Avaliação do Potencial Antioxidante”
Branca Maria Cardoso Monteiro da Silva, UP, Jun de 2005
Orientadores: M A Ferreira, R Seabra e P Andrade
“Desenvolvimento de métodos de biologia molecular para a identificação de castas e clones de videira –
aplicação ao estudo da autenticidade de mostos e vinhos”
Miguel Ângelo Rodrigues Pinto de Faria, FFUP, Dez. de 2005
Supervisor: Margarida A. Ferreira
Laboratório Associado para a
Química Verde
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“Epidemiologia da resistência a antibióticos em Enterococcus spp portugueses de origem humana, animal
e ambiental”
Carla Novais, UP, Dez de 2005
Supervisors: L. V. Peixe and T. C. Coque.
“Ulilização de um produto natural (cortiça) como adsorvente de pesticidas piretróides em água”
Valentina Maria Fernandes Domingues
Faculdade de Engenharia da Universidade do Porto, 22 Dezembro de 2005
Supervisor: Cristina Maria Fernandes Delerue Alvim Matos
“Filmes Poliméricos do Tipo [M(salen)] Funcionarizados para o Reconhecimento de Catiões
Representativos”
Andrea da Conceição Ferreira Carneiro, UP, Dezembro, 2005
Supervisor: C. Freire
Laboratório Associado para a
Química Verde
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M. Sc. Dissertations
“Classificação geográfica automática de petróleos brutos”
Ana Margarida Pereira da Fonseca, UNL 2005, (Mai 2005)
Supervisors: J. Aires-de-Sousa, Ana M. Lobo , J. L. Biscaya
“Avaliação do Efeito de Poluentes Orgânicos na Membrana Celular”
Joana Moutinho de Veiga Marcelino, UP, Jan de 2005
Orientador (es): Carla Matos e Salette Reis
“Avaliação da Actividade Captadora de Espécies Reactivas de Oxigénio e de Azoto por Antagonistas BetaAdernégicos”
Ana Maria de Carvalhais Mendes Gomes, UP, Jan de 2005
Orientador (es): Eduarda Fernandes e Costa Lima
“Determinação de Parâmetros Físico-Químicos em Xantonas”
Ana Bela Ferreira Gomes
Orientador: Carlos Afonso e Salette Reis, UP, Abr de 2005
“Determinação de ião brometo em águas através de um sistema automático baseado em multi-seringa”
Liliana Sara Melo Oliveira, UP, Dez de 2005
Orientador: Marcela Segundo
“Hidrólise Enzimática de Concentrados de Proteínas do Soro: Separação e Caracterização de Péptidos –
Contribuição para a Valorização da Fracção Proteica do Soro”
Maria Virgínia Custódio Moreira Teixeira da Mota, FFUP, Jul. de 2005
Supervisor: IMPLVO Ferreira
“Estudo Comparativo da Fracção Protéica de Maltes, Mostos e Cervejas Provenientes das Variedades de
Cevada Scarlett e Prestige”
José Filipe Campos da Silva, FFUP, Set. de 2005
Supervisor: IMPLVO Ferreira
“Validação de um método analítico para a quantificação de Ocratoxina A em cerveja” Sofia Catarina Cunha
Silva Nogueira, FFUP, Nov. de 2005
Supervisors: MBPP Oliveira and Arminda Alves
“Contributo para a Caracterização do Queijo Terrincho: Estudo da Proteólise e Avaliação da Autenticidade
por HPLC/UV”
Carla Beatriz Rodrigues Vieiros, FFUP, Nov. de 2005
Supervisor: IMPLVO Ferreira
“Estudo do efeito da exposição de geotêxteis a soluções de catiões metálicos, por voltametria de
redissolução anódica”
Marta Sofia Roma Pires, FCUP, 2005.
Supervisor: Paulo Joaquim Ferreira de Almeida.
“Gelificação Térmica de Hidrolisados Enzimáticos de Proteínas do Soro do Leite Bovino. Comportamento
de Sistemas Aquosos Mistos Péptidos-Polissacarídeos”
Duarte Paulo Martins Torres, UMinho, Mai de 2005.
Supervisors: Maria do Pilar F. Gonçalves and José A. C. Teixeira
Laboratório Associado para a
Química Verde
A - 49
“Fermi Resonance Coupling in the C–H Stretching Region of Methoxide Adsorbed on Clean Ru(0 0 1)”
Ana Sofia Pinto, UP, Mar de 2005
Supervisor: Natália Cordeiro
“Estudo Computacional da Decomposição de Energia em Processos de Associação Molecular”
Alexandre Rodrigues Faria de Carvalho, UP, Mar de 2005
Supervisor: André Melo
“Líquidos Iónicos como Solventes Neotéricos para a Epoxidação Asimétrica de Alcenos Catalisada por
Complexos de [Mn(III)salenX]”
Fernando Jorge de Sousa Teixeira, UP, Outubro, 2005
Orientadora: C. Freire
Laboratório Associado para a
Química Verde
A - 50
C
Patents
Laboratório Associado para a
Química Verde
A - 51
Patents
“Ultra-fast protein enzymatic digestion by high intensity focused ultrasound”
J. L. Capelo Martinez; I. Moura; J. Vasquez and D. Lopez;
Patent Nº: 23848, code: 0198, 2005/07/07-16:12:38 (103303 C)
“Método para a obtenção de um concentrado natural rico em hidroxitirosol a partir de resíduos da produção
de azeite utilizando tecnologias limpas”, submission of Portuguese patent, process nº 103326 K.
Inventors: Manuel Nunes da Ponte, Catarina Duarte, João Goulão Crespo, Svetlozar Velizarov, Ana Matias,
Ana Nunes, José Santos.
“New Catalysts for Producing N-phosphonomethylglycine”
P.B. Correia
Patent PCT 2005 3773
“Novel Higher Eficiency Formulation of Glyphosate with Lower Attending Time Before Rain”
P.B. Correia
National Patent, filed, 2005
“Process for Producing N-phosphonomethylglycine”
P.B. Correia
Patent PCT IB 2005 003201
“Glyphosate Containing Mixtures with Cell Membrane Disrupture Compounds and Ammonium Sulphate for
Herbicide Application in Normal Crops and in Genetic Modified Crops”
P.B. Correia
Patent WIPO, filed, 2005
Laboratório Associado para a
Química Verde
A - 52
ANNEX III
RESEARCH PROJECTS
Laboratório Associado para a
Química Verde
A - 53
International funding
“New approach to waste recovery into selective adsorbents of heavy metals”
Project NATO Science for Peace Program nº 977984, 2002-2006
SUPERGREENCHEM Green Chemistry in Supercritical Fluids: “Phase Behaviour, Kinetics and Scale-up”
Marie Curie Research Training Network (MCRTN-2004-504005)
“Membrane bioreactor technology (MBR) with an EU perspective for advanced municipal wastewater treatment
strategies for the 21st century”
EUROMBRA,European Commission,2005-2008
European Research Network TMR: (HPRN-CT-1999-00084) - “The Mechanism, Specificity and Inhibition of
Enzymes belonging to the Xantine Oxidase family: Medical and Industrial Applications”
EA-BIOFILMS: “Electrochemical control of biofilm-forming micro-organisms : screening, identification, and
design of new knowledge-based technologies”
NEST – 508866 (STREP) (2005 – 2008)
COST Action D31 - “Organizing Non-Covalent Chemical Systems with Selected Functions”
Internal Project N. D31/0016/05
“Non-Covalent Interactions between Functional Abiotic Receptors and Ion Pairs”
COST Action D31 - “Organizing Non-Covalent Chemical Systems with Selected Functions”
Internal Project N. D31/0011/04
“Supra-Biomimetics: Towards Bio-Inspired Photoadressable Supramolecular Systems. Synthesis, LightEmission, Dynamics, Biomedical Applications”
Funded by FCT
“Natural Vegetal Antioxidants”
Ref. POCTI/QUI/47343/2002
Coordinator: Abel José de Sousa Costa Vieira
“Hopanoid composition of sediments from Lower Tagus Basin”
POCTI/QUI/45720/2002
Coordinator: Angela M. S. Relva
“New carbohydrate-based compounds from ethnopharmacological plants: bioactivity against pathogenic yeasts”
POCTI/1999/FCB/35863
Coordinator: Elvira Maria Mendes Sardão Monteiro Gaspar
“Volatile phenol yeast producers: a great concern in wine industry to be understood” POCTI/AGR/56771/2004
Ciências Agrárias e Florestais.
Coordinator: Manuel Malfeito Ferreira, Departamento de Botânica e Engenharia Biológica, Instituto Superior
de Agronomia. REQUIMTE participation: M.D.R. Gomes da Silva.
“Chemical and Sensorial Characterization of Malolactic Fermentation impact in Wines”,
POCTI/AGR/55432/2004 - Ciências Agrárias e Florestais
Coordinator: Ana Costa Freitas, Dep. Fitotecnia, U. Evora. REQUIMTE participation: M.D.R. Gomes da Silva
and Angela M.S. Relva.
Laboratório Associado para a
Química Verde
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“EnvironMetalControl: Fast monitoring in environmental matrices of total and speciation of elements and its
control by a new method based on enzyme probe sonication”
POCTI/AMB/58045/2004
Coordinator: Luis Fonseca; IST, Universitade Técnica de Lisboa.
Participation: J. L. Capelo Martinez.
“Towards New Supramolecular Devices: from new synthetic methods to multifunctional applications”,
POCTI/QUI/55519/
Coordinator: Carlos Lodeiro Espinho, FCT-UNL, REQUIMTE.
Participation: J. L. Capelo Martinez.
“Sensores químicos baseados em receptores poliamínicos ligados a unidades fluorescentes”
Projecto POCTI/47357/QUI/2002
Coordinator: Fernando Jorge da Silva Pina
“Photochromism of Flavylium Compounds in Water/Ionic Liquid Biphasic Systems”
POCTI/QUI/57735/2004
Coordinator: Fernando Jorge da Silva Pina
POCI/QUI/55519/2004
“Towards New Supramolecular Devices: from new synthetic methods to multifunctional applications” .
Coordinator: Carlos Lodeiro Espiño
“Alcalóides Naturais como sintões quirais”
POCTI/QUI/44501/2002
Coordinator: Prof. A. M. Lourenço
“Volatile phenol yeast producers: a great concern in wine industry to be understood.”
POCTI/AGR/56771/2004
Coordinator: Prof. Manuel Malfeito Ferreira (Instituto Superior de Agronomia)
Coordinator: Prof. João Aires de Sousa
“Algicidas naturais contra microalgas nocivas”
POCI/AMB/60351/2004
Coordinator: Dr. P. Pereira (Instituto Nacional de Saúde)
Coordinator: Prof. A. M. Lobo
POCTI/QUI/37423/2001
“New Synthetic methodology towards chiral gamma-amino acids useful in the design of new drugs”
Coordinator: Prof. M. Pereira
“Towards New Supramolecular Devises: from new synthetic methods to multifuntional applications”
POCTI/QUI/55519/2004.
“Tailored Synthesis of Biopolymers by mixed microbial cultures from molallse”,
Fundação para a Ciência e Tecnologia, POCI/BIO/55789/2004
“Saccharose as a Water Soluble Chiral Auxiliary and a Linker for Solid Phase Chemistry”
Fundação para a Ciência e a Tecnologia, POCTI/QUI/47973/2002.
“Rationalising Stereoselectivity in Organic Chemistry: From Theory to Practice”
POCTI/QUI/42983/2001
Laboratório Associado para a
Química Verde
A - 55
“High-Pressure NMR Spectroscopy of Polymers and biopolymers in CO2 Emulsions”
POCTI/QUI/42313/2001
“Catalytic Depolymerisation Of Current Plastics Residues”
POCTI/EQU/37946/2001
“Mechanisms Of Drug Controlled Release From Microsphere”
POCTI/EQU/46715/2002
“Technology For Obtention of Edible Films and Coatings for Foods Obtained From Low Value National
Resources”
POCTI/EQU/45595/2002
“Transesterification Of Vegetable Oils. Application To The Production of Biodiesel”
POCTI/EQU/48879/2002
“Development Of Catalytic Polymeric Membranes For The Oxidation Of Terpenic Olefins”
POCTI/CTM/45449/2002
“Technology to produce edible films and coatings for foods obtained from low value national resources”
POCTI/EQU/45595/2002, 2003-2006.
“Removal of Trace Heavy Metals from Drinking Water Supplies in an Ion Exchange Membrane Bioreactor”
POCI/AMB/57356/2004, 2005-2008
“Bioremediation of soil and water contaminated with pesticides”
POCI/AMB/59836/2004, 2005-2008.
“Enzymatic reduction of perchlorate”
POCTI/QUI/55435/2004, 2005-2008
“New biodegradable and environmental friendly chelating agents for industrial applications”
POCTI/QUI/57891/2004, 2005-2008.
“Metabolism and characterization of mixed cultures in wastewater processes for simultaneous removal of
nitrogen and phosphorus”
POCTI/AMB/56075/2004, 2005-2008
“Síntese de biopolímeros “á medida” por culturas microbianas mistas a partir de melaços”
POCTI/BIO/55789/2004, 2005-2008
“Advanced Bioprocess Automation Based on Hybrid Grey-Box Systems”
POCTI/BIO/56571/2004, 2005-2008
“Advanced Control of BHK cultivation processes based on intracellular metabolic fluxes”
POCTI/BIO/57927/2004, 2005-2008
“Production and characterisation of a PDLC. New strategies for polymerisation and incorporation in
supercritical CO2”
POCTI/CTM/47363/2002
Laboratório Associado para a
Química Verde
A - 56
“Static mixers as heat exchangers in supercritical fluid extraction processes - computational fluid
dynamics and process simulation studies”
POCTI/EME/61713/2004
“Gas separation by cyclic process combining pressure swing adsorption and membrane permeation”
POCTI/EQU/45102/2002
“Viscous fluid mixing and chaotic heat transfer in spatially and time-periodic flows”
POCTI/EME/40057/2001
“Chromatographic separation of anthocyanins by simulated moving bed”
POCTI/EQU/39391/2001
“Process integration of supercritical fluid extraction and membrane separation to recover “vegetal” squalene
from olive oil residues”
POCTI/EQU/61550/2004.
“Estudo por espectroscopia dieléctrica das alterações na dinâmica molecular durante a formação de um
cristal líquido disperso numa matriz polimérica”
POCTI/CTM/37435/2001
“RenH2 – Stand-Alone Energy System Supported by Totally Renewable Hydrogen Production”
POCTI/ENR/59623/2004
“Bacterial cytochrome c peroxidases- Activation, Enzymatic Mechanism And Structure”
POCTI/QUI/42309/2001
Coordinator: Isabel Moura
“Enzimas chave aa reduçâo do Nitrato. Redutase do Óxido Nítrico e Redutase do Óxido Nitroso - As duas
enzimas terminais”
POCTI/BME/42265/2001
Coordinator: Isabel Moura
“High Pressure NMR spectroscopy of polymers and biopolymers in CO2 emulsions”
POCTI/QUI/42313/2001
Coordinator: Maria dos Anjos Macedo
“Estudos Electroquímicos Dinâmicos em Proteínas de Transferência Electrónica”
POCTI / QUI / 42277 / 2001
Coordinator: José J. G. Moura
“NMR structural studies of the active center of 5-aminolevulinate synthase, the first enzyme of the
mammalian heme biosynthetic pathway”
POCTI/BME/39184/2001
Coordinator: Maria dos Anjos Macedo
“Orientation of Proteins By Liquid Crystals”
POCTI/QUI/42279/2001
Coordinator: Francisco Jorge Caldeira
Laboratório Associado para a
Química Verde
A - 57
“Structural and mechanistic studies of fatty acid desaturases. Looking for reactivity of diiron clusters”
POCTI/QUI/37413/2001
Coordinator: Pedro Tavares
“Mechanistic and Structural Studies of Iron Oxidation and Storage by Fast Ferritins”
POCTI/QUI/47273/2002
Coordinator: Alice S. Pereira
“Protease de Plasmodium chabaudi como alvo na terapia da malária”
POCTI/43637/BME/2000
Coordinator: Jorge Lampreia
“As proteases de parasitas da malária como alvo na quimioterapia”
POCTI/ESP/42223/2001,
Coordinator:Jorge Lampreia
“Mechanisms and Energetics of Electron Transport in Metalloproteins by Dynamic Voltammetry”
POCT/QUI/55743/2004
Coordinator:José J. G. Moura (FCT-UNL) and Margarida Santos (IST-UTL)
“Transient Protein Complexes – Soft Docking and NMR”
POCT/QUI/57741/2004
Coordinator:José J. G. Moura
“Development of enzyme biosensors for multi-parametric control and monitoring of environmental samples”
POCTI/QUI/58026/2004
Coordinator:M. Gabriela Almeida.
“New molybdo and copper cofactors in proteins isolated from sulphate reducers”
POCTI/QUI/55350/2004
Coordinator:Isabel Moura
“Cobalt in metabolism of sulfate reducers. Noncorrin Co/Zn ATPS, AK, and a new Co-corrinoid protein”
POCTI/QUI/59119/2004
Coordinator:Sergey Bursakov
“(Per)chlorate enzymatic reduction”
POCTI/QUI/55435/2004
Coordinator:Cristina Costa
“Spectroscopy of Oriented Proteins on Conductive Polymers”
POCTI/QUI/58973/2004
Coordinator:Francisco Caldeira
“Studying kinetics and mechanism of superoxide reduction”
(POCTI/QUI/57475/2004)
Coordinator:Alice Pereira
“Análise estrutural e funcional de um novo citocromo multihémico: um modelo para redutases do fumarato”
POCTI/QUI/42902/2001
Coordinator:C. Salgueiro
Laboratório Associado para a
Química Verde
A - 58
“Caracterização Bioquímica de Proteínas com Actividade Bioremediativa”
POCI/QUI/60060/2004
Coordinator:C. Salgueiro
“Caracterização de CymA: uma proteína chave na respiração anaeróbia de Shewanella”
POCI/BIA-PROQUI/58722/2004
Coordinator:C. Salgueiro
“Desenvolvimento de biossensores enzimáticos para controlo e monitorização multiparamétrica de
amostras ambientais”
(POCI/QUI/58026/2004).
Coordinator: M. Gabriela Almeida.
“Structural and Functional Characterization of Nitrate Reductases and Formate Dehydrogenases”
POCTI/QUI/57641/2004
“Molecular determinants of ligand specificity in carbohydrate-binding modules and cohesin-dockerin complexes”
POCTI/BIA-PRO/59118/2004
(with Prof Carlos Fontes and Prof. José Prates, FMV-UTL)
“Bacterial Cytochrome c Peroxidases- Activation, Enzymatic Mechanism and Structure”.
POCTI/QUI74230972001
“Development and Application of in Vitro Methodologies for Evaluation of Anti-Inflammatory and Antioxidant
Mechanisms of Non-Esteroidal Anti-Inflamatory Drugs”
FCT (POCTI/FCB/47186/2002)
Coordinator: Salette Reis
“Assessing the dietary exposure of Portuguese consumers to acrylamide. Improvemrnt, development and
validation of rapide screening procedures and confirmatory analytical methodologies”
FCT (POCTI/AGR/61543/2004)
Coordinator: José Fernandes e João L. M. Santos
“Síntese de cromonas e avaliação in vitro das propriedades antioxidantes e anti-inflamatórias”
FCT (POCTI/QUI/59284/2004)
Coordinator: José L.F.C. Lima
“Desenvolvimento de equipamentos automáticos e metodologias expeditas para controlo ambiental na perpectiva
de um desenvolvimento sustentado”
FCT/GRICES/CNPq (Projecto Bilateral Portugal/Brasil)
Coordinator: José L.F.C. Lima
“Desenvolvimento de biossensores enzimáticos para a determinação de nitratos e nitritos em amostras
complexas”
FCT (POCTI/QUI/58026/2004)
Coordinator: M. Conceição Montenegro
“Caracterização fitoquímica e actividade antioxidante de culturas in vivo e in vitro de Brassica oleracea var
costata”
POCTI/AGR/57399/2004
Coordinator: Paula Andrade
Laboratório Associado para a
Química Verde
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“Tecnologias para a valorização de bio-produtos da Salvia sp. (SalvaBiotech)”
POCTI/AGR/62040/2004
Coordinator: Manuel Ferreira (Universidade do Minho)
“Stress Oxidativo na Doença Cardíaca: Acção das Catecolaminas”
POCI/SAU-OBS/55849/2004
Coordinator: Fernando Manuel Gomes Remião
“Estudos farmacogenéticos para avaliação da influência do metabolismo na expressão da toxicidade de
derivados anfetamínicos usados correntemente como drogas de abuso”
POCI/SAU-FCF/57187/2004
Coordinator: Maria de Lourdes P.A.S. Bastos
“Indicators of swine industry pollution: antibiotics and antibiotic-resistant bacteria”
POCI/AMB/61814/2004
Coordinator: Luísa Peixe
“Study of factors implicated in the stability and dissemination of multi-resistant Enterococcus spp in
distincts metagenomes”
POCTI/SAU-ESP/61385/2004
Coordinator: Luísa Peixe
“Biological Markers of contamination by hospital wastewater”
POCTI/ESP/39885/2001
Coordinator: Helena Maria Neto Ferreira
“Metodologias para controlo de solos contaminados com pesticidas”
POCTI/AGR/44491/2002
Coordinator: Simone Barreira Morais
“Bioremediation of organic pollution in soil by augmentation with robust methylotrophic bacteria”
POCI/AMB/57353/2004
Coordinator: Paolo De Marco (IBMC/UP)
“Experimental Protocols for Estimating the Design Parameters of Permeable Reactive Barriers”
POCI/ECM/59779/2004
Coordinator: António Fiúza
“Respirometry of Rock Acid Drainage and the Usage of Oxygen Consuming Coatings as a Mean of
Reducing Environmental Emissions from Tailings Disposals”
POCI/ECM/60438/2004
Coordinator: António Fiúza (FEUP)
“Previsão do tempo de remediação de solos contaminados utilizando a extracção de vapor”
POCI/AMB/61315/2004
Coordinator: Maria da Conceição M. Alvim Ferraz
“Study of geosynthetics durability in order to obtain a better definition of their safety factors”
POCTI/ECM/42822/2001
Coordinator: Maria de Lurdes Lopes (FEUP)
Laboratório Associado para a
Química Verde
A - 60
“Tecnologia para a obtenção de filmes e revestimentos comestíveis para alimentos a partir de recursos
nacionais de baixo valor”
POCTI/EQU/45595/2002
Coordinator: Alberto M. Sereno
COMFOOD: “Controling the microstructure of Food products by rheo-optical methods”
POCTI/EQU/58064/2004
Coordinator: Loic Hilliou
“Tailored synthesis of biopolymers by mixed microbial cultures from molasses”
POCTI/BIO/55789/2004
Coordinator: Loic Hilliou
“Interaction between metal ions and buffers for the environmental and biological pH ranges”
POCTI/QUI/39950/2001
Coordinator: Helena Soares
“Development of a clean technology for heavy metals removal and recovery from industrial effluents”
POCTI/CTA/47875/2002
Coordinator: Helena Soares
“New potentially biodegradable chelating agents for industrial and domestic applications environment-friendly”
POCI/QUI/57891/2004
Coordinator: Helena Soares
POCTI/ESP/39885/2001
Coordinator: Helena Maria Neto Ferreira
Luísa Peixe, Diana Tuna Silva, Carla Novais
“Experimental Protocols for Estimating the Design Parameters of Permeable Reactive Barriers”
POCI/ECM/59779/2004
Coordinator: António Fiúza
“Respirometry of Rock Acid Drainage and the Usage of Oxygen Consuming Coatings as a Mean of
reducing Environmental Emissions from Tailings Disposals”
POCI/ECM/60438/2004
Coordinator: António Fiúza
“Previsão do tempo de remediação de solos contaminados utilizando a extracção de vapor”
POCI/AMB/61315/2004
Coordinator: Maria da Conceição M. Alvim Ferraz
“Metodologias para controlo de solos contaminados com pesticidas”
POCTI/AGR/44491/2002
Coordinator: Simone Barreira Morais
“Tecnologia para a obtenção de filmes e revestimentos comestíveis para alimentos a partir de recursos
nacionais de baixo valor”
POCTI/EQU/45595/2002
Coordinator: Alberto M. Sereno
Laboratório Associado para a
Química Verde
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COMFOOD: “Controling the microstructure of Food products by rheo-optical methods”
POCTI/EQU/58064/2004
Coordinator: Loic Hilliou
“Tailored synthesis of biopolymers by mixed microbial cultures from molasses”
POCTI/BIO/55789/2004
Coordinator: Loic Hilliou
“Interaction between metal ions and buffers for the environmental and biological pH ranges”
POCTI/QUI/39950/2001
Coordinator: Helena Soares
“Development of a clean technology for heavy metals removal and recovery from industrial effluents”
POCTI/CTA/47875/2002
Coordinator: Helena Soares
“New potentially biodegradable chelating agents for industrial and domestic applications environment-friendly”
POCI/QUI/57891/2004
Coordinator: Helena Soares
“Ácidos Gordos Trans em Bolachas: Contribuição para a Ingestão Total de Gorduras Trans da População
Portuguesa e Consequente Risco Cardiovascular”
Segurança Alimentar
Susana Isabel Pereira Casal Vicente (FFUP)
“Study of geosynthetics durability in order to obtain a better definition of their safety factors”
POCTI/ECM/42822/2001
Maria de Lurdes Lopes (FEUP)
Bilateral cooperations
Acção Integrada Luso-Britânica-2006 Nº B-16/06.
“Towards novel flexible fluorescence chemosensors for metal ion detection”
Portuguese Coordinator: Carlos Lodeiro Espiño
British Coordinatior: Dr. Emília Bértolo Pardo
Department of Geographical and Life Sciences
Canterbury Christ Church Univeristy
2005-2007
Acção Integrada Luso-Espanhola Nº E-68/05
“Multistate/Multifunctional Systems Trapped in Polymer Hydrogel Matrices”
Portuguese Coordinator: António Jorge Dias Parola
Spanish Coordinator: Prof. Santiago V. Luis
Departamento de Quimica Inorganica y Organica
Universidade Jaume I de Castellón, Spain
2005-2006
Acção Integrada Luso-Alemã A-11/04
“Computer Prediction of Biological Activity: Chirality in QSAR Applications”
Coordinator of Portuguese team: Prof. João Aires de Sousa (Universidade Nova de Lisboa, Portugal)
Coordinator of German team: Prof. Johann Gasteiger (University of Erlangen-Nürnberg, Germany)
Laboratório Associado para a
Química Verde
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Acção Integrada Luso-Espanhola E-59/06
“Compostos Ogânicos azotados: desenho de moléculas com actividade biológica baseado em sugestões
da natureza (actividade e biotransformações)
Coordinator of Portuguese team: Prof. A. M. Lobo (Universidade Nova de Lisboa, Portugal)
Coordinator of the Spanish team: Prof. M. Amat (Univ. Barcelona, Espanha)
“SciencEduc” project
http://www.cienciaviva.pt/projectos/scienceduc/
SciencEduc is a European project aimed at improving science education in primary education schools by
creating a network which will stimulate and support the development of good practices in Science teaching
at this level of education and will foster interaction between the project partners at a national and crossborder level.
The project is coordinated by the Ecole Normale Supérieure (Paris) in partnership with the following institutions:
University of Tartu (Estonia), La main à la pâte – Académie des sciences (France), Apor Vilmos Catholic
College (Hungary), Ciência Viva (Portugal), NTA – and the Royal Swedish Academy of Sciences (Sweden).
Advisory of the Portuguese Partner: Prof. P. Mata
“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, Programa 2003-2004 (J.J.G.Moura e Carlos Brondino)
“Thermodynamic Structural Studies of The Temperature Induced Unfolding of Plant Thermophylic and
Bacterial Mesophylc Enzymes”
Acçoes Itegradas Luso-Espanholas 2006
Action N0 E-62/06
Sergy Bursakov
CRUP
“Study of a multihemic nitrite reductase by direct electrochemistry and electronic paramagnetic
ressonance spectroscopy (EPR)”
M. Gabriela Almeida and Bruno Guigliarelli.
CQFB, Dept. Química, FCT/UNL, Monte da Caparica and CNRS/Université de Provence, Aix-Marseille I
(France)
“Nanoenssambled based electroactive biointerfaces for environmental and food analysis of nitrates and
nitrites”
M. Gabriela Almeida and Serge Cosnier.
CQFB, Dept. Química, FCT/UNL and ICM, Grenoble, UMR CNRS 5630
GRICES
“Desenvolvimento de equipamentos automáticos e metodologias expeditas para controlo ambiental na perpectiva
de um desenvolvimento sustentado”
FCT/GRICES/CNPq (Projecto Bilateral Portugal/Brasil)
Responsável: José L.F.C. Lima
“Influence of NSAIDs on the activity of enzymes at interfaces”
CRUP (Acção Integrada Luso-Alemã A-6/06)
Responsável: Salette Reis
“Novos Compostos para um Futuro Ambientalmente Limpo”
Acção Integrada Luso-Britânica
Laboratório Associado para a
Química Verde
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Acções Luso-Alemãs
Ref. A-5/04
“Metabolism and toxicity of the designer drugs 4-MTA and 2C-B: identification of hypersensitive individuals
by pharmacogenetic studies”
Investigador Responsável pela equipa portuguesa: Maria de Lourdes P.A.S. Bastos
“Ecology and evolution of local genetic elements implicated in the antibiotic resistance of enterococci: study
of factors determining the stability and spread of multiresistant bacteria in distinct metagenomes”
Acções integradas Luso-Espanholas (CRUP). Acção nº 52/05.
Coordinator: Luísa Peixe
Collaborators: Carla Novais, Teresa Coque
Biological Markers of contamination by hospital wastewater
“Rheological behaviour and morphology of protein-polysaccharide mixed systems”
GRICES/ CAPES (Portugal/ Brazil)
Maria do Pilar Gonçalves
“Frutas tropicais de alta humidade: processamento, embalagem sob atmosfera modificada e avaliação da
qualidade”
GRICES/CAPES (Portugal/ Brazil)
Alberto M. Sereno
“Tecnologia de películas biodegradaveis para alimentos en ibero-américa” “Technology for food grade
biodegradable films in Ibero-America”
CYTED project XI.20
Alberto M. Sereno
International Co-ordinator: Dr. Paulo José do Amaral Sobral (Brazil)
“Characterization of biopolymer mixtures for food applications”
GRICES/DAAD (Portugal/Germany) 2005/2006
Loic Hilliou
Projects financed by “Agência de Inovação”
BEERVOLT (3 years project submitted to Agência de Inovação, which was signed in November 2004 and
initiated in April 2004). Proposing Institution – Unicer, Bebidas de Portugal, SGPS S. A. Other institutions
involved: Faculty of Science of the University of Porto, Carlsberg S/A and CAI, Controle e Automação
Industrial, L.da. Project Director – Aquiles Barros.
Projects financed by “Fundação Calouste Gulbenkien”
Projecto Referência FCG 10/04
“Efeitos comportamentais e bioquímicos resultantes da exposição à MDMA (ecstasy) durante a
adolescência”,
Entidade Financiadora: Fundação Calouste Gulbenkian
Data de início do Projecto: 4/Janeiro/2005
Investigador Responsável: Professor Doutora Maria Amélia Ferreira e Doutora Teresa Summavielle
Laboratório Associado para a
Química Verde
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Food safety and public health: definition of the risks associated to E. coli (VTEC), Listeria monocytogenes e
Salmonella spp.
Fundação Calouste Gulbenkian. 2002.
Coordinator: Xavier Malcata
Collaborators: Luísa Peixe, Patrícia Antunes, Paula Teixeira, Nazaré Pestana
Laboratório Associado para a
Química Verde