KATOWICE – September, 9 – 12 - Chemistry towards Biology

Transcription

Chemistry towards Biology
7th Central Europe Conference
Under the auspicies of:
KATOWICE – September, 9 – 12th 2014
7th Central Europe Conference – Katowice, September 9 – 12th 2014
Chemistry towards Biology
7th Central Europe Conference
University of Silesia, Institute of Chemistry
Katowice, Poland
September, 9 – 12th 2014
Dear Colleques,
This conference is a continuation of a long-term cooperation among scientists from
Central Europe sharing a scientific interest in chemical biology and biological chemistry. The
previous conference of this series was held in Triest, Italy in September 2013. The 7th Central
European Conference "Chemistry towards Biology" will specifically address the following
research topics:
Carbohydrate chemistry and biochemistry
Chemistry of cell biology
Nucleic acids chemistry
Structure, function and interactions of proteins
New/engineered enzymes
Drug design and drug delivery
Molecular pharmacology, medicine and biomaterials
Catalysis in organic synthesis, pharmacy and environmental protection
A major objective of the conference will be to develop a platform for mutual scientific
contacts between researchers from the European and other countries and to foster future
collaborations between scientists in these countries. In view of this perspective we specifically
encourage younger scientists to participate. We hope to welcome you at the conference!
Best wishes
International Steering Committee:
András Perczel, Budapest, HUNGARY (Chair)
Jarosław Polański, Katowice, POLAND
Dusan Berek, Bratislava, SLOVAKIA
Piotr Cysewski, Bydgoszcz, POLAND
Lucia Gardossi, Trieste, ITALY
Silvano Geremia, Trieste, ITALY
Pavel Hobza, Prague, CZECH REPUBLIC
Robert Konrat, Vienna, AUSTRIA
Henryk Kozlowski, Wroclav, POLAND
Vladimir Kral, Prague, CZECH REPUBLIC
Gábor Náray-Szabó, Budapest, HUNGARY
Botond Penke, Szeged, HUNGARY
Ladislav Petrus, Bratislava, SLOVAKIA
Janez Plavec, Ljubljana, SLOVENIA
Vilim Simanek, Olomouc, CZECH REPUBLIC
Grazyna Stochel, Krakow, POLAND
Sanja Tomić, Zagreb, CROATIA
Robert Vianello, Zagreb, CROATIA
Walter Keller, Graz, AUSTRIA
Bela Gyurcsik, Szeged, HUNGARY
Local Steering Committee:
Prof. Wiesław Banyś, Katowice, Poland
Prof. Alicja Ratusza, Katowice, Poland
Prof. Michał Daszkowski, Katowice, Poland
Prof. Josef Jampilek, Brno, Czech Republic
Local Organising Committee:
Dr Krystyna Jarzembek (Coordinator), Katowice, Poland
Dr Barbara Podeszwa, Katowice, Poland
Dr Monika Pyrkosz-Bulska, Katowice, Poland
Dr Violetta Kozik, Katowice, Poland
Dr Andrzej Bąk, Katowice, Poland
Dr Maciej Serda, Katowice, Poland
Ph.D. student Maciej Kapkowski, Katowice, Poland
Ph.D. student Mateusz Korzec, Katowice, Poland
Ph.D. student Jacek Bogocz, Katowice, Poland
Organizing Secretariat:
Krystyna Jarzembek
Monika Pyrkosz-Bulska
University of Silesia
Institute of Chemistry
Faculty of Organic Synthesis
ul. Szkolna 9
40-006 Katowice
Poland
tel. +48 32 359 1336
e-mail: [email protected]
University of Silesia
Institute of Chemistry
Faculty of Organic Chemistry
ul. Szkolna 9
40-006 Katowice
Poland
tel. +48 32 359 1399
Thanks to the sponsors:
CONFERENCE PROGRAM
TUSEDAY 09/09/2014
13.30
14.30
14.45
15.25
16.05
16.45
17.15
17.55
18.10
18.30
Registration
Opening
Chairperson: Andras Perczel, Robert Vianello
PL1: Henryk Kozłowski, Wrocław, POLAND
METAL ION BINDING, NEURODEGENERATION AND BACTERIAL
INFECTIONS-CHALLENGE FOR BIOINORGANIC CHEMISTRY
PL2: Elżbieta Gumienna-Kontecka, Wrocław, POLAND
CAN WE DECEIVE BACTERIA WITH FERRICHROME BIOMIMETIC
ANALOGS?
PL3: Paola Cescutti, Trieste, ITALY
STRUCTURE-FUNCTION OF BACTERIAL POLYSACCHARIDES
Coffee break
PL4: Miloš Hricovíni, Bratislava, SLOVAKIA
DFT CALCULATIONS OF STRUCTURE AND NMR PARAMETERS IN
HEPARIN OLIGOSACCHARIDES
O1: Bela Gyurcsik, Szeged, HUNGARY
NOVEL ZINC FINGER NUCLEASES BASED ON COLICIN E7 NUCLEASE
DOMAIN
O2: Karolina Krzywoszyńska, Wrocław, POLAND
CYSTEINE-RICH PEPTIDES AS TEMPTING LIGANDS FOR BIOLOGICALLY
RELEVANT METAL IONS
Welcome Buffet
9.00
9.40
10.20
11.00
11.30
11.50
12.10
12.30
13.00
15.00
15.40
16.00
16.20
18.00
WEDNESDAY 10/09/2014
Chairperson: Henryk Kozłowski, Roberto Rizzo
PL5: Marian Hajduch, Olomouc, CZECH REPUBLIC
PRACTICAL ASPECTS OF ACADEMIC DRUG DISCOVERY: FROM
MOLECULES TO PROOF-OF-CONCEPT CLINICAL TRIALS
PL6: Ton Rijnders, Tipharma, NETHERLANDS
CROWDSOURCING BIOLOGICAL TARGETS AND CHEMICAL SCAFFOLDS
A LARGE-SCALE, CONTROLLED EXPERIMENT
PL7: Beata Walczak, Katowice, POLAND
CHALLENGES OF DATA ANALYSIS IN OMICS STUDIES
Coffee break
Chairperson: Piotr Cysewski, Peter Szalay
O3: Daniel Enriz, San Luis, ARGENTINA
DESIGN OF NEW MIMETIC PEPTIDES INHIBITORS OF Aβ-AGGREGATION
FOR STRUCTURAL BASES. MOLECULAR SIMULATIONS USING
MONOMERIC, DIMERIC AND PENTAMERIC MODELS
O4: Małgorzata Brindell, Cracow, POLAND
CELLULAR IMAGING AND CYTOTOXIC PROPERTIES OF NONINTERCALATIVE RUTHENIUM(II) POLYPYRIDYL COMPLEXES
O5: Josef Jampilek, Brno, CZECH REPUBLIC
SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF
HYDROXY(AZA)NAPHTALENECARBOXAMIDES
O6: Valery Andrushchenko, Prague, CZECH REPUBLIC
ACCURATE MODELING OF MOLECULAR OPTICAL PROPERTIES BY A
COMBINATION OF MOLECULAR DYNAMICS AND QUANTUM
CHEMISTRY
Lunch
PL8: Żaneta Wojnarowska, Katowice, POLAND
THE ROLE OF PROTON TRANSFER REACTION ON THE
PHYSICOCHEMICAL PROPERITIES OF AMORPHOUS
PHARMACEUTICALS
O7: Tomasz Frączek, Łódź, POLAND
DESIGN OF NOVEL TRIAZOLE HIV-1 REVERSE TRANSCRIPTASE
INHIBITORS
O8: Tea Pavkov-Keller, Graz, AUSTRIA
STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF TWO
NOVEL ENZYMES WITH PROMISCOUS ENE-REDUCTASE ACTIVITY
Chairperson: Beata Walczak, Marian Hajduch
Poster session with coffee break
Social party
9.00
9.40
10.20
11.00
11.30
12.10
12.30
15.00
15.20
15.40
18.0022.00
THURSDAY 11/09/2014
Chairperson: Miloš Hricovíni, Primoz Sket
PL9: Péter Szalay, Budapest, HUNGARY
CAN QUANTUM CHEMISTRY METHODS BE USED TO DESCRIBE
EXCITED STATES OF THE BUILDING BLOCKS OF DNA?
PL10: Ivo Piantanida, Zagreb, CROATIA
SENSING THE SHAPE OF THE DS-DNA/RNA STRUCTURES BY NONCOVALENT INTERACTIONS
PL11: Jurij Lah, Ljubljana, SLOVENIA
WHAT DRIVES BINDING-COUPLED FOLDING OF G-QUADRUPLEX DNA?
Coffee break
Chairperson: Aidan Coffey, Zbigniew Kamiński
PL12: Primoz Sket, Ljubljana, SLOVENIA
GUANINE – RICH DNA OLIGONUCLEOTIDES IN SOLUTION
O9: Viktor Víglaský, Košice, SLOVAKIA
GENERAL ASPECTS OF DNA G-QUADRUPLEX MULTIMERIZATIONS
Lunch
O10: Alicja Urbaniak, Poznań, POLAND
SYNTHESIS OF NOVEL AROMATIC ESTERS OF RESVERATROL
O11: Małgorzata Szelag, Poznań, POLAND
COMPARATIVE SCREENING AND VALIDATION ASA NOVEL TOOL TO
IDENTIFY STAT-SPECIFIC INHIBITORS
Chairperson: Teresa Kowalska, Walter Keller
Poster session with coffee break
Guided tour to the museum of mining history: the Guido coal mine Zabrze.
The bus will take participants from the lecture hall in Chorzow at 16.45
9.00
9.40
10.20
10.40
11.20
11.40
12.00
12.20
13.00
FRIDAY 12/09/2014
Chairperson: Ivo Piantanida, Daniel Enriz
PL13: Tamás Martinek, Szeged, HUNGARY
PEPTIDE FOLDAMERS: STRUCTURE AND APPLICATIONS
PL14: Teresa Kowalska, Katowice, POLAND
ARE PEPTIDE NANO- AND MICROSTRUCTURES STABLE ENOUGH FOR
BIOTECHNOLOGY?
Coffee break
PL15: Piotr Cysewski, Bydgoszcz, POLAND
PREDICTION OF CRYSTAL STABILITIES OF ACTIVE PHARMACEUTICAL
INGREDIENTS BY MEANS OF QUANTUM CHEMISTRY COMPUTATIONS
O12: Zbigniew Kamiński, Łódź, POLAND
APPLICATION OF N-LIPIDATED PEPTIDES IMMOBILIZED ON SOLID
SUPPORTS AS MOLECULAR RECEPTORS
O13: Aidan Coffey, Cork, IRELAND
CHARACTERIZATION AND APPLICATIONS OF A CYSTEINE- HISTIDINEDEPENDENT AMIOHYDROLASE/PEPTIDASE ENZYME TARGETING
STAPHYLOCOCCAL CELL WALLS.
O14: Walter Keller, Graz, AUSTRIA
STRUCTURES OF ENVIRONMENTAL ALLEGENTS AND STRUCUREBASED EPITOPE MAPPING
O15: Beata Kolesińska, Łódź, POLAND
APPLICATION OF CELL PENETRATING PEPTIDES FOR DRUG DELIVERY
Conference closure
ABSTRACTS OF
SCIENTIFIC
CONTRIBUTIONS
Planar session
Oral session
METAL ION BINDING, NEURODEGENERATION AND BACTERIAL
INFECTIONS-CHALLENGE FOR BIOINORGANIC CHEMISTRY
H. Kozlowski a, A. Hecel a, P. Kolkowska a
a
Department of Chemistry, University of Wrocław
e-mail of presenting author: [email protected]
1. Introduction
In the last few years, many experiments and studies have been providing evidence that
copper, iron and zinc ions are involved in the pathogenesis of major neurodegenerative
disorders. Likewise, the homeostasis of Ni2+ is a crucial ion for the survival of Helicobacter
pylori, bacterium that colonizes the gastric mucosa in humans. Therefore the detailed
understanding of interactions of these metal ions with peptides and proteins involved in metal
homeostasis and biology of proteins as well as their manipulation and redistribution are of
great importance for medicinal bioinorganic chemistry.
PL1
2. Results and Discussion
As largely discussed Cu, Fe and Zn play key roles in many neurodegenerative diseases.
Many reports support the use of compounds that modulate metal binding to neurodegenerative
proteins as promising therapeutic strategy for neurodegeneration (especially for AD). The use
of metal chelators results in regulation of metal-induced Aβ aggregation and neurotoxicity
both in vitro and in vivo [1]. Several attempts were made to obtain efficient chelators with
moderate affinity towards the metal ions such as Cu2+, Zn2+ or Fe3+ that participate of the
amyloid aggregation, in order to prevent the formation of plaques [2]. Recently, copper and
iron chelators have resulted effective in both inhibiting Aβ fibrils aggregation and potentially
capable to cross the blood–brain barrier (BBB) [3].
Recently, numerous findings were described on the homeostasis of nickel in H. pylori, a
Gram-negative bacterium that is the causative agent of acute and chronic gastritis, peptic ulcer
disease, gastric carcinoma, and gastric lymphoma [4]. The homeostasis of nickel ions is
crucial for the survival of this bacteria in the extremely acidic environment of the stomach.
Many accessory proteins are involved in nickel metabolism. They consist of long poly-his
sequences and cysteine rich sequences. Zinc often plays a structural or regulatory role in
nickel chaperones. The disruption of proper nickel distribution could be a tool for bacteria
eradication [5].
[1]. V.B. Kenche, K.J. Barnham, Br. J. Pharmacol., 2011, 163, 211.
[2]. H. Kozlowski, M. Luczkowski, M. Remelli, D. Valensin, Coord. Chem. Rev., 2012, 256,
2129.
[3]. A.S. DeToma, S. Salamekh, A. Ramamoorthy, M.H. Lim, Chem. Soc. Rev., 2012, 41,
608.
[4]. D. Witkowska, M. Rowinska-Zyrek, G. Valensin, H. Kozlowski, Coord. Chem. Rev.,
2012, 256, 133.
[5]. M. Rowinska-Zyrek, J. Zakrzewska-Czerwinska, A. Zawilak-Pawlik, H. Kozlowski,
Dalton Trans., 2014, 43, 8976.
PL1
CAN WE DECEIVE BACTERIA WITH FERRICHROME BIOMIMETIC
ANALOGS?
E. Gumienna-Kontecka a, A. Szebesczyk a, J. Besserglick b, E. Olshvang b, A. Shanzer b
a
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland.
Weizmann Institute of Science, 234 Herzl Street, 76100 Rehovot, Israel.
b
1. Introduction
At the time of increasing number of severe and often lethal infections caused by
multiresistant bacterial strains and fungi, the research in the field of iron transport in
microorganisms seems to be of great importance. The difficulties in synthesis of structurally
complicated natural siderophores, has prompted research in the direction of artificial
siderophores used as structural probes of microbial iron uptake processes [1].
PL2
Our current research is focused on characterization of novel biomimetic compounds,
artificial iron-carriers, in terms of iron complex formation and stability. Here we will present
a new series of ferrichrome analogues, based on a tripodal template, where the asymmetric
hexapeptide ring of the natural ferrichrome is replaced by a much simpler C3 symmetric
template, anchored to a quaternary carbon. Three identical arms are comprised of a spacer
containing an amino acid and terminated by hydroxamate metal binding moiety. Various
aminoacid residues are studied, in order to determine the possibility of hydrogen bonds
formation and the role of side groups. Preliminary data show that Fe(III) binding properties of
studied analogs are close to natural ferrichrome. Moreover, growth promotion studies show
that these biomimetic compounds are able to transfer iron to Pseudomonas putida as
efficiently as natural ferrichrome, and therefore act like siderophores.
Acknowledgment:
Financial support by the Polish National
2011/03/B/ST5/01057) is gratefully acknowledged.
Science
Centre
(NCN,
UMO-
[1]. Shanzer A, Felder CE, Barda Y, Natural and biomimetic hydroxamic acid based
siderophores. In The Chemistry of Hydroxylamines, Oximes and Hydroxamic Acids, Rapport
Z, Liebman JF, Eds. A John Wiley & Sons, Chichester (2009) 2: 751-815.
PL2
STRUCTURE-FUNCTION OF BACTERIAL POLYSACCHARIDES
P. Cescutti a
a
Department of Life Sciences, University of Trieste, Italy
PL3
Bacteria are able to synthesise a wide number of structurally different polysaccharides [1],
usually constituted of common pentoses, hexoses, amino sugars and uronic acids, although
occasionally, less common sugars are detected. In addition, non-carbohydrate substituents like
N- and O-acetyl, pyruvic, phosphoric, lactic and succinic acids, amino acids, glycerol, ribitol,
may be present, consequently changing the polymer physico-chemical properties. These
polysaccharides are mainly located facing the surrounding environment, and are classified in
O-antigens, capsular and extracellular polysaccharides. Their position indicates that their
biological functions are involved in interactions with the outside world, which can be water,
surfaces of various nature, as well as living organisms, like micro-organisms, plants, animals
and humans. The functions of the polysaccharides can be distinguished in general and
specific. Polysaccharides can provide an anti-dessication milieu and a physical barrier
towards potential enemies, preventing the recognition and attachment to epitopes exposed on
the underneath layer. When polyanionic, a further charge effect adds to the simple barrier,
capturing positively charged species and repulsing negatively charged molecules, like serum
components and cells with similar surface charge. For example, the lytic action of cationic
antimicrobial peptides can be impaired by anionic polysaccharides, through the formation of
stable complexes [2]. Polysaccharides were reported to inhibit neutrophil chemotaxis and
production of reactive oxygen species (ROS); some polymers exhibited also scavenging
effects against ROS, with a concurrent protecting action towards bacterial cells [3].
In addition, polysaccharides can promote the adherence of bacteria to surfaces as well as to
each other, facilitating the formation of biofilms and the colonization of different ecological
niches. In fact, independently from the surrounding environment, bacteria prefer to live in
biofilms which are structured communities enclosed in a self-produced polymeric matrix and
adherent to an inert or living surface. The matrix is constituted of macromolecules like
proteins, DNA, and polysaccharides, together with other relatively small molecules (water,
nutrients and molecular signals). The matrix not only protects bacterial cells, but can also be
dispersed when cells migrate to another site of colonisation. Therefore, understanding which
are the structural features of the polysaccharides involved in matrix architecture and their
interactions with the other components would aid in the development of novel efficacious
anti-biofilm agents.
[1]. Cescutti P. Bacterial capsular polysaccharides and exopolysaccharides. in “Microbial
Glycobiology Structures, Relevance and Application”, (Eds. Moran A. P., Brennan P. J., Holst
O., and von Itzstein M.) Academic Press: New York (2009) pp 93-108.
[2]. Foschiatti M. et al., (2009) Mol. Microbiol. 72(5), 1137–1146.
[3]. Cuzzi B. et al., (2012) Innate Immun. 18(4) 661–671.
PL3
DFT CALCULATIONS OF STRUCTURE AND NMR PARAMETERS IN
HEPARIN OLIGOSACCHARIDES.
M. Hricovíni a
a
Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovakia
Heparan sulphate, a highly negatively charged glycosaminoglycan, participates in a
number of biological processes. The molecular basis of heparan sulphate-protein interactions
in solution has been analysed by biophysical methods, mainly by NMR spectroscopy and
molecular modelling. Theoretical analysis is thus an important part of our understanding of
biological properties of heparin and heparan sulphate oligosaccharides. In the present
contribution, results of calculations using density functional theory (DFT) methods are
discussed. Geometry optimizations, evaluating explicit solvent molecules, were performed by
B3LYP/6−311+G(d,p) methods. Optimized molecular geometries in heparin trisaccharide and
pentasaccharide allowed calculations of NMR parameters, such as chemical shifts and
coupling constants. Comparison of theoretical and experimental NMR data showed that DFT
method using B3LYP functional and the 6−311+G(d,p) basis set, combined with explicit
solvent model, can yield sufficiently accurate structural and NMR data for these
oligosaccharides.
PL4
PL4
PRACTICAL ASPECTS OF ACADEMIC DRUG DISCOVERY: FROM
MOLECULES TO PROOF-OF-CONCEPT CLINICAL TRIALS
M. Hajduch a
a
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry,
Palacky University and University Hospital in Olomouc, Hnevotínská 5, CZ-775 15 Olomouc,
Czech Republic
e-mail of presenting autor: [email protected]
PL5
PL5
CROWDSOURCING BIOLOGICAL TARGETS AND CHEMICAL SCAFFOLDS - A
LARGE-SCALE, CONTROLLED EXPERIMENT
T. Rijnders a
a
Top Instute Pharma, Leiden, the Netherlands
The EU Lead Factory is the first pharmaceutical and life sciences partnership of its kind.
It has been designed to create unrivalled opportunities for the discovery of new drug lead
molecules. Academics and SMEs now have access to an ‘industry‐like’ discovery platform.
Scientists with innovative biology target and chemistry scaffold owners are welcome to
participate in the European Lead Factory.
PL6
PL6
Seven large pharmaceutical companies have joined forces and contributed proprietary
compounds to the core Joint European Compound Library (JECL). Targets from public
partners are screened at the top‐modern, industry‐standard European Screening Centre (ESC)
at no upfront cost, as funding is provided by the IMI. Further exploitation of generated hits is
subject to a number of conditions.
This unique large‐scale experiment, combining open innovation, crowdsourcing and
several established pharmaceutical companies, is now producing its first scientific results.
Acknowledgment:
The research leading to these results has received support from IMI under grant agreement
n° 115489 and EFPIA companies’ in-kind contribution.
CHALLENGES OF DATA ANALYSIS IN OMICS STUDIES
B. Walczak a
a
Institute of Chemistry, The University of Silesia, Szkolna Street 9, 40-006 Katowice, Poland
Instrumental signals of complex biological samples can be treated as their respective
fingerprints and used to solve numerous calibration, classification and discrimination
problems. This approach requires application of modern chemometrics methods, which allow
signals enhancement, preprocessing and modeling. Analysis of multivariate data is a
multistage process. All its elements are equally important and decide about final results and
conclusions. There are strict rules concerning sampling (in a case of natural samples),
experimental design, data modeling, and results validation. There are not, however, any
established rules concerning data pre-processing. The preprocessing step usually involves
preprocessing of individual signals (signals enhancement via signals de-nosing and
background elimination), as well as preprocessing of the signal set (signal transformations,
normalization, alignment, etc.). All these aspects of data preprocessing will be discussed and
illustrated for the simulated and real data sets [1-3].
PL7
[1]. K. Kaczmarek, B. Walczak, S. de Jong, B.G.M. Vandeginste, Feature based fuzzy
matching of 2D gel electrophoresis images, Journal of Chemical Information and
Computer Sciences, 42 (2002) 1431-1442
[2]. K. Kaczmarek, B. Walczak, S. de Jong, B.G.M Vandeginste, Preprocessing of twodimensional gel electrophoresis images, Proteomics, 4 (2004) 2377-2389
[3]. M. Daszykowski, I. Stanimirova, A. Bodzon-Kulakowska, J. Silberring, G. Lubec, B.
Walczak, The start-to-end processing of two-dimensional gel electrophoretic images,
Journal of Chromatography A, 1158 (2007) 306-317
PL7
THE ROLE OF PROTON TRANSFER REACTION
IN THE PHYSICOCHEMICAL PROPERTIES OF AMORPHOUS PHARMACEUTICALS
Z. Wojnarowska a, M. Paluch a
a
Institute of Physics, University of Silesia, Uniwersytecka 4, Katowice, Poland
1. Introduction
There are a number of chemical compounds that readily convert to other isomers when
their crystalline structure is lost (e.g., during melting or dissolution). This phenomenon
involving the proton transfer reaction is known as tautomerization reaction. The proton
migration becomes an important problem especially in pharmaceutical industry because there
is a growing interest in amorphous drugs, characterized by better solubility and
bioavailability. Recently, it has been shown that preparation of the amorphous
pharmaceutical, in which tautomerization takes place, leads to the state which is chemically
different than its crystalline counterpart. The proton transfer reaction can strongly affect the
molecular dynamics of drug, its re-crystallization tendency and biological activity.
PL8
Herein, we present the effect of tautomerization on the molecular dynamics, physical
stability and solubility of hypoglycemic agents from sulfonylurea group studied by means of
experimental (dielectric spectroscopy, rheological measurements, infer-red technique, HPLC,
and differential scanning calorimetry) and computational methods. The valuable information
on the isomerization kinetics of pharmaceuticals, coming from dielectric experiments,
concern the energy barrier of proton transfer reaction. Intuitively, the higher is the activation
energy necessary for proton hopping the lower is the probability of tautomerization process.
Consequently, based on the value of Ea it is possible to pre-identify the isomerization abilities
of pharmaceutical materials. Herein, it is worth noting that the experiments of tautomerization
kinetics performed so far for various chemical compounds clearly indicate that generally the
values of activation energy of given isomeric conversion determined from BDS measurements
are in good agreement with theoretical predictions as well as pressure-temperature-volume
(PVT), rheological, infrared experimental data and refractive index measurements.
Acknowledgments
Authors Z.W. and M.P. are deeply thankful for the financial support received from the
National Science Center within the framework of the project OPUS 3 entitled “Proton transfer
reaction in condensed materials at ambient and elevated pressure”.
PL8
CAN QUANTUM CHEMISTRY METHODS BE USED TO DESCRIBE EXCITED
STATES OF THE BUILDING BLOCKS OF DNA?
P. G. Szalay a
a
Laboratory of Theoretical Chemistry, Institute of Chemistry, Eötvös Loránd University,
Budapest, Hungary
1. Introduction
What happens with the DNA after UV irradiation? This question is asked not only by
scientist, but even public interest is high due to health risks related to increased UV impact on
earth surface. To understand all details of this process, the most logical route, both
theoretically and experimentally, should start with the investigation of the nucleobases, the
principal building blocks of DNA responsible for the absorbance of UV photons, and then
continue with a systematic build-up of the natural DNA with sugar residues, hydrogen-bonded
Watson-Crick pairing, π stacking, solvent effects, etc. If DNA is split in to small building
blocks, even the highest level methods of quantum chemistry can be used to understand the
basic processes [1].
PL9
Our prime interest is excited states,
simulation of excitation spectra. We have
calculated the excitation energy of nucleobases
with the highest level methods available today.
Then we added water, as well the sugar residue
to see how the environment changes the
property of the excited states. In the next step
the dimers of nucleobases have been
investigated: Watson-Crick pairs, and stacked ππ complexes. Here two types of excited states
could be identified: a) local excitations on the
nucleobases and b) charge transfer states. The
former states are very much like those the
monomers, while in the latter process an
electron is transferred from one basis to the
other. The excitation energy of these excitations
are surprisingly low. Our ability to describe
these processes with high level quantum
chemical methods allows the investigation of
charge transfer processes.
And then very fascinating questions can be asked: can DNA conduct electric charge? I will
not be able to answer this question now, but I will show our first results on the mechanism of
charge transfer between neighbouring nucleobases.
[1]. Szalay, P. G. Can Coupled-Cluster Methods Be Used to Describe Excited States of the
Building Blocks of DNA?, Int. J. Quantum Chem, 113(14), 1821–1827, (2013).
PL9
SENSING THE SHAPE OF THE DS-DNA/RNA STRUCTURES BY NON-COVALENT
INTERACTIONS
I. Piantanida a
a
Laboratory for study of interactions of biomacromolecules, Division of Organic Chemistry
and Biochemistry; Ruđer Bošković Institute; Zagreb, Croatia,
Small molecules targeting DNA and RNA have attracted significant scientific interest not
only because of their biomedicinal applications, but also due to widespread use of
spectrophotometric markers in the related scientific research – for instance, fluorescent
techniques significantly developed during the last two decades and now represent about 60%
of the detection enabling technologies used in molecular biology and medicine.
PL10
PL10
Scheme 1. Single small organic molecule interacting with various DNA/RNA structures and reporting the
structural specificity of each polynucleotide by sensitive and biologically applicable spectrometric method.
Small molecules targeting double stranded (ds)-DNA/RNA often rely on the one dominant
non-covalent binding mode for their interaction (e.g., intercalation, minor or major groove
binding, external electrostatic binding). However, the combination of different binding modes
with additional steric and structural features controlling three-dimensional recognition were
intensively explored within the last decades. Our research was particularly focused on small
organic molecules binding via non-covalent interactions (logKs > 5 M-1) to the most of
naturally occurring ds-DNA and ds-RNA secondary structures and yielding different signals
for each of the most common secondary structures (e.g. Scheme 1; A-DNA/RNA, B-DNA,
subtypes of DNA characterized by narrower minor groove, etc.) by sensitive and biologically
applicable methods (UV/Vis, CD, fluorescence, SERS).i The development of such single
molecule sensors, able to report simultaneously the presence of several different secondary
structures of DNA / RNA varieties by different spectroscopic signals could replace the
necessary application of several dyes, each one specific for one DNA or RNA target.
[1]. M. Radić Stojković et al; Org. Biomol. Chem., 11, (2013), 4077; T. H. Rehm et al, Chem.
Sci., 3, (2012), 3393; L.-M. Tumir et al, Chem., Eur. J., 18 (2012), 3859; J. Wu et al, J. Am.
Chem. Soc. , 134 (2012), 1958; K. Klemm et al; Chem., Eur. J., 18 (2012) 1352.
WHAT DRIVES BINDING-COUPLED FOLDING OF G-QUADRUPLEX DNA?
M. Bončina a, I. Prislan a, J. Lah a
a
Faculty of chemistry and chemical technology, University of Ljubljana, Aškerčeva 5, 1000
Ljubljana, Slovenia,
Guanine rich DNA sequences can fold into complex structures called G-quadruplexes in
which four guanines form square planar structure stabilized with eight Hoogsteen hydrogen
bonds, stacking interactions between the neighboring G-quartets and coordinated cations.
They can be found in telomeres where they decrease the activity of the enzyme telomerase,
which is responsible for maintaining the length of telomeres and is involved in around 85% of
all cancers [1]. Formation of stable G-quadruplexes in the region of the telomeric singlestranded overhangs has been found to inhibit telomerase activity. Therefore, telomeric Gquadruplexes are emerging as promising targets for anticancer agents able to inhibit the
telomerase activity by binding to G-quadruplexes and thus stabilizing them.
Folding/unfolding pathways of the telomeric human (ht) DNA fragment in the presence of
+
K and Na+ ions were investigated by calorimetric and spectroscopic methods. Experimental
data may be described as a monomolecular equilibrium three-state process that involves
folded, intermediate and unfolded state [2]. Understanding the driving forces of ht-DNA
folding into G-quadruplex structures sets the basis for experimental and theoretical studies of
its recognition by small molecules. In this light the mechanisms, structural features and
driving forces of binding of various ligands will be discussed.
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[1]. Neidle S.; Balasubramanian S. Quadruplex Nucleic Acids, 2006, Cambridge.
[2]. Bončina, M.; Lah, J.; Prislan, I.; Vesnaver, G. J. Am. Chem. Soc. 2012, 134, 9657-9663.
GUANINE – RICH DNA OLIGONUCLEOTIDES IN SOLUTION
P. Šket a,b, J. Plavec a,b,c
a
Slovenian NMR center, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana,
Slovenia
b
EN-FIST Centre of Excellence, Trg OF 13, SI-1000 Ljubljana, Slovenia
c
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva cesta 5,
SI-1000 Ljubljana, Slovenia
1. Introduction
G-quadruplexes are higher order secondary structures formed by guanine-rich DNA
sequences that can be found in biologically significant regions of the genome such as
telomeres, immunoglobulin switch regions and promoter regions of eukaryotic cells. The
main building blocks of G-quadruplex structures are stacks of square-planar arrays of
G-quartets, consisting of four guanines that are linked together by eight Hoogsteen hydrogen
bonds. The presence of cations such as K+ or Na+ seems to be essential for the formation of
G-quadruplexes due to their role in reducing repulsions amongst guanine carbonyl oxygen
atoms within G-quartets and additionally enhancing base-base stacking interactions.
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With the use of solution-state NMR spectroscopy and other experimental techniques
(PAGE, TDS, UV, CD, DSC) we have studied the behaviour of guanine-rich sequences and
explored their features in an environment almost completely free of G-quadruplex promoting
cations [1]. Experimental data has
shown the formation of a new
structure, which can be considered
as an intermediate on the way to
folding into G-quadruplexes. It is
interesting to note that the guanine
bases are not held together by
Hoogsteen hydrogen bonds like in
G-quartets but rather by alternative
base pairing. Guanine-rich DNA
sequences that are pre-organized in
the way shown here could fold
very rapidly and not by chance
into a large number of structurally diverse G-quadruplex structures with mechanisms of
varying complexity. G-quadruplex formation by telomeric repeats and gene regulatory G-rich
regions could simply go through populating various intermediate states, which are generally
unstable and hard to detect. This kind of studies where intermediate states can be
characterized structurally, thermodynamically and kinetically provide an important step in the
quest to elucidate general principles by which G-quadruplexes adopt their native folds.
[1]. Ceru, S.; Sket, P.; Prislan, I.; Lah, J.; Plavec, J., Angew. Chem. Int. Ed. Engl. 2014, 53,
4881-4884.
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PEPTIDE FOLDAMERS: STRUCTURE AND APPLICATIONS
T. A. Martinek a
a
Institute of Pharmaceutical Analysis, University of Szeged
1. Introduction
Solvent exposed flat regions are responsible for many of the known protein–protein
interaction interfaces [1]. It is challenging however to construct artificial folded structures
(foldamers) that are able to cover these relatively large flat surfaces, and have programmable
anchor points to stabilize the interaction. Short peptidic foldamer helices have been shown to
be good candidates to inhibit protein-protein interactions, [2] but the localized nature of the
surface features is a limiting factor when a generic protein surface is targeted. We addressed
this problem in three ways (Figure ).
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First, construction of multivalent
foldameric helix arrays can provide large
interaction
surface
by
forming
contiguous binding area or separated
anchor patches (A). We have
successfully synthesized multivalent
helical foldamer-dendrimer conjugates
that recognize and inhibit the neurotoxic
properties of β-amyloid [3]. Second, we
explored the possibilities of synthesizing
foldamer helices with increased diameter
(B). This approach aims to increase the
radius of curvature in a single helix approach. Two large-diameter helices, β-H18 [4] and βH18/20 [5] have been discovered recently. Third, water soluble β-sandwich mimicking
foldamers would offer ligands with sufficiently flat surface, but their stabilization in water is a
great current challenge (C). Here we present the design, main structural features and
biological effects of the foldameric analogs of anginex, a 33-mer antiangiogenic peptide with
a tendency to form β-sandwich. The effects of the β3-amino acid substitutions in this βsandwich tertiary structure will be discussed [6].
[1]. Li, B.; Turuvekere, S.; Agrawal, M.; La, D.; Ramani, K.; Kihara, D. Proteins 2008, 71,
670.
[2]. Wilson, A. J. Chem Soc Rev 2009, 38, 3289.
[3]. Fulop, L.; Mandity, I. M.; Juhasz, G.; Szegedi, V.; Hetenyi, A.; Weber, E.; Bozso, Z.;
Simon, D.; Benko, R.; Kiraly, Z.; Martinek, T. A. Plos One 2012, 7.
[4]. Szolnoki, E.; Hetenyi, A.; Martinek, T. A.; Szakonyi, Z.; Fulop, F. Org Biomol Chem
2012, 10, 255.
[5]. Szolnoki, E.; Hetenyi, A.; Mandity, I. M.; Fulop, F.; Martinek, T. A. Eur J Org Chem
2013, 3555.
[6]. Hegedus, Z.; Weber, E.; Kriston-Pal, E.; Makra, I.; Czibula, A.; Monostori, E.; Martinek,
T. A. J Am Chem Soc 2013, 135, 16578.
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ARE PEPTIDE NANO- AND MICROSTRUCTURES STABLE ENOUGH FOR
BIOTECHNOLOGY?
A. Godziek a, A. Maciejowska a, M. Sajewicz a, T. Kowalska a
a
Department of General Chemistry and Chromatography, Institute of Chemistry, University
of Silesia, 9 Szkolna street, 40-006 Katowice, Poland
1. Introduction
Nowadays, peptide nano- and microfibers as promising new candidates in biotechnology
attract an increasing interest. Especially promising seem the short, linear self-assembling
peptides which characterize with a praiseworthy stability rightfully awaited from the building
blocks for nanodevices (like, e.g., the nanocarriers for drug delivery, or scaffolds for tissue
engineering). An informative review on this subject is provided in paper [1]. Particular
attention is paid to the self-assembling properties of diphenylalanine, which is a fragment of
the Alzheimer’s β-amyloid protein (e.g., [2]). As stability of peptide nano- and
microstructures is essential for biotechnology, topical studies on this important issue are
needed, which should start from a thorough scrutiny of the model abiotic systems.
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In the course of the past decade or so, we have focused our attention on the phenomena of
spontaneous oscillatory chiral conversion and spontaneous oscillatory condensation of the low
molecular weight carboxylic acids (in the first instance, profen drugs, hydroxyl acids, and
amino acids; e.g., [3-5]), when dissolved in abiotic aqueous and non-aqueous systems. As a
result, vast experimental evidence has been collected, basically with aid of liquid
chromatography (TLC and HPLC), mass spectrometry, and polarimetry, which pointed out to
striking instability of chiral carboxylic acids in a homogenous solution. Recently, pulsating
instability was revealed in the two-phase systems composed of spontaneously formed lower
peptides dissolved in abiotic solvents and spontaneously formed higher peptides, which
cannot further dissolve and hence, precipitate. Pulsation manifests itself by a visually
perceptible sequential increase and decrease of the peptide nano- and microstructure amounts
freely floating in an agitated solution. This phenomenon was investigated with aid of liquid
chromatography, mass spectrometry, scanning electron microscopy, and turbidimetry for a
selection of biologically important amino acids. Characteristics of this pulsation were
provided in quantitative terms and a graphical form, and the results obtained are going to be
discussed in the course of our lecture. The phenomenon seems rather general and inherent of
most peptides. Hence, the question mark can be posed regarding peptide stability, although
peptide instability can also prove an asset for biotechnology.
[1]. A. Lakshmanan, S. Zhang, C.A.E. Hauser, Trends Biotechnol., 30, 155-165 (2012).
[2]. M.J. Wang, L.J. Du, X.L. Wu, S.J. Xiong, P.K. Chu, ACS Nano, 5, 4448–4454 (2011).
[3]. M. Sajewicz, M. Matlengiewicz, M. Leda, et al., J. Phys.Org. Chem., 23, 1066-1073
(2010).
[4]. M. Sajewicz, M. Dolnik, D. Kronenbach, et al., J. Phys. Chem. A, 115, 14331-14339
(2011).
[5]. M. Sajewicz, M. Dolnik, T. Kowalska, I.R. Epstein, RSC Advances, 4, 7330-7339 (2014).
PL14
PREDICTION OF CRYSTAL STABILITIES OF ACTIVE PHARMACEUTICAL
INGREDIENTS BY MEANS OF QUANTUM CHEMISTRY COMPUTATIONS
P. Cysewski a
a
Physical Chemistry Department, Collegium Medicum of Bydgoszcz, Nicolaus Copernicus
University in Toruń, Kurpińskiego 5, 85-950 Bydgoszcz, Poland;
1. Introduction
The Cambridge Crystallographic Database offers unique information about structure of
many organic crystals including active pharmaceutical ingredients (API). Many of them can
exhibit intriguing structural diversity adopting distinct polymorphic forms. Unfortunately data
obtained by different authors, with divers of equipment and measurement conditions, with
variety refinement protocols and corresponding errors suffer from serious incongruences.
Some CIF’s are even deficient from geometric parameters and often position of hydrogen
atoms are ambiguously determined. That is why optimization of molecular geometries are to
be performed for consistent analysis. The obtained structural data can be used for detailed
characteristics of energy contributions. The aim of this project is to characterize origin of
crystal stabilization of selected API using energy decomposition analysis (EDA). Besides,
some methodological issues are addressed.
PL15
The application of DFT methods for IIE estimation
is sufficiently accurate what was verified against
benchmark comprising 132 pairs taken form S66, S26
and X40 reference sets [1]. The pairwise addictive
model for crystals energies was used for computing of
the lattice energy This value can also be defined by
the difference between crystal energy and isolated
monomer in the gas phase. Since the sublimation
enthalpy is a direct measure of the lattice energy these
data are used for accuracy of additive model
verification [2]. The advantage of additive model is
discussed in terms of contributions coming from three
alternative EDA schemes.
PL15
Figure 1. The difference between
computed and experimental values of
lattice energy estimated for benzoic acid,
aspirin and salicylic acid crystals.
[1]. Jurečka, P., Šponer, J., Černy, J., Hobza, P., Phys.
Chem. Chem. Phys. 2006, 8, 1985
[2]. Gavezzotti, A, Filippini, G., Energetic aspects of crystal packing: experiment and
computer simulations. InA. Gavezzotti (ed)., The Molecular Solid State: Theoretical Aspects
and Computer Modeling, Wiley, New York, 1997, p. 61Y98;
NOVEL ZINC FINGER NUCLEASES BASED ON COLICIN E7 NUCLEASE DOMAIN
E. Németh a, C. Oostenbrink b, M. Asaka c, K. Nagata c, B. Gyurcsik a
a
Department of Inorganic and Analytical Chemistry, MTA-SzTE Bioinorganic Chemistry
Research Group, University of Szeged, Dóm tér 7, 6720 Szeged, Hungary
b
University of Natural Resources and Life Sciences, Institute of Molecular Modeling and
Simulation, Muthgasse 18,1190 Wien, Austria
c Nagata Special Laboratory, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575,
Japan
1. Introduction
Zinc-finger/FokI chimeric nucleases (ZFN-FokI) with designed DNA recognition are a
useful tool of targeted cleavage of genomic DNA. However, the presently available ZFN-s
have too high toxicity to be applied as therapeutic agents for monogenetic diseases.
Our research is focused on the design of controlled ZFNs, that are inactive in absence of
the substrate, while binding to the specific target DNA induces their active conformation.
Instead of the commonly used FokI domain, in a novel approach, we fuse NColE7 to the zinc
fingers. The C- and N-terminal sequence of NColE7 have been separated and linked to
different ends of the zinc finger array to achieve the substrate dependent intramolecular
allosteric control.1
C
O1
O1
N
N
C
Figure 1. The designed zinc finger/NColE7 nucleases: zinc fingers in blue, the C-terminal
and N-terminal part of NColE7 are in green and red, respectively.
Computational methods including database search, protein stability estimation and MD
simulations were carried out to design the linkers, as well as the division of NColE7 to C-and
N-terminal units. The genes encoding four designed proteins have been constructed and the
proteins were expressed in E. coli cells, without any cytotoxic effect. The protein folding,
DNA-binding affinity and specificity, as well as, the nuclease activity were studied.
[1]. E. Németh, G.K. Schilli, G. Nagy, C. Hasenhindl, B. Gyurcsik, C. Oostenbrink (2014)
J. Comp-Aid. Mol. Des., submitted
CYSTEINE-RICH PEPTIDES AS TEMPTING LIGANDS FOR BIOLOGICALLY
RELEVANT METAL IONS.
K. Krzywoszyńska a, H. Kozłowski a
a
Department of Chemistry, University of Wrocław
1. Introduction
The multifunctionality of thiol group present in the side chain of cysteine residue places
this amino acid on the top of the most important molecules in biological systems. The
significant role of this amino acid in biochemistry of metal ions and their complexes is
undeniable. Cysteine-rich proteins are widespread in the nature and are responsible for the
maintenance of homeostasis of essential metal ions like it is for Zn2+ or Cu+ and participate in
the detoxication processes of toxic metal ions, like Cd2+or Ni2+ [1-3].
O2
The binding ability of cysteine rich peptides containing sequentional motif CXXC (where
C - cysteinyl residue, X - random α-aminoacid residue) towards Zn2+, Cd2+ and Ni2+ ions was
studied with potentiometric and spectroscopic methods. The main aim of this project was to
evaluate the impact of the number of cysteine residues on the specificity of binding metal ions
to such peptides. We have studied as first three peptides with sequences: AcGCASCDNCRACKK-NH2 (C4), Ac-GCASCDNCRAAKK-NH2 (C3) and AcGCASCDNARAAKK-NH2 (C2). All of them differ by the number of thiol groups able to
coordinate metal ion. We have shown that the higher number of Cys present in sequence
results the formation of more stable peptide complexes with studied metal ions. Peptide AcGCASCDNCRACKK-NH2, containing four cysteine residues was proved to be the most
efficient ligand among others for studied metal ions [4]. The next step of studies on the
CXXC motif was focused on the evaluation of the influence of the cyclization on the peptide
binding mode as well as the specificity of the peptide - metal ion interactions when compared
to linear Ac-GCASDNCRACKK-NH2 analogue. The obtained results for cyclic
GCASDNCRACKK clearly indicate that in the case of Cd2+ and Ni2+ cyclization increases
distinctly the complex stability in whole pH range when compared to the systems with linear
peptide. Surprisingly different results were received for Zn2+complex.
[1]. J.R. Nuttall, P.I. Oteiza, Genes Nutr., 2014, 9(1), 379.
[2]. M.A. Garcia-Sevillano, T. Garcia-Barrera, F. Navarro and J. L. Gomez-Ariza,
Metallomics, 2014, 6, 672.
[3]. G. Roesijadi, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol., 1996, 113(2),
117.
[4]. K. Krzywoszynska, M. Rowinska-Zyrek, D. Witkowska, S. Potocki, M. Luczkowski and
H. Kozlowski, Dalton Trans., 2011, 40, 10434.
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DESIGN OF NEW MIMETIC PEPTIDES INHIBITORS OF AΒAGGREGATION FROM STRUCTURAL BASES. MOLECULAR
SIMULATIONS USING MONOMETRIC, DIMETRIC AND
PENTAMETRIC MODELS
E. B. Guisasola a,b, R. Salcedo a,b, E. Hubin c, K. Broersen c, S. Andujar a,b, R. D. Enriz a,b,
A. M. Rodríguez a,b
a
Departamento de Química, Universidad Nacional de San Luis, Chacabuco 915, 5700 San
Luis, Argentina
b
IMIBIO-SL (CONICET), Chacabuco 915, 5700 San Luis, Argentina;
c
MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and
Technology, University of Twente, 7500 AE Enschede, The Netherlands
1. Introduction
Alzheimer disease (AD) is a steadily increasing threat all over the world with a growing
percentage of old individuals suffering this pathology and is predicted to double within the
next 50 years.
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We obtained a new series of mimetic peptides possessing a significant Aβ antiaggregant
effect. Such compounds were obtained based on a molecular modeling study which allowed
us to perform a structure-based virtual screening. For the MD simulations we used an Aβ
pentamer model previously developed by our group1 where the
A
compounds were docked using a blind docking technique. To
analyze the dynamic behavior of the complexes we performed
extensive MD simulations in explicit water and measured
parameters that allowed us to quantify the potential inhibitory
effect of Aβ aggregation (Figure A). We also simulated these
mimetic peptides using monomeric and dimeric Aβ models,
showing remarkable changes in terms of secondary structures,
B
interchain interactions and binding free-energies profiles (Figure
B). Monitoring Aβ aggregation by ThT fluorescence and TEM
revealed that fibril formation is significantly decreased in
presence of the active compounds. In turn, dot blot analysis
showed a decrease of soluble oligomers strongly associated with
cognitive decline in AD. We believe that our results may be
helpful providing a guide in the design of new antiaggregant
ligands.
[1]. Masman, M. F.; Eisel, U. L. M.; Csizmadia, I. G.; Penke, B.; Enriz, R. D.; Marrink, S.
J.; Luiten, P. G.M. . J. Phys. Chem. B. 2009, 113, 11710–11719.
O3
CELLULAR IMAGING AND CYTOTOXIC PROPERTIES OF NON-INTERCALATIVE
RUTHENIUM(II) POLYPYRIDYL COMPLEXES
M. Brindell a, O. Mazuryk a, K. Magiera a,b
a
Department of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University
Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University
b
1. Introduction
Recently the development of the ruthenium complexes toward their application in optical
imaging of cells in hypoxia or as cytotoxic agents selectively activated in hypoxic cells show
a new direction in design and great potency of this type of compounds. One of the most
interesting research aspects is a dual imaging and therapeutic application of ruthenium
polypyridyl complexes. In this context the cytotoxicity and luminescence properties are two
important parameters which have to be tuned to obtain such hybrid complexes [1-3].
O4
In this study we show the multifaceted relationship between lipophilicity, protein
interaction and luminescence properties of ruthenium(II) complexes affecting cell imaging
and cytotoxic properties. The system chosen for this study as illustrated, is the family of the
ruthenium(II) complexes comprising two dip ligands and one bpy ligand, which, in turn,
possesses various substituents at 4 and 4’ positions.
To demonstrate the interplay between physicochemical/photophysical properties and
biological activity we analyze the
cytotoxicity and uptake of the studied
compounds using 4T1 breast cancer cell
line as well as the luminescence emitted
by cells arising from ruthenium complex
accumulation. We also show that the
luminescence properties of these
ruthenium complexes strongly depend
on the interaction with albumin, which
suggests that in cells the interaction with
proteins can alter their imaging
properties as well. Our results point out
that complex with 1,3-dicyclohexyl-1carbonyl-urea substituent exhibits the best staining properties in spite of the lowest quantum
yield of luminescence in aqueous solution.
[1]. H. Komatsu, K. Yoshihara, H. Yamada, Y. Kimura, A. Son, S. Nishimoto, K. Tanabe,
(2013), Chem. Eur. J., 19: 1971-1977.
[2]. O. Mazuryk, M. Maciuszek, G. Stochel, F. Suzenet, M. Brindell, (2014), J. Inorg.
Biochem., 134: 83[3]. Y. Chen, M.-Y. Qin, L. Wang, H. Chao, L.-N. Ji, A.-L. Xu, (2013), Biochimie, 95: 20502059.
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SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF HYDROXY
(AZA)NAPHTHALENECARBOXAMIDES
J. Jampilek a, J Kos a, I Zadrazilova a, T Gonec a, M Hruba a, M Moravcova a, M Stujber b,
T Liptaj b
a
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic;
b
Department of NMR Spectroscopy and Mass Spectrometry, Faculty of Chemical and Food
Technology, Slovak University of Technology in Bratislava, Radlinskeho 9, 812 37 Bratislava,
Slovakia
1. Introduction
Hydroxybenzamide based compounds have a broad spectrum of antimicrobial activity [1];
therefore a special attention is paid to them at research and designing of new drugs. Promising
results of biological screening of some hydroxyquinoline derivatives [2-4] inspired us to
prepare and evaluate ring-substituted hydroxy(aza)naphthalenecarboxanilides as potential
antimicrobial agents, because the increasing number of bacterial, mycobacterial and
associated fungal infections evokes the necessity of searching for new antimicrobial
chemotherapeutics [5].
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Several
series
of
ring-substituted
hydroxynaphthalenecarboxanilides
and
hydroxyquinolinecarboxanilides were prepared by microwave-assisted synthesis. Primary in
vitro antimicrobial screening of the synthesized compounds was performed against wide
spectrum of Gram-positive and Gram-negative bacteria, against resistant clinical isolates, and
also against mycobacterial species. Generally it can be summarized that compounds
substituted by lipophilic and/or electron-withdrawing moieties proved to have significant
biological activity.
Acknowledgment:
This study was supported by the IGA VFU Brno 37/2014/FaF, 52/2014/FaF and the Slovak
Grant Agency VEGA, Grants No. 1/0972/12.
[1]. Pauk, K.; Zadrazilova, I.; Imramovsky, A.; Vinsova, J.; Pokorna, M.; Masarikova, M.;
Cizek, A.; Jampilek, J. Bioorg. Med. Chem. 2013, 21, 6574.
[2]. Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469, 483.
[3]. Jampilek, J.; Dolezal, M.; Kunes, J.; Buchta, V.; Silva, L.; Kralova, K. Med. Chem. 2005,
1, 591.
[4]. Musiol, R.; Jampilek, J.; Buchta, V.; Silva, L.; Niedbala, H.; Podeszwa, B.; Palka, A.;
Majerz-Maniecka, K.; Oleksyn, B.; Polanski, J. Bioorg. Med. Chem. 2006, 14, 3592.
[5]. Cieslik, W.; Musiol, R.; Nycz, J.E.; Jampilek, J.; Vejsova, M.; Wolff, M.; Machura, B.;
Polanski, J. Bioorg. Med. Chem. 2012, 20, 6960.
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ACCURATE MODELING OF MOLECULAR OPTICAL PROPERTIES BY A
COMBINATION OF MOLECULAR DYNAMICS AND QUANTUM CHEMISTRY
V. Andrushchenko a, P. Bouř a
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic
1. Introduction
Spectroscopic techniques have been successfully used for conformational and structural
studies of nucleic acids for several decades. However, due to complexity of biomolecular
spectra, their clear and straightforward interpretation is often a difficult task. Theoretical
calculations of biomolecular spectra can significantly simplify this task, provide reliable
spectra-structure connection and explain experimentally observed spectral features. Spectra of
certain nucleic acid functional groups, such as the phosphate group, are strongly influenced by
presence and dynamics of solvent, ions and its own conformational flexibility. This makes it
extremely difficult to reliably model spectra of such groups.
O6
Nucleic acid phosphate group is the most difficult for spectral modelling. Its vibrational
spectra are very sensitive to the hydrogen bonding with solvent molecules and to presence of
ions. Simple increasing of the level of theory cannot improve the results of the calculations.
Instead, the calculations should account for the solvent and ion dynamics and molecular
flexibility to correctly represent the real system. We developed a computational procedure
allowing to account for these factors. The methodology combines molecular dynamics (MD)
simulations with quantum chemistry (QM) spectra calculations. The MD step provides
realistic water molecule and ion distribution around the phosphate group and their dynamics
as well as accounts for the phosphate group flexibility. Subsequent QM calculations of spectra
for a large set of MD snapshots allow to obtain a reliably modelled averaged spectrum closely
reproducing the experiment. The spectral feature assignments and understanding of spectral
changes based on the first principles becomes then easily achievable, which allows to
establish a dependable spectra-structure relationship.
O6
DESIGN OF NOVEL TRIAZOLE HIV-1 REVERSE TRANSCRIPTASE INHIBITORS
T. Frączek a, P. Paneth a
a
Department of Chemistry, Lodz University of Technology, Zeromskiego 116,
90-924 Lodz, Poland
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Reverse transcriptase (RT) is one of the key enzymes for HIV virus replication. Because
the enzyme has no counterpart in a human organism, it is an attractive target for antiretroviral
drugs. Current standard for HIV infection treatment is to combine several drugs with a
different mechanism of action. Most of the used drug combinations comprise one of five
currently available non-nucleoside reverse transcriptase inhibitors (NNRTIs). Because of a
high mutation rate of HIV, it quickly develops a resistance to most of the currently used
inhibitors, therefore it is necessary to find new effective drugs. Among tens of chemically
distinct groups of reverse transcriptase inhibitors, one of the most promising class of
compounds are 1,2,4-triazoles, for example a clinical candidate RDEA-806 [1].
By employing the Computer Aided Drug Design we
are trying to design novel triazole NNRTIs. One of our
ideas was to explore derivatives at the position C5 of the
triazole core (Fig. 1). In order to reduce the number of
compounds to synthesize, we looked for a fast
computational method to help us select the most
Fig. 1. Triazole NNRTIs
promising structures out of thousands of potential
inhibitors. We found that a combination of selected molecular docking methods and scoring
functions is able to discern between more and less active inhibitors [2]. Using this
methodology we chose a small number of most promising compounds which we synthesized
and tested for the RT inhibitory activity. Most of the tested compounds showed a moderate
activity which proves the validity of our approach. Currently we are using a more rigorous
computational and experimental methods in order to develop our initial compounds into
highly active reverse transcriptase inhibitors.
Acknowledgment:
The reported studies are supported by the grant 2011/02/A/ST4/00246 (2012-2017) from
the Polish National Center for Science (NCN).
[1]. G. Moyle, M. Boffito, A. Stoehr, A. Rieger, Z. Shen, K. Manhard, B. Sheedy, V.
Hingorani, A. Raney, M. Nguyen, T. Nguyen, V. Ong, L. T. Yeh, B. Quart. Antimicrob.
Agents Chemother. 2010, 54, 3170-3178.
[2]. T. Frączek, A. Siwek, P. Paneth, J. Chem. Inf. Model. 2013, 53, 3326-3342
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STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF TWO NOVEL
ENZYMES WITH PROMISCOUS ENE-REDUCTASE ACTIVITY
T. Pavkov-Keller a, A. Binter a, G. Steinkellner a, C. C. Gruber a, K. Steiner a, C. Winkler c, H.
Schwab d, K. Faber c, P. Macheroux e, K. Gruberb
a
ACIB GmbH, Petersgasse 14, Graz, Austria;
Institute of Molecular Biosciences, Humboldtstraße 50, Graz, Austria;
c
Department of Chemistry, University of Graz, Heinrichstraße 28, Graz, Austria;
d
Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, Graz,
Austria;
e
Institute of Biochemistry, Graz University of Technology, Petersgasse 12, Graz, Austria
b
1. Introduction
An approach using three-dimensional motifs reflecting specific active site arrangements
(catalophore) was developed in our group. It does not depend on overall protein similarity and
therefore enables the search across enzyme families and the detection of potential catalytic
promiscuity.
O8
This catalophore approach led to the discovery of two novel enzymes with ene-reductase
activity [1]. Enzymes of this family have recently been shown to possess a great potential for
(industrial) biotransformations. Neither the amino acid sequence of these two enzymes nor
their overall structure is related to those of the well known old yellow enzymes (OYE). These
two flavoproteins contain FMN as a cofactor and exist as homodimers. We cloned, expressed
and purified both enzymes and subjected them to crystallization trials. Obtained crystals were
soaked with putative substrates/inhibitors. The enzymatic characterization was pursued by
stopped-flow and difference titration experiments. Additionally, several typical OYE
substrates (i.e. alkenes bearing an electron-withdrawing activating group) were tested to
assess the biocatalytic performance. The analysis showed some salient features of typical
OYEs as well as some striking differences, i.e. a stereocomplementary behaviour. In
conclusion, the two novel enzymes can be described as NADPH-dependent quinone
reductases with significant OYE-like side activities [1].
[1]. Steinkellner G, Gruber CC, Pavkov-Keller T, Binter A, Steiner K, Winkler C, Lyskowski
A, Schwamberger O, Oberer M, Schwab H, Faber K, Macheroux P, Gruber K.; Identification
of promiscuous ene-reductase activity by mining structural databases using active site
constellations., 2014, Nature Communications 5: 4150.
O8
GENERAL ASPECTS OF DNA G-QUADRUPLEX MULTIMERIZATIONS
V. Víglaský a, P. Tóthová a, P. Krafčíková a
a
Department of Biochemistry,Institute of Chemistry, Faculty of Sciences, P. J. Šafárik
University, Košice, Slovakia,
1. Introduction
Guanine-rich (G-rich) DNA and RNA sequences have the propensity to form Gquadruplex structures. In addition, these molecules, depending on their sequence and
condition, can also form high-ordered multimeric structures, Figure. Multimeric forms obtain
certain specific properties: increase of the CD signal in specific wavelength regions, change
of the ligand binding affinity, decrease of electrophoretic mobility, increase of light scattering,
etc. Our aim is to present a simple and cost effective method which can verify the formation
of G-quadruplex motifs from oligomeric DNA sequences and a way to determine the
stoichiometry of these structures. This methodology could be of great use in the prediction of
G-quadruplex assembly and the basic principles of our techniques can be extrapolated for any
G-rich DNA sequences.
O9
CD spectroscopy is applied to
detect the distinctive signals in
wavelength
regions
which
are
characteristic for G-quadruplexes (220
- 320 nm), and UV-Vis spectroscopy is
used to obtain the thermal difference
spectra and to determine DNA
concentrations for adjustments to
molar dichroism intensity [1]. The
intensity of the CD signal of various
G-rich sequence was compared with
Figure 1: Multimerization of G-quadruplex subunits
that of the d(G3T2A)3G3) oligomer.
and corresponding CD spectra of these structural
Standard electrophoresis was also
forms.
applied in order to confirm the
coexistence of different conformers
under given conditions and to estimate the molecularity of these structures. The application of
this set of techniques is simple and cost efficient, and would be highly suitable for use as a
preliminary screening method prior to the use of more expensive techniques for determining
molecular structure.
Acknowledgments:
This work was supported by the Slovak Research and Development Agency under
contracts No. APVV-0280-11 and Slovak Grant Agency (1/0504/12).
[1]. V. Víglaský, K. Tlučková, L. Bauer (2011) The first derivative of a function of circular
dichroism spectra: biophysical study of human telomeric G-quadruplex. Eur Biophys J. 40:
29-37.
[2]. P.Tóthová, P. Krafčíková, V. Viglasky (2014) Formation of high–ordered multimers in
G-quadruplexes submitted to Biochemistry
O9
SYNTHESIS OF NOVEL AROMATIC ESTERS
OF RESVERATROL
A. Urbaniak a, K. Kacprzak a
a
Laboratory of Bioorganic Chemistry, Adam Mickiewicz University in Poznań, Poland,
1. Introduction
Resveratrol is one of the most active antioxidant of natural origin. Its free radical
scavenging and anticancer properties have been demonstrated in many in vitro and in vivo
studies [1-3]. Since its water solubility and cell membranes penetration is limited a lot of
research have been dedicated to its modification improving its activity [4-7]. One of the most
promising resveratrol derivatives are its esters due to easy modulation of its polarity and acyl
moieties (adding to the overall biological activity). Surprisingly very little is known about the
synthesis of aromatic or more sophisticated resveratrol esters.
O10
Although
the
classical
esterification of resveratrol
with acid chlorides has been
already reported, we found
that this condition led to
incomplete conversion of
resveratrol and despite long
reaction time gave only low
yields of products. For this
reason we screened three alternative esterification methodologies by employing microwave
and pressure microwave heating as well as solvent-free condition in ball mill. We found that
combination of microwave heating under pressure is the most convenient alternative for the
synthesis of aromatic resveratrol esters, providing good yield and reducing the reaction time
to the minutes. Other methodologies were found also applicable to the synthesis of particular
esters, such as resveratrol tricinnamate. In some cases crystallization of resveratrol esters was
found as an alternative to the column chromatographic purification. The results of the
synthetic work and comparison between these methods will be presented in communication.
[1]. Z. Ovensa, K. Kozics, Y. Bader, P. Saiko, N. Handler, T. Erker, T. Szekeres, Oncol Rep
16, 617 (2006).
[2]. B. Olas, B. Wachowicz, J. Saluk-Juszczak, T. Zieliński, W. Kaca, A. Buczyński, Cell Biol
Toxic 17, 117-125 (2001).
[3]. M. Athar, J. H. Back, X. Tang, K. H. Kim, L. Kopelovich, D. R. Bickers, A. L. Kim,
Toxicol. Appl. Pharmacol. 224(3), 274-283 (2007).
[4]. G.R. Pettit, N. Melody, A. Thornhill, J.C. Knight, T.L. Groy, C.L. Herald, J Nat Prod 72,
1637-1642 (2009)
[5]. J. Das, S. Pany, A. Majhi, Bioorg Med Chem 19, 5321-5333 (2011)
[6]. B. Sun, J. Hoshino, K. Jermihov, L. Marler, J.M. Pezzuto, A.D. Mesecar, M. Cushman
Bioorg Med Chem 18, 5352-5366 (2010)
[7]. A.V. Moro, F.S.P. Cardoso, C.R.D. Correia, Tetrah Lett 49, 5668-5671 (2008)
O10
COMPARATIVE SCREENING AND VALIDATION AS A NOVEL TOOL
TO IDENTIFY STAT-SPECIFIC INHIBITORS
M. Szelag a, A. Czerwoniec b, J. Wesoly c, H. A. R. Bluyssen a
a
Department of Human Molecular Genetics,
Bioinformatics Laboratory and
c
Laboratory of High Throughput Technologies, Institute of Molecular Biology and
Biotechnology, Adam Mickiewicz University in Poznan, Poland
b
1. Introduction
Signal transducers and activators of transcription (STATs) facilitate action of cytokines,
growth factors and pathogens. STAT activation is mediated by a highly conserved SH2
domain, which interacts with phosphotyrosine (pTyr) motifs for specific STAT-receptor
contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus
by binding to specific DNA-response elements of target genes. Abnormal activation of STAT
signaling pathways is implicated in many human diseases, like cancer, inflammation and
auto-immunity. Searches for STAT-targeting compounds, exploring the pTyr-SH2 interaction
area, yielded many small molecules for STAT3 but sparsely for other STATs. However, many
of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and
selection strategies of SH2 domain-based STAT inhibitors.
O11
We generated new 3D structure models for all human (h)STATs (1, 2, 3, 4, 5A, 5B and 6)
and applied a comparative in silico docking strategy to obtain further insight into STAT-SH2
cross-binding specificity of previously identified STAT3 inhibitors. Indeed, by primarily
targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds
exhibited similar binding affinity and tendency scores for all STATs. By comparative
screening of a natural compound library and using the “comparative STAT3 and STAT1
affinity and binding tendency scores” as a selection criterion, we provided initial proof for the
possible existence of STAT3 as well as STAT1-specific inhibitors. In silico screening of a
multi-million “clean lead” compound library for binding of all STATs, likewise identified
potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on
comparative virtual screening and docking validation, we developed a novel STAT inhibitor
screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds.
This could increase our understanding of the functional role of these STATs in different
diseases and benefit the clinical need for more drugable STAT inhibitors with high
specificity, potency and excellent bioavailability.
Acknowledgments:
This research was supported by grants: UMO-2012/07/B/NZ1/02710 (to HB), UMO2012/07/N/NZ2/01359 (to MS) from National Science Center Poland and No 128 from the
Poznan Supercomputer Center (PCSS) (to MS). This research was supported in part by PLGrid Infrastructure.
O11
APPLICATION OF N-LIPIDATED PEPTIDES IMMOBILIZED ON SOLID SUPPORTS AS
MOLECULAR RECEPTORS
J. Fraczyk a, Z. Kamiński a
a
Institute Organic Chemistry, Lodz University of Technology, Zeromskiego 116,
90-924 Lodz, Poland
1. Introduction
Molecular recognition consists noncovalent bonding determining selectivity of specificity
interaction between molecules. In biological systems molecular recognition is observed in
between receptor-ligand, antigen-antibody, DNA-protein, sugar-lectin, RNA-ribosome, etc. In
the other application areas, relatively weak binding forces and decrease of entropy in the
system which favours competitive character of the process should be evaluated as
advantageous in designing biosensors, chemometric and diagnostic devices, etc. In all cases,
however, rational design of the structure of both interacting ligand/receptor partners still
remains a challenging task due to the complex nature of engaged interactions.
O12
In our approach to design entire structure of synthetic molecular receptor we incorporated
into binding pockets most of the structural attributes responsible for intermolecular
interactions. These include hydrogen-bond donors and acceptors, as well as lipophilic and
hydrophilic fragments supplemented with π-donors and π-acceptors. These elementary
fragments were allocated inside the linear N-lipidated peptide structure anchored via aromatic
linker on solid support and forming the regular matrix of podands. Moreover, the flexible
peptide fragment was separated from the support by relatively rigid, aromatic rings. Thus, a
bonding “pocket” located between the podands, was composed from the tethered “walls”
constructed from aromatic rings and expanded with a diversity of interactions offered by
flexible peptide fragments, and finally closed with a “zipper” of hydrophobic chains of lipidic
fragments [1]. The binding process was found to be reversible and competitive. This enabled
convenient monitoring of colorless ligands docking by using reporter dye approach. Recently,
we attempts to use arrays of chemical receptors as a new tool in medical diagnostic.
Acknowledgement:
This work was supported by MSE Poland N N 405 669540.
[1]. a) Frączyk, J.; Malawska, B.; Kamiński, Z.J., J. Comb. Chem., 2009, 11, 446-451.b)
Frączyk, J.; Kolesińska, B.; Czarnecka, A.; Malawska, B.; Więckowska, A.; Bajda M.;
Kamiński, Z.J., QSAR & Comb. Sci. 2009, 11; 728-736.
O12
CHARACTERIZATION AND APPLICATIONS OF CYSTEINEHISTIDINE- DEPENDENT AMINOHYDROLASE/PEPTIDASE
ENZYME TARGETING STRAPHYLOCOCCAL CELL WALLS
A. Coffey a
a
Centre for Research in Advanced Therapeutic Engineering & Department of Biological
Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland.
Staphylococcus aureus is a major cause of infection in humans and animals causing a
wide variety of conditions from local inflammations to fatal sepsis. The bacterium is
commonly multi-drug resistant and thus many front-line antibiotics have been rendered
practically useless for treating human infections. We sequenced the genome of antistaphylococcal phage K, which has a broad host range among staphylococci. We then cloned
the gene for the phage endolysin. This enzyme, named LysK, was found to have a modular
organisation with three domains, a cysteine/histidine-dependent amido hydrolase peptidase
(CHAPk), an amidase, and thirdly a cell-wall binding domain. The latter facilitates attachment
of the enzyme to the bacterial cell wall, while former two domains catalyse the degradation of
the peptidoglycan, mediating rapid bacterial cell death. Deletion analysis of the enzyme
showed that full lytic activity against live antibiotic-resistant staphylococci was retained when
the endolysin was truncated to its CHAPk (peptidase) domain. The enzyme was purified by
ion-exchange chromatography and characterized in detail including elucidation of its 3-D
structure. Addition of the enzyme to a turbid bacterial MRSA culture resulted in elimination
of turbidity. The peptidase was used in in-vivo studies in mouse models where it successfully
eliminated MRSA colonization without adverse effects on the animals; and furthermore, exvivo studies confirmed a low immunogenicity.
O13
O13
STRUCTURES OF ENVIRONMENTAL ALLEGENTS AND STRUCUREBASED EPITOPE MAPPING
W. Keller a, F. Dall'Antonia b, S. C. Devanaboyina,c, R. Valenta d
a
Institute of Molecular Biosciences, University of Graz, Humboldtstr. 50/III, 8010 Graz,
Austria
b
European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany
c
Department of Immunology, University of Texas Southwestern Medical Center, Dallas, USA
d
Division of Immunopathology, Department of Pathophysiology and Allergy Research,
Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna,
Vienna, Austria
The structure determination of major allergens is a prerequisite for the conformational
analysis of surface exposed areas of the allergen and for mapping conformational epitopes.
These may be determined by experimental methods including crystallographic and NMRbased approaches or predicted by computational methods [1]. In this paper we present newly
determined structures of allergens and summarize the existing structural information on
allergens and their classification in protein fold families. We developed a program “SPADE”
which uses the structural similarity of homologous allergens combined with immunological
cross-reactivity data for IgE epitope prediction [2]. Furthermore we present recent
applications of the program SPADE and the experimental verification of the predicted IgE
epitopes by mutagenesis studies and immunological cross-reactivity assays [3].
O14
[1]. Dall’Antonia, F., T. Pavkov-Keller, K. Zangger, W. Keller (2014) Methods 66, 3–21.
[2]. Dall’Antonia, F., A. Gieras, S.C. Devanaboyina, R. Valenta, W. Keller (2011) J Allergy
Clin Immunol. 128, 872-879.e8.
[3]. Devanaboyina, S. C., C. Cornelius, C. Lupinek, K. Fauland, F. Dall’Antonia, A. Nandy,
S. Hage, S. Flicker, R. Valenta, W. Keller, Allergy. 2014 Aug 14. doi: 10.1111/all.12511.
[Epub ahead of print]
O14
APPLICATION OF CELL PENETRATING PEPTIDES FOR DRUG DELIVERY
B. Kolesińska a, Z. Kamiński a, D. Serbach b, D. Drozdowska c, J. Frączyk a
a
Institute of Organic Chemistry, Lodz University of Technology
Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences (DCHAB), Swiss Federal Institute of Technology Zürich (Eidgenössische Technische
Hochschulen (ETH Zürich), Switzerland
c
Department of Organic Chemistry, Medical University, Bialystok, Poland
b
1. Introduction
The application of cell-penetrating peptides (CPPs) for drug delivery currently consists one
of the fastest developing field in medicinal chemistry [1]. Both, natural CPPs as well as
synthetic polyelectrolytic peptides have been found useful to carry cargo into eukaryotic [2],
and prokaryotic cells. The most widespread non-natural CPPs are oligo-arginines [3],
delivering broad range of cargos including peptides, proteins, nucleic acid derivatives and
drug molecules. The characteristic features of cell-penetrating peptides are a net positive
charge and amphipathic structure. There are many ways for the preparations of CPPconjugates, the most important are: (i) conjugation by a chemically or enzymatically labile
covalent bond to release the cargo inside the target cell; (ii) formation of non-covalent
polyion-counterion complexes; (iii) attachment by a stable covalent bond in cases where the
presence of the CPP does not prevent the activity of the transported cargo.
O15
The most fundamental problem for in vivo application of CPPs as transporters is their
peptidolytic degradation and potential toxicity (immunogenicity). The enzymatic degradation
can be prevented by employing D-Aaa derivatives or by the use of βh-Aaa derivatives.
Designing the structure of CPPs it has to be also considered the rate of delivery and releasing
of cargo at the target site. Herein there are presented results of our studies on tuning of
functional properties (permeation, penetration rate, resistance to proteolytic enzymes) of R8
derivatives by incorporation of unnatural amino acids into chain. An alternative to polyionic
transport system has been developed and applied to cargo delivery based on neutral oligoprolines transport system.
[1]. Ü. Langel, "Handbook of Cell-Penetrating Peptides", CRC Press, Boca Raton, 2007; Ü.
Langel, "Cell-Penetrating Peptide: Methods and Protocols", Humana Press, New York, 2011.
[2]. F. Kamena, B. Monnanda, D. Makou, S. Capone, K. Patora-Komisarska, D. Seebach,
Chem. Biodiv. 8, 1, 2011; b) X. Sisquella, K. de Pourcq, J. Alguacil, J. Robles, F. Sanz, D.
Anselmetti, S. Imperial, X. F.Busquets, FASEB J. 24, 4203, 2010.
[3]. a) P. Wender, W. Galliher, E. Goun, L. Jones, T. Pillow, Adv. Drug Delivery Rev. 60,
452, 2008; b) I. Nakase, Y. Konishi, M. Ueda, H. Saji, S. Futaki, J. Controlled Release. 159,
181, 2012.
O15
POSTER SESSIONS
I POSTER SESSION
Chemistry of cell biology
Carbohydrate chemistry and biochemistry
Nucleic acids chemistry
New/engineered enzymes
Drug design and drug delivery
II POSTER SESSION
Structure, function and interactions of proteins
Molecular pharmacology, medicine and biomaterials
Catalysis in organic synthesis, pharmacy and environmental protection
ENZYMATIC SYNTHESIS OF Β-D-2-DEOXYGLUCOSIDES BY Β-GLUCOSIDASE IN
THE PRESENT OF IONIC LIQUIDS
P. Hahn a, Anna Kasprzycka a, Wiesław Szeja a, Agnieszka Drożdż b, Anna Chrobok b
a
P1
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of
Chemistry, Silesian University of Technology, Gliwice, Poland
b
Department of Chemical Organic Technology and Petrochemistry, Faculty of Chemistry,
Silesian University of Technology, Gliwice, Poland
1. Introduction
Synthesis of carbohydrate complex with high stereoselectivity is still a challenge for many
research
teams.
2-Deoxyglycosides
occur
widely
in
natural
products,
2-deoxyglycosyl moieties are present in many biologically active compounds like
anthracycline antitumour antibiotics, aureolic acids, highly potent antimicrobial agents or
cardiac glycosides.
Ionic liquids as a novel cosolvent using in the enzymatic synthesis can affect by increasing
the final reaction yield
This communication describes the use of ionic liquids as cosolvent in the glycosylation
reaction catalysed by β-glucosidase from Aspergillus niger K0440. During the investigation
we were examined the influence of eight different ionic liquids on the final reaction efficiency
measured by HPLC system. Finally we received unexpected results in the increased reaction
efficiency three times.
3. Acknowledgments
P. Hahn was supported by a DoktoRIS fellowship.
[1]. “Ionic liquids in biotransformations and organocatalysis: solvents and beyond”, red.
Pablo Dominguez de Maria, John Wiley & Sons, New Jersey, 2012.
[2]. van Rantwijk F, Sheldon R.A., „Biocatalysis in Ionic Liquids”, Chem. Rev., 2007, 107,
2757-2785
[3]. van Rantwijk F., Lau R., Sheldon R.A., „Biocatalytic transformations in ionic liquids”,
Trends Biotechnol, 2003, 21, 131-138
[4]. Lang M., Kamrat T., Nidetzky B., „Influence of ionic liquid cosolvent on
transgalactosylation reactions catalyzed by thermostable β-Glycosylhydrolase CelB from
Pyrococcus furiosus”, Biotechnol. Bioeng., 2006; 95, 1093
[5]. van Rantwijk F., Oosterom M.W.V., Sheldon R.A., „Glycosidase-catalysed synthesis of
alkyl glycosides”, J. Mol. Catal. B, 1999, 6, 511–532
P1
ARE DIMERIC URIDINE DERIVATIVES
PROMISING CANDIDATES FOR CHITIN
SYNTHASE INHIBITION?
K. Kral a , P. Hahn a, T. Bieg a, J. Paszkowska a, U. Nawrot b, I. Wandzik a
a
P2
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian
University of Technology, Gliwice, Poland
b
Department of Microbiology of Medicine Academy, Wrocław, Poland.
1. Introduction
Chitin synthase (CS) is essential for fungal cell wall biosynthesis and due to its absence in
mammalian cells, this enzyme is an attractive medicinal target. Dimeric uridine derivatives
were proposed as potential inhibitors for CS. Since it is believed that chitin synthase has two
active sites in close proximity [1], novel compounds were designed to show additional proves
for this hypothesis.
In this study we have synthesized dimeric uridine derivatives with linkers of different
length between uridine parts (Scheme 1). Synthesized compounds 1-3, are expected to
coordinate to metal ions in enzyme active site and thus exhibiting bivalent inhibition.
O
O
HN
O
O
N
O
OH OH
NH
NH
O
NH
NH
NH
N
HN
O
O
O
O
O
1
O
O
OH OH
N
O
O
OH OH
HN
NH
2
N
O
O
O
OH OH
NH
O
N
NH
O
O
O
NH
O
NH
NH
O
OH OH
O
NH
3
N
O
O
OH OH
In our current study we have isolated chitin synthase from Saccharomyces cerevisiae
(strain X2180) according to published procedure [2]. Synthesized compounds were tested as
potential CS inhibitors in a nonradioactive assays [3].
As a part of our research programme we have tested synthesized compounds for
antifungal properties against Aspergillus fumigatus and Candida albicans. The synthetic
procedure and biological evaluation of synthesized compounds will be presented.
[1]. A. R. Yeager, N. S. Finney „The first direct evaluation of the two-active site mechanism
for chitin synthase”, J.Org.Chem., 2004, 69, 613-618.
[2]. L.B. Crotti, T. Drgon, E. Cabib, "Yeast cell permeabilisation by osmotic shock allows
determination of enzymatic acyivities in situ", Anal. Biochem., 2001, 292, 8 – 16.
[3]. H.A. Lucero, M.J. Kuranda, D.A. Bulik, "A Nonradioactive, high throughput assay for
chitin synthase activity", Anal. Biochem., 2002, 305, 97 – 105.
P2
THE EFFECT OF POLYANION STRUCTURE ON INDUCED CIRCULAR DICHROISM OF
DYES
J. Kudláčová a, M. Antalík a,b
a
P3
Department of Biochemistry, University of Pavol Jozef Šafárik in Košice, Slovakia
Institute of Experimental Physics, Slovak Academy of Sciences, Slovakia
b
1. Introduction
Study on self-assembly of molecules has become a hot topic in recent years. The selfaggregation of dyes as well as assembly of chromophores on biomacromolecules was
extensively studied and significantly applied in disease treating, photosynthesis and nanodevices construction [1].
The self-assembly of nonchiral organic dye and material having a chiral structure create a
chiral supermolecule which exhibits induced CD signal (ICD) in dye´ spectrum. This ICD
signal is free of overlaps and very diagnostic of a dye binding mode. Investigations reveal that
binding affinities and sequence specificity of small molecules towards biomacromolecules are
controlled by various structural and electronic factors [2].
In this work, the interactions between safranin T (ST), thiazole orange (TO) and 9aminoacridine (9-AA) with different polyanions (heparin, poly-D-glutamic acid,
polygalacturonic acid, polyadenylate, pentosan polysulfate and calf thymus DNA) were
investigated using both UV-vis and CD spectroscopic techniques. In the case of heparin, a
blue shift in the spectrum of the ST and TO takes place when dye is bound to the polymer.
Such a phenomenon is known as metachromasy [3]. This effect has not been observed in the
absorption spectrum of 9-AA. Interaction between ST and heparin led to production of a
biphasic signal with one positive peak at 475 nm and one negative peak at 450 nm in induced
CD spectrum suggesting that dye-dye interaction is occurring. Different shape of the ICD
spectrum was observed after interaction of heparin with TO and 9-AA.
Acknowledgments:
Poster is the result of the Project implementation: VEGA 2/0025/12, APVV-0280-11,
VVGS-2013-127, VVGS-PF-2013-114, VVGS-PF-2014-454, KVARK - quality education
and skills development for doctoral and post-doctoral students of Pavol Jozef Šafárik
University in Košice, ITMS: 26110230084, supported by the Research & Development
Operational Programme funded by the ESF.
[1]. Zhang, S. et al. Spectrochim Acta A 2002, 58, 2613-2619.
[2]. Sarkar, D. et al. J. Phys. Chem. B 2008, 112, 9243-9249.
[3]. Zsila, F. et al. Biochem Biophys Res Commun 2006, 346, 1267-1274.
P3
EXOPOLYSACCHARIDES IN BIOFILMS OF BURKHOLDERIA
MULTIVORANS STRAIN C1576
P. Cescutti a, S. Dolfi a, R. Rizzo a
a
P4
Department of Life Sciences, University of Trieste, Via Licio Giorgieri 1, 34127 Trieste –
Italy
1. Introduction
Burkholderia multivorans is a member of the Burkholderia cepacia Complex (BCC)
which includes eighteen bacterial species of environmental origin. Many of these species are
also opportunistic pathogens of individuals affected by the genetic disease Cystic Fibrosis
(CF). CF causes alterations of many epithelial secretions among which the most serious are
those producing a viscous mucus on the surface of lungs. The high viscosity impedes a
correct mucociliary clearance and the abnormal physico-chemical conditions favour the set up
of serious bacterial infections which are the main cause of death in CF patients. Sometimes
BCC infections lead to the so-called “cepacia syndrome”, an acute pulmonary deterioration
with bacteremia, necrotizing pneumonia, and death within weeks to months [1].
Exopolysaccharides (Epol) produced by these bacteria are considered one of the factors
favoring the infection set up and maintenance. In addition Epol are also involved in the
structure of the matrix of biofilms. These are communities of bacteria embedded in a
hydrophilic gelling matrix which, when involved in infections, exhibit increased antibiotic
resistance.
In our laboratory, the primary structure of numerous Epol produced by many BCC
species where unraveled and recently an investigation of Epol involved in BCC biofilm
matrix formation started [2]. Quite surprisingly, Epol engaged in biofilms resulted to be
structurally different from those obtained in non-biofilm conditions and, in addition, cepacian,
which is the most common Epol produced by many of the BCC species, seems not to be
associated with biofilm matrix formation.
Burkholderia multivorans strain C1576 was able to produce a mixture of two
polysaccharides when cultured in non-biofilm conditions: cepacian and a novel one.
However, in biofilm forming conditions, C1576 biosynthesized only the novel one, as showed
by NMR spectra. This evidence prompted us to study the primary structure of this Epol
which resulted to be a rhamno-mannan with an equimolar ratio Rha-to-Man and the linkage
analysis revealed the presence of 2- and 3-linked rhamnose residues together with 2- and 3linked mannose residues. The monosaccharides sequence was investigated by 1D and 2D
NMR which showed also the presence of methoxy groups non-stoichiometrically substituted
on C-3 of the 2-linked rhamnose units. This finding, together with the presence of the D
enantiomer of the Rha sugars, are not common structural motifs of bacterial polysaccharides.
[1]. G. Zahariadis, M. H. Levy , J. L. Burns. 2003. Can J Infect Dis 14(2) p. 123-125
[2]. B. Cuzzi, Y. Herasimenka, A. Silipo, R. Lanzetta, G. Liut, R. Rizzo, P. Cescutti. 2014
PLOS ONE 9(4) e94372 p. 1-10
P4
STUDIES ON POTENTIAL SMART CONTRAST AGENTS BASED ON
FE(III) COMPLEXES
M. Wysocka a, N. Kuźnik a
a
P5
University of Technology
1. Introduction
Magnetic resonance imaging (MRI) is a method of diagnosis in common medical practice.
The main advantages of this method are: non-invasive, high spacial resolution of images and
the ability to obtain a three-dimensional image. In order to obtain better quality of image and
to distinguish healthy from changed tissues contrast agents are used [1]. Currently gadolinium
preparations are commonly used however, they are far from ideal solutions. Gadolinium is not
a natural metal to organism. In case of renal failure gadolinium is accumulated in the body
and causes damage to central nervous system [2]. The increasing concentration of gadolinium
in surface waters is of a serious concerns [3]. We studied the synthesis of new, bioactivable
contrast agents based on Fe(III) complexes. Iron is a natural element, thus its biochemistry is
well known both in the human body and the environment.
We synthesized pentadentate
amino-phenolate ligands. This
synthesis
was
carried
by
reductive amination. For selected
models carbon chain was
extended (from two-, to three and
four-carbon chain) to reduce
tension between atoms and
improve geometry and stability
of compounds. The resulting
complexes with Fe3+ were
Figure 1 Principle action of smart contrast agent
obtained, purified and tested for
the use as contras agents. Relaxivity measurements confirm that the pentadentate complexes
can achieve high relaxivity r1, particularly in an environment of high viscosity (i.e. serum).
Higher r1 values were observed for the compounds with additional methoxy groups. The
results are very promising as for potential positive contrast agents. We performed
functionalization of the selected ligands by introdaction of a bioactivable fragment. The
bioactivation will transform the obtained compounds from the inactive “off” form into an
active “shining” smart contrast. The activation will be achieved by enzymatic hydrolysis. This
model may serve as a molecular probes in vitro studies - in imaging biological materials (e.g.,
tissue sections) or in vitro testing on smaller animals.
[1]. Lauffer, R.B., Chem. Rev., 1987, 87, 901.
[2]. Witkowicz J., Nephrol. Dial. Pol., 2009, 13, 10.
[3]. Kummerer K., Helmers E., Environ. Sci. Technol., 2000, 34, 573.
P5
SYNTHESIS AND PRELIMINARY EVALUATION OF ANTICANCER ACTIVITY
OF 2(5H)-FURANONE DERIVATIVES
A. Byczek a, A. Kasprzycka a, K. Walczak a
a
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of
Chemistry, Silesian University of Technology
P6
1. Introduction
2(5H)-Furanone (Fig. 1) is a lead structure in many
biologically active substances of natural origin. They
include compounds such as penicillin acid, basidalin or
mucohalic acids (Fig. 1). The derivatives of 2(5H)furanone are currently a large group of compounds
showing varied biological activity: anti-inflammatory
[1,2], antioxidative [2,3], antiviral [4], antibacterial [5],
antifungal [6] and anticancer [3]. Our earlier research
confirmed biological activity of mucohalic acid
derivatives in research on protozoa [5] and cancer cells
[6,7].
The research began with functionalization of the 5hydroxyl group of 3,4-dichloro-5-hydroxy-2(5H)furanone by transforming it into good leaving group
(ester group or chloride ion), and then obtaining amino
derivatives of mucochloric acid (Fig. 2).
The results acquired so far allow us to say that 3,4dichloro-5-(2’-hydroxyethyloamino)-2(5H)-furanone
presents the lowest cytotoxicity in relation to regular
cells with simultaneous inhibition of cancer cell
(HCT 116 wt) growth at 1,78 µM.
P6
O
O
O
O
OH
X
2(5H)-furanone
O
O
X: Cl, Br
X
mucohalic acid
OH
CH2
O
O
O
CH3
OCH3
NH2
penicillin acid
basidalin
Fig. 1
O
O
O
O
Cl
NHR
NH 2R
R
Cl
R3
ArNH 2
Cl
O
O
R
R = Cl, C(O)OCH 2CH 3
1
R = aminoalkil
Cl
NHAr
Fig. 2
[1]. M. Alam et al., Eur. J. Med. Chem., 2009, 44, 2636-2642
[2]. V. Weber et al., Bioorg. Med. Chem., 2005, 13, 4552-4564
[3]. F. Bailly et al., Eur. J. Med. Chem., 2008, 43, 1222-1229
[4]. D. Iannazzo et al., Bioorg. Med. Chem., 2008, 16, 9610-9615
[5]. E. Gondela, K. Walczak, Eur. J. Med. Chem., 2010, 45(9), 3993-3997
[6]. M. Skonieczna, E. Gondela, K.Z. Walczak, Badania aktywności w odniesieniu do linii
czerniaka, 2011 (non-published data)
[7]. A. Byczek, A.E. Kasprzycka, K.Z. Walczak, Synthesis and rating of biological activity of
3,4-dichloro-2(5H)-furanone derivatives, 2014 (non-published data)
1
ANTIPROLIFERATIVE EFFECTS OF NEW PALLADIUM(II)
COMPLEXES WITH CURCUMIN ANALOGUES ON COLORECTAL
CANCER CELLS
N. Miklášová a, E. Fischer-Fodor b, R. Mikláš a, J. Kožíšek c, O. Soritau b, J. Valentová a,
F. Devínsky a
a
P7
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University in
Bratislava, Slovakia
b
Radiobiology and Tumor Biology Department, I. Chiricuţă Cancer Institute, Cluj-Napoca,
Romania
c
Department of Physical Chemistry, Faculty of Chemical and Food Technology, Slovak
University of Technology, Bratislava, Slovakia
1. Introduction
Transition metal complexes of curcumin and its analogues exhibit various biological
interests. Our work is focused on palladium complexes with curcuminoids and thier use as
potential antiproliferative agents. Palladium complexes with curcumin [1, 2] exhibit
antitumoral and oxidant properties on human prostate cancer cells. Our studies [3] on
palladium complexes with curcuminoids having significant antineoplastic effects, encouraged
us to investigate their activity on colorectal cancer cell lines, where platinum-based
chemotherapy has limited efficiency.
Our research is conducted on the synthesis and
biochemistry of palladium(II) complexes containing
diarylheptanoids. Complexes were synthesized by
reacting the curcuminoid with a corresponding
precursor palladium complex in an equimolecular ratio.
The biological evaluations of palladium(II) complexes
performed in vitro on human cell lines (DLD-1 and
HT-29 colorectal cancer cells) proved their significant
inhibitory effect against human colorectal cancer cell
growth, being able to trigger early and late apoptotic
processes in the cells. Synthesized palladium
complexes are able to diminish the CD133 (prominin1) in HT-29 cancer cell line and inhibit the stem cell
factor in the treated cancer cell lines.
Acknowledgement
We gratefully acknowledge financial support from the Slovak Research and Development
Agency under the contract No. APVV-0516-12
[1]. A. Valentini, F. Conforti, A. Crispini, A. De Martino, R. Condello, C. Stellitano, G.
Rotilio, M. Ghedini, G. Federici, S. Bernardini, D. Pucci, J. Med. Chem. 52 (2009) 484-491.
[2]. D. Pucci, R. Bloise, A. Bellusci, S. Bernardini, M. Ghedini, S. Pirillo, A. Valentini, A.
Crispini, J. Inorg. Biochem. 101 (2007) 1013-1022.
[3]. Natalia Miklášová, Roman Mikláš, Ferdinand Devínsky, PCT/SK2014/050004.
P7
STYRYLQUINOLINE DERIVATIVES AS A BIOEFFECTORS AND FLUORESCENT DYE
E. Spaczyńska a, W. Cieślik a, A. Mrozek-Wilczkiewicz b, R. Musioł a
a
Department of Organic Chemistry, University of Silesia in Katowice, Poland
Department of Solid State Physics, University of Silesia in Katowice, Poland
b
P8
1. Introduction
Quinoline moiety is present in many compounds with broad biological activities. A
number of them have been widely investigated and clinically used as antifungal or
antibacterial agents [1,2]. Styrylquinoline derivatives have gained strong attention recently
due to their activity as perspective HIV integrase inhibitors [3]. Some quinoline-based
compounds have been reported as potent antitumor agents [4].
Novel styrylquinoline derivatives were tested
for their in vitro antitumor activity. Cellular
proliferation was determined using MTS assay
against the human colon carcinoma (Hct116)
cell lines with wild type p53 (p53+/+) and with
a p53 deletion (p53-/-). The compounds were
also tested for their cytotoxicity against murine
melanoma cell line (B16-F10) and normal human dermal fibroblasts (NHDF). Several
styrylquinoline analogues were found to have markedly greater antiproliferative activity than
doxorubicin. Styrylquinoline derivatives inhibit the proliferation of tumor cells and thus can
be used as anticancer drugs. Moreover, spectroscopic characterization for the quinoline was
performed. Several quinoline derivatives were found to have interesting fluorescent
properties. A group of quinoline analogues showing good potential as candidates for imaging
agents.
Acknowledgements:
Ewelina Spaczyńska appreciates the support of the DoktoRIS studentship.
[1]. Roth H.J., et al., Arzneistoffe, 2000, 51-114.
[2]. Harris C.R., et al., Current Medicinal Chemistry, 2004, 11, 2213-2243.
[3]. Polanski J., et al., Journal of Medicinal Chemistry, 2002, 45, 4647-4654.
[4]. Musiol R., et al., Bioorganic Medicinal Chemistry, 2007, 15, 1280-1288.
P8
STUDIES ON THE INTERACTIONS BETWEEN
4-HYDROXY-TEMPO AND ESCULETIN
K. Żamojć a, M. Zdrowowicz a, D. Wyrzykowski a, D. Jacewicz a, L. Chmurzyński a
a
Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk
P9
1. Introduction
4-hydroxy-TEMPO is a stable membrane permeable nitroxide radical, which effectively
protects cells and tissues from damages associated with oxidative and nitrosative stress
conditions. It has been proven that 4-hydroxy-TEMPO interacts with nitrogen dioxide (one of
the compounds responsible for an oxidative stress) and decreases its concentration in cells
even 500 times [1, 2]. Profluorescent nitroxides have been reported previously as probes for a
detection of free radicals as well as damages mediated by these species [3, 4]. On the other
hand there is no literature data on fluorescent sensors (which display the fluorescence
enhancement) used for the detection of nitroxides, such as 4-hydroxy-TEMPO. We report
herein esculetin (6,7-dihydroxycoumarin) as a selective fluorescent sensor for 4-hydroxyTEMPO.
2. Results and discussion
The UV absorption and fluorescence emission spectra of different coumarins were
recorded in aqueous solution at the temperature of 25 °C in the absence of 4-hydroxy-TEMPO
and in the presence of the increasing amounts of that radical. In case of nine coumarins
studied (7-hydroxy-4-methylcoumarin, 5,7-dimethoxycoumarin, 7-amino-4-methyl-3coumarinylacetic
acid,
7-ethoxy-4-methylcoumarin,
7-methoxycoumarin,
7hydroxycoumarin,
7-hydroxy-4-methyl-3-coumarinylacetic
acid,
6,7-diacetoxy-4methylcoumarin, 7-amino-4-methylcoumarin) the same effect under the action of 4-hydroxyTEMPO was observed – the fluorescence intensity decreased. Only in case of 6,7dihydroxycoumarin after an addition of 4-hydroxy-TEMPO its fluorescence intensity
increased and a small batochromic shift was observed. We state that the unique fluorescence
enhancement and the slight red shift attribute to the formation of 6,7-dihydroxycoumarin
dimer. The product was determined with the use of HPLC coupled with mass spectrometry.
As 6,7-dihydroxycoumarin was found to interact with 4-hydroxy-TEMPO selectively and
specifically it may be applied in measurements of that TEMPO derivative concentration.
Acknowledgements:
This work was supported by the Polish National Science Center (NCN) under the Grant
No. 2012/07/B/ST5/00753 and by grant for Young Scientists 2014 from University of Gdansk
(538-8232-B342-14).
[1]. Dąbrowska, A.; Jacewicz, D.; Chylewska, A.; Szkatuła, M.; Knap, N; Kubasik-Juraniec,
J.; Woźniak, M.; Chmurzyński, L. Curr. Pharm. Anal. 2008, 4, 183.
[2]. Dąbrowska, A.; Jacewicz, D.; Łapińska, A.; Banecki, B.; Figarski, A.; Szkatuła, M.;
Lehman, J.; Krajewski, J.; Kubasik-Juraniec, J.; Woźniak, M.; Chmurzyński, L. Biochem.
Biophys. Res. Comm. 2005, 326, 313.
[3]. Blinco, J.P.; Keddie, D.J.; Wade, T.; Barker, P.J.; George, G.A.; Bottle, S.E. Polym.
Degrad. Stab. 2008, 93, 1613.
[4]. Aspée, A.; Maretti, L.; Scaiano, J.C. Photochem. Photobiol. Sci. 2003, 2, 1125.
P9
COMPUTATIONAL STUDY OF THE HCV RNA
M. G. Branilović a, S. Tomić a
a
Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Bijenička 54
P10
1. Introduction
The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA drives
noncanonical initiation of protein synthesis necessary for viral replication. In order to fulfill
its role in HCV translation its domain II should adopt an L-shaped conformation. We
performed detailed molecular dynamics (MD) simulations for the ligand free RNA and its
(native and mutated) complexes with the potential HCV inhibitors, with and without the
presence of magnesium ions.
We have shown that upon ligand removal the
IRES IIa subdomain conformation changed
from extended into the L-shaped one during
several tens of ns MD simulation. Differently,
binding of the benzimidazole translation
inhibitors locked IIa in the extended
conformation (Figure 1). We also performed
MD study for the IIa in complex with newly
discovered
translation
inhibitor
diaminopiperidine (DAP) for which is
experimentally suggested that locks the IIa
RNA switch in the bent conformation and
inhibits IRES function perhaps by preventing
ribosome releas [1].
The starting points for our calculations were the three-dimensional structures of the IRES
subdomain IIa determined by X-ray diffraction (34 nucleotides) (PDB ID code 3TZR) [2] and
by NMR spectroscopy (38 nucleotides) (PDB ID code 2KTZ) [3]. In both of them the
subdomain IIa is complexed with a benzimidazole translation inhibitor 2. Since there are no
crystallographically determined structure of the complexes with ligand 1, analog 3 and
inhibitor DAP, we constructed them and performed molecular modeling calculations. Besides
MD, we determined changes in the inhibitor structure and electronic charge distribution at the
presence of magnesium cations and RNA using QM/MM approach.
[1]. S. M. Dibrov, J. Parsons, M. Carnevali, S. Zhou, K. D. Rynearson, K. Ding, E. Garcia
Sega, N. D. Brunn, M. A. Boerneke, M. P. Castaldi and T. Hermann, J. Med. Chem., 2014,
57, 1694-1707.
[2]. S. M. Dibrov, K. Ding, N. D. Brunn, M. A. Parker, B. M. Bergdahl, D. L. Wyles and T.
Hermann, P Natl Acad Sci USA, 2012, 109, 5223-5228.
[3]. R. B. Paulsen, P. P. Seth, E. E. Swayze, R. H. Griffey, J. J. Skalicky, T. E. Cheatham, 3rd
and D. R. Davis, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 7263-7268.
P10
DNA-BINDING PROPERTIES AND ANTICANCER EFFECTS OF SELECTED TACRINE
DERIVATIVES
J. Janočková a, S.Hamuľaková b, D. Kučerová c, J. Kovaľ c, R. Jendželovský c, P. Fedoročko c,
J. Kašpárková d. V. Brabec d, P. Kristian b, M.Kožurková a
a
Department of Biochemistry,
Department of Organic Chemistry, Institute of Chemistry,
c
Department of Cell Biology, Institute of Biology and Ecology, Pavol Jozef Šafárik University
in Košice, Slovakia,
d
Department of Molecular Biophysics and Pharmacology, Institute of Biophysics in Brno,
Academy of Sciences of the Czech Republic
b
P11
P11
1. Introduction
One of the main molecular targets in the design of anticancer drugs is DNA. A number of
studies have shown that many anticancer agents could bind to DNA or prevent the proper
relaxation of DNA through the inhibition of topoisomerases. Interactions between small
molecules and DNA lead to DNA-damage in cancer cells and the increased inhibition of
cancer cells thereby resulting in apoptosis or cell death [1]. Several acridines and tacrines
have been shown to intercalate into DNA bases in order to slow down DNA replication and
also to poison topoisomerase I/II [2], and so our study analyses tacrine derivative in order to
establish whether this compound exhibits similar effects.
This research work focuses on the study of the binding properties of selected bis-tacrine
derivatives and their anticancer effects. A series of novel bis-tacrine derivatives consisting of
two tacrines linked with urea and various length of an alkyl chains (ethyl-, propyl-, butyl-,
pentyl-, hexyl-) was synthesized. Drug-DNA interactions can be classified into two main
categories – intercalation between base pairs and minor/major groove binding [2].
Spectroscopic techniques (UV-Vis, fluorescence, circular and linear dichroism) and
viscosimetry were used to establish the types of DNA interactions which had occurred.
Binding constants were determined from UV-Vis spectroscopic titrations (K = 0.5×106 –
3.5×107 M-1). The experimental results show that the studied derivatives bind to DNA through
intercalation. The biological activities of the derivatives were determined using MTT-assay
and flow cytometric methods (detection of mitochondrial membrane potential, measurement
of cell viability) after 24, 48 and 72h incubation with human leukemic cancer cell line HL60.
Pentyl- (4) and hexyl- (5) derivatives proved to be the most cytotoxic of the studied
compounds.
Acknowledgments:
Poster is the result of the Project implementation: KVARK - quality education and skills
development for doctoral and post-doctoral students of P. J. Šafárik University in Košice,
ITMS: 26110230084, supported by Operational Programme Education funded by the ESF,
VVGS-PF-2014-435, MediPark Košice (ITMS: 26220220185), and VEGA 1/0001/13.
[1]. Sirajuddin M., Ali S., Badshah A., J. Photochem. Photobiol., B, 2013, 214, 1-19.
[2]. Mansouri A., et al., Hepatology 2003, 38, 715-725.
NOVEL ACRIDINE-NUCLEOSIDE CONJUGATES AND THEIR BIOLOGICAL ASSAYS
TOWARDS ANTICANCER AND ANTIMALARIAL ACTIVITIES
N. Kleczewskaa, F. Ramiandrasoa b, S. Pethe b, L. Celewicz a
a
Department of Chemistry, Adam Mickiewicz University Poznań, Poland
Laboratory of Bioorganic and Bioinorganic Chemistry, ICMMO,Paris-Sud XI University,
France
b
P12
1. Introduction
Recently reports are showing a significant role of the application of the ‘click chemistry’ as
a pathway to obtain drugs or substances with interesting biological features. A revelatory
approach to combine two scaffolds which each of them expose interesting mechanism of
action. Nucleoside component incorporates directly into the DNA strand and acridine
derivatives can be define as potential intercalators [1,2].
Figure 1
An approach based on the copper(I) catalyzed Huisgen azide–alkyne cycloaddition enable
to obtain series of conjugates which were prepared employing 3’-azido-3’-deoxynucleoside
(AZT 1) as the azide component and 2-methoxy-6,9-dichloroacridine propargylamine
derivatives (2 a-d) as the alkyne components [3]. All obtained conjugates were tested for
cytotoxic and antimalarial activity.
[1]. Celewicz L., Jóźwiak A., Ruszkowski P., Laskowska H., Olejnik A., Czarnecka A.,
Hoffmann M., Hładoń B. Bioorg. Med. Chem., 19, 6375 (2011).
[2]. Guetzoyan L., Xiao-Min Y., Ramiandrasoa F., Pethe S., Rogier Ch., Pradines B., Cresteil
T., Perrée-Fauvet M., Mahy.J. P., Bioorg. Med. Chem. 17, 8032 (2009).
[3]. Meldal M., Tornoe C. V., Chem. Rev. 108, 2952 (2008).
P12
SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE NEW
TOCOPHEROL ANALOGUE: DNA-TOCOPHEROL INTERACTIONS
J. Janočková a, G.V. Donchenko b, O.I. Kuzmenko b, M. Kožurková a
a
Department of Biochemistry, Institute of Chemistry, P.J. Šafárik University in Košice,
Slovakia,
b
Department of Vitamins and Coenzymes Biochemistry, Palladin Institute of Biochemistry of
National Academy of Sciences of Ukraine, Kiev, Ukraine
Novel tocopherol analogues were synthesized and their binding affinity with calf thymus
DNA was investigated using a variety of spectroscopic techniques including UV-Vis,
fluorescence and CD spectroscopy. The fluorescence spectra of exhibited a decrease in
fluorescence following the addition of calf thymus DNA to tocopherol analogue 1 (Fig.).
Upon addition of ctDNA, the fluorescence of samples 2, 3 and 4 was quenched and the results
became much weaker, indicating that no direct interaction between DNA and compounds had
occurred. CD spectra were measured at fixed
300
concentrations of ctDNA. When compounds 1-4 were
incubated with ctDNA, the intensity of the CD signal
of ctDNA underwent visible changes in both the
200
negative and positive bands without any significant
change in wavelength. These results therefore suggest
100
that size of the side chain of tocopherol molecules and
hydroxyl group availability regulates its affinity to
DNA. This specificity should be taken into
350
400
450
consideration in the creation of drugs designed to
Wavelength [nm]
modulate, inhibit or activate DNA function.
Intensity [a.u.]
P13
1. Introduction
The search for new pharmacologically active drugs has led to the discovery of a wide
range of new DNA-binding drugs [1]. Tocopherol is a naturally occurring chemical element
which is found in a variety of foods. Pre-clinical cell culture and animal model studies suggest
that certain vitamin E forms show considerable potential as anticancer agents [2]. The aim of
this research is to study the interaction of a series of synthesized shortened side chain
tocopherol analogues (1-phenolic, 2-acetate forms) with DNA; α-tocopherol (3-phenolic, 4acetate forms) served as a control.
Figure 1
Acknowledgments:
This study was supported by Slovak Research and Development Agency (VEGA grants
No. 1/0001/13, APVV-0280-11) and NASU (grant No. 0112U002625).
[1]. Gurova K., Future Oncol. 5, 1685, 2009.
[2]. Yu W. et al., Mol. Nutr. Food Res. 53, 1573, 2009.
P13
THE LIGAND DRIVEN TOPOLOGICAL CHANGE OF G-QUADRUPLEX
P. Krafčíková a, P. Tóthová a, V. Víglaský a
a
Department of Biochemistry,Institute of Chemistry, Faculty of Science, University of P.J.
Safarik, Moyzesova 11, Sk-040 01, Košice, Slovak Republic
P14
1. Introduction
G-quadruplexes are four-stranded motifs located in G-rich nucleic acids. G-rich sequences
which are able to form G-quadruplex structures were found in human telomeres and also in
some other important regions of human genome (gene promoters). This structural motifs show
a high potential for use in therapeutic and diagnostic areas [1]. G-quadruplex structures can be
extremely stable, although the topology and stability of such structures depend on many
factors, including the length and sequence composition of the quadruplex motif, the size of
the loops between the guanines, strand stoichiometry and the presence of the cations [2].
Recently, there has been extensive research on the development of G-quadruplex ligands
performed throughout the world. Small molecule ligands that promote and stabilize Gquadruplex structures are used as potential anticancer agents [3].
In this work are used low molecular
weight ligands (e.g. thiazol orange,
BRACO, quinacrine) that interact with a
high affinity with G-quadruplexes. We
describe the effect of selected specific
ligands on the topology and stability of
various topological variant of Gquadruplexes. The main aim of this work
is to clarify the effect of different
ligands, as well as the mode of binding
with the DNA.
Our results were obtained by using UV-Vis absorption spectroscopy and circular
dichroism. Circular dichroism provides information of ligands interaction and their influence
on the topology of G-quadruplexes. In addition, the method allows analyzing the stabilization
and destabilization effects of various ligands with G-quadruplexes.
Acknowledgments:
This work was supported by the Slovak Research and Development Agency under
contracts No. APVV-0280-11, Slovak Grant Agency (VEGA 1/0504/12, VVGS-PF-2013108) and Project implementation: KVARK - quality education and skills development for
doctoral and post-doctoral students of Pavol Jozef Šafárik University in Košice, ITMS:
26110230084, supported by the Operational Programme Education funded by the ESF.
[1]. Parkinson GN, Lee MP, Neidle S. Crystal structure of parallel quadruplexes from human
telomeric DNA. Nature, 2002, 417: 876-80.
[2]. Hardin CC, Perry AG, White K. Thermodynamic and kinetic characterization of the
dissociation and assembly of quadruplex nucleic acids. Biopolymers, 2000, 56: 147–194.
[3]. Kerwin SM. G-quadruplex DNA as a target for drug design. Current Pharma Design,
2000. 6: 441-471.
P14
DNA BINDING INTERACTION OF NOVEL BIOLOGICAL
INTERESTING CU(II) COMPLEX
D. Sabolová a, J. Kudláčová a, J. Kuchár b
a
Department of Biochemistry,
Department of Inorganic Chemistry, Institute of Chemistry, P. J. Šafarik University,
Moyzesova 11, 041 054 Košice, SK.
b
P15
1. Introduction
DNA plays a key role in the synthesis of proteins (gene expression) as well as its own
replication making it a potential target for drugs, especially for antiviral, antibiotic and
anticancer action. Thus, favorable DNA interaction patterns based on the study of small
molecules that bind to nucleic acids is one of the most important parameters in the screening
design for new drugs and development processes [1]. There are several types of sites in the
DNA molecule where binding of small complexes can occur: (i) between two base pairs
(intercalation), (ii) in the minor and major groove, (iii) on the outside of the helix [2]. The
design and construction of new drugs for use as structural probes in biological systems has
been an active area of research during the last 20 years [3]. Understanding the binding of
small molecules to DNA is potentially useful in developing design principles to guide the
synthesis of new improved drugs which can recognize a specific site or conformation of DNA
and to provide a good tool for biotechnology and molecular biology. In studying these
interactions, metal complexes are flexible reagents which offer the opportunity of exploring
the effect of the central metal atom, the ligands and the coordination geometry on the binding
event.
In this work, a new [Cu(phen)2Cl]Cl·CH3OH·4.5H2O complex has been synthesized and
characterized on the basis of elemental analysis, IR spectra and X-ray crystallography
method. Moreover, the DNA-binding properties of this Cu(II) complex were investigated by
electronic absorption, fluorescence, CD spectra and thermal denaturation studies.
Electronic absorption and fluorescence spectra are initially employed to determine the
binding mode of complexes to DNA. The intercalation of the chromophore into the double
helix usually results in the hypochromism. The former phenomenon can be ascribed to the
decrease of the π→π * transition energy as π * orbital of the intercalated compound couples
with the π orbital of the base pairs. The absorption spectrum of Cu(II) complex consist of one
well-resolved band with the maximum λmax=269 nm. Upon the addition of CT-DNA (calf
thymus DNA) the band showed moderate hypochromic effect and slight blue shift of his
maximum about 3nm. The KSV constant obtained from fluorescence displacement EB
(Ethidium bromide) from the DNA-EB complex is given by the slope of the Stern-Volmer
plot (2.2 × 104 M-1) . The observed quenching of DNA-EB fluorescence suggests that
investigated drug is able to interact with CT-DNA by the intercalative mode.
The melting of DNA is a phenomenon observed when the double-stranded DNA
molecules are heated and separate into two single strands; it occurs due to the disruption of
the intermolecular forces such as π - π stacking and hydrogen- bonding interactions between
the DNA base pairs. DNA melting experiments revealed that the Tm of calf thymus DNA
(68°C) increases in the presence of Cu(II) complex to within the range 81°C. An increase in
the helix stabilization is due to intercalation of Cu(II) complex into DNA.
P15
The experimental results show that the Cu(II) complex can bind to CT-DNA by
intercalation mode. Results obtained from our study will be helpful to understand the
mechanism of interaction of new complex with nucleic acids and should be useful in the
development of potential probes of DNA.
Acknowledgements:
This work was supported by the Slovak Research and Development Agency under the
contract No. APVV-0280-11, by VEGA 1/0001/13 and by VVGS-2013-127
[1]. P. Jaividhya, R. Dhivya, M.A. Akbarsha, M. Palaniandavar, J. Inorg. Biochem. 114
(2012) 94–105.
[2]. H.K. Liu, P.J. Sadler, Acc. Chem. Res. 44 (2011) 349–359.
[3]. M. Morshedi, H. Hadadzadeh, J. Fluoresc. 23 (2013) 259–264.
P15
P15
PHOTODEGRADATION OF DOUBLE STRANDED DNA LABELED
WITH 8-BROMO-2’-DEOXYGUANOSINE
M. Zdrowowicz a, M. Żyndul a, J. Rak a
a
Department of Chemistry, University of Gdańsk
P16
1. Introduction
Nucleoside analogs are potential, non-toxic candidates for the effective sensitizers of DNA
damage induced by UV and/or ionizing radiation. The structural modifications of nucleosides
should rely on the introduction of suitable substituents to nucleobases that increase
nucleosides’ sensitivity to degradation induced by photons or solvated electrons, being one of
the main products of water radiolysis. Thus, the incorporation of such modified nucleosides
into genome should lead to increase in the photo- and/or radiosensitivity of tumor cells.
The halogen derivatives of nucleobases are the most widely studied group of sensitizing
compounds. One of the best known DNA sensitizer is 5-bromo-2'-deoxyuridine. Also other
halogen derivatives of nucleosides like 5-bromo-2’-deoxycytidine, 8-bromo-2’dexyguanosine, and 8-bromo-2’-deoxyadenosine should possess similar properties. In the
current project, we show the photosensitizing ability of 8-bromo-2’-deoxyguanosine (8-BrdG)
using a double stranded 80 base pairs DNA fragment.
In order to synthesize the sensitized material, we employed an isothermal
amplification of DNA (using Bst 2.0 polymerase), where in the reaction mixture the standard
2’-deoxyguanosine triphosphate, dGTP, was substituted with 8-BrdGTP. Such prepared and
purified material has been irradiated in aqueous solutions using near-UV photons. The photoinduced damage of the labeled oligonucleotide was analysed by means of native and
denaturing electrophoresis, enzymatic digestion coupled to HPLC and mass spectrometry.
Our studies demonstrate unequivocally a significant photosensitivity of DNA labeled with 8BrdG.
Acknowledgements:
This work was supported by the Polish National Science Center (NCN) under the Grant
No. 2012/07/N/ST5/01877 (M.Z.)
P16
POSSIBLE ROLES OF CYSTEINE RESIDUES (CYS139 AND CYS320) IN THE
ACTIVITY OF AUXIN-AMIDOHYDROLASE BRILL2 FROM B.RAPA
A. Smolko a, F. Šupljika
B. Salopek-Sondi a
b
, J. Martinčić
a
, N. Jajčanin-Jozić
b
, I. Piantanida
b
,
a
Division of Molecular Biology, Ruđer Bošković Institute
Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute
b
P17
1. Introduction
Auxin-amidohydrolases (AAHs) from Chinese cabbage (Brassica rapa L.) (BrILL2 and
BrIAR3) hydrolase amino acid conjugates (AACs) of plant hormones auxins, thereby
releasing free active auxins. The mode of action of free auxins and their accumulation at sites
of physiological action are still not well established, but it is evident that AAHs play a role in
auxin homeostasis. So far, we established that BrILL2 and BrIA3 preferentially cleave longchain AACs (IPAala and IBAala), while Arabidopsis AAHs have higher specificity for
IAAala [1]. Furthermore, a potential active site of the auxin amidohydrolase BrILL2 was
proposed by modelling. AAHs possess two conserved cysteines of which one, Cys 139 is
suggested to be a part of the binding pocket that coordinates metal binding. Mn2+ was found
to be the only metal ion with cofactor activity, although number and positioning of the
cofactor in the binding pocket is still unclear. Herein we investigated the role of Cys residues
at positions 139 and 320 in the regulation of the enzyme activity.
The 3D structure of BrILL2 was modeled by using the
X-ray structure of A.thaliana IAA-AAH [2] as a template
(Figure 1.); the only amidohydrolase crystallized so far
with high primary sequence similarity (76%) to BrILL2.
The readily recognizable architecture of both AAHs is
characterized by two perpendicular domains, with the
larger catalytic domain bearing a proposed binuclear metal
center, and the smaller ‘‘satellite’’ domain, usually
functioning as as a polymerization site. Enzymes, wt and
mutants Cys139Ser, Cys320Ser, and Cys139,320Ser were
Figure 1.
generated and produced in E. coli as previously described
[1,3]. The preservation of structure of mutants in
comparison to wt enzyme was confirmed by circular
dichroism. Results of kinetics analysis show that mutant Cys320Ser remains active, but less
than wt. Cys139Ser mutation leads to a complete inactivation of the enzyme, probably due to
its role in the coordination of one of the Mn2+ ions. Further experiments by isothermal
titration calorimetry will give insight into the metal binding and the possible role of Cys139.
[1]. Savić, B., Tomić, S., Magnus, V., Gruden, K., Barle, K., Grenković, R., Salopek-Sondi,
B. (2009). Plant & Cell Physiology, 50(9), 1587–99.
[2]. Bitto, E., Bingman, C. A., Bittova, L., Houston, N. L., Phillips, G. N. J. (2009). Proteins,
74(1), 61–71.
[3]. Schuller, A, Ludwig-Müller, J. (2006). The New Phytologist, 171, 145–158.
P17
RAPESEED AMINOPEPTIDASES – ISOLATION AND PARTIAL
CHARACTERIZATION
J. Tolarczyk a, D. Gillner a, A. Sierant a
a
1. Introduction
Plant aminopeptidases play crucial role in several physiological processes such as protein
turnover, maturation and degradation. It has been proven that leucine aminopeptidases are
involved in the processes of aging, germination, plant defense and in meiosis [1].
Characteristics of enzymes isolated from cereals and other plants is a popular subject of the
latest research. Due to the key role rapeseed plays in polish economy the characterization of
isolated aminopeptidases from this crop is very important.
P18
Isolation and partial characterization of aminopeptidases from oilseed rape seeds has been
described. Purification procedure involved several steps including extraction with cold
hexane, precipitation with (NH4)2SO4, ion exchange chromatography and gel filtration.
Isolated aminopeptidase was characterised including temperature optima, pH optima, and
thermal stability. The enzyme isolated form rape seeds has a broad pH optimum range of 6.5 8.0, and optimum activity at 37-45°C, it is rapidly inactivated above this temperature.
Substrate specificity studies were also performed. Isolated enzyme cleaves preferentially AlapNA and Phe-pNA.
[1]. Hartl, M.; Merker, H.; Schmidt, D.; Baldwin, I. Optimized virus-induced gene silencing
in Solanum nigrum reveals the defensive function of leucine aminopeptidase against
herbivores and the shortcomings of empty vector controls. New. Phytol. 2008, 179, 356–365.
P18
MODIFICATION OF TRANSDERMAL PENETRATION OF DIFFERENT DRUGS WITH
ALAPTIDE AS POTENTIAL ENHANCER
A. Cernikova a, J. Jampilek a
a
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic;
P19
1. Introduction
Application of per-oral dosage forms faces some problems, e.g., irritation of
gastrointestinal tract by drugs, interactions with other simultaneously applied pharmaceutics
or food, incomplete absorption or presystemic elimination of the applied drug. Transdermal
administration of drugs represents an excellent alternative to conventional pharmaceutical
dosage forms. Drugs can be administered in the form of gels, creams, ointments and or via
transdermal patches. However, in case of transdermal drug delivery the problem of
insufficient or no penetration of active pharmaceutical substances through the skin arises.
Thus the process of permeation of drugs to the body is limited by the permeability of drugs
through the skin barrier, especially through the epidermis. Drug permeation can be improved
by using chemical permeation enhancers for transdermal drug delivery. These are compounds
facilitating the drug transfer through the skin, so their development is a subject of extensive
research in recent decades [1].
Alaptide (8-methyl-6,9-diazaspiro[4.5]decane-7,10-dione) was identified to be a potential
compound modifying transdermal penetration of drugs [2-4]. Alaptide is a cyclic dipeptide
that is capable to interact with components of the skin. It is an original Czech compound
prepared in the 80s of the 20th century [5]. Alaptide showed significant skin curative effect,
while any toxic, teratogenic or embryotoxic effects were not observed [2,6]. The aim of the
project was to investigate how both alaptide enantiomers affect the permeation of different
types of drug substances from various media, including final semi-solid pharmaceutical
compositions, through full-thickness pig ear skin using static Franz cells. Samples were
withdrawn at pre-determined time intervals and were immediately analysed by HPLC
methods. Based on the calculated characteristics (cumulative amounts of permeated drugs,
steady state fluxes and enhancement ratios) it can be concluded that both enantiomers
specifically enhanced permeation of administered drugs.
Acknowledgment:
This study was supported by the Czech Science Foundation – GACR P304/11/2246.
[1]. Jampilek, J.; Brychtova, K. Med. Res. Rev. 2012, 32, 907.
[2]. Jampilek, J.; Opatrilova, R.; Coufalova, L.; Cernikova, A.; Dohnal J. WO/2013/020527
A1, 2013.
[3]. Opatrilova, R.; Cernikova, A.; Coufalova, L.; Dohnal, J.; Jampilek, J. Sci. World J. 2013,
2013, Article ID 787283.
[4]. Opatrilova, R.; Jampilek J. ADMET 2014, 2, 56.
[5]. Kasafirek, E.; Vanzura, J.; Krejci, I.; Krepelka, J.; Dlabac, A.; Valchar, M. CS Pat.
231227, 1986.
[6]. Radl, S.; Kasafirek, E.; Krejci, I. Drugs Fut. 1990, 15, 445.
P19
NOVEL AMINO-Β-LACTAM CHOLESTEROL ABSORPTION
INHIBITORS
T. Dražića, K. Molčanov a, M. Malnar a, S. Hećimović a, V. Sachdev b, J. V. Patankar b,c, S.
Obrowsky b, S. Levak-Frank b, D. Kratky b, I. Habuš a
a
Ruđer Bošković Institute, Bijenička c. 54, HR-10002 Zagreb, Croatia,
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz,
Austria,
c
present address: Centre for Molecular Medicine and Therapeutics, Department of Medical
Genetics, University of British Columbia, Vancouver, Canada
b
OH
OH
OH
OH
H
N
P20
F
H
N
N
F
O
1
F
N
P20
O
2
F
Figure 1. The structure of compounds 1 and 2.
1. Introduction
Cardiovascular heart disease (CHD) is the leading cause of death in the world with high
blood cholesterol level being one of the major risk factors for its development. Therefore,
lowering serum cholesterol is among the main goals in CHD prevention and clinical
treatment. One of the possible pharmacological approaches for the treatment of CHD is the
application of cholesterol absorption inhibitors (CAI). Ezetimibe (Zetia, Ezetrol), approved in
2002, is the main representative of CAI [1]. As ezetimibe is the only CAI on the market
today, there is an increasing need for identifying novel active compounds. Here we report two
new ezetimibe bioisosteres 1 and 2 (Figure 1) and their diastereoisomeric mixtures 1/2 (70:30)
and 2/1 (85:15) which exhibit strong in vitro and in vivo CAI activity.
We prepared compounds 1 and 2 from enantiomerically pure trans-(3R,4R)-3-amino-βlactam, which was synthesized applying the chiral ester enolate-imine cyclocondensation.2
For the synthesis of 1 and 2 we pursued two approaches: 1) N-alkylation of amino-β-lactam
followed by stereoselective reduction of the side chain carbonyl group with methyl-CBScatalyst and 2) stereoselective reduction preceding N-alkylation.
The employment of the first synthetic pathway yielded diastereoisomeric mixtures 1/2
(70:30) when (R)-methyl-CBS-catalyst was used and 2/1 (85:15) with (S)-methyl-CBScatalyst. Pure diastereoisomere 2 was obtained by recrystallization of mixture 2/1 (85:15) in
dichloromethane, while pure 1 was not obtained with the first method. When the second
approach was applied, pure diastereoisomeres 1 and 2 were obtained in moderate yields.
pKa values of 1 and 2 were measured using spectrophotometric titration and were
determined to be 9.35 for both compounds. This finding indicates that they are in the form of
ammonium salt in the small intestine and blood.
We determined the cytotoxicity of compounds 1, 2 and 1/2 (70:30), as well as that of
ezetimibe in Madin-Darby Canine Kidney wild type (MDCKIIwt), MDCKII stably expressing
human NPC1L1 protein (hNPC1L1/MDCKII), and HepG2 cell lines using MTT cell
proliferation assay. New compounds had LC50 values higher than 100 µM and were
considered non-toxic, whereas LC50 values of ezetimibe were >100, 62 and 70 µM,
respectively.
In vitro cholesterol inhibitory activity of the new compounds was tested using
[ H]cholesterol in hNPC1L1/MDCKII cells. The compounds showed significant in vitro
activity. Maximum cholesterol uptake inhibition 50-55% was obtained at 120 µM
concentration. The IC50 values for compounds 1, 2, 1/2 (70:30) and 2/1 (85:15) were 72, 80,
60, and 71 µM, respectively.
3
P20
In addition, we determined in vivo acute cholesterol absorption inhibition in C57/BL6
mice using [3H]cholesterol. Mice were gavaged with 10 or 20 mg/kg/day of compounds 1, 2,
1/2 (70:30) or ezetimibe for 2 days, after which radioactivity was measured in plasma, liver
and small intestine. Novel compounds showed significant cholesterol absorption inhibition,
which was comparable to that of ezetimibe. Reductions of [3H]cholesterol in plasma for our
compounds were 50%, 32% and 64%, respectively, whereas ezetimibe caused 60% reduction.
New compounds had lower average [3H]cholesterol reduction in the small intestine (22%,
44% and 46%, respectively) compared to ezetimibe (58%). Radioactivity values in liver for 1
and 2 were decreased by 44% and 47%, respectively, and were comparable to ezetimibe
(52%). On the other hand 1/2 (70:30) decreased radioactivity in the liver by 70%.
Administration of 20 mg/kg/day dose of the compound did not further improve inhibition.
Both in vitro and in vivo results show that our new compounds 1 and 2 are strong
cholesterol absorption inhibitors. Furthermore, it appears that diastereomeres 1 and 2 improve
their inhibitory activity when they act as diastereoisomeric mixture (e.g. 1/2 (70:30)).
[1]. Burnett, D. A. β-Lactam cholesterol absorption inhibitors. Curr. Med. Chem. 2004, 11,
1873-1887.
[2]. Ojima, I.; Habuš, I. Asymetric synthesis of β-lactams by chiral ester enolate-imine
condensation. Tetrahedron Lett. 1990, 31, 4289-4292.
P20
PREPARATION AND IN VIVO PERMEATION OF DRUGS IN SILICA GEL-BASED
NANOCARRIERS THROUGH BLOOD–BRAIN BARRIER
J. Jampilek a, M. Oravec b, K. Zaruba c, P. Ulbrich c, P. Babula a, M. Kunes a, P. Suchy a
a
Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3,
612 42 Brno, Czech Republic;
b
Global Change Research Centre AS CR, Belidla 986/4a, 603 00 Brno, Czech Republic
c
Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic
e-mail of presenting author: e-mail [email protected]
P21
1. Introduction
The drugs that are targeted to the central nervous system are characterized by a very high
rate of failure due to the presence of the blood-brain barrier, which limits the entry of
xenobiotics into the brain. To minimise drug failure considerable efforts are now made to
design and prepare nanoscale materials with the aim of increasing drug bioavailability. Thus
engineered nanoparticles are at the leading edge of the rapidly developing field of
nanotechnology. Their unique size-dependent properties make them suitable for a wide range
of biomedical applications, since drugs can permeate through cell walls/membranes to cells
and tissues including brain. The application of techniques of nanoparticle preparation or
nanoparticle drug delivery allows to deliver many pharmacological agents to the site of action
[1-3].
Nootropics are drugs that purportedly improve mental functions, and brain is their
supposed site of action, nevertheless only some of them can reach it. To solve this problem,
clinically used drugs from the class of nootropics were processed to silica gel based
nanoparticles. The size of nanoparticles was evaluated by transmission electron microscopy,
and the content of drug substances was determined by elemental analysis. In vivo perfusion
experiments on rats were performed using bulk materials and silica gel-based nanoparticles.
Samples of brain tissues were analysed histologically (by microscope), and the concentration
of the drug substances in tissues was determined by means of UHPLC-DAD/HRMS LTQ
Orbitrap XL. It was found that the drug substances in silica gel-based nanoparticles permeated
through the blood brain barrier to brain tissue, whereas bulk materials were not detected in
brain in practice.
Acknowledgments:
This study was supported by GACR P304/11/2246 and by the Project CzechGlobe Reg.
No. CZ.1.05/1.1.00/02.0073.
[1]. Rao, C.; Muller, A.; Cheetham, A.K. The chemistry of nanomaterials, synthesis,
properties and applications; Wiley-VCH: Weinheim, Germany, 2005.
[2]. Bitar, A.; Ahmad, N.M.; Fessi, H.; Elaissari, A. Drug Discov. Today 2012, 17, 1147.
[3]. Passeleu-Le Bourdonnec, C.; Carrupt, P.A.; Scherrmann, J.M.; Martel S. Pharm. Res.
2013, 30, 2729.
P21
DISCOVERY OF RKI-123, NOVEL SYNTHETIC DERIVATIVE OF
GENISTEIN WITH PROAPOPTOTIC ACTIVITY IN COLON CANCER
CELLS
R. Kitel a, A. Rusin b, W. Szeja a
a
Department of Organic, Bioorganic Chemistry and Biotechnology, Silesian University of
Technology, Gliwice, Poland,
b
Center for Translational Research and Molecular Biology of Cancer, Maria SkłodowskaCurie Memorial Center and Institute of Oncology, Gliwice, Poland
e-mail of corresponding author: [email protected]
1. Introduction
Apoptosis, or programmed cell death, is multistage process leading to elimination of
unhealthy cells from organism. Evasion of apoptosis is a hallmark of many cancer types and
often plays crucial role in their resistance to conventional therapy [1,2]. Therefore
development of compounds triggering apoptosis of cancer cells represents an attractive
approach for novel anticancer therapy [3].
P22
In our drug discovery program we have screened small library of synthetic genistein
derivatives against their ability to promote apoptosis in HCT-116 cell line. This led us to
discovery of novel compound, shortly named RKI-123, which caused cell death of HCT-116
cancer cell line at concentrations significantly lower than genistein. RKI-123 increased
content of sub-G1 cells and caused dissipation of mitochondrial membrane potential as
indicated by FACS experiments. To gain insights into molecular mechanism of proapoptotic
action of RKI-123 we have performed proteomic profiling using antibody arrays against
kinases involved in cellular signaling. We found that induction of apoptosis by RKI-123 is a
result of activation of the c-Jun terminal kinase signaling axis. This results suggest that RKI123 exerts different mechanism of action than other genistein derivatives previously
investigated by us [4].
Taken together we have discovered novel proapoptotic small molecule compound RKI-123
able to triggering cell death in HCT-116 cell line. Our results clearly indicate that proper
derivatization of natural compounds may lead to more potent derivatives with novel
mechanism of action.
[1]. Fernald, K., Kurokawa M., Trends in Cell Biology 23 (2013) 620-633
[2]. Kasibhatla, S., Tseng B., Molecular Cancer Therapeutics 2 (2003) 573-580
[3]. Ghobrial, I.M., Witzig, T.E., Adjei, A.A., CA Cancer Journal for Clinicans 55 (2005)
178-194
[4]. Rusin A., Zawisza-Puchałka J., Kujawa K., Gogler-Pigłowska, A., Wietrzyk, J.,
Świtalska, M., Głowala-Kosińska, M., Gruca, A., Szeja, W., Krawczyk, Z., Grynkiewicz, G.,
Bioorganic and Medicinal Chemistry 19 (2011) 295-305.
Acknowledgement
Project was supported by the Ventures Program (VENTURES/2012-9/6) of the
Foundation for Polish Science, run by the Foundation thanks to the acquisition of EU funds
under Operation 1.2 "Improvement of science human potential" Innovative Economy
Operational Program 2007-2013.
P22
GRAMINE DERIVATIVES - SYNTHESIS, PHYSICO-CHEMICAL PROPERTIES AND
BIOLOGICAL ACTIVITY
W. Kozanecka a, K. Hamze a, B. Jasiewicz a, L. Mrówczyńska b
a
Department of Chemistry,
Department of Biology, A. Mickiewicz University,
b
1. Introduction
Gramine is a natural indole alkaloid [1] and it exhibits wide pharmaceutical activities
similar to Ephedrine such as: relaxation of bronchial smooth muscle, vasorelaxation, blood
pressure elevation, relief of bronchitis nephritis and bronchial asthma [2]. Up to now, gramine
has been widely used as a pharmaceutical lead scaffold for constructing various biologically
active indole-containing compounds [3] because gramine analogues show effective
antibacterial, antiviral and anticancer properties [4]. Hence a few series of derivatives of
gramine were synthesized and the biological activity of these compounds was examined.
P23
In our research, gramine (1) and its derivatives: Nmethylgramine iodide (2) and (1-acetyl-1H-indol-3yl)methyl acetate (3) were the starting materials for
synthesis new functional analogues 4-12 (Figure 1).
All obtained gramine derivatives were characterized
by spectroscopies analysis (NMR, MS) and the
antioxidative properties have been investigated for
free-radical scavenging assay (DPPH model system).
The results indicated that the obtained derivatives
have showed the biological activity but compounds
9, 10 and 11 have the highest
antioxidative
activity (Figure 2).
The research is being continued and the cytotoxicity
activity of obtained compounds will be also
determined. The discovery and identification of new
highly effective agents with biological activity are
still an important endeavor in medicinal chemistry.
P23
Figure 1
[1] P. M. Dewick, Medicinal Natural Products (…),
Wiley, London 2002, pp. 346-349.
[2] S. Iwata, S. Saito, K. Kon-ya, Y. Shizuri, Y.
Ohizumi, Eur. J. Med. Chem., 432 (2001), 63–70.
[3] A. R. Katritzky, L. Khelashvili, M. A. Munawar,
J. Org. Chem., vol. 73 (2008), 9171–9173.
[4] S. Ke, L. Shi, X. Cao, Q. Yang, Y. Liang, Z.
Yang, Eur. J. Med. Chem., 54 (2012), 248-254.
Figure 2
NOVEL STILBAZOLIUM SALTS: SYNTHESIS AND
ANTIPROTOZOAL ACTIVITIES AGAINST ACANTHAMOEBA SPP.
M. Lukáč a, M. Garajová b, M. Mrva b, P. Chomanič a, L. Timko a, F. Ondriska c,
J. Karlovská d, F. Devínsky a
a
Department of Theory of Drugs, Faculty of Pharmacy, Comenius University, Bratislava,
Slovakia
b
Department of Zoology, Faculty of Natural Science, Comenius University, Bratislava,
Slovakia
c
HPL (Ltd.)Department of Parasitology, Microbiological Laboratory, Bratislava, Slovakia
d
Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University,
1. Introduction
Stilbazolium salts represent an interesting group of quaternary ammonium compounds.
They have been studied for their fluorescence and optical properties they also possess
antimicrobial activities [1].
P24
A series of stilbazolium salts
(Figure 1) bearing (E)-4-{2-[4-(N,N–
dimethyl-amino)phenyl]etenyl}pyridinium moiety has been prepared.
Quaternary nitrogen is conected with
alkyl
(cationic
compound)
or
[(alkylphosphinato)oxy]etyl
(zwitterionic
compound)
groups.
The prepared zwitterionic compounds represent alkylphosphonatocholines a new types of
organophosphorous alkylphosphocholines [2]. Alkyl chains are represented by methyl,
benzyl, and decyl groups. Physico-chcemical properties of prepared compounds were studied
by fluorescence spectroscopy and fluorescence microscopy. Their antiprotozoal activities
were studied on parasites of the genus Acanthamoeba.
Acknowledgment:
This work was supported by Grants APVV-0516-12, UK/123/2014, UK/263/2014 and
VEGA 1/0796/12.
[1]. Chanawanno, K. et al.: Eur. J. Med. Chem. 45 (2010) 4199–4208.
[2]. Lukáč, M. et al.: J. Fluorine Chem. 164 (2014) 10–17.
P24
SYNTHESIS OF NEW ARYLCARBONYLOXYAMINOPROPANOL
DERIVATIVES AS POTENTIAL CARDIOVASCULAR DRUGS
P. Marvanová a, P. Mokrý a, K. Odehnalová a, T. Padrtová a
a
Department of Chemical drugs, University of Veterinary and Pharmaceutical sciences,
Palackého 1/3, 612 42 Brno
1. Introduction
Antagonists of β-adrenergic receptors are widely used in therapy of many cardiovascular
indications because of their positive effects on cardiovascular system. Moreover, newly
synthesized compounds have also other beneficial effects such as vasodilatory activity,
antioxidant activity and ultrashort effect. These features can improve haemodynamic and
metabolic profile, prevent atherosclerosis complication or ischaemia-reperfusion injury and
can be used for treatment of urgent cases [1].
P25
The aim of this study was synthesis and evaluation
OH
of new (arylcarbonyloxy)aminopropanol derivatives
R1
O
O
NH
as potential β-blockers for treatment of cardiovascular
O
diseases and their complications.
R1: 2‐O‐CH3, 4‐O‐CH3, 2,6di‐O‐CH3
We synthesized 6 compounds via multi-step
R: ‐CH3, ‐CH2‐CH 3
synthesis. The compounds were hydrochlorides or
Fig.1
fumarates
of
3-[(2-aryloxyethyl)amino]-2O
R
O
hydroxypropyl 4-alkoxybenzoates (Fig. 1). The
synthesis of the compounds started from hydroxybenzoic acid through reaction with methoxyto ethoxyethyltosylate or methoxy- to ethoxyethylchloride giving appropriate substituted
acids. The following products, oxiran-2ylmethyl-4-(alkoxycarbonylamino)benzoate
intermediates, were prepared either via reaction of oxiran-2-ylmethanol with the acylchlorides
formed by thionylchloride treatment, or via the reaction of glycidyltosylate with the potassium
salt. The oxirane ring was then opened by addition of methoxyphenoxyethylamines prepared
by
Gabriel
synthesis
via
appropriate
2-bromoethoxybenzene
and
N(phenoxyethyl)phthalimide intermediates. Obtained bases were transformed to hydrochloride
or fumarate salts. The structure of final compounds was confirmed and the purity was verified
by the available methods of instrumental analysis (NMR, IR, HPLC, TLC). The final products
have not been described in literature so far and they are expected to have β-adrenolytic, αadrenolytic and antioxidant activity and ultrashort effect. The compounds will be tested for
their biological activity.
[1]. Tengler, J. et al. Synthesis of New (Arylcarbonyloxy)aminopropanol Derivatives and the
Determination of Their Physico-Chemical Properties. Cent. Eur. J. Chem. 2013, 11, 17571767
P25
EVALUATION OF ADHESIVE AND PRO- ANGIOGENIC PROPERTIES OF RUNITROIMIDAZOLE CONJUGATE IN CANCER AND ENDOTHELIAL CELLS.
O. Mazuryk a, F. Suzenet b, C. Kieda c, M. Brindell a
a
Department of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University,
Ingardena 3, 30-060 Krakow, Poland
b
Institute of Organic and Analytical Chemistry, University of Orléans, UMR-CNRS 7311, rue
de Chartres, BP 6759, 45067 Orléans Cedex 2, France
c
Centre de biophysique moléculaire, CNRS, rue Charles Sadron, 45071, Orléans Cedex 2,
France
P26
1. Introduction
Ruthenium polypyridyl complexes were firstly developed as structure- and site-specific
DNA probes and nucleus imaging agents in biological systems [1]. But despite their high
DNA binding constant (> 106 M-1) they demonstrate cellular internalization (staining of
cytoplasm) with limited nuclear accumulation in live cells [2]. Recent increase of interest in
those compounds is caused by a combination of their high cytotoxic properties and the unique
optic features, which make possible simultaneous visualisation and therapy of the cancer. By
attaching of the 2-nitroimidazole to the ruthenium polypyridyl unit a new theranostic agent
[Ru(dip)2(bpy-NitroIm)]Cl2 for treatment and imaging the hypoxic tissues was designed [3].
The exact mechanism of the action of the ruthenium complexes is remaining unknown. The
influence of ruthenium complexes on cell adhesion and mobility was checked to evaluate a
possible use of ruthenium complexes in treatment of metastasis [4].
The effect of nitroimidazole-ruthenium polypyridyl conjugate on the adhesive properties of
cells was examined using cancer 4T1 and endothelial MLuMEC cell lines. As a model system
for checking if at the primary site cells are able to detach from other cells to be transported
away, the resistance to trypsin has been measured. The exposure of 4T1 cells to
[Ru(dip)2(bpy-NitroIm)]2+ significantly increases percentage of adherent cells after trypsin
treatment, while no major increase is observed on endothelial cells. On the other hand, the
ability of treated cells to attach to new surface was inhibited both for cancer and endothelial
cell lines. Treatment with ruthenium complex also induced tube-like organization of
endothelial cells, that can be explained by pro-angiogenic properties of the investigated
ruthenium-nitroimidazole complex. This complex also caused decrease in mobility of
MLuMEC cells in wound-scratching assay. All these findings indicate significance of
ruthenium-nitroimidazole conjugate towards inhibition of metastasis.
[1]. Gill, M. R.; Derrat, H.; Smythe, C. G. W.; Battaglia, G.; Thomas, J. A. ChemBioChem
2011, 12, 877.
[2]. Puckett, C. A.; Barton, J. K. J. Am. Chem. Soc. 2007, 129, 46.
[3]. Mazuryk, O.; Maciuszek, M.; Stochel, G.; Suzenet, F.; Brindell, M. J. Inorg. Biochem.
2014, 134, 83.
[4]. Mazuryk, O.; Suzenet, F.; Kieda, C.; Brindell, M. in preparation 2014.
P26
PHOTOPHYSICAL AND BIOLOGICAL EVALUATION OF SERIES OF HOMOLEPTIC
POLYPYRIDYL RUTHENIUM(II) COMPLEXES.
M. Łomzik a, O. Mazuryk a, P. C. Gros b, M. Brindell a
a
Department of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University,
Ingardena 3, 30-060 Krakow, Poland
b
SOR, SRSMC, Nancy-Université, CNRS, Boulevard des Aiguillettes,
54506 Vandoeuvre-lès-Nancy, France.
P27
1. Introduction
Polypyridyl ruthenium complexes are very interesting class of compounds which by a
proper designing can combine properties needed for the treatment of the cancer as well its
visualization. The unique optical features (intense and long lifetime luminescence strongly
depending on the molecular oxygen concentration, emission close to near-infrared) together
with favourable biological properties (ability to pass cellular membrane, reasonable solubility
in aqueous media) give rise to their application in an optical imaging [1]. Recently many
ruthenium(II) complexes with bipirydyl derivatives ligands were tested as potential anticancer
agents, and their cytotoxicity varies greatly with IC50 from 0.7 µM for [Ru(tpy)(Nh)3]2+ (tpy
is 2,2':6',2″-terpyridine, Nh is Norharman) to more than 200 µM for ruthenium-coumarin
derivatives [2,3]. Therefore it is important to understand how to tune their activity and
recognise the most important parameters influencing their biological effect.
The studied ruthenium(II) complexes are presented in the Scheme. In general, to tune
electronic properties of the metal complexes 2,2'-bipyridyl ligands were modify by
pyrrolidine or pyrrol functional groups.
The impact of this modification on molar
absorption coefficient, luminescence properties
as well as lipophilicity has been investigated
and will be presented. Activity towards
biomacromolecules such as human serum
albumin and DNA were tested and it was found
that all the studied complexes possess very low
affinity toward DNA while they exhibit
relatively high affinity to albumin. The
cytotoxicity of these complexes strongly
depends on their uptake by cells and this
correlates with thier lipophilicity. The biological
effect of these complexes will be also disscused.
[1]. O. Mazuryk, M. Maciuszek, G. Stochel, F. Suzenet, M. Brindell, J. Inorg. Biochem. 134
(2014) 83-91.
[2]. Y. Chen, M.-Y. Qin, L. Wang, H. Chao, L.-N. Ji, A.-L. Xu, Biochimie 95 (2013) 20502059.
[3]. M.J. Li, K.M.C. Wong, C. Yi, V.W.W. Yam, Chem. Eur. J. 18 (2012) 8727-8730.
P27
SYNTHESIS OF A NOVEL 1,2,3-TRIAZOLES BASED ON A QUINOLINE SCAFFOLD
J. Mularski a, W. Cieślik a, R.Musioł a
a
Department of Organic Chemistry, Institute of Chemistry, University of Silesia, Szkolna 9,
40-006 Katowice, Poland
1. Introduction
Rational drug design could be a challenging discipline. Despite the recent impressive
growth of technology, a receipt for efficient drug discovery has not been found. Screening of
many drug candidates would be pointless if performed randomly. On the other hand,
struggling with a complexity of few fine selected molecular scaffolds is time consuming and
could be a dead end either. We stand for a midway, variance approach.
If pharmacophore is a source of bioinformation, then molecular scaffold is its
interpretation. Since the information could be encoded by means of diverse structures, a
toolbox of facile synthesis methods could be engaged. Proper stereoelectronic features of
bioactive molecule would be reached, as long as an optimized molecular recognition has not
been crushed.
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We investigated antifungal activity of quinoline derivatives of structures presented below.
An altered 1.3-dipolar cycloaddition protocol with copper catalyst [1], appeared effective for
synthesis of 1.4-disubstituted 1,2,3-triazoles.
R
1
R
R
R
2
R
3
N
4
R
N3
1
2
R
3
N
N
R
4
N
N
The triazole scaffold would be a bioisosteric to (E)-styrylquinolines analogues, which have
revealed antifungal activity [2]. Strong point of those bioisosteric approach is apparently
better pharmacokinetics and synthetic availability of the triazoles. Thus our aim was
exploration of bioactivity landscape and harvesting data for a computational processing in
order to facilitate the development process.
Acknowledge:
Research was supported by the NCN grant 2012/07/N/NZ7/02110.
[1]. Rostovtsev, V.; Green, L.; Fokin. V,; Sharpless, B. Angew. Chem. Int. Ed. 2002, 41,
2596-2599
[2]. Cieslik, W.; Musiol, R.; Nycz, J.; Jampilek, J.; Vejsova, M.; Wolff, M.; Machura, B.;
Polanski, J., Bioorg. Med. Chem. 2012, 20, 6960-6968
SYNTHESIS OF QUATERNARY AMMONIUM DERIVATIVES – POTENTIAL
ANTIARRHYTHMIC DRUGS
T. Padrtová a, P. Mokrý a, K. Odehnalová a, P. Marvanová a
a
Department of Chemical Drugs, University of Veterinary and Pharmaceutical Sciences Brno
1. Introduction
Antagonists of β-adrenergic receptors belongs thanks their positive cardiovascular effects
to the treatment of a number of cardiovascular indications. In the treatment of acute
conditions with the use of so-called ultra-short-acting β-blockers, the very short plasma halflife reduces the risk of adverse effects of conventional β-blockers [1]. In addition the βadrenolytic action of these compounds also exhibit antiarrhythmic effect. It remains even after
the conversion of these substances on the quaternary ammonium salt, while β-adrenolytic
activity is significantly reduced or completely lost, which ultimately leads to reducing the
adverse effects associated with β-adrenolytic activity [2].
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The aim of the project was the synthesis and structural analysis of a number of new
arylcarbonyloxyaminopropanol derivatives with potential β-adrenolytic and antiarrhythmic
activity with ultrashort action and their conversion to quaternary ammonium derivatives.
In the first part of the synthesis, the
preparation of tertiary amines was carried
out according to the Mokrý et al [3]. In the
second part, it was focused on the
conversion of tertiary amines to the
quaternary ammonium salts. There was
the N-alkylation of tertiary amine with
appropriate alkyljodide. The reaction
carried out in a microwave reactor with
the using of solvent. Within the present
project,
a series of new compounds of
type quaternary ammonium derivatives of
arylcarbonyloxyaminopropanols
have
been prepared. It was managed to optimizing the individual steps of the synthesis. The
structure of all final compounds was confirmed, and the purity was verified by available
methods of instrumental analysis (NMR, IR, HPLC). The synthetised compounds will be
tested for their biological activity.
[1]. BODOR, N. and P. BUCHWALD. Soft drug design: general principles and recent
applications. Medicinal Research Reviews. 2000, 20(1), 58-101.
[2]. FOKEN, H. et al. Synthese und pharmakologische Eigenschaften von Pranolium und
seinen optischen Isomeren. Pharmazie. 1990, 45(3), 180 – 183.
[3]. MOKRÝ, P. et al. Synthesis and pharmacological evaluation of novel potential ultrashortacting β-blockers
P29
BIOLOGICAL ACTIVITY OF CHITOSAN-SILVER NANOCOMPOSITES
A. Regiel-Futyra a, M. Kus-Liśkiewicz b, M. Arruebo c,d, G. Stochel a, A. Kyzioł a
a
Facultyof Chemistry, Jagiellonian University.
Faculty of Biotechnology, Biotechnology Centre for Applied and Fundamental Sciences,
University of Rzeszów.
c
Department of Chemical Engineering and Nanoscience Institute of Aragon (INA),
University of Zaragoza.
d
Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBERBBN, 50018 Zaragoza.
b
P30
1. Introduction
Nowadays, there is an urgent need for searching innovative biomaterials exhibiting
antibacterial activity against multi-drug resistant and biofilm forming bacterial strains [1].
Antibiotic resistance is forcing researchers to explore new alternative bactericidal agents
using materials with desirable properties as biocompatibility, biodegradability and nontoxicity against human cells. Among a number of nanotechnological strategies, the synthesis
of polymer-based metal nanoparticles and further bionanocomposite preparation draws a lot
of attention [2].
In the present investigation chitosan (CS) based silver
nanoparticles (AgNPs) and further composites were prepared.
Different variables have been analyzed in order to optimize the
bactericidal properties of chitosan films loaded with silver
nanoparticles. The main goal was to achieve a complete
bactericidal effect against antibiotic-resistant and biofilm forming
bacterial strains with simultaneous maintenance of low cytotoxic
towards mammalian cell lines.
The best results were obtained for materials
based on chitosan with the medium molecular weight and the
highest deacetylation degree where a fast reduction of silver
ions was favored (leading to smaller nanoparticle formation
and a homogenous NPs dispersion across the membrane) [3].
High antibacterial activity (Fig.1) and low cytotoxic effect
towards human keratinocytes were obtained.
Acknowledgements:
This work was supported by the National Science Centre through the PRELUDIUM
(2012/07/N/ST5/00157) project.
[1]. WHO. http://www.who.int/mediacentre/factsheets/fs194/en/ 2014.
[2]. Monica Potara, E. J., Annette Damert, Octavian Popescu, Valentin Canpean and Simion
Astilean. Nanotechnology 2011, 22, (135101 (9pp)).
[3]. Regiel, A.; Irusta, S.; Kyzioł, A.; Arruebo, M.; Santamaria, J. Nanotechnology 2013, 24,
(1), 015101.
P30
CAFFEINE DERIVATIVES – SYNTHESIS, SPECTROSCOPIC ANALYSIS AND
ANTIOXIDANT CAPACITY
A. Sierakowska a, B. Jasiewicz a, L. Mrówczyńska b
a
Department of Chemistry, A. Mickiewicz University, Umultowska 89b, 61-614 Poznań
Department of Biology, A. Mickiewicz University, Umultowska 89, 61-614 Poznań
b
1. Introduction
Coffee is well-known as a rich source of antioxidants, which can prevent an oxidative
stress. This kind of stress is induced by free radicals like reactive oxygen species. They
exhibit hard damages in human cells such as cancer, diabetes, cardiovascular and
neurogenerative diseases (e.g. Parkinson’s disorder). One of the main compounds present in
coffee is caffeine [1]. Caffeine, a central nervous system stimulant, enhances the effect of
certain anticancer drugs. This compound and its derivatives can modulate drugs against lung,
liver, uterine cervix and breast cancer [2]. Different modifications of caffeine are the best way
to discover new drugs. For example 8-substituted caffeine derivatives are very important
chemical compounds that possess pharmacological activity [3]. Moreover, antioxidant
capacity of caffeine created new way on treatment of compound from coffee brews.
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P31
The aim of our study was the
synthesis of a series of new 8-alkylthioand 8-alkylaminocaffeine derivatives in
order to determine the effect of
substituent on the antioxidant potencies.
C-8 substituted caffeine derivatives were
synthesised
by reaction
of 8bromocaffeine with an appropriate
diamine or sodium alkylthiolate (Scheme
1). The structure of new compounds
were verified by NMR and MS analysis.
The antioxidant potential of new
caffeine derivatives was evaluated by
DPPH assay.
Moreover, the capacity erythrocytes
of new caffeine derivatives to protect
human erythrocytes from oxidative hemolysis was evaluated. We hope that research on this
kind of caffeine derivatives should support to better understand the genesis of cancer or
Parkinson’s disorder.
Scheme 1. Two-step synthesis.
[1]. J. Bravo, L. Arillaga, M. Paz De Pena,
C. Cid, Food Chem. Toxicol. 60 (2013),
397-403;
[2]. S. Miwa, S. Yano, et al., J. Cell. Biochem. 114 (2013), 2454-2460
[3]. H. P. Booysen, C. Moraal, G. Terre’Blanche, A. Petzer, J. J. Bergh, Bioorg. Med. Chem.
20 (2012), 7507-7518
IDENTIFICATION OF NOVEL ALDOSE REDUCTASE INHIBITORS BASED ON
CARBOXYMETHYLATED MERCAPTO-TRIAZINO-INDOLE SCAFFOLD
M. Šoltésová Prnová a, I. Miláčková a, M. Staško a, M. Májeková a, M. Štefek a
a
Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences,
1. Introduction
The etiology of diabetic complications involves the polyol pathway which degrades
excessive glucose by aldose reductase (ALR2). On the basis of our previous work [1], where
we observed significant inhibition of ALR2 by 1-indole acetic acid, we searched for novel
ARIs (aldose reductase inhibitors) in databases of purchasable compounds. The experimental
set consisted of 19 compounds, of which 15 compounds had indole-1-acetic acid moiety as a
common fragment in their structure.
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Among the compounds studied, 5-carboxy-3-mercapto-1,2,4-triazino-[5,6-b]indole
(compound 13) was identified as the most promising inhibitor of ALR2 with IC(50) in
submicromolar region and selectivity factor relative to ALR1 around 400. Analysis of the
enzyme kinetics for 13 showed uncompetitive inhibition. Molecular "docking" identified key
interactions with specific amino acid residues of the ALR2 binding site. At the organ level,
compound 13 attenuated sorbitol accumulation in isolated rat eye lenses as a result of its
inhibition of glucose flux through the polyol pathway. This finding indicates that the drug is
readily taken up by the eye lens and interferes with cytosolic ALR2. The partition coefficient
logD of 13, recorded in the system of water/octanol at pH 7.4, gave value around -1.90.
Uptake of compound 13 into rat RBC seems good membrane penetration.
Molecular obesity indices, in addition to the structural parameters fitting „the rule of
three“, along with water solubility, points to an excellent „lead-likeness“ of compound 13,
with prospects of further structure optimizations to obtain eventually pharmacologically
applicable agents for prevention of diabetic complications.
[1]. Juskova M1, Majekova M, Demopoulos V, Stefek M.: Substituted derivatives of indole
acetic acid as aldose reductase inhibitors with antioxidant activity: structure-activity
relationship. Gen Physiol Biophys. 2011 Dec;30(4):342-9.
P32
SYNTHESIS OF STRUCTURAL ANALOGUES OF HEXADECYLPHOSPHOCHOLINE
AND THEIR AMOEBICIDAL AND FUNGICIDAL ACTIVITIES
L. Timko a, M. Garajová b, M. Mrva b, F. Ondriska c, M. Bukovský d, M. Lukáč a,
F. Devínsky a
a
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University,
b
Department of Zoology, Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia
c
HPL (Ltd), Department of Parasitology, Microbiological Laboratory, Bratislava, Slovakia
d
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius
University, Bratislava, Slovakia
1. Introduction
Hexadecylphosphocholine (miltefosine, HPC) is a lead compound of alkylphosphocholines
(APCs), which structurally belong to zwitterionic surfactants. APCs possess antineoplastic
[1], antifungal [2] and antiprotozoal [3] activities.
P33
A series of twelve alkylphosphocholines was synthesised. Prepared compounds are
structural derivatives of hexadecylphosphocholine. The compounds contain two alkyl chains
and have the same molecular formula as HPC. The series was prepared to determine how the
position and the length of alkyl chain influence biological activity. Synthesis was based on
phosphorylation of primary alcohol with POCl3 and subsequent reaction with choline
derivative.
Antiprotozoal and antifungal activities of prepared compounds were studied and compared
to activity of miltefosine. Studied protozoans were Acanthamoeba lugdunensis and
Acanthamoeba quina, which belong to genotype T4. Investigated compounds showed similar
or better activity than HPC. The most effective compound on both strains was the one with
chain of 15 carbons linked to amminium nitrogen. Fungicidal activity of the series was
studied on yeast Candida albicans. None of the compounds was more active than miltefosine.
Similar activity was observed only in compound with chain of 15 carbons linked to phosphate
group. These findings suggest possibly different mode of action of prepared compounds
against protozoans and yeast.
Acknowledgments:
This work was supported by the Slovak Research and Development Agency under the
contract No. APVV-0516-12 and grants UK/123/2014, UK/263/2014 and VEGA 1/0796/12.
This work is the result of the project implementation: Centre of Excellency in Security
Research supported by the Research and Development Operational Programme funded by the
ERDF Grant number: ITMS 26240120034.
[1]. Lukáč, M. et al.: Eur. J. Med. Chem. 44 (2009) 4970–4977
[2]. Obando, D. et al.: Bioorg. Med. Chem. 15 (2007) 5158–5165
[3]. Avlonitis, N. et al.: J. Med. Chem. 46 (2003) 755-767
P33
DEVELOPMENT OF (NON)COVALENT USP2 INHIBITORS BY
TRUNCATION-COVALENT DELETION STRATEGY
M. Tomala a, K. Magiera a, K. Guzik a, K. Kubica a, B. Musielak a, M. Leś a, T. A. Holak a
a
Department of Chemistry, Jagiellonian University
1. Introduction
The aim of the project is to design and synthesize covalent USP2 small molecular
inhibitors. The C-terminal sequence of ubiquitin a natural binder for USP2 will form the
starting point of the design. Analysis of the structure of ubiquitin bound to USP2 can identify
hydrogen bonds which are the key to design of specific and potent inhibitors.
P34
The USP2 hydrolase debiquitinates
polyubiquinated target proteins such as
MDM2, MDM4 and CCND1. USP2
deubiquitinates MDM2 without reversing
MDM2-mediated p53/TP53 ubiquitination,
therefore indirectly promotes p53/TP53
s
degradation, decreasing its activity. USP2
prevents MDM2 mediated degradation of
MDM4. USP2 promotes cyclin D1
stabilization by antagonizing ubiquitindependent degradation USP2 plays a role in
the G1/S cell cycle progression in normal
and cancer cells. Moreover, USP2 plays
Figure 1 Comparison of docked compound and
role
in
regulation
of
myogenic
ubiquitine-USP2 complex
differentiation of embryonic muscle cells
[1]. The design of inhibitors has been based
on the information available for complex of ubiquitin and USP2. The synthesis of the scaffold
compound comprises of three steps. The first is synthesis of isocyano-acetic acid methyl ester;
second the ammonium salt formation of cyanoacetic acid and finally Ugi-4CR reaction of
formaldehyde along with the substrates [2]. The library of compounds can be envisioned by
simple aminolysis with primary/secondary amines and α,β-aminoacids. The activity of
potential inhibitors against USP2 can be deterred in assay experiments by measuring level of
Gly-AMC fluorescence [3]. The marker is released from the complex of Ub-Gly-AMC as a
product of enzymatic reaction catalyzed by USP2. Ub-Gly-AMC complex essential for the
binding affinity tests, can be obtained by reacting an aldehyde derivative of ubiquitin with
compound HCl·Gly-AMC. The most active compounds will also be tested with application of
NMR spectroscopy to be finally examined on living cell lines also according to their eventual
toxicity.
[1]. Shan, J.; Zhao, W.; Gu, W. Molecular Cell 2009, 469, 36.
[2]. Dömling, A. Chemical Reviews 2005, 106, 17.
[3]. Bozza, W.P.; Liang, Q.; Gong, P.; Zhuang, Z. American Chemical Society 2012, 10075,
S1.
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DEVELOPMENT OF SMALL-MOLECULE INHIBITORS OF THE MDM2-P53
PROTEIN-PROTEIN INTERACTION: THREE-SUBSTITUTED DERIVATIVES OF
1,5-DIHYDRO-2H-PYRROLE-2-ONE
E. Wrona a, A. Hogendorf a, M. Tomala a, T. A. Holak a
a
Jagiellonian University, Faculty of Chemistry, Ingardena st. 3, 30-060 Cracow,
1. Introduction
The MDM2 and p53 proteins and their mutual interaction have been studied extensively in
terms of their potential for a non-genotoxic anticancer therapy. Damage or inactivation of p53
protein could cause cancer cells growth. In about 50% of cancer cells it is the overexpression
of MDM2 protein that inactivates the p53. Inhibiting the MDM2-p53 protein-protein
interaction restores the activity of p53 creates opportunity of a non-genotoxic anticancer
therapy development [1].
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P35
Figure 1. Structure of exemplary 1,5-dihydro-2H-pyrrole-2-one inhibitor for MDM2-p53 interaction
The five-membered heterocyclic ring that contains nitrogen atoms are amongst several
small-molecule inhibitors of the p53-MDM2 interaction known so far [2]. The structure of
presented inhibitors was established by structural analysis of the p53-MDM2 interaction
(Figure 1) [3].
Desired compounds was obtainted in 5 stage synthesis with overwall yield in range up to
45 %.1H-15N HSQC 2D NMR titration of the 15N labeled proteins with small molecule,
characterized these compounds as medium strengt inhibitors with KD in the range of 7-26 µM.
[1]. Chène, P. (2004) Mol. Cancer Res., 2, 20-28
[2]. Zak, K., Pecak, A., Rys, B., Wladyka, B., Dömling, A., Weber, L., Holak, T.A. and
Dubin, G. (2013), Expert Opin. Ther. Patents, 23, 425-448
[3]. Popowicz, G.M., Dömling, A. and Holak, T.A. (2011) Angew. Chem. Int. Ed., 50(12),
2680-2688
A COMPARATIVE STUDY OF DOXAZOSIN BINDING TO A- AND N-FORM OF HUMAN
SERUM ALBUMIN
M. Chudzik, J. Równicka-Zubik, J. Pożycka, A. Sułkowska
a
Medical University of Silesia, Faculty of Pharmacy, Departmeny of Physical Pharmacy,
Jagiellońska 4, 41-200 Sosnowiec, Poland,
1. Introduction
Doxazosin (DOXA) is a postsynaptic α1- adrenoreceptor antagonist used for the
treatment of hypertension and benigin prostatic hyperplasia. DOXA has gradual onset of
hypotensive effect and a longer half-life, which makes it possible to take once a day1. The
differences in the binding of doxazosin to N- and A-form of human serum albumin are an
important factor, which determines therapeutic dosage and toxicity of the test drug. To
indicate these differences fluorescence spectroscopy was used.
The analysis of fluorescence quenching HSA
and AHSA by doxazosin points that both forms of
albumin form a complex with doxazosin. The
Scatchard curves indicates the existence of two types
of cooperative - negative cooperativity for the
DOXA-HSA (λex 295 nm) and positive for systems
DOXA - HSA (λex 275 nm) and DOXA - AHSA
(λex 275 nm, λex 295 nm). The association constant
(Ka) obtained from fluorescence quenching of
albumin by DOXA demonstrated that doxazosin has
higher affinity for AHSA than for HSA. A grater red
shift of fluorescence emission maximum of AHSA
Figure 1
than HSA in the presence of DOXA was observed.
This suggests that the binding of DOXA to AHSA was accompanied by a stronger increase in
polarity around the fluorophores in comparison to HSA. The molecular docking experiment
indicate sites IIA and IB as binding sites of doxazosin to human serum albumin (Figure 1).
P36
Acknowledgements:
This work was supported by the grants of Medical University of Silesia: KNW-1001/K/4/0
[1]. Prys-Roberts, C., Farndon, J. R. World Journal of Surgery, 26(8), 1037-1042, 2002.
P36
X-RAY CRYSTAL STRUCTURES OF LYSOZYME AND CYTOCHROME C WITH A
OCTAANIONIC SULFONATE CALIX[4]ARENE
G. Brancatelli a, S. Geremia a
a
Department of Chemical and Pharmaceutical Sciences, University of Trieste,
1. Introduction
Small molecules that recognize protein surfaces specifically are important tools for modifying
protein interactions [1]. Supramolecular ligands, such as cucurbituril [2] and calixarene [3],
have been proved to interact efficiently with the protein surface, thank to their ability of
hosting the target molecule (or residue) in an hydrophobic cavity through non-covalent
interactions. In particular the first crystallographic structure of cytochrome c with a tetraanionic calix[4]arene reported by Crowley et al. in 2012 [4] has shown that the artificial
receptors binds the positively charged lysine residues present on the protein surface. In order
to gain some more structural information about the host-guest supramolecular recognition
mechanism of surface residues, we started a crystallographic study the complexes between
proteins, such as lysozyme and cytochrome c, with an octaanionic sulfonate calix[4]arene
(C4T4).
Rod-like crystals of lysozyme with the receptor C4T4 have
been obtained by co-crystallization of the protein in presence of a
tenfold excess of ligand and NaCl as precipitant at pH 4.5. In the
case of cytochrome, crystals were obtained with the same excess
of ligand, but by using PEG3350 as precipitant at pH 5.5. Data
collections carried out at the XRD1 beamline of the Elettra
synchrotron (Trieste, Italy) provided datasets with a resolution of
2.4 and 2.8 Å for lysozyme and cytochrome crystals,
respectively.
The crystallographic analysis revealed that the receptor binds
to the protein surface on different positively charged residues: in
the structure of lysozyme it hosts the Arg14, whereas the
favourite residue on cytochrome surface is the Lys87.
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A
B
Figure 1. A) Structure of lysozyme
with C4T4 hosting Arg14 and B)
Structure of cytochrome with C4T4
hosting Lys87.
[1]. L.-G. Milroy, T. N. Grossmann, S. Hennig, L. Brunsveld, C. Ottmann, Chem. Rev. 2014,
114, 4695−4748.
[2]. J. M. Chinai, A. B. Taylor, L. M. Ryno, N. D. Hargreaves, C. A. Morris, P. J. Hart, A. R.
Urbach, JACS 133, 2011, 8810–8813.
[3]. O. Danylyuk, K. Suwinska, Chem. Comm. 2009, 5799–5813.
[4]. R. E. McGovern, H. Fernandes, A. R. Khan, N. P. Power, P. B. Crowley, Nature
Chemistry 4, 2012, 527–533.
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REDESIGNING AN ANTIANGIOGENIC β -SHEET PEPTIDE, ANGINEX TO  α /β
PEPTIDE FOLDAMERS
Z. Hegedüs a,b, N. Imre a,b, A. Hetényi c, I. Makra d, É. Monostori d, T. Martinek a,b
a
Institute of Pharmaceutical Chemistry, University of Szeged, Hungary
Institute of Pharmaceutical Analysis, University of Szeged, Hungary
c
Department of Medical Chemistry,University of Szeged, Hungary
d
Lymphocyte Signal Transduction Laboratory, Institue of Genetics Biological Research
Center of the Hungarian Academy of Sciences, Hungary
b
1. Introduction
β-sheets have a potentially large interaction
surface, which can be useful to target solvent
exposed
protein-protein
interactions.
Unfortunately, their tendency to aggregation, poor
pharmacokinetics and degradation by proteases
limit their application.
Introducing β-amino acids in a peptide
sequence increases the stability against proteases
[1]. Our earlier results showed that, β [3] amino
acid substitution decreased the folding propensity
and aggregation of the β-sheet of the 33mer
antiangiogenic anginex, but biological activity
was maintained [2]. The decreased folding could
be explained by improper side chain orientation
and packing leading to lack of stabilizing
hydrophobic interactions.
Our new approach was to fix the conformation
with cyclic β-amino acid substitution and enhance
the overlap of side chains to promote hydrophobic
interactions. Various substitution patterns were
used to test these conformational effect, and
influence on the biological activity (Figure 1).
P38
Figure 1
Circular dichroism and NMR based structural
analysis revealed that substitution by cyclic β-amino acid is favourable to β-sheet formation
and folding propensity is dependent on the substitution position relative to the β-sheet core.
Hydrophobic interactions also can be scaled up and biomimetic behaviour can be retained.
Some of the analogs maintained biological activity measured by using endothel
proliferation assay.
[1]. Hook, D. F.; Bindschadler, P.; Mahajan, Y. R.; Sebesta, R.; Kast, P.; Seebach, D.
Chemistry & biodiversity 2005, 2, 591.
[2]. Hegedus, Z.; Weber, E.; Kriston-Pal, E.; Makra, I.; Czibula, A.; Monostori, E.; Martinek,
T. A. Journal of the American Chemical Society 2013, 135, 16578.
P38
STRUCTURAL INVESTIGATION OF SHORT-SEQUENCE
PEPTAIBOL MOLECULES
Z. Násztor a,b, J. Horváth a, B. Leitgeb a,c
a
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences,
Department of Medical Chemistry, University of Szeged,
c
Department of Microbiology, University of Szeged,
b
1. Introduction
Based on the sequence length of peptaibols, these molecules can be divided into two
distinct groups, namely the short-sequence peptaibols and the long-sequence ones. The
trichobrachin A (TB A), trichobrachin B (TB B), hypomurocin A (HM A) and trichorozin
(TZ) peptides, composed of 11 amino acid residues, are the representative members of the
group of short-sequence peptaibols. In this theoretical study, a comprehensive structural
investigation was performed by molecular dynamics methods for the peptaibol molecules
mentioned above.
In the case of short-sequence peptaibols, the backbone conformations were studied, and
different secondary structural elements (i.e. types I and III β-turns, as well as β-bend ribbon
spiral) were identified in certain tetrapeptide units of molecules. Nevertheless, the side-chain
conformations of amino acid residues were also examined, and the preferred rotamer states of
side-chains were determined. For the peptides, two types of distances were measured, as
follows: (1) the end-to-end distance between the N atom of the backbone NH group of Xaa1
amino acid and the C atom of the backbone CH2 group of Xaaol11 amino alcohol; (2) the
residue-residue distances between all the residue pairs of molecules. Based on the data
obtained by these structural investigations, the three-dimensional structure of short-sequence
peptaibols were characterized in detail, as well as they were compared to one another. Our
results indicated that the TB A, TB B, HM A and TZ peptides possessed characteristic
structural properties, however, it could be concluded that both conformational similarities and
dissimilarities could be observed for these peptaibol molecules.
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Acknowledgements:
For Balázs Leitgeb, this research was supported by the European Union and the State of
Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/211/1-2012-0001 „National Excellence Program – Elaborating and operating an inland student
and researcher personal support system convergence program”. This research was supported
by the Hungarian Scientific Research Fund (OTKA K 106000).
P39
STUDYING THE FOLDING PROCESSES OF LONG-SEQUENCE PEPTAIBOL
MOLECULES
Z. Násztor a,b, J. Horváth a, B. Leitgeb a,c
a
c
b
1. Introduction
Based on the sequence length of peptaibols, these molecules can be divided into two
distinct groups, namely the short-sequence peptaibols and the long-sequence ones. The
hypomurocin B (HM B) peptides consisting of 18 amino acid residues, as well as the
trichobrachin IIb (TB IIb) peptides composed of 20 amino acids belong to the group of longsequence peptaibols. In this study, molecular dynamics (MD) calculations were carried out on
the peptaibol molecules mentioned above, in order to explore their folding processes
comprehensively.
Based on the MD trajectories, the formation of various helical structures (i.e. α-, 310- and
left-handed α-helix) was studied, in the course of which, the alterations of helicity values were
investigated considering not only the entire sequence of peptides, but also each amino acid
residue. Furthermore, the formation of different intramolecular H-bonds evolved between the
backbone NH donor and CO acceptor groups was examined. For the H-bonds, the following
two groups were distinguished: (1) local H-bonds (i.e. the i←i+4 and i←i+3 H-bonds); nonlocal H-bonds (i.e. the i←i+n H-bonds, where n > 4; as well as all the i→i+n H-bonds). The
evolution of these H-bonds was studied with regard to the average numbers of H-bonds, as
well as to each i←i+4 and i←i+3 H-bonds, respectively. The results derived from the MD
simulations revealed that the appearance of local H-bonds was in agreement with the presence
of helical structures. Nevertheless, it was observed that the non-local H-bonds did not produce
important effects on the evolution of helical conformations. On the whole, the folding
processes were explored comprehensively for the HM B and TB IIb peptides, as well as the
typical structural and folding features of these long-sequence peptaibols were identified.
P40
Acknowledgements:
P40
CRYSTALLOGRAPHIC AND CALORIMETRIC STUDIES OF NEW Β-LACTOGLOBULIN
VARIANTS POSSESSING MUTATIONS AT N-TERMINUS AND INSIDE Β-BARREL
J. Loch a, A. Łach a, E. Ludwin a, M. Tworzydło b, P. Bonarek b, A. Polit b, M. Jabłoński a,
K. Lewiński a
a
Faculty of Chemistry, Department of Crystal Chemistry and Crystal Physics, Jagiellonian
University in Kraków,
b
Faculty of Biochemistry, Biophysics and Biotechnology, Department of Physical
Biochemistry, Jagiellonian University in Kraków
1. Introduction
Bovine β-lactoglobulin (βLG) is a protein belonging to lipocalin family. It has ability to
bind wide range of hydrophobic ligands and, as other lipocalins, βLG can be re-engineered to
gain high specificity to selected drugs. Two lactoglobulin variants possessing mutation inside
β-barrel: V43N and V92Q, have been designed and produced. Polar residues have been
introduced to the binding site to act as donors or acceptors of hydrogen bonds. Moreover, all
expressed βLG variants, wide type (WT) and both mutants, have modifications at N-terminus
(L1AI2S) that facilitate cleavage of N-terminal Met.
Recombinant βLG and its V43N and V92Q mutants were expressed in Origami B(DE3)
cells and purified by different protocols including several chromatography steps combined
with salting-out and SDS-PAGE electrophoresis. WT variant with mutation L1AI2S was
correctly folded, however as showed ITC studies, its ability to bind ligands was significantly
reduced. The stoichiometry of binding model ligand SDS for L1AI2S was in range 0.15 –
0.30. Crystal structure of L1AI2S revealed that binding site is occupied by tightly bound
endogenous aliphatic ligand that prevents binding other compounds. However, most of the
correctly folded protein had blocked β-barrel, it was possible to isolate proteins with empty
binding site that can form complexes with exogenous ligands.
Polar residues introduced to β-barrel destabilize lactoglobulin structure. βLG variants with
polar residues inside β-barrel were also susceptible for proteinase cleavage. However, we
were able to isolate the small amounts of correctly folded V43N variant, it was not possible
for V92Q. Interestingly, the obtained previously V92F variant was correctly folded, however
it also had reduced affinity to aliphatic ligands.
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P41
INFLUENCE OF THE PRESENCE OF NAF AND NACLO4 SALTS
ON THE THERMAL DESTABILIZATION OF TRP-CAGE MINIPROTEIN
Z. Násztor a,b, B. Leitgeb a,c, F. Bogár d
a
c
d
Supramolecular and Nanostructured Materials Research Group, University of Szeged,
b
1. Introduction
The Trp-cage miniprotein (TC5b) is a widely investigated short-sequenced polypeptide,
generally known for its temperature sensitive, protein-like spatial structure. Its fold contains
various secondary structure elements, a hydrophobic core with two aromatic rings of Tyr3 and
Trp6 "buried" within and a salt bridge is formed between the Asp9 and Arg16 residues. Its
compact and stable structure is originated from a set of stabilizing interactions which are
differently altered by temperature, pH change and Hofmeister-active salt addition. In this
study, we investigated the influence of kosmotropic F- and chaotropic ClO4- ions on the
stabilizing interactions and structural changes of the miniprotein during its thermal
destabilization.
P42
In our theoretical study, the Amber ff99SB-ILDN force field together with the TIP3P water
model were applied in three 600-ns-long REMD simulations for the miniprotein immersed in
neat water, in water with NaF and NaClO4 in 1 M final concentration. The REMD simulations
proved to be a suitable tool for the characterization of structural stability alterations at
different temperatures caused by the cosoluted salts. The folding-state of the Trp-cage
miniprotein was mainly characterized by the root mean square deviation (RMSD) of the
backbone heavy atoms and by the side chain heavy atom distances of residues participating in
essential structure stabilizing interactions. The helix content and RMSD values were
calculated for each residue separately, in the presence and in the absence of ions. The average
values of these quantities experienced consequent shifts in the presence of Hofmeister-active
salts, which were in accordance with the positions of the ions in the Hofmeister series. The
correlation of the identified structural changes were also investigated in the case of the two
salts and compared to the neat water case. As a result, significant differences were identified
between the thermal and the chaotropic ClO4- ion-induced destabilization processes.
Acknowledgements:
This work was funded by the grants: OTKA No. K101825, K101821, TAMOP-4.2.2.A11/1/KONV-2012-0047 and TAMOP-4.2.2.C-11/1/KONV-2012-0010.
P42
PARAMETERIZATION OF THE NON-STANDARD RESIDUES
OF PEPTAIBOLS
J. Horváth a, Z.Násztor a,b, B. Leitgeb a,c
a
c
b
1. Introduction
Peptaibols are fungal peptide antibiotics belonging to the family of antimicrobial peptides.
These molecules show a broad spectrum of biological activities comprising not only
antibacterial, antifungal and antiviral effects, but also immunosuppressive and neuroleptic
properties. The peptaibol molecules are composed of 5-20 amino acid residues, and they
contain several non-proteinogenic amino acids (e.g. α-aminoisobutyric acid and isovaline).
The N-terminal residues of these peptides are usually acetylated, as well as an amino alcohol
(e.g. leucinol, phenylalaninol and valinol) is linked at their C-terminus. In order to study the
three-dimensional structure and folding processes of peptaibols by means of different
theoretical methods, the above-mentioned non-standard residues should be parameterized.
In the course of present study, the following non-standard residues of peptaibols were
parameterized using quantum chemical calculations: (1) non-proteinogenic amino acids:
α-aminoisobutyric acid, β-alanine, isovaline, β-hydroxyleucine, hydroxyproline,
methylproline and pipecolic acid; (2) amino alcohols: alaninol, isoleucinol, leucinol,
phenylalaninol, prolinol, serinol, tryptophanol and valinol. For the parameterization, a
detailed quantum chemical protocol was developed to calculate accurately the partial atomic
charges with regard to the non-standard residues of peptaibol molecules. Subsequently,
applying the parameterized non-standard residues, various molecular dynamics simulations
were carried out on peptaibols, in order to assess the suitability of these residues for the
theoretical calculations. On the basis of results, it could be concluded that the partial atomic
charges, calculated for the non-proteinogenic amino acids and amino alcohols, proved to be
suitable to study the structural and folding properties of peptaibol molecules using molecular
modelling methods.
P43
Acknowledgements:
P43
Β-SANDWICH FOLDAMERS: A PROTEIN-SIZED MODEL
G. Olajos a, A. Hetényi b, T. A. Martinek a
a
Department of Pharmaceutical Analysis., University of Szeged
Department of Medicinal Chemistry, University of Szeged
b
1. Introduction
β-sheets and β-sandwiches play important roles in protein-protein interactions. Foldameric
α/β peptides were shown to successfully mimic monomeric β-sheet structures, but foldameric
β-sandwiches were not reported. Using betabellin 14, a 64-mer β-sandwich model protein as a
template, [1] we designed α/β peptidic foldamers with β-amino acid mutations in the
hydrophobic core of the structure.
P44
We substituted two central residues in each chain to β3 homologs and different alicyclic
amino acids in the monomeric and dimeric analogs of betabellin 14. (Figure 1.)
CD spectra analysis of the analogs showed decreased β-sheet content compared to
betabellin. The dimerization resulted in higher folding propensities, suggesting that the
inducible β-sandwich formation is maintained. Structural data at the residual level was
obtained through 1H, 15N, 13Cα and 13Cβ NMR secondary chemical shifts. Secondary
structure propensities were scored using the SSP software. In line with the CD data, the
analogs displayed increasing β-sheet content with dimerization, but the overall β-sheet content
was higher in the parent betabellin 14. The SSP score values at the mutation sites suggested
local destructuring effects for the β-residues.
MD simulations in explicit water revealed that the distorting effect was due to the
destabilization of the hydrogen bond network and the increased solvent exposure of the core.
The compatibility of the β-residues with the β-sheet structures depended on the side-chain
properties, and both simulations and experimental data suggested the cyclohexane side-chain
as the best choice.
[1]. Yan, Y. and B.W. Erickson. Protein Sci., 1994, 3, 1069-73.
P44
INTERACTIONS OF VINCA ALKALOID ANTICANCER DRUGS WITH
HUMAN SERUM ALBUMIN STUDIED BY MOLECULAR DOCKING
B. Pawełczak a, J. Równicka-Zubik a, A. Sułkowska a
a
Medical University of Silesia, Faculty of Pharmacy with Division of Laboratory Medicine,
Department of Physical Pharmacy, Jagiellońska 4, 41-200 Sosnowiec, Poland
1. Introduction
Vinca alkaloid derivatives are mitotic inhibitors widely used in the treatment of cancers
such as acute lymphoblastic leukaemia, nephroblastoma and burkitt’s lymphoma. The binding
of drugs to human serum albumin (HSA) is the one of many factors, that determines the
pharmacological actions and the side effects of drugs. The aim of the present study was to
compare the binding mechanism of natural (vinblastine, VLB) and semisynthetic (vinorelbine,
VIN) vinca alkaloid to HSA.
The interactions between vinca alkaloid
anticancer drugs and HSA (PDB ID: 1AO6 [1])
have been studied by means of molecular docking
simulation. Docking experiment was carried out
using computer programs including Marvin
Sketch [2], Molegro Virtual Docker [3] and
Discovery Studio [4]. Presented results shows,
that both vinblastine and vinorelbine may interact
with HSA mainly via hydrogen bonds and
hydrophobic interactions (Fig 1.). The lower
binding energy of VIN (–144,87 a.u.) than VLB
(–119,06 a.u.) suggested, that semisynthetic vinca
alkaloids binds to HSA stronger than natural. This
phenomenon can be explained by electrostatic Figure 1. Docking view showing interactions
interactions, which occur mainly between HSA
between vinblastine and HSA
and VIN.
P45
Acknowledgement:
This work was supported by grant KNW-1-001/K/4/0 from Medical University of Silesia,
Katowice, Poland.
[1]. S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Protein Eng., 1999, 12,
439–446.
[2]. Marvin 6.3.1, 2014, ChemAxon (http://www.chemaxon.com).
[3]. Molegro Virtual Docker (MVD) 2008.3.0. Molegro ApS.
[4]. Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.0, San
Diego: Accelrys Software Inc., 2013.
P45
NEW EPITOPES OF H. PYLORI UREASE
I. Relich a, B. Kolesińska a, J. Frączyk a, I. Konieczna b, M. Kwinkowski b, W. Kaca b,
A. Kaczmarek c, D. Timler c, Z. Kamiński a
a
Department of Organic Chemistry, Lodz University of Technology,
The Jan Kochanowski University (JKU) in Kielce,
c
Nicolas Copernicus Hospital in Lodz
b
1. Introduction
Bacterial infections are associated with numerous autoimmune diseases including
rheumatoid arthritis (RA), although, the mechanism of this processes still remains unclear.
The particular attention has received infections involving H. pylori because it has been
implicated in both organ specific and non-organ specific autoimmune disease [1]. It is
postulated that development of RA is the consequence of an erroneous immune response
against self-antigens that results in the damage and eventual dysfunction of synovial joints.
This phenomenon is called molecular mimicry and means cross-reactivity of immune reagents
both with host ‘self’ antigens and microbial determinants [2].
To establish a direct link between H. pylori infections and the development of rheumatoid
arthritis [3], we focused the studies on bacterial urease which was selected as an exocrine
pathogenic antigen produced in relatively large amount. The library of decapeptides,
overlapping the whole H. pylori urease consisting of α and β subunits was prepared according
to the SPOT methodology, immobilized on the cellulose with C-terminus via isocyanuric
linker. Peptides reacting with antibodies against Jack bean urease were identified and then
epitope mapping procedure selected the collection of epitopes which are able to form strong
immunological complex with antibodies against Jack bean urease. A set of new 20 epitopes
from α subunits and 15 from β subunits was identified. Library of this epitopes was used in
tests with antinuclear antibodies and anti–citrullinated protein antibodies present in RA
patients sera. In both cases, for all synthetic peptides it has been found significantly stronger
reactions for RA patients in comparison with healthy blood donors. Based on this preliminary
studies it is expected to develop a set of synthetic epitopes which would be helpful to
diagnosis RA and prognosis a risk of the disease.
P46
Acknowledgments:
This work was supported by MSE Poland NCN UMO-2012/05/N/ST5/01460
[1]. D. S Smyk, A. L Koutsoumpas, M. G Mytilinaiou, E. I Rigopoulou, L. I Sakkas, D. P
Bogdanos. World J. Gastroenterol. 20, 613 (2014)
[2]. M. C. Levin, S. Min Lee, F. Kalume, Y. Morcos, F. Curtis Dohan, K. A. Hasty, J. C.
Callaway, J. Zunt, D. M. Desiderio, J.M. Stuart, Nat. Med. 8, 509 (2002)
[3]. a) M. Arabski, I. Konieczna, D. Sołowiej, A. Rogoń, B. Kolesińska, Z.J. Kamiński, W.
Kaca, Clin. Biochem. 43, 115 (2010) b) I. Konieczna, P. Żarnowiec, M. Kwinkowski, B.
Kolesińska, J. Frączyk, Z. Kamiński, W. Kaca, Curr. Protein Pept. Sc., 789 (2012)
P46
BEGINNING OF DEORPHANIZATION OF SELECTED DHRS ENZYMES
L. Skarydova a, H. Stambergova a, T. Lundova a, A. Skarka a, B. Malcekova a, M. Šafr b,
V. Wsol a
a
Department of Biochemical Sciences, Faculty Of Pharmacy in Hradec Kralove, Charles
University in Prague, Czech republic
b
Institute of Legal Medicine, Faculty of Medicine, Charles University and University
Hospital in Hradec Kralove, Czech Republic
e-mail of presenting author:[email protected]
1. Introduction
Although human genome was solved more than 10 years ago, there is still significant
fraction of gene products that remain uncharacterized to date. Some of these proteins are
found also in a short-chain dehydrogenase/reductase (SDR) superfamily that contains many
patho/physiologically important enzymes that play a role in the development of some cancers
(e.g. 17β-HSD1), metabolic syndrome (e.g. 11β-HSD1) or biotransformation of drugs (e.g.
CBR1) [1].
75 SDR genes were identified in the human genome from them 17 are dhrs genes and
roughly half of corresponding proteins are regarded as orphan. There is lack of information
about them or these information are inadequate compared to other SDR enzymes. However,
there are initial indications and results of possible significance of DHRS enzymes. These
preliminary results along with information from databases (e.g. BioGPS) may be good
indication for necessity of deeper study of DHRS proteins.
The aim of the study is basic characterization of selected members of DHRS group DHRS3
and DHRS7.
P47
The recombinant forms of DHRS3 and DHRS7 were successfully prepared in Sf9 cells
using baculovirus expression system and their predicted localization in microsomes was
experimentally confirmed. Sf9 microsomes were used for determination of both activities
towards carbonyl/hydroxyl-bearing compounds and membrane topology. Sf9 microsomes
were solubilized by detergents and subsequently DHRS enzymes were purified and
reconstituted into the liposomes. These forms will be used for further characterization.
Moreover, expression of these enzymes was also determined at mRNA and protein level in
the panel of 16 human tissues.
In this study bright new properties of DHRS enzymes were determined. Results are quite
interesting, so this investigation could be a basis for determination of the role of DHRS in
humans. The project will be continued with deeper insight to properties of these proteins and
with characterization of other DHRS enzymes.
Acknowledgment:
This work was supported by Grant Agency of Charles University
677012/C/2012).
[1]. Hofmann F. and Maser. E (2007) Drug Metab Rev 39, 87-144.
(GAUK
P47
CRYSTAL STRUCTURE OF AVIDIN COMPLEX
WITH BIOTIN-LINKER-FERROCENE CONJUGATE
P. Strzelczyk a, G. Bujacz a
a
Institute of Technical Biochemistry, Lodz University of Technology,
1. Introduction
Avidin is the fatty-acid binding protein that belongs to the calycin protein superfamily as
well as to the lipocalins. This protein is the object of various biochemical investigations
because of its unique binding properties with biotin (vitamin H) that is one of the strongest
interactions in nature. Homotetramer of avidin binds up to four molecules of vitamin H in
non-covalent interactions with high affinity (Kd ≈ 10-15 M) [1].
The aim of the study was to determine the crystal structure of avidin, the glycosylated
protein from Hen egg white, in the form of complex with the 6-(6biotinamidohexanamido)hexanoylferrocene (B2hexFc). This new vitamin H derivative
exhibits therapeutically important properties that may open new possibilities in the treatment
of cancer [2].
Crystals of this complex were obtained by the hanging-drop vapor-diffusion method. Xray diffraction data were collected on the 14.2 beam line at the BESSY synchrotron in Berlin,
Germany. Diffraction data were processed up to resolution of 2.0 Å. The crystal belongs to
orthorhombic space group P21212, with two protein molecules in the asymmetric unit [2].
P48
We have determined the three dimensional X-ray structure of the avidin complex with
B2hexFc. Avidin is organized in an eight-stranded antiparallel β-barrel, characterized by the
conventional right-handed twist. The barrel provides a well-defined cavity for biotin
derivative binding, which is roughly oriented along the barrel axis. The interactions of biotinlinker-ferrocene conjugate with avidin appear to be different in the two monomers. The biotin
bicyclic moiety of the B2hexFc is located in the deepest part of the binding pocket in both
monomers and most of the interactions found for the ligand in the canonical avidin-biotin
complex are maintained. Due to the flexibility of the aliphatic 6-aminohexanoic linkers and
the crystal packing the interactions of the ferrocene moiety differ in both monomers.
A spatial displacement of the L3-4 loop takes place as a result of this flexibility. In this
context, the crystal structure of avidin-B2hexFc complex is considered as important for better
understanding of how the ligand binds to the protein molecule and initiates protein
conformational changes upon ligand binding. The specific structural properties of the ligand
and its interactions with the protein may be useful in biochemical and medical applications[2].
[1]. N. M. Green, Adv. Prot Chem., 29 (1975) 85-133.
[2]. D. Plażuk, J. Zakrzewski, M. Salmain, A. Błauż, B. Rychlik, P. Strzelczyk, A. Bujacz, G.
Bujacz, Organometallics 32 (2013) 5774-5783.
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THE EFFECT OF IN VITRO GLYCATION ON HUMAN SERUM ALBUMIN STRUCTURE
A. Szkudlarek-Haśnik a, J. Równicka-Zubik a, M. Maciążek-Jurczyk a, A. Sułkowska a
a
Medical University of Silesia, Faculty of Pharmacy, Department of Physical Pharmacy;
1. Introduction
Human serum albumin (HSA) undergoes structural changes that can affect its drug binding
capacity. One of them is glycation leading to formation of Advanced Glycation End-Products
(AGEs). Accumulation of AGEs plays an important role in pathogenesis of diabetic
complications. Studies were undertaken to reveal the impact of glycation with fructose (FRC)
on the structure of HSA and to define potential disorders in binding tolbutamide (TB) and
losartan (LOS) to glycated albumin (gHSAFRC).
Quenching of the fluorescence of HSA and
gHSAFRC by TB and LOS revealed the nature of the
drug-protein interactions. The energy transfer from
tryptophan residue (Trp-214) (λex 295 nm) and the
tryptofan and tyrosine residues (λex 275 nm) to TB and
LOS chromophores as well as drug-gHSAFRC
complexes formation were confirmed on the basis of
fluorescence quenching of HSA. Greater values of both
association (Ka) and the Stern-Volmer (KSV) constants
allow for suggestion that glycation increases the protein
binding capacity of TB and reduces the free drug
concentration. It has been shown that the affinity of
LOS to HSA and gHSAFRC is greater than the affinity
of TB and it can negatively influence on TB binding to
HSA in the presence of LOS. Our studies suggest the
validity of monitoring the multidrug therapy of diabetes
and coexisting diseases. Location of main glycation
sites for a) lysine, b) arginine and cysteine residues in
the HSA structure is shown in Figure 11.
P49
Acknowledgements:
This work was supported by grant KNW-1001/K/4/0 from Medical University of Silesia,
Katowice, Poland.
[1]. P Rondeau, E Bourdon. The glycation of albumin: Structural and functional impacts.
Biochimie 93 (2011) 645-658.
P49
MAPPING OF ‘HOT SPOTS’ OF INSULIN FRAGMENTS
M. Świontek a, B. Kolesińska a, J. Frączyk a, Z. J. Kamiński a
a
Institute of Organic Chemistry, Lodz University of Technology (TUL), Zeromskiego 116,
90-924 Lodz, Poland
1. Introduction
Defective protein conformational homeostasis resulting in protein-misfolding cascades and
the accumulation of insoluble protein fibrils leads to severe pathogenic processes. Formation
of cytotoxic oligomers and amyloid fibrils is linked to cell damage and considered as
molecular trigger in etiology of at least 25 diseases named as conformational diseases [1]
including Alzheimer’s disease, type 2 diabetes and others. There are several evidences that
aggregation process is associated with short amyloidogenic fragments of proteins, so called
“hot spots” responsible for the triggering of the aggregation process. This phenomene relates
also to insulin, the hormone which regulates the physiological glucose level in human blood
and has so far been the most important pharmaceutical peptide for diabetes treatment.
However, aggregation of insulin [2] occurs readily in almost every biopharmaceutical process,
ranging from production, purification, storage, transportation, delivery, to in vivo utilization
at the terminal [3]. Despite a lot of efforts [4] the molecular mechanism of insulin aggregation
is still unknown.
In our studies we systematically synthesized all short fragments covering complete
structure of Insulin (1-10 A, 6-10 A, 11-21 A, 16-21 A; 1-10 B, 6-10 B, 11-20 B, 16-20 B, 2130B, 26-30 B) and selected the new, shorter then those already known aggregating fragments,
capable to initiate thermodynamically unfavorable, self-assembly process, leading finally to
formation of deposits. Based on the systematic studies on the aggregation of fragments of
insulin as well as on theoretical studies evaluating ability of insulin fragments to aggregation
[5], four short peptides were selected as the hot spots of insulin aggregation. In our studies we
synthesized and mapped fragments (A 13-19), (A 13-18), (B 11-18), (B 12-17) in order to
determine the smallest amyloidogenic fragments of insulin. The aggregation of synthetic
fragments was systematically studied using UV-VIS spectrophotometric method with Congo
red and using fluorescence spectra with Thioflavin T.
P50
[1]. C. M. Dobson, Trends Biochem. Sci., 24, 329 (1999)
[2]. a) D. F. Waugh, J. Am. Chem, Soc.,68, 247 (1946)
[3]. Y.Shikama, J. Kitazawa, N. Yagihashi, O. Uehara, Y. Murata, N. Yajima, R. Wada,
S.Yagihashi, Intern Med., 49, 397 (2010)
[4]. a) M. I. Smith, J. C. Sharp, C. J. Roberts, Biophys. J., 95, 3400 (2008); b) S.
Grudzielanek, A. Velkova, A. Shekula et al., J. Mol. Biol., 370, 372 (2007); c) M. R. H.
Krebs, E. H. C. Bromley, S. S. Rogers, A. M. Donald, Biophys. J., 88, 2013 (2005)
[5]. M. I. Ivanova, S. A. Sievers, M. R. Sawaya, J. S. Wall et al. (2009) Proc. Natl. Acad.
Sci., 106: 18990 (2009)
P50
EXAMINATION THE ß-CYCLODEXTRIN AS AN EFFECTIVE FUNCTIONAL
MONOMER FOR MOLECULAR IMPRINTED POLYMERS WITH SELECTED
METABOLITES OF CATECHOLAMINES
B. Dubrawska a, A. Korytkowska-Wałach a
a
1. Introduction
Molecular imprinting (MI) technique enables to obtain materials mimicking biological
receptors with their natural selectivity. Nevertheless function of such synthetic receptors is
somewhat defective when it comes to water solution because of no possibility to recognize the
target molecule precisely due to hydrogen bond formation. The water-compatible molecularly
imprinted polymers for binding of homovanillic acid (HVA) and vanillylmandelic acid
(VMA) were designed. Catecholamines are biogenic amines which play an important role in
the nervous system. HVA and VMA as their metabolites take significant part in the diagnosis
of neuroblastoma and pheochromocytoma. Moreover excretion rates of VMA with urine is
also informative in characterization of the many drugs effects [1].
P51
In our research ß-cyclodextrin (ß-CD), which often
form an inclusion complexes with many compounds, was
tested as the potential, functional monomer for binding
VMA and HVA respectively in aqueous solution (Figure
1). The stoichiometry and association constants of the
pre-polymerization complexes were determined in D2O
using 1H NMR technique. The experiments were
Figure 2 Chemical structure of
performed using VARIAN 600 MHz or VARIAN
a) VMA, b) HVA, c) ß-CD
Unity/INOVA 300 MHz spectrometers. The total
concentrations of the reagents during experiments were kept constant [2]. The stoichiometry
of 1:1 for ß-CD−VMA and ß-CD−HVA was proved, evaluated according to the Job’s method.
The successive increase in titrant concentrations (VMA or HVA) resulted in noticeable
changes in chemical shift of ß-CD protons. The biggest changes were observed for protons H1,
H3 and H5 of ß-CD, suggesting strong interaction. The chemical shifts changes are greater in
the case of H5 than H3 suggesting that total inclusion takes place [3]. Linearization methods of
Benesi-Hildebrand and Scatchard were used for association constants determination.
From the forgoing studies it is evident that some interactions between illustrated monomer
and templates exist. The binding constants are on the satisfactory level. The synthesis of MIPs
demands a good understanding of chemical equilibria, molecular recognition theory and
polymer chemistry in order to ensure a high level of success.
[1]. N. W. Barnett, P. S. Francis, S. W. Lewis, K. F. Lim, Anal. Commun., 1999, 36, 131-134
[2]. L. Fielding,Tetrahedron, 2000, 56, 6151-6170
[3]. D. Greatbanks D., Pickford R., Magn. Reson. Chem., 1987, 25, 208-215
P51
PRELIMINARY ATTEMPS TO APLLICATION OF N-LIPIDATED PEPTIDES
IMMOBILIZED ON SOLID SUPPORTS AS ORGANOCATALYSTS
J. Fraczyk a, Z. Kamiński a, M. Walczak a, B. Kolesinska a
a
Institute Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz,
Poland
e-mail of presenting author: e-mail [email protected]
1. Introduction
The term organocatalysis define the acceleration of chemical reactions through the addition
of a substoichiometric quantity of an organic molecules predominantly composed of C, H, O,
N, S and P [1]. Organocatalysts efficiency, in terms of specificity and selectivity is steadily
increasing and recently it is comparable to natural enzymes. Nowadays, organocatalysis are
becoming powerful tools in the construction of complex molecular skeletons.
L
L
L
We found that monolayer of Ni
i
i
p
p
p
i
lipidated peptides immobilized on solid
i
i
d
d
d
support via aromatic linker are able to
L
L
create
highly
organised,
dynamic
i
i
Peptide
Peptide
Peptide
p
p
i
structures of binding pockets, which very
i
d
d
efficiently recognize the shape, size,
Π−donor
Π−
Π−donor
Π−
Π−donor
Π−
chirality and polarity of ligands [2]. Due to
Peptide
Peptide
Π−acceptor
Π−
the fact that recognition of ligands
Π−acceptor
Π−
Π−acceptor
Π−
depends mainly on the structure of
Π−donor
Π−
Π−donor
Π−
Carbohydrate
Carbohydrate
Carbohydrate
immobilized peptide fragments, we
redesigned the structure of the binding
Π−acceptor
Π−
Π−acceptor
Π−
pocket by introducing amino acid residues
Carbohydrate
Carbohydrate
typical for the enzyme active centre. In the
preliminary studies, we mimicked the
proteolytic enzymes and found, that the
Figure 1
presence of Asp(Glu), His, Ser residues in
the peptide fragment leads to the system catalysing hydrolysis of ester and amide bonds.
Recently, the second type of organocatalyst was endowed with properties of enzymes capable
of catalysis of carbon-carbon formation (aldolases mimetic active site). This has been
achieved by incorporation of proline and basic amino acid residues into a peptide fragments.
P52
Acknowledgement:
This work was supported by MSE Poland N N 405 669540.
[1]. P. I. Dalko, L. Moisan, Angew. Chem. Int. Ed. 2004, 43, 5138.
[2]. Frączyk, J.; Malawska, B.; Kamiński, Z.J., J. Comb. Chem., 2009, 11, 446-451.Frączyk,
J.; Kolesińska, B.; Czarnecka, A.; Malawska, B.; Więckowska, A.; Bajda M.; Kamiński, Z.J.,
QSAR & Comb. Sci. 2009, 11; 728-736.
P52
DETERMINATION OF MOLECULAR SIZE DISTRIBUTION AS A
QUALITY CONTROL TEST FOR HUMAN ALBUMIN IN
PHARMACEUTICAL PREPERATIONS
Omar Al- Sayed Omar M. Khalifa a
a
Drug and Food Quality control laboratories ,Ministry of Health , Kuwait
1. Introduction
Pharmaceutical preparation of human albumin is a solution containing human
plasma protein of which at least 96% is human albumin . Human albumin is useful in
restoration and maintenance of circulating blood volume where volume deficiency
has been demonstrated in patient . The concentration of the albumin in the
pharmaceutical preparation , dosage and the infusion rate should be adjusted to the
patient ,s individual requirements . Determination of molecular size destruction for human
albumin presents one of most important test for quality .This work describes the
determination of the molecular size distribution in human albumin pharmaceutical
preparation . For that purpose an analytical procedure has been elaborated and validated
in our laboratories based on HPLC – Size Exclusion Chromatography ( HPLC-SEC )
under conditions according to Euro . Pharma [1]. The separated fractions were detected
and quantified at the column exit by means of photometry at 280nm.
In this study when albumin pharmaceutical
preparation pass through SEC column (TSK-gel , G3000
SW. 300X7.5mm , particle size 10 um, mean pore size
250 A0 , TOSOH Bioscience ) , the protein molecules
appear in different distribution zones depending on
the molecule . size and pore size distribution of the
used stationary phase . As seen in Figure 1 the largest
molecule ( polymer albumin ) migrate through the
column most rapidly followed by dimmer and finally
followed by monomer albumin . Human albumin samples
(500 samples of pharmaceutical preparation) were
tested in our laboratories using this method and the
results showed that 99.1% of the investigated samples
was full fill the requirements of Euro. Pharma [1].
P53
Figure 3
[1]. Euro . Pharma .( 2011) : European Pharmacopoeia , Vol. 1,7 ed, European Directorate
for the Quality of medicines and Health Care ,Council of Europe, Strasbourg .
P53
THE BIOLOGICAL ACTIVITY AND MECHANISM OF ACTION OF
THIOSEMICARBAZONES IN CANCER RESEARCH.
K. Malarz a, M. Rams-Baron b, M. Serda a, A. Mrozek-Wilczkiewicz b, R. Musioł a
a
Department of Organic Chemistry, University of Silesia, Szkolna 9, 40-006 Katowice,
Poland
b
Department of Solid State Physics, University of Silesia, Uniwersytecka 4, 40-007, Katowice,
Poland
e-mail of presenting author: e-mail: [email protected]
1. Introduction
Iron is indispensable for proper cell proliferation. The essential character of elemental iron
is due to its involvement in DNA synthesis, mitochondrial electron transport, and its
participation in the Fenton reaction leading to reactive oxygen species (ROS) generation [1].
ROS together with antioxidants may also play a key role in anti-cancer therapies that exploit
oxidative stress [2]. The generation of ROS by drugs is associated with loss of intracellular
redox potential. Glutathione plays a central role in maintaining redox homeostasis. Decreases
in cellular GSH levels leads to cell death, through apoptosis [3].
Thiosemicarbazones are compounds, which exhibit an extensive range of biological effects
including the antiviral, antifungal, antibacterial, antiproliferative activities. The most possibile
mechanism of action includes generation of reactive oxygen species within cancer cells, iron
chelation or inhibition of the enzyme ribonucleotide reductase (RR) necessary in the synthesis
of DNA [1].
To understand the overall mechanism of action of these compounds we focused on their
ability to induce oxidative stress, DNA damages and apoptosis. Obtained series of novel
derivatives thiosemicarbazones, showing the highest antiproliferative activity against human
colon carcinoma (HCT116 +/+) have been tested for effects on mitochondrial GSH. Selected
derivatives thiosemicarbazones exhibited ability to induce cell death through formation of
reactive oxygen species and reduced level of GSH. The next step was to determine, caspases
3/7 activation and their participation on the apoptosis pathway. Furthermore, the binding
ability of compounds in calf thymus DNA was studied by UV–Vis absorption.
Thiosemicarbazones exhibited ability to DNA intercalation, due to flat, heteroaromatic ring
systems in their structures.
P54
[1]. Richardson D. R., Kalinowski D. S. et. al., Biochimica et Biophysica Acta, 2009; 1790:
702-17
[2]. Manda G.,Nechifor M. T., Neagu T.-M., Current Chemical Biology, 2009; 3: 342-366
[3]. Circu M. L., Aw T. Y., Free Radical Biology & Medicine, 2010; 48: 749–762
P54
METALLACROWN COMPLEXES WITH NOVEL STRUCTURAL AND
MAGNETIC PROPERTIES AS TOOLS FOR BIO- OR
NANOTECHNOLOGICAL DEVICES
M. Olędzka a, E. Gumienna-Kontecka a, I. Golenia b, I. O. Fritsky b
a
Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie, 50-383 Wroclaw, Poland
Department of Chemistry, National Taras Shevchenko University, 01601Kiev, Ukraine
b
1. Introduction
Metallacrowns (MCs) are self-assembled metallamacrocycles defined as a repeat unit -[M-NO]n- in a cyclic arrangement. They are the inorganic analogues of crown ethers [1]. In the
MTs structure metals of almost all groups can be represented together with a large variety of
both inorganic and organic ligands [2].
Every year, the number of MTs grows, together with the number of their newly identified
structural, magnetic and optical features, or host–guest capabilities. MCs are recognized as
appropriate compounds in a number of research fields, from bioinorganic chemistry to
catalysis and molecular recognition and in the development of systems with innovative
magnetic properties [2].
Most of the reported MCs were synthesized using aminohydroxamic acids as ligands.Copper
metallacrowns with hydroxamic acid derivatives were studied in recent years for their
capabilities to bind anions of biological interest, and to encapsulate metal ions [1].
Main directions in metallacrowns research undertaken by us in recent years include:
• studies of thermodynamics of self-assembly and host–guest equilibria of
metallacrowns of Cu2+ and hydroxamate derivatives in solution [3]
• use of hydroxamate ligands for preparation of metallacrown as promising objects for
molecular magnetism, functional fluorescent materials and probes, and metal complex
catalysis [4]
The key information obtained in our recent studies will be presented in the view
of their applications for various biological and inorganic purposes.
Acknowledgements:
The authors gratefully acknowledge the funding from EU FP7 (International Research
Staff Exchange Scheme, PIRSES-GA-2013-611488).
P55
[1]. M. Tegoni, M. Remelli, Coordination Chemistry Reviews 256 (2012), 289-315.
[2]. G. Mezei, C.M. Zaleski, V.L. Pecoraro, Chem. Rev. 107 (2007) 4933.
[3]. E. Gumienna-Kontecka, I.A. Golenya, A. Szebesczyk, M. Haukka, R. Krämer, I.O.
Fritsky, Inorg. Chemistry 52 (2013), 7633−7644.
[4]. I.A. Golenya, E. Gumienna-Kontecka, A.N. Boyko, M. Haukka, I. O. Fritsky, Inorg.
Chemistry 51 (2012), 6221-6227.
P55
ALGINATE/CHITOSAN CORE-SHELL PARTICLES WITH BIOACTIVE
FUNCTIONALITIES
A. Regiel-Futyra a, A. Mazgała a, J. Michna a, G. Stochel a, A. Kyzioł a
a
Facultyof Chemistry, Jagiellonian University.
1. Introduction
Multidrug-resistant (MDR) strains of pathogenic bacteria emerge as one of the most
important problem [1]. Drug resistance is the main cause of prolonged infections which
enhances the risk of dangerous illnesses such as wound infections, osteomyelitis, septic
arthritis, endocarditis, etc.. As a consequence, death rate related to MDR-bacteria is rising
quickly. In general, acute infections are known to be caused by planktonic bacteria, which are
usually treated with antibiotics. In contrast to planktonic bacterial infections, most of cases of
biofilm forming bacterial infections show up to be untreatable and usually develop into
chronic [2]. Thus, new strategies to overcome bacteria resistance are required. Application of
polymeric nano/microparticles is now one of the most popular and promising approach
mainly due to many enticing properties as biocompatybility, biodegradability and noncytotoxicity towads mammalian cells.
Present study focuses on alginate/chitosan submicroparticles constructed of polymeric core
loaded with antibiotic (e.g. ciprofloxacin) and polymeric shell prone for further lytic enzyme
immobilization. Several synthetic approaches were conducted in order to obtain stable and
uniform core-shell beads. Physicochemical characterization of obtained materials was
performed. Size and zeta potential were determined by dynamic light scattering technique
(DLS). Particles shape and morphology were investigated via scanning electron microscopy
(SEM). Drug loading efficiency and cumulative drug release profiles were evaluated with
UV-Vis spectrophotometry. Finally, preliminary biological tests were also performed.
Alginate/chitosan core-shell particles loaded with ciprofloxacin appears to be a promising
alternative for ineffective antibiotics. Preliminary biological tests encourages for further
investigation of those hybrid materials.
Acknowledgements:
This work was supported by Polish Foundation of Science within POMOST project
“Alginate/chitosan core-shell beads with bioactive functionalities” (POMOST/2013-7/7).
P56
[1]. Høiby, N.; Bjarnsholt, T.; Givskov, M.; Molin, S.; Ciofu, O., Antibiotic resistance of
bacterial biofilms. International Journal of Antimicrobial Agents 35 (4), 322-332.
[2]. Bjarnsholt, T., The role of bacterial biofilms in chronic infections. APMIS 2013,121, 158.
P56
NATURAL COMPOUNDS AS INHIBITORS OF AKR1B ENZYMES
L. Skarydova a, J. Hofman a, J. Havrankova a, L. Cahlikova b, V. Wsol a
a
Department of Biochemical Sciences, Faculty Of Pharmacy in Hradec Kralove, Charles
University in Prague, Czech republic
b
Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy in Hradec
Kralove, Charles University in Prague, Czech Republic
1. Introduction
Aldo-keto reductase (AKR) superfamily contains 13 human members that participate in the
reduction of compounds with carbonyl groups and thus participate in many important
processes in the body [1]. AKR1B family consists of three enzymes - AKR1B1, AKR1B10
and AKR1B15. AKR1B1 (also known as aldose reductase) reduces glucose to sorbitol and is
responsible for the development of diabetic complications. Recently, the role of AKR1B1in
the development of some cancers through metabolism of retinoids and oxidative stress was
described [2]. AKR1B10 participates in the metabolism of retinal, glyceraldehyde and several
xenobiotics and is overexpressed in many cancers, such as lung, liver and colon. A role of
AKR1B15 has not been elucidated to date [3]. AKR1B1 and AKR1B10 can be targets for the
prevention and treatment of above mentioned types of disease and, therefore, the search for
new inhibitors is of a great interest.
The aim of the present study is to test inhibitory potencies of diverse natural compounds
from plants toward AKR1B1 and AKR1B10 in vitro and the detailed characterization of the
strongest ones.
Almost sixty diverse plant secondary metabolites were tested as potential inhibitors of
AKR1B1 and AKR1B10 using physiologically catalysed reaction, the conversion of all-trans
retinal to all-trans retinol. It was proved that several groups of alkaloids are poor inhibitors of
both enzymes whereas some types of polyphenols show high inhibitory potency. Flavons
(apigenin, 7-hydroxyflavone) or flavanol quercetin belong to the strongest inhibitors of both
enzymes. Moreover, AKR1B1 is inhibited by diverse structural type, such as flavanolignin
silibinin or by chlorogenic acid. Values of IC50 were determined for the strongest inhibitors.
Cytotoxicity and functionality on the cellular level were also evaluated for selected
substances.
It seems that natural compound are strong but only poorly selective inhibitors of AKR1B
enzymes. However, they can serve as leading structures for the development of analogs with
better characteristics.
P57
Acknowledgements:
This work was co-financed by the European Social Fund and by the state budget of the
Czech Republic (project no. CZ.1.07/2.3.00/30.0061) and supported by Charles University in
Prague (UNCE 204026/2012).
[1]. Ramana K.V. (2011) Biomol concepts 2, 103-114.
[2]. Salabei J.K. et al. (2011) Chem Biol interact. 191(1-3), 177-8.
P57
ANTIBACTERIAL PROPERTIES OF RESVERATROL ANALYZED BY
BIOAUTOGRAPHY
M. Skorek a, W. Jesionek b, B. Majer-Dziedzic c, M. Sajewicz a, T. Kowalska a, I. Choma b
a
of Silesia, Katowice, Poland
b
Department of Chromatographic Methods, Faculty of Chemistry, Maria Curie Sklodowska
University, Lublin, Poland
c
Department of Veterinary Microbiology, University of Life Sciences, Lublin, Poland
e-mail presenting author: [email protected]
1. Introduction
In the investigations carried out both in vivo and in vitro, polyphenols demonstrate a wide
variety of bioactivities, including anti-oxidative, anti-inflammatory, antifungal, anti-tumor,
etc. These are the reasons, why polyphenols are used in prophylaxis and therapy of many
health problems. Different fruits (chokeberries, blueberries, pomegranates, etc.) and drinks
(green tea, red wine, coffee, etc.) are very rich in polyphenols.
Many cosmetic and pharmacological products (e.g., dietary supplements and drugs sold
over the counter) make use of polyphenols-rich plant extracts. In traditional medicines,
unprocessed botanical materials are often utilized for further dispensing. Cosmetics which
contain botanical extracts standardized for the contents of polyphenols act as the free-radical
scavengers, and show antibacterial and antifungal properties as well.
Currently, chemical compounds are not only studied for their chemical and physical
properties, but also for biological ones. For this purpose, bioautography appears as a
convenient analytical technique, which enables studying antibacterial and antifungal
properties of the compounds of interest [1-4].
In this study, samples of wine and the aqueous or aqueous ethanolic extracts from the
grape vine underwent the procedure of solid phase extraction (SPE). Then the samples of
resveratrol’s solution in ethyl acetate were analyzed by bioautography on TLC plates. Surface
areas on the bioautogram printouts were measured with use of a planimeter and the average
values in [cm2] of the bacterial growth inhibition zones are presented in the respective tables.
Samples of resveratrol have similar bacterial growth inhibition zones. The results obtained are
presented in the form of bioautograms, the discussion is provided and relevant conclusions are
drawn.
P58
[1]. Tyihák E., Botz L., Nagy S. et al.: In “Proc. Intern. Symp. on Planar Separations, Planar
Chromatography“ (Nyiredy Sz. Ed.), Budakalász 2001, 3.
[2]. Choma I.M., Grzelak E. M. J. Chromatogr. A 2011, 1218, 2684-2691.
[3]. Tyihak E., Moricz A.M., Ott P.G. Med. Chem. 2012, 8, 75-84.
[4]. Grzelak E.M., Jesionek W., Majer-Dziedzic B. et al.: JAOAC 2013, 96, 1167-1174
P58
SYNTHESIS, INVESTIGATIONS OF THE ABSORPTION SPECTRA AND APPLICATION
OF DYES DERIVED FROM HYDROXYAZOQUINOLINES
M. Szala a, J. E. Nycz a, A. Świtlicka-Olszewska a, A. Maroń a, K. Czyż a, R. Strzelczyk b,
J. G. Małecki a, R. Podsiadły b, B. Machura a
a
Institute of Chemistry, University of Silesia, Szkolna 9, 40-007 Katowice, Poland
Institute of Polymer and Dye Technology, Lodz University of Technology, Stefanowskiego
12/16, 90-924 Łódź, Poland
b
1. Introduction
The quinolines are part one of the most important compounds among N-heterocycles found
their broad application in pharmaceutical and agrochemical industries [1]. These conjugated
systems have been widely employed in components for molecular electronic devices [2].
Many azo dyes are utilized as acid-base indicators due to the protonation of the azo group and
its effect on the absorbance of the azo dye. The azo group is easily protonated to form the
azonium ion, which generally causes a bathochromic shift in its absorption. The protonation
of an azo dye is reversible and the azo form is regenerated by the addition of base.
Hydroxyazo dyes are popular coloring materials due to their application in various fields of
science and technology such as biological and medical studies [3, 4].
Nine novel hydroxyazoquinoline dyes were
synthesized and analyzed by 1H NMR, 13C
NMR, FTIR, MS, HRMS, UV-Vis and single
crystal X-ray characteristics. The photophysical
properties of hydroxyazoquinoline dyes were
investigated by UV-Vis spectroscopy. Visible
absorption spectra of the dyes are discussed; the
effects of substituent, varying pH and solvent
upon
the
absorption
ability
of
hydroxyazoquinolines. In addition, synthesized
hydroxyazoquinoline were used for dyeing
polyester fibers and were tested for color
fastness to light. The results will be presented on poster.
P59
[1]. Sridharan V, Suryavanshi PA, Menendez JC; Chem Rev 111 (2011) 7157–7259.
[2]. Tang CW, VanSlyke SA, Appl Phys Lett 51 (1987) 913–915.
[3]. Gilani G, Moghadam M, Zakerhamidi MS, Moradi E: Dyes and Pigments 92 (2012)
1320–1330.
[4]. Samanta S, McCormick TM, Schmidt SK, Seferos DS., Woolley GA; Chem Commun 49
(2013) 10314–10316.
P59
APPLICATION OF SIMPLEX METHOD IN DRUG DESIGN
J. Bogocz a, A. Tkocz a, J. Polański a
a
Department of Organic Chemistry, Institute of Chemistry, University of Silesia, Katowice,
Poland
1. Introduction
The development of new drugs with potential therapeutic applications is one of the most
difficult and expensive process in the pharmaceutical industry. Different approaches are
currently used to facilitate and accelerate the process involved in creating new drugs. In this
study we considered the application of simplex method in drug design. Simplex approach has
potential advantage for structural chemical data and has impact in chemistry and drug
development. Furthermore, using this method allows us to find interesting patterns.
Simplex approach is focused on the fact that every molecule can be represented as a
system of different fragments with fixed composition, structure, chirality, symmetry [1]. The
simplex representation of molecular structure for biologically active substances allows
unifying the description of spatial structure of compounds with saving of the complete
stereochemical information [2].
CH3 O
OH
Dolutegravir (DTG) is an integrase inhibitor
O
F
F
known as Tivicay. DTG demonstarates activity
N
against some strains of HIV that are resistant to
N
NH
other integrase inhibitors such as raltegravir and
O
H
elvitegravir [3]. DTG has been represented as a
O
system of different simplexes. The Tanimoto
Figure 1 Dolutegravir
coefficient similarity, topological polar surface
area (TPSA) and ADMET properties was used as
descriptor in our further analyses. Our results were presented and visualized using PCA
method, which proved efficient for working with a large number of variables (simplex
descriptors).
P60
[1]. Kuz’min V. E., et. al.: Hierarchic System of QSAR Models (1D-4D) on the Base of
Simplex Representation of Molecular Structure. Journal of Molecular Modelling. 2005, 11,
456-467.
[2]. Kuz’min V. E., et. al.: The analysis of structure-anticancer and antiviral activity
relationships for macrocyclic pyridinophanes and their analogues on the basis of 4D QSAR
models (simplex representation of molecular structure). Acta Biochimica Polonica. 2002, 49,
157-168.
[3]. Kobayashi M., Yoshinaga T., Seiki T., et. al.: In Vitro Antiretroviral Properties of
S/GSK1349572, a Next-Generation HIV Integrase Inhibitor. Antimicrob Agents Chemother.
2011, 55(2), 813-821.
P60
HPLC-ELSD, HPLC-MS, AND LC-MS INVESTIGATION OF SPONTANEOUS
OSCILLATORY REACTIONS OF L-HISTIDINE
A. Godziek a, A. Maciejowska a, M. Sajewicz a, T. Kowalska a
a
Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland
1. Introduction
In oue previous studies, it has been shown that the low molecular weight chiral compounds
belonging to the group of profen drugs [1], amino acids [2], and hydroxy acids in solution can
undergo spontaneous oscillatory condensation [3]. These reactions are characteristic of single
compounds, or of the mixtures thereof in aqueous or non aqueous solutions. For example, in a
study on a mixture of L-proline and L-phenylalanine dissolved in 70% aqueous acetonitrile
solution, it was found out that these compounds can undergo the oscillatory peptidization
reaction, resulting also in heteropeptides.
To check, if another amino acid (L-histidine) can undergo spontaneous oscillatory
peptidization, we used the achiral high performance liquid chromatography with light
scattering detection (HPLC-ELSD) and the mass spectroscopy detection (LC-MS). The choice
of this amino acid was due to its important functions in human body. L-Histidine (L-His) is
essential in formation of the red and white blood cells. It is also a starting material for the
production of a neurotransmitter histamine, which plays a key role in the body defense
reactions.
The obtained results demonstrate the oscillatory instability of L-His in water solution. As
a result of the HPLC-ELSD analysis, we observe non-monotonic changes of the peak heights,
which point out to the oscillatory nature of the polycondensation process. An outcome of the
HPLC-MS and LC-MS experiments confirms that the spontaneous peptidization process has
really taken place.
Acknowledgement
One author (A.G.) acknowledges the financial support of the DoktoRIS project, co-financed
by the European Union within the European Social Found.
P61
[1]. Sajewicz, M., Piętka, R., Pieniak, A., Kowalska, T.; Application of thin-layer
chromatography (TLC) to investigating oscillatory instability of the selected profen
enantiomers; Acta Chromatographica, (2005); 15: 131- 149.
[2]. Sajewicz, M., Gontarska, M., Kowalska, T., HPLC/DAD evidence of the oscillatory
chiral conversion of phenylglycine; Journal of Chromatographic Science, (2014); 52: 329333.
[3]. Sajewicz, M., Kronenbach, D., Gontarska, M., Wróbel, M., Piętka, R., Kowalska, T.;
TLC in search for structural limitations of spontaneous oscillatory in-vitro chiral conversion.
α-hydroxybutyric and mandelic acids; Journal of Planar Chromatography – Modern TLC,
(2009); 22: 241-248.
P61
SiO2-, Cu-, AND Ni-SUPPORTED AU NANOPARTICLES FOR SELECTIVE
GLYCEROL OXIDATION IN THE LIQUID PHASE
M. Kapkowski a, M. Słota a, J. Polański a
a
University of Silesia in Katowice, Institute of Chemistry, University of Silesia, 9 Szkolna
Street, 40-007 Katowice, Poland
1. Introduction
Glycerol yielded as a byproduct in biodiesel production is one of the most widelyavailable biosourced chemicals, making it an attractive target of investigations. The oxidation
of glycerol could yield a variety of C1 to C3 oxygenates, which are potentially valuable
chemicals in chemical and pharmaceutical applications or intermediates in organic synthesis.
This has triggered a growing interest in new methods for this process. Although there has
been tremendous progress in recent years in this area, a number of problems remain to be
solved [1, 2].
We assumed that a high catalyst reactivity accompanied by the support wettability and
availability should be of major importance in viscous glycerol solutions. Thus, we
investigated as a potential catalyst for glycerol processing SiO2-supported Au NPs that should
be compatible with the polar reactant, oxidant, and solvent, i.e., glycerol, H2O2, and H2O,
respectively. Such a catalyst has not been tested previously in glycerol processing. In fact,
tested Au/SiO2 systems appeared more reactive than Au/C systems.
We observed that, within the complex network of possible reactions, a process of deep
glycerol oxidation proceeded on Au/SiO2 catalysts preferentially to acetic acid. The
selectivity of glycerol oxidation to C3 products was investigated thoroughly. It is not a
coincidence, because only C3 oxygenates can form the single carbonous products of glycerol
processing, while any oxidation to C2 must also yield the C1 oxygenate (C3 → C2 + C1). This
means that, although the process can be fully selective, we obtain at least two products.
Thus, the reactivity to C3 products has been well described, in contrast to the C1 and C2
products. Herein, we described the systems providing high selectivity to acetic acid, the C2
glycerol oxygenate. High conversions (100%) and acetic acid yields (90-99%) were observed
for the best catalysts in the diluted aqueous glycerol solutions. Although in a relatively
viscous liquid phase these values decreased to ca. 40% and 20%, the addition of acetonitrile
could improve the acetic acid yield to ca. 40%, while surfactants were found to be capable of
a many-fold enhancement of the catalyst activity. However, this was relatively low at the
room temperature highly-viscous liquid phase.
In summary, SiO2-supported Au NPs can form an interesting catalytic system for deep
selective glycerol oxidation to acetic acid in undiluted viscous liquid solutions. This seems
especially interesting for the processing of glycerol wastes.
P62
Acknowledgments:
The research was co-financed by the National Research and Development Center
(NCBiR) under Grant ORGANOMET No: PBS2/A5/40/2014. Maciej Kapkowski received a
grant for the project DoktoRIS - Scholarship program for innovative Silesia co-financed by
the European Union in the framework of the ESF.
[1]. N. Dimitratos, J. A. Lopez-Sanchez, G. J. Hutchings, Top Catal. 52 (2009) 258.
[2]. M. Besson, P. Gallezot, C. Pinel, Chem. Rev. 114 (2014) 1827.
P62
NANOCATALYST Pd / Cu IN THE SYNTHESIS OF THE CHEMICALS
AND PHARMACEUTICALS OR THEIR BUILDING BLOCKS
M. Korzec a, P. Zenderowska a, R. Rzycka a, J. Polański a
a
University of Silesia, Institute of Chemistry, Departament of Organic Chemistry, Szkolna 9
street, 40-006 Katowice.
1. Introduction
The Pd-catalyzed Sonogashira C–C cross-couplings can be performed under both
heterogeneous and homogenous conditions. A major problem in the synthesis of
pharmaceuticals is the residual contamination of a toxic Pd(I) species [1]. The studies focus
on the development of a more efficient, less costly, and more simply formulated catalyst to
chemistry of alkynes.
We present an innovative method for
the preparation and
application of
bimetallic catalyst Pd/Cu. In our
investigations we showed that the
heterogeneous catalyst consisting of an
active catalytic Pd species supported on
the copper is active enough to perform
the Sonogasira reaction under mild
conditions.
The reactions with the highly
effective Pd/Cu system could provide
even quantitative conversions and
subsequently quantitative yields of the
coupling products (Fig. 1). Indeed, a Pd
loading as low as 0.035 mol% was sufficient to activate the Sonogashira reaction in our
catalytic system. Another important advantages of the use Pd/Cu catalyst it selectivity and
lower contamination of the final products [2].
Acknowledgement:
This study was supported by the Grant ORGANOMET No: PBS2/A5/40/2014 from the
Polish National Research and Development Center. Paulina Zenderowska appreciates the
support of the FORSZT fellowships. Mateusz Korzec received a grant for the project
DoktoRIS - Scholarship program for innovative Silesia co-financed by the European Union in
the framework of the ESF
P63
[1] R. Chinchilla, C. Najera. Chemical Reviews, 107 (2007) 874.
[2] M. Korzec, P. Bartczak, A. Niemczyk, J. Szade, M. Kapkowski, P. Zenderowska, K. Balin
J. Lelątko, J. Polański. Journal of Catalysis, 313 (2014) 1.
P63
FRAGMENT-BASED DRUG DISCOVERY APPROACHES IN A SEARCH FOR NOVEL
ANTI HIV-1 CHEMOTYPES
A. Kurczyk a, J. Polanski b
a
Institute of Automatic Control, Silesian University of Technology, 16 Akademicka Street, 44100 Gliwice, Poland
b
1. Introduction
A concept of privileged structures (PS) was introduced by Evans [1]. The theory of PS
evolved from a pragmatic tendency to simplify the complexity of drug design through
fragmentation and has already been used in explaining the structure activity relationships in
diverse groups of drugs. If PS prove to be true, then molecular motifs that enrich biological
activity can be used when designing new drugs. Especially some molecular fragments, as
quinoline, were claimed privileged among anticancer, antifungal, antimicrobial and also as
HIV integrase inhibitors.
We attempted to test the attractiveness of the different azanaphthalene molecular fragments
in chemical space [2]. Hence, we analyzed a number of the PubChem registered compounds
having a given azanaphthalene structural motif. Quinoline appeared the most frequent hit.
What is the origin of this popularity: practical applications, synthetic availability or else? To
test the different possibilities, we considered two parameters: range of interest and b-value,
representing respectively the number of compounds tested to all hits and active to tested ratio
(b-value), which are the simplest measures of attractiveness and drug-likeness.
In a search for new anti HIV-1 chemotypes we developed a multistep ligand-based virtual
screening (VS) protocol combining machine learning (ML) methods with PS concept [3]. In
its learning step we based our VS protocol on the HIV integrase inhibitors fetched from
ChEMBL database. The performance of various ML methods and PS weighting scheme was
evaluated and applied as VS filtering criterion. Finally, using our multistep ligand-based VS
cascade methodology, database of 1.5 million commercially available compounds was
screened and 13 unique structures were indicated and bioactivity of these compounds was
tested by measuring the inhibition of HIV replication in infected cells. This approach allowed
us to discover two novel chemotypes with antiretroviral activity.
[1]. Evans B, Rittle K, Bock M, DiPardo R, et al.; J Med Chem (1988), 2235–46.
[2]. Polanski J, Kurczyk A, Bak A, Musiol R; Curr Med Chem (2012), 1921–45.
[3]. Kurczyk A, Warszycki D, Musiol R, Kafel R, Bojarski A, Polanski J; J Chem Inf Model,
sent.
P64
P64
A STUDY OF SELF-ORGANIZATION OF THE PROTEINOGENIC AMINO ACID (LMET) BY CHROMATOGRAPHIC METHODS.
A. Maciejowska a, A. Godziek a, M. Sajewicz a, T. Kowalska a
a
1. Introduction
Methionine (Met) is one of two sulfur-containing proteinogenic amino acids. Its derivative
S-adenosyl methionine (SAM) serves as a methyl donor. Met is an intermediate in the
biosynthesis of cysteine, carnitine, taurine, lecithin, phosphatidylcholine, and other
phospholipids. Improper conversion of Met can lead to atherosclerosis [1].
Spontaneous self-organization is understood as formation of peptides as a result of an
oscillatory response. Oscillating reactions are a special group of physical and chemical
processes, which include the essential characteristics of living organisms. Previous studies of
low molecular weight chiral compounds belonging to the group of profen drugs [2] hydroxy
acids [3] and amino acids have shown their structural instability.
Observation of the oscillatory reactions of L-Met was possible by using high performance
liquid chromatography (HPLC) and liquid chromatography coupled with mass spectrometry
(LC-MS).
HPLC analysis of L-Met with continuous registration allows observation of a spontaneous
process. This spontaneous process consists of the appearance and disappearance of an
additional peak in the chromatogram. An additional peak appearing in the HPLC analysis is
the product of the process of oscillatory peptidization. The height changes of the observed
peaks furnish an additional confirmation of the occurrence of oscillatory process. The results
of the HPLC-MS analysis confirm an appearance of oligopeptides.
Acknowledgement:
One author (A.G.) acknowledges the financial support of the DoktoRIS project, cofinanced by the European Union within the European Social Found.
P65
[1]. H. Refsum , P. M. Ueland, O. Nygård, S. E. Vollset; Ueland; Nygård, Homocysteine and
Cardiovascular Disease, Annual review of medicine 49 (1): 31–62 (1998)
[2]. M. Sajewicz, D. Kronenbach, M. Gontarska, M. Wróbel, R. Pietka, T. Kowalska, TLC in
search for structural limitations of spontaneous oscillatory in-vitro chiral conversion.
Hydroxybutyric and mandelic acids. J. Planar Chromatogr. - Modern TLC, 22, 241-248
(2009)..
[3]. M. Sajewicz, M. Gontarska, D. Kronenbach, and T. Kowalska, On the spontaneous
abiotic peptization of phenylglycine in an aqueous medium, Acta Chromatographica,. 21,
151–160 (2009).
P65
DETERMINATION OF ANTIOXIDANT PROPERTIES OF DIFFERENT THYME SPECIES
BY MEANS OF „DOT BLOT” TEST
M. Orłowska a, G. Szymczak b, M. Waksmundzka-Hajnos c, T. Kowalska a, M. Sajewicz a
a
Institute of Chemistry, University of Silesia, Katowice, Poland
Botanical Garden, Maria Curie-Sklodowska University, Lublin, Poland
c
Department of Inorganic Chemistry, Medical University of Lublin, Poland
b
1. Introduction
Thyme (Thymus L.) is an aromatic and medicinal plant of a growing importance, both in
gardening and the economy. Nowadays, the thyme genus is known to embrace ca. 350 species
which belong to the mint (Lamiaceae) family. Polyphenols are a group of compounds which
are produced in the different parts of a plant, and they can be categorized as, e.g., phenolic
acids, flavonoids, and lignins. Polyphenols exert anti-oxidative, anti-inflammatory,
antibacterial, pain-killing, and also other curative effects [1-3].
Plant material investigated in this study originates from Botanical Garden of the Maria
Curie-Sklodowska University in Lublin. The eighteen thyme (Thymus L.) species were
collected in July, 2012, and dried under adequate conditions.
The analysis of the obtained extracts was performed by means of the “dot blot” test based
on the reduction of the DPPH• radical. Purely qualitative analysis of the “dot blot” test
showed that all investigated thyme samples exerted antioxidant activity. In quantitative terms,
antioxidant activity of individual thyme samples recalculated to GA appeared in the range
from 11.94 to 60.16 mg GA / 1 g dry herbal matter. As none of the antioxidant activity
quantification methods reported in the literature and referring to the thyme species is based on
the “dot blot” test alone and moreover, as the majority of the DPPH• radical based tests
employ UV/Vis spectrophotometry, there was no possibility to compare the “dot blot” results
obtained in this study with those originating from earlier reports.
Acknowledgement:
One author (MO) acknowledges the financial support of the DoktoRIS project, cofinanced by the European Union within the European Social Found.
P66
[1]. S. Y. Oh, J.W. Ko, S.Y. Jeong, J. Hong, Journal of Chromatography A, 1205 (2008) 117127
[2]. B. Nickavar, N. Esbati, Journal of Acupuncture and Meridian Studies, 5(3), (2012), 119125
[3]. B. Boros, S. Jakabová, G. Horváth, Z. Pluhár, F. Kilár, A. Felinger, Journal of
Chromatography A, 1217, (2010) 7972-7980
P66
THIOSEMICARBAZONES LIGANDS. COORDINATION PROPERTIES
IN SOLUTION.
M. Pyrkosz-Bulska a, M. Serda a, E. Gumienna-Kontecka b
a
Department of Organic Chemistry, Institute of Chemistry, University of Silesia Szkolna
Street 9,40-006, Katowice, Poland
b
Faculty of Chemistry, University of Wrocław,F. Joliot-Curie 14,50-383 Wrocław, Poland
1. Introduction
Triapine® as the most prominent representative is currently undergoing phase I / II trials
acting as effective inhibitor of the DNA enzyme ribonucleotide reductase [1]. Their copper
and iron complexes also exert antitumor effect, which have higher antiproliferative effect than
the ligands alone in many cases [2,3]. In connection with the anticancer applications of TSCs
there have been proposed several mechanisms of action, but many questions still remain
unanswered. Information on the speciation of metal complexes, particularly at physiological
pH, can be the first step towards elucidation of the cytotoxic mechanism of TSCs.
In this work, we characterize novel thiosemicarbazone ligands, in terms of complex
formation with Cu(II) and Fe(III) ions and their stability constants in a water/methanol
mixture. Knowledge of the actual chemical forms of these complexes in the aqueous solutions
strongly contributes to the structure-stability-activity relationship studies.The data reveal the
formation of copper(II) complexes with only a 1:1 metal/ligand stoichiometry such as
[CuLH]2+, [CuL]+ and [CuLH–1], and with iron(III) formation of bis-complexes such as
[FeL2H], [FeL2]- and [FeL2H–1]2- was observed. The Uv-Vis titration indicate that L1 is a very
efficient Cu(II) and Fe(III) chelator since complex formation processes start at as low as pH≈
2. The metal ions are tridentately coordinated by L3: with an O–,N1,S donor set and with the
proton presumably located at the non-coordinating hydrazine N2 atom, and [FeL2] with an O–
,N1,S–-donor set. In case of L1-L2 ligands most probably coordinate to the metal ions via the
(N(pyr),N1,S-) donor set. We succeed to achieve the study goal of creating several molecular
entities with high anti-cancer activity with good selectivity to human normal fibroblasts
similar to referential compound Dp44mT. The IC50 values of these compounds is comparable
to the highest activity level of the previously reported TSC analogues.
[1]. J. Kolesar, R.C. Brundage, M. et al., Cancer Chemother. Pharmacol. 67 (2011) 393−400.
[2]. M.N.M. Milunovic, E.A. Enyedy, et al., Inorg. Chem. 51 (2012) 9309-9321.
[3]. E. Gumienna-Kontecka, M. Pyrkosz-Bulska, A. Szebesczyk, A.Oledzka Curr Med.
Chem, 2014 Jul 6
P67
P67
FRAGTAL – A PROCEDURE FOR PROBING A CHEMICAL SPACE FOR
FRAGMENTAL TOPOLOGY-ACTIVITY LANDSCAPES: APPLICATION FOR DIKETO
ACID AND CATECHOL HIV INTEGRASE INHIBITORS
A. Bak a, T. Magdziarz a, V. Kozik a, K. Jarzembek a, M. Rojkiewicz a, A. Kurczyk a, K.
Serafin a, J. Polanski a
a
Institute of Chemistry, University of Silesia, 40-006 Katowice, Poland
P68
The nature of the drug-target interactions ruled by inter- and/or intra-molecular forces is a
tremendously complex and divergent process that still lacks a universal approach. Hence,
several approaches to drug discovery have been developed in the quest to identify the
differences between drug- and non-drug molecules (chemicals). Most often, these methods are
related to the drug-likeness concept originating from the idea that drug compounds differ from
other molecules in their physicochemical properties.
In reality, molecular descriptors quantifying drug-like properties (DP) are easily available
from molecular formulae, e.g. the number of hydrogen bond donors (HBD) or acceptors
(HBA). The emergence of the quantitative property-based Lipinski Ro5 ‘sieve’ is focused on
the importance of restricting the chosen in silico molecular parameters of preclinical drug
candidates in the pharmacokinetic-friendly property space. The discrimination Ro5 model
restrains the ‘drug-like property space’ through a set of threshold values which are necessary
but are not sufficient boundary guidelines of the preferred ‘drug architecture’ that is especially
useful in the computer-driven process of filtering chemical libraries; however, this concept is
still elusive and ambiguous. Fragment-based drug design or fragonomics, which has recently
been getting more and more widespread appeared to offer an alternative way for identifying
advantageous drug structures. This methodology assumes the existence of a preferred
molecular ‘core’, namely a framework that works as an ‘anchor’ for the ‘ornamenting’ blocks
– privileged motifs. Generally, the problem of identifying relevant molecular fragments is a
complex issue that is related to the so-called frequent subgraph mining (FSM). More practical
FSM approaches to drug design are still a matter of future developments that need some
sophisticated algorithms for massive in-silico database mining and data processing.
Fragmental topology-activity landscapes (FRAGTAL), a new concept for encoding
molecular descriptors for fragonomics into the framework of the molecular database records
is presented. Thus, a structural repository containing biological activity data was searched in a
substructure mode by a series of molecular fragments constructed in an incremental or
decremental manner. The resulted series of database hits annotated with their activities
construct FRAGTAL descriptors encoding a frequency of the certain fragments among active
compounds and/or their activities. Actually, this method might be interpreted as a simplified
adaptation of the frequent subgraph mining (FSM) method. The FRAGTAL method
reconstructs the way in which medicinal chemists are used to designing a prospective drug
structure intuitively. A representative example of the practical application of FRAGTAL
within the ChemDB Anti-HIV/OI/TB database for disclosing new fragments for HIV-1
integrase inhibition is discussed. In particular, FRAGTAL method identifies ethyl malonate
amide (EMA) as the diketo acid (DKA) related arrangement. Since new molecular constructs
based on the EMA fragment are still a matter of future investigations we referred to this as an
the DKA offspring.
P68
DETERMINATION OF HCH ISOMERS AND CHROMATOGRAPHIC
EVALUATION OF BIOREMEDIATION OF WATER ENVIRONMENTS
CONTAMINATED WITH PESTICIDES
D. Szeremeta a, M. Knaś a, T. Kowalska a, M. Sajewicz a
a
of Silesia in Katowice,40-006 Katowice, Szkolna Street 9, Poland
1. Introduction
Pesticides have been applied for decades in preventing, repelling or mitigating the effects
of pests. Although most of organochlorine pesticides (OCPs) including
hexachlorocyclohexane (HCH) isomers, have been banned in many countries because of
mutagenic and carcinogenic effects [1], they and their metabolites are still present in the
environment [2]. Most of these organic compounds have a tendency to bioaccumulate and
present low rates of biodegradation and consequently, they could represent a risk to
environmental and human health.
In our studies, we have focused on determination of HCH isomers in water samples by gas
chromatography and chromatographic evaluation of the effectiveness of bioremediation of
water environments contaminated with organochlorine pesticides and their degradation
products.
Pesticides are present in the environment at concentrations of the order of mg/kg or ng/kg,
and therefore chromatographic analysis usually follows the tedious sample preparation to
extract the pollutants from environmental matrices and to enrich the analyte.
Chromatographic techniques have been considered as the best methods to determine OCPs in
varied sample matrices [3].
Analytical procedure used in our research involved the selection of the gas
chromatographic working conditions for the determination of organochlorine pesticides and
their degradation products, preparation of calibration curves of the test compounds, and the
choice of extraction technique that allows for satisfying recovery of the analytes from
environmental samples. Purified extracts were analyzed by capillary gas chromatography
equipped with mass spectrometer (MS) and electron capture detector (ECD). Further stage of
our research will be chromatographic monitoring of the process of bioremediation aided by
biostimulation and bioaugmentation. We also wish to draw wider attention to the observation
of the natural self-purification of the contaminated environment.
P69
Acknowledgements:
One author (D.Sz.) is the scholarship recipient within the framework of the “DoktoRIS
Scholarship Programme for the Innovative Silesia”, subsidized by the European Social Fund
of the European Union.
[1]. G.R. van der Hoff, P. van Zoonen, J. Chromatogr. A, 843, 1999, 301-322.
[2]. M.T. Gonullu, et. al, Fresen. Environ. Bull. 12, 2003, 1457-1464.
[3]. F.J. Santos, M.T. Galceran, Trends Anal. Chem., 23, 2004, 672-685.
P69
SEPARATION OF TRANSFERRIN FORMS WITH DIFFERENT IRON SATURATION AND
ANALYSIS OF IRON BINGING MECHANISM BY MICELLAR ELECTROKINETIC
CHROMATOGRAPHY
K. Śpiewak a, P. Nowak b, M. Woźniakiewicz b, G. Stochel a, M. Brindell a
a
Department of Inorganic Chemitry, Faculty of Chemistry, Jagiellonian University,
Department of Analytical Chemitry, Faculty of Chemistry, Jagiellonian University,
b
1. Introduction
Efficient separation of iron-free and iron-saturated forms of transferrin (Tf) by using
chromatographic and electromigration techniques was a problematic task, due to their barely
subtle difference in a mass-to-charge ratio. However, micellar electrokinetic chromatography
(MEKC) occurred to be suitable technique for that purpose, and allowed us to separate these
similar proteins. MEKC is a chromatography technique. It is a modification of capillary
electrophoresis (CE), where the samples are separated by differential partitioning between
micelles (pseudo-stationary phase) and a surrounding aqueous buffer solution (mobile phase).
This technique allowed us to investigate the mechanism of iron binding by transferrin.
Separation of apo- and holotransferrin
was
successfully
performed.
The
established BGE (Background Electrolyte)
composition was: 50 mM Tris buffer, pH
8.5, 0.2% (w/v) SDS [1].
Then, the method has been further
tuned to disclose the presence of predicted
monoferric forms of Tf, but previously
undetected by our method. New
conditions optimized by a Doehlert
experimental design and surface response
methodology consisted in the addition of
22.5% (v/v) methanol and increased SDS
concentration up to 17.5 mM.2 This
Fig.1 Electropherogram presenting the separation of the
method allowed us to separate four
mixed sample containing h-Tf, a-Tf, and HSA,
different forms of Tf, including: iron-free,
N-terminal monoferric, C-terminal monoferric, and differic forms (Fig. 1), with excellent
selectivity and total separation time 4 min. The experiment assuming the preparation of
samples with gradually increasing concentration of iron revealed continuously changing
contribution of monoferric forms, and was consistent with theoretical assumptions [2].
P70
[1]. Nowak P. Śpiewak K. Brindell M. Woźniakiewicz M. Stochel G. Kościelniak P. J.
Chrom. A. 2013
[2]. Nowak P. Śpiewak K. Nowak J., Brindell M. Woźniakiewicz M. Stochel G. Kościelniak
P. J. Chrom. A. 2014
P70
TRANSFERRIN AS SPECIFIC DRUG CARRIER. AFFINITY OF NAMI-A TO
TRANSFERRIN COMPONENT IN PLASMA MODELS AND HUMAN SERUM
K. Śpiewak a, G. Stochel a, M. Brindell a
a
Department of Inorganic Chemitry, Faculty of Chemistry, Jagiellonian University,
1. Introduction
A new and attractive strategy to enhance therapeutic properties of drugs is to deliver these
agents specifically to defined target cells thus keeping them away from healthy cells. Recent
studies focus on the transferrin receptor (TfR), carrier protein for transferrin (Tf) which is
taking part in cellular iron uptake [1]. Due to overexpression of TfR in primary proliferating
malignant cells [2], this receptor is an attractive molecule for the targeted cancer therapy. TfR
can be targeted in two ways: the delivery of a drug into malignant cells or blocking the
function of receptor leading to cancer cells death. This finding leads to concept that the
transferrin uptake pathway could be used for delivery of chemotherapeutic drugs or other
molecular cargo to cancer cells [3]. The transferrin targeted delivery is proposed as a
mechanism of uptake for antitumor ruthenium complex NAMI-A [4]. Since NAMI-A is
administrated to systemic blood circulation binding of drug to transferrin should be examined.
This information will give an answer what percentage of administrated complex can be
specifically transported into cancer cells.
Our research shows that holo-transferrin binds NAMI-A complex in about 1:1 (Ru:Tf)
molar ratio in simplified serum model in presence of albumin as competitive binding target.
Addition of small molecule components of serum like amino acids, lactate, citrate and oxalate
and others increases NAMI-A binding to transferrin up to 2:1.What is interesting the presence
of metal ions (Ca2+, Mg2+, Zn2+) increases amount of ruthenium bound to transferrin even
more to 3:1 molar ratio. Finally, ruthenium content in transferrin isolated from human serum
incubated with NAMI-A was checked. Ruthenium was bound to transferrin in 0.65:1 molar
ratio. That outcome suggests that only about 3 % of administered NAMI-A complex can be
specifically transported by transferrin, even though most of the Ru in blood plasma is
accumulated in the protein-bound form (>97%)4. Our results indicate that only a certain
amount of administrated NAMI-A is bound to transferrin. To avoid drug side effects, NAMIA can be administrated as a transferrin bound adduct and effectiveness of this form should be
checked.
Acknowledgment:
Grant K/PBM/000241, 2014-2016, from Polish Ministry of Science and High School
Education is gratefully acknowledged.
P71
[1]. Daniels, T.R., et al., Biochim. Biophys. Acta, 2012. 1820(3): p. 291-317.
[2]. Ding Cheng, Y., et al., 2001. 21(1 B): p. 541-549.
[3]. Ali, S.A., et al., J. Biol. Chem. 1999. 274(34): p. 24066-24073.
[4]. J.M. Rademaker-Lakhai, et al., Clin. Cancer Res. 10 (2004) 3717–3727.
P71
RUTHENIUM COMPLEXES WITH PHOSPHANES POSSESSING
MORPHOLINE OR PIPERAZINE RINGS
K. Śpiewak a M. Płotek a,b, R. Starosta c, U. Komarnicka c, A. Skórska-Stania a, G. Stochel a,
A. Kyzioł a
a
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow
Faculty of Conservation and Restoration of Works of Arts, Jan Matejko Academy of Fine
Arts in Krakow, Lea 27-29, 30-052 Krakow
c
Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw
b
1. Introduction
Cancer is still the second most common cause of death in the world [1]. Therefore a search
for other metal complexes with selected cytotoxicity against cancer cells seems to be one of
the most important challenges of the twenty-first century. Anticancer activity of cisplatin,
proved by Rosenberg, resulted in searching for other metal complexes with potential activity
in antitumor therapies [2]. Currently, ruthenium complexes are one of the biggest group of
investigated coordination compounds. It was shown that ruthenium compounds could mimic
iron in biological systems and thus be easily transported into cells by transferrin [3].
Synthesis and physicochemical analysis of ruthenium compounds with phosphanes
possessing morpholine or piperazine ring was the main aim of the presented research. The
general formula of presented complexes is Ru(phosphane)2Cl2. Our research revealed that
discussed phosphanes coordinate to ruthenium(II) ions as a chelating bidentate agents using
phosphorus and nitrogen atoms. Studied complexes showed cytotoxic activity in vitro against
human lung adenocarcinoma cell line (A549). Moreover interaction with human serum
albumin was confirmed.
P72
[1]. American Cancer Society, Global Cancer – Facts&Figures 2007, p. 1.
[2]. Pizarro A.M., Sadler P.J., Biochimie 91 (2009) 1198-1211.
[3]. Allardyce C.S., Dorcier A., Scolaro C., Dyson P.J., Appl. Organomet. Chem. 19 (2005)
1-10.
P72
NEW THIOPHENE DERIVATIVES – [2+1+2+1] CYCLOADDITON REACTIONS
A. Tkocz a, S. Kula a, A. Szłapa a, J. Bogocz a, S. Krompiec a
a
Institute of Chemistry, University of Silesia, Szkolna 9, 40-006 Katowice, Poland
1. Introduction
The rhenium catalyzed [2 + 1 + 2 + 1] cycloaddition reactions of disubstituted alkynes to
β-keto esters provides a very strightforward and efficient strategy with which to obtain
thiophene derivatives [1-3]. Disubstituted alkyne (1,2-bis(2,2`-bithiophene-5-yl)ethyne) [4]
allows the preparation of 2-pyranone which could be used as a building block in the synthesis
of polysubstituted benzene derivatives with a 2,2- bithiophene motif. Thus, mentioned
approach allows to synthesize new monomers especially for conductive polymers, which are
typically used in organic electronics. The results indicate that the heteroarene and poli
(heteroarene) containing thiophene moiety shows good thermal and chemical stability.
In the first part, the preparation of 5-iodo-2,2-bithiophene was carried out. Afterwards, the
synthesis of 1,2-bis(2,2`-bithiophene-5-yl)ethyne was achieved through Sonogashira coupling
reaction between 5-iodo-2,2-bithiophene and acetylene. Next, the formation of 2-pyranone
derivative with 2,2’-bithiophene motif was observed in [2 + 1 + 2 + 1] cycloaddition reaction
of 1,2-bis(2,2`-bithiophene-5-yl)ethyne with ethyl acetoacetate. Subsequently, we carried out
a Diels-Alder [4 + 2] cycloaddition reaction with a conjugated diethyl acetylenedicarboxylate
which gave pentasubstituted benzene derivative with two 2,2-bithiophene substituents. The
structure of all final compounds have been characterized by using HRMS and spectroscopic
methods. Furthermore, LUMO and HOMO orbitals energy was calculated using DFT method.
Hereby, width of energy gap and localization of orbitals have been established.
Acknowledgments:
The research was co-financed by the National Research and Development Center
(NCBiR) under Grant ORGANOMET No: PBS2/A5/40/2014. Agata Szłapa received a grant
for the project DoktoRIS - Scholarship program for innovative Silesia co-financed by the
European Union in the framework of the ESF. Sławomir Kula appreciates the support of the
FORSZT fellowships subsidized by the European Social Fund of the European Union..
[1]. Y. Kuninobu, A. Kawata, M. Nishi, H. Takata, K. Takai, Chem. Commun., 2008, 6360–
6362.
[2]. Y. Kuninobu, H. Takata, A. Kawata, K. Takai, Org. Lett., 10, 2008, 3133-3135.
[3]. Y. Kuninobu, M. Nishi, A. Kawata, H. Takata, Y. Hanatani, S. Yudha, A. Iwai, K. Takai,
J. Org. Chem., 2010, 75, 334–341.
[4]. S. Krompiec, M. Filapek, I. Grudzka, S. Kula, A. Słodek, Ł. Skórka, W. Danikiewicz, P.
Ledwon, M. Lapkowski, Synthetic Metals, 165, 2013, 7–16.
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THE STUDY OF THE INTERACTIONS OF 1,2-DIMYRISTOYL-SNGLYCERO-3-PHOSPHOCHOLINE WITH MYRICETIN BY IR
SPECTROSCOPY
D. Vojta a, K. Dominković a, G. Baranović a
a
Division of Organic Chemistry and Biochemistry, Ruđer Boškovic Institute, Bijenička 54,
10000 Zagreb
1. Introduction
Myricetin (MY) is a herbal compound that belongs to the class of flavonols and expresses
various beneficial effects for animal organisms [1]. As the mechanism of its actions is still
vague, its elucidation primary requires the research of its pharmacological activities. It is
essential to explore MY interactions with biological and, very often, model membranes [2]. In
this work, the interactions between 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as
model lipid (Fig. 1a) and MY (Fig. 1b) are explored by using IR spectroscopy.
Pure DMPC and the mixtures of DMPC and MY, containing various concentrations of
MY, were recorded as liposomes in phosphate buffer (PBS). From the position of the band
attributed to the symmetric CH2 stretching, DMPC melting temperature (Tm) was determined.
It is 24 °C for pure DMPC, while for DMPC+MY mixtures, Tm decreases with MY
concentration. For molar ratio n(DMPC):n(MY)=1: 0.35, Tm is 19 °C .
Fig. 1. a) DMPC, b) MY
Due to the lowering of melting point of DMPC in the presence of MY, the incorporation
of the latter is unambigously proved. Their interaction pattern, obtained by recording
multilayered films of DMPC and MY, hydrated with PBS, suggest the preferential
associations of MY with the polar-apolar interface of DMPC and, respectively, with its polar
head groups. The findings obtained here will serve as the platform for the study of MY effect
on the neural cells [3] and, hopefully, will provide an insight into its regenerating ability.
P74
P74
[1]. Y. S. Tarahovsky et al., Biochim. Biophys. Acta, 2014, 1838, 1235.
[2]. M. Arczewska et al., Biochim. Biophys. Acta, 2013, 1828, 213.
[3]. M. Jazvinšćak Jembrek et al., J. Mol. Neurosci., 2012, 47, 286.
SYNTHESIS OF NEW THIOTEREPHTALAMIDES
A. Jędrzejowska a, P. Dybał a, V. Kozik a, M. Matuszek a, K. Jarzembek a, A. Bąk a, P. Kuś a
a
University of Silesia, Institute of Chemistry, Szkolna 9 street, 40-006 Katowice, Poland
e-mail of presenting author:
The aim of this study are to synthesizing new thioterephtalic acid diamides
(thioterephtalamides) from thioterephthaloyl chloride, phosphorus pentasulfide with carrier
compound and methyl esters of chosen aminoacids, investigating the physicochemical
properties of the new compounds and trying to find their crystallographic structure. Chemical
structure obtained intermediates products and thioterephtalamides was determined by 1H
NMR, 13C NMR spectra. Solubility and melting point were determined for new molecules.
R
S
NH
NH
S
R
R = methyl ester aminoacid
[1]. Polshettiwar, V.; Kaushik, M. P. Tetrahedron Lett. 2006, 47, 2315.
[2]. Curphey, T. J. Tetrahedron Lett. 2002, 43, 371.
P75
P75
AUTHOR INDEX
Andrushchenko V.
Andujar S.
Antalík M.
Arruebo M.
Asaka M.
Babula P.
Baranović G.
Bąk A.
Besserglick J.
Bieg T.
Binter A.
Bluyssen H.A.R.
Bogár F.
Bogocz J.
Bonarek P.
Bončina M.
Bouř P.
Brabec V.
Brancatelli G.
Branilović M.G.
Brindell M.
Broersen K.
Bujacz G.
Bukovský M.
Byczek A.
Cahlikova L.
Celewicz L.
Cernikova A.
Cescutti P.
Chmurzyński L.
Choma I.
Chomanič P.
Chrobok A.
Chudzik M.
Cieślik W.
Coffey A.
Cysewski P.
Czerwoniec A.
Czyż K.
Dall'Antonia F.
Devanaboyina S.C.
O6
O3
P3
P30
O1
P21
P74
P68, P75
PL2
P2
O8
O11
P42
P60, P73
P41
PL11
O6
P11
P37
P10
O4, P26, P27,
P70, P71
O3
P48
P33
P6
P57
P12
P19
PL3, P4
P9
P58
P24
P1
P36
P8, P28
O13
PL15
O11
P59
O14
O14,
Devínsky F.
Dolfi S.
Dominković K.
Donchenko G.V.
Dražić T.
Drozdowska D.
Drożdż A.
Dubrawska B.
Dybał P.
Enriz R.D.
Faber K.
Fedoročko P.
Fischer-Fodor E.
Frączek T.
Frączyk J.
Fritsky I.O.
Garajová M.
Geremia S.
Gillner D.
Godziek A.
Golenia I.
Gonec T.
Gros P.C.
Gruber Ch.C.
Gruber K.
Guisasola E. B.
Gumienna-Kontecka E.
Guzik K.
Gyurcsik B.
Habuš I.
Hahn P.
Hajduch M.
Hamuľaková S.
Hamze K.
Havrankova J.
Hecel A.
Hećimović S.
Hegedüs Z.
Hetényi A.
Hofman J.
Hogendorf A.
P7, P24, P33
P4
P74
P13
P20
O15
P1
P51
P75
O3
O8
P11
P7
O7
O12, O15, P46,
P50, P52
P55
P24, P33
P37
P18
PL14, P61, P65
P55
O5
P27
O8
O8
O3
PL2, P55, P67
P34
O1
P20
P1, P2
PL5
P11
P23
P57
PL1
P20
P38
P38, P44
P57
P35
Holak T.A.
Holak T.A.
Horváth J.
Hricovíni M.
Hruba M.
Hubin E.
Imre N.
Jabłoński M.
Jacewicz D.
Jajčanin-Jozić N.
Jampilek J.
Janočková J.
Jarzembek K.
Jasiewicz B.
Jendželovský R.
Jesionek W.
Jędrzejowska A.
Kaca W.
Kacprzak K.
Kaczmarek A.
Kamiński Z.
Kapkowski M.
Karlovská J.
Kašpárková J.
Kasprzycka A.
Keller W.
Khalifa M.
Kieda C.
Kitel R.
Kleczewska N.
Knaś M.
Kolesińska B.
Kolkowska P.
Komarnicka U.
Konieczna I.
Korytkowska-Wałach A.
Korzec M.
Kos J.
Kovaľ J.
Kowalska T.
Kozanecka W.
P34
P35
P39, P40, P43
PL4
O5
O3
P38
P41
P9
P17
O5, P19, P21
P11, P13
P68, P75
P23, P31
P11
P58
P75
P46
O10
P46
O12, O15, P46,
P52, P50
P62
P24
P11
P1, P6
O14
P53
P26
P22
P12
P69
O15, P46, P50,
P52
PL1
P72
P46
P51
P63
O5
P11
PL14, P58,
P61, P65, P66,
P69
P23
Holak T.A.
Horváth J.
Hricovíni M.
Hruba M.
Hubin E.
Imre N.
Jabłoński M.
Jacewicz D.
Jajčanin-Jozić N.
Jampilek J.
Janočková J.
Jarzembek K.
Jasiewicz B.
Jendželovský R.
Jesionek W.
Jędrzejowska A.
Kaca W.
Kacprzak K.
Kaczmarek A.
Kamiński Z.
Kapkowski M.
Karlovská J.
Kašpárková J.
Kasprzycka A.
Keller W.
Khalifa M.
Kieda C.
Kitel R.
Kleczewska N.
Knaś M.
Kolesińska B.
Kolkowska P.
Komarnicka U.
Konieczna I.
Korytkowska-Wałach A.
Korzec M.
Kos J.
Kovaľ J.
Kowalska T.
P35
P39, P40, P43
PL4
O5
O3
P38
P41
P9
P17
O5, P19, P21
P11, P13
P68, P75
P23, P31
P11
P58
P75
P46
O10
P46
O12, O15, P46,
P52, P50
P62
P24
P11
P1, P6
O14
P53
P26
P22
P12
P69
O15, P46, P50,
P52
PL1
P72
P46
P51
P63
O5
P11
PL14, P58,
P61, P65, P66,
P69
Makra I.
Malarz K.
Malcekova B.
Malnar M.
Małecki J.G.
Maroń A.
Martinčić J.
Martinek T.
Martinek T.A.
Marvanová P.
Matuszek M.
Mazgała A.
Mazuryk O.
Michna J.
Mikláš R.
Miklášová N.
Miláčková I.
Mokrý P.
Molčanov K.
Monostori É.
Moravcova M.
Mrozek-Wilczkiewicz A.
Mrówczyńska L.
Mrva M.
Mularski J.
Musielak B.
Musioł R.
Nagata K.
Násztor Z.
Nawrot U.
Németh E.
Nowak P.
Nycz J.E.
Obrowsky S.
Odehnalová K.
Olajos G.
Olędzka M.
Olshvang E.
Ondriska F.
Oostenbrink C.
Oravec M.
Orłowska M.
Padrtová T.
P38
P54
P47
P20
P59
P59
P17
P38
PL13, P44
P25, P29
P75
P56
O4, P26, P27
P56
P7
P7
P32
P25, P29
P20
P38
O5
P8, P54
P23, P31
P24, P33
P28
P34
P8, P28, P54
O1
P39, P40, P42,
P43
P2
O1
P70
P59
P20
P25, P29
P44
P55
PL2
P24, P33
O1
P21
P66
P25, P29
Malarz K.
Malcekova B.
Malnar M.
Małecki J.G.
Maroń A.
Martinčić J.
Martinek T.
Martinek T.A.
Marvanová P.
Matuszek M.
Mazgała A.
Mazuryk O.
Michna J.
Mikláš R.
Miklášová N.
Miláčková I.
Mokrý P.
Molčanov K.
Monostori É.
Moravcova M.
Mrozek-Wilczkiewicz A.
Mrówczyńska L.
Mrva M.
Mularski J.
Musielak B.
Musioł R.
Nagata K.
Násztor Z.
Nawrot U.
Németh E.
Nowak P.
Nycz J.E.
Obrowsky S.
Odehnalová K.
Olajos G.
Olędzka M.
Olshvang E.
Ondriska F.
Oostenbrink C.
Oravec M.
Orłowska M.
Padrtová T.
P54
P47
P20
P59
P59
P17
P38
PL13, P44
P25, P29
P75
P56
O4, P26, P27
P56
P7
P7
P32
P25, P29
P20
P38
O5
P8, P54
P23, P31
P24, P33
P28
P34
P8, P28, P54
O1
P39, P40, P42,
P43
P2
O1
P70
P59
P20
P25, P29
P44
P55
PL2
P24, P33
O1
P21
P66
P25, P29
Skarydova L.
Šket P.
P47, P57
PL12
Skorek M.
Skórska-Stania A.
Słota M.
Smolko A.
Šoltésová Prnová M.
Soritau O.
Spaczyńska E.
Stambergova H.
Starosta R.
Staško M.
Štefek M.
Steiner K.
Steinkellner G.
Stochel G.
P58
P72
P62
P17
P32
P7
P8
P47
P72
P32
P32
O8
O8
P30, P56, P70,
P71, P72
P48
P59
O5
P21
P36, P45, P49
P17
P26
P59
PL9
PL2
P1, P22
O11
P69
P49
P73
P66
P70, P71, P72
P50
P59
P24, P33
P46
P60, P73
P18
P34, P35
P10
O9, P14
P41
Strzelczyk P.
Strzelczyk R.
Stujber M.
Suchy P.
Sułkowska A.
Šupljika F.
Suzenet F.
Szala M.
Szalaya P.G.
Szebesczyk A.
Szeja W.
Szelag M.
Szeremeta D.
Szkudlarek-Haśnik A.
Szłapa A.
Szymczak G.
Śpiewak K.
Świontek M.
Świtlicka-Olszewska A.
Timko L.
Timler D.
Tkocz A.
Tolarczyk J.
Tomala M.
Tomić S.
Tóthová P.
Tworzydło M.
Šket P.
PL12
Skorek M.
Skórska-Stania A.
Słota M.
Smolko A.
Šoltésová Prnová M.
Soritau O.
Spaczyńska E.
Stambergova H.
Starosta R.
Staško M.
Štefek M.
Steiner K.
Steinkellner G.
Stochel G.
P58
P72
P62
P17
P32
P7
P8
P47
P72
P32
P32
O8
O8
P30, P56, P70,
P71, P72
P48
P59
O5
P21
P36, P45, P49
P17
P26
P59
Strzelczyk P.
Strzelczyk R.
Stujber M.
Suchy P.
Sułkowska A.
Šupljika F.
Suzenet F.
Szala M.

KATOWICE – September, 9 – 12 - Chemistry towards Biology

Transcription

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