2 - Biotransformacje

2nd Symposium on Biotransformations for
Pharmaceutical and Cosmetic Industry
Programme and Abstracts
October, 23rd - 24th 2014
Warsaw, Poland
Institute of Organic Chemistry
Polish Academy of Sciences
Warszawa 2014
Programme and Abstracts
2nd Symposium on Biotransformations for Pharmaceutical and Cosmetic Industry
Cover design: Marcin Staśko
Editors:
Anna Brodzka
Szymon Kłossowski
Dominik Koszelewski
Małgorzata Zysk
Copyright © by Institute of Organic Chemistry,
Polish Academy of Sciences
ISBN: 978-83-913306-9-2
Warszawa 2014
2
Committees
Organizing Committee
Ryszard Ostaszewski – Head of Organizing Committee
Dominik Koszelewski - Secretary
Szymon Kłossowski
Anna Brodzka
Małgorzata Zysk
Scientific Committee
Prof. Ryszard Ostaszewski
Dr. Dominik Koszelewski
Dr. Stanisław Berłożecki
3
Sponsors
The Organizing Committee gratefully acknowledges the sponsorship of the Symposium
by following companies and organizations:
4
Biotransformations useful for pharmaceutical and cosmetic industries
Five years ago a consortium of seven Polish academic institutions decided to join the efforts
and experience in order to build up the project devoted to the use of biotransformations in
such a way that they could be useful as a mean for pharmaceutical and cosmetic industries.
The first step of these efforts was consolidation of the research carried out in Poland in that
respect and to build the platform for further collaboration. This task was fully achieved and
gave a new dimension to Polish studies within the frame of “white biotechnology”.
The following institutions have been collaborating within the scope of the title project:
Jerzy Haber Institute of Catalysis and surface Chemistry Polish Academy of Sciences
in Kraków (leader: prof. Maciej Szaleniec);
Institute of Organic Chemistry Polish Academy of Sciences (leader: prof. Ryszard
Ostaszewski)
Łódź University of Technology (leader: prof. Tadeusz Antczak);
Silesian University of Technology (leader: prof. Andrzej Jarzębski);
Warsaw University of Technology (leader: prof. Maria Bretner);
Wrocław University of Environmental and Life Sciences (leaders: prof. Czesław
Wawrzeńczyk and dr Edta Kostrzewa-Susłow);
Wrocław University of Technology (leaders: prof. Jolanta Bryjak, prof. Ewa
Żymańczyk-Duda and prof. Stanisław Lochyński);
The research was carried out within the scope of 12 research tasks considering purely basic
studies to studies devoted to introduction new technologies and new biocatalysts. The results
of the projects could be illustrated by 65 papers published in internationally respected
journals, by 90 issued patent and 22 patent applications.
5
Conference venue
The 2nd Symposium on Biotransformations for Pharmaceutical and Cosmetic Industry will
take place at Institute of Organic Chemistry Polish Academy of Sciences.
Institute of Organic Chemistry PAS
Kasprzaka 44/52, 01-224 Warsaw
Public transport
BUS – Szpital Wolski
105, 178 - from Świętokrzyska Metro Station
109 – from Central Railway Station
BUS - Krzyżanowskiego
103 – from West Railway Station
6
General Information
Registration Desk, Institute of Organic Chemistry Polish Academy of Sciences
Opening hours:
Thursday, 23rd of October: 10.00am –5.00pm
Friday, 24th of October: 9:00am –1:00pm
Please register upon arrival in order to receive your congress materials and personal name
badge.
Conference Dinner: Thursday, 23rd of October, Institute of Organic Chemistry PAS
Time: 6.30pm –10.00pm
Information for the Speakers:
Plenary lectures will take 45 minutes, lectures of invited speakers -30 minutes and oral
presentations -15 minutes, including discussion. Speakers are kindly requested to prepare
their oral presentations in .pdf, .ppt and .pptx file formats, compatible with Windows 7. If
using a Macintosh computer, please ensure that it has a VGA socket for external signal.
Speakers are asked to contact the registration desk at least 30 minutes prior to the session start
to load their presentations onto the conference computer and to preview them in advance.
Posters:
The suggested poster size is in a portrait format with a dimension of A0 size. All the material
necessary (pins and tacks) for attaching the poster to the poster board will be provided by the
organizers.
The best posters will be awarded.
Lunches:
Lunch on 24th of October is included in the conference fee, and Lunch Voucher will be
provided in the conference package. There will be the opportunity to purchase lunches in our
canteen on the other days.
7
8
Programme
9
10
The Conference is dedicated to Prof. Paweł Kafarski on the occasion of his
65th birthday.
Thursday, October 23rd, 2014
10:00 – 14:00
Conference registration
14:00 – 14:10
Opening ceremony
Prof. Ryszard Ostaszewski
Prof. Sławomir Jarosz (Director of the Institute of Organic Chemistry,
Polish Academy of Sciences)
Session chair: Prof. Czesław Wawrzeńczyk
14:10 – 14:40
14:40 – 15:25
Honour Lecture
Prof. Paweł Kafarski (Wroclaw University of Technology)
My Way
Plenary Lecture
Prof. Kurt Faber (Austrian Centre of Industrial Biotechnology)
Stereo-Complementary Biotransformations for the Pharmaceutical and
Cosmetic Industry
15:25 – 15:45
Coffee break
15:45 – 16:45
Poster session
Session chair: Prof. Piotr Kiełbasiński
16:45 – 17:15
17:15 – 17:30
17:30 – 17:45
17:45 – 18:00
Invited Lecture
Dr Anna Fryszkowska (Dr. Reddy’s Laboratories Ltd)
Application of Biocatalysis in Active Pharmaceutical Ingredients
Synthesis: from Reaction to Process
Oral Communication
Dr Anna Gliszczyńska (Wroclaw University of Environmental and
Life Sciences)
Biotransformations of Isoprenoids and Their Practical Applications
Oral Communication
Dr Aleksandra Grudniewska (Wroclaw University of Environmental
and Life Sciences)
Biotransformation
of
Terpenoid
Lactones
with
the p-Menthane System
Oral Communication
Dr Witold Gładkowski (Wroclaw University of Environmental and
Life Sciences)
Microbial Transformations of Halolactones
18:00 – 18:30
Free time
18:30 – ……
Conference dinner
11
Friday, October 24th, 2014
Session chair: Prof. Kurt Faber
9:15 – 10:00
10:00 – 10:15
10:15 – 10:30
10:30 – 10:45
10:45 – 11:00
11:00 – 11:20
Plenary Lecture
Prof. Stefano Servi (The Protein Factory, Polimi, Uninsubria)
Systems Biocatalysis: Organizing Multiple Enzymatic Catalysts for
Improved Synthetic Functions
Oral Communication
Szymon Kłossowski (Institute of Organic Chemistry PAS)
The Application of Lipases Promiscuity for Enzymatic
Multicomponent Reaction With Isocyanides
Oral Communication
Dr Rama Krishna Gudiminchi (Austrian Centre of Industrial
Biotechnology)
Sucrose Phosphorylase – a Potential Biocatalyst for Industrial
Glycosylation Reactions
Oral Communication
Małgorzata Cieńska (Wroclaw University of Technology)
Diphenolic Compounds Production by Tyrosinase from Agaricus
bisporus
Oral Communication
Natalia Kmiecik (Wroclaw University of Technology)
Biotransformation of Aminophosphonic Acids Using Fungi
Coffee break
Session chair: Prof. Stefano Servi
11:20 – 11:50
11:50 – 12:05
12:05 – 12:20
12:20 – 12:35
12:35 – 12:50
Invited Lecture
Prof. Piotr Kiełbasiński (Centre of Molecular and Macromolecular
Studies, Polish Academy of Sciences)
Hydrolytic Enzyme-Based Syntheses of Enantiopure Heteroatom
Derivatives as Precursors to Chiral Catalysts
Oral Communication
Anna Żądło (Institute of Organic Chemistry PAS)
Mixed Carbonates as a New Class of Substrates for Fluorogenic Assays
Oral Communication
Dr Illa Ramakanth (VŠB - Technical University of Ostrava)
Stimuli Responsive CPC Gels: Effect of pH on Gel Formation
Oral Communication
Mara Ana Naghi (Babes-Bolyai University)
Lipase-Mediated Kinetic Resolution Towards Enantiomerically
Enriched α- and β-Hydroxy Acids Derivatives
Oral Communication
Zofia Hrydziuszko (Wroclaw Univeristy of Technology)
Screening of Carriers for Lipase Immobilization for Reactions in Water
System
12
12:50 – 14:10
Lunch
Session chair: Dr Anna Fryszkowska
14:10 – 14:40
14:40 – 14:55
14:55 – 15:10
15:10 – 15:25
15:25 – 15:45
Invited Lecture
Dr Sylvia Glueck (Austrian Centre of Industrial Biotechnology)
Decarboxylases
as
Promising
'Green'
Biocatalytic
Tools
for Synthetic Applications
Oral Communication
Catalina Wiśniewska (Institute of Organic Chemistry PAS)
t-Butyl Acetoacetate as a Substrate for Enzymatic Transesterification
Oral Communication
Monika Górak (Wroclaw University of Technology)
Cyanobacteria as Biocatalyst for the Reduction of β Oxoalkylphosphonates
Oral Communication
Jarosław Popłoński (Wroclaw University of Environmental and Life
Sciences)
Filamentous Fungi as a Tool for Obtaining Hops Minor
Prenylflavonoid Glycosides
Coffee break
Session chair: Prof. Tadeusz Antczak
15:45 – 16:15
16:15 – 16:30
16:30 – 16:45
16:45 – 17:00
17:00 – 17:15
Invited Lecture
Dr Maciej Szleniec (Jerzy Haber Institute of Catalysis and Surface
Chemistry, Polish Academy of Scences)
Synthesis of Chiral Alcohols by Ethylbenzene Dehydrogenase – from
Modeling to Application
Oral Communication
Dr Katarzyna Szymańska (Silesian University of Technology)
High Performance Continuous Microreactors for Enzymatic
Biotransformations
Oral Communication
Paweł Borowiecki (Warsaw University of Technology)
First Chemoenzymatic Stereodivergent Total Synthesis of Both
Enantiomers of Promethazine and Ethopropazine
Oral Communication
Agnieszka Rugor (Jerzy Haber Institute of Catalysis and Surface
Chemistry, PAS)
Selective Modifications of Sterols Performed by Enzymes from
Sterolibacterium denitrificans
Closing remarks
13
14
Lectures
15
16
L01
My Way
Paweł Kafarski
Faculty of Chemistry, Department of Bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland –
[email protected]
Keywords: people, research, science
I was born in Gdańsk (Poland) on January 13th, 1949. I had finished high school in Zielona Góra, where I had a
good luck to meet real teacher – Dr. Zbigniew Czrnuch. I studied chemistry at Wrocław University of Technology where my
scientific adventure started with M.Sc. Thesis, completed in 1971 under the supervision of Prof. Przemysław Mastalerz. Prof.
Mastalerz subsequently supervised my scientific career for many years.
To my surprise this Symposium is organized to honor my birthday and I was asked to present some short resume of
my career. Thus, this presentation will present my research way from the beginning up to today. However, my biggest
success is seen at the Figure.
Figure
Acknowledgements
I would like to thank all the people I had a chance, pleasure and honor to work with.
17
L02
Stereo-Complementary Biotransformations for the Pharmaceutical and Cosmetic
Industry
Karl Gruber,2 Georg Steinkellner,1,2 Melanie Hall,3 Verena Resch,1,3 Michael Toesch,3 Nikolaus Turrini,3 Kurt
Faber1,3
1
2
3
Austrian Centre of Industrial Biotechnology (ACIB) c/o
Institute of Molecular Biosciences, University of Graz, Graz, Austria,
Department of Chemistry, University of Graz, Graz, Austria – [email protected]
Keywords: asymmetric bioreduction, ene-reductase, stereo-convergent sulfatase
Despite the clear advantages of biocatalysts, such as high catalytic efficiency, environmental compatibility and
mild reaction conditions, the most striking drawback is the unavailability of mirror-image proteins, which makes the
production of enantiomeric products a difficult task [1]. In order to circumvent this limitation, several strategies have been
devised, which are based on (i) choice of enzymes with opposite stereo-preference, and (ii) the modification of the substrate
[2]. These concepts are demonstrated by examples from the stereo-complementary asymmetric bioreduction of activated
alkenes using ene-reductases [3] and the enantio-convergent hydrolysis of sec-sulfate esters using sulfatases [4].
Prochiral alkenes bearing an electron-withdrawing group can be reduced by ene-reductases yielding the
corresponding trans-hydrogenation products in excellent yields and e.e.s. The stereochemical outcome of the bioreduction
can either be controlled by the E/Z-configuration of the substrate or by choice of the enzyme. The applicability of this method
was demonstrated by the synthesis of α-methylcinnamaldehyde derivatives used in the fragrance industry (lily-of-the-valley,
LilialTM) and precursors for pharma-targets (GABA-analogs, PregabalinTM) [5]. A computer-based three-dimensional search
protocol for stereo-complementary enzymes was recently developed [6].
In a complementary approach, the deracemization of sec-alcohols was achieved by stereo-convergent hydrolysis of
the corresponding racemic sulfate esters using a pair of sulfatases acting with retention or inversion of configuration, which
yields a single enantiomeric product in 100% theoretical yield [7]. The validity of this protocol was demonstrated by the
chemo-enzymatic asymmetric total synthesis of the anti-leukemic agent Lasiodiplodin [8].
Acknowledgements: This work has been supported by the FWF (projects W9 and P22722) and the BMWFJ,
BMVIT, SFG, Standortagentur Tirol and ZIT through the FFG-COMET funding program.
References
[1] P. Mugford, U. Wagner, Y. Jiang, K. Faber, R. Kazlauskas, Angew. Chem. Int. Ed. 2008, 47, 8782.
[2] G. Oberdorfer, K. Gruber, K. Faber, M. Hall, Synlett 2012, 1857.
[3] C. K. Winkler, G. Tasnadi, D. Clay, M. Hall, K. Faber, J. Biotechnol. 2012, 162, 381.
[4] P. Gadler, K. Faber, Trends Biotechnol. 2007, 25, 83; M. Toesch, M. Schober, K. Faber, Appl. Microbiol. Biotechnol.
2014, 98, 1485.
[5] C. Stueckler, N. J. Mueller, C. K. Winkler, S. M. Glueck, K. Gruber, G. Steinkellner, K. Faber, J. Chem. Soc., Dalton
Trans. 2010, 39, 8472; C. K. Winkler, et al. J. Org. Chem. 2013, 78, 1525.
[6] G. Steinkellner, et al. Nat. Commun. 2014, DOI: 10.1038/ncomms5150.
[7] M. Schober, M. Toesch, T. Knaus, G. A. Strohmeier, B. van Loo, M. Fuchs, F. Hollfelder, P. Macheroux, K. Faber,
Angew. Chem. Int. Ed. 2013, 52, 3277.
[8] M. Fuchs, M. Toesch, M. Schober, C. Wuensch, K. Faber, Eur. J. Org. Chem. 2013, 356.
18
L03
Application of Biocatalysis in Active Pharmaceutical Ingredients Synthesis: from
Reaction to Process
Anna Fryszkowska, Justine Peterson, Colin Dewar, Toni Fleming, Matthew Bycroft, Neil Triggs, Pieter de
Koning, Ian Taylor, Srikanth Sarat Chandra Gorantla, Christel Kronig, Karen Holt-Tiffin
Dr. Reddy’s Laboratories Ltd, Chirotech Technology Centre, 410 Science Park, Milton Road, Cambridge, CB4 0PE, UK –
[email protected]
Keywords: biocatalysis, screening, stereoselectivity, regioselectivity, oxidoreductases, racemases, amino acids, statins, steroids, assay
development
In 2000, 35% of APIs were chiral compounds,[1] while in the last 10 years, approximately 80% of the smallmolecule drugs approved by the Food and Drug Administration were chiral, of which nearly 75% were approved as single
enantiomer products. Biocatalysts are increasingly prevalent in the large-scale synthesis of enantiomerically pure
intermediates used in the synthesis of active pharmaceutical ingredients (APIs).
Recent advances in the development of both experimental and computational protein engineering tools have
enabled a number of further successes in the development of biocatalysts ready for large-scale applications; however the first
hurdle in every process R&D is the selection of the right enzyme/lead to develop a scalable biotransformation. In this talk I
would like to present two case studies and I will focus on the journey from the reaction discovery to a robust and efficient
process, enabled by using a key enzymatic step [2-3].
Fig. 1 Chiral intermediates obtained using chemoenzymatic reactions.
References
[1] D. J. Pollard, J. M. Woodley, Trends Biotechnol. 2007, 25, 66 – 73
[2] Patent application WO2012140507A1
[3] Patent application PCT/IB2014/061590
19
L04
Biotransformations of Isoprenoids and Their Practical Applications
Anna Gliszczyńska, Czesław Wawrzeńczyk
Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland - [email protected]
Keywords: microbial transformations, isoprenoids
Microbial biosynthesis of natural products is an emerging area of metabolic engineering and industrial
biotechnology. It offers significant advantages over conventional chemical methods or extraction from biomass. It is an
alternative way for chemical synthesis to produce new, active compounds in mild reaction conditions without risk of the
environment contamination.
One of the most diverse and largest family of natural products are isoprenoids [1,2,3]. They are widespread in the
nature and occur in plants, fungi and animals. They are the group of compounds with noteworthy biological and therapeutic
activities e.g. anti-inflammatory, antitumor, hypotensive, sedative so they are part of many important drugs [4,5]. As carriers
of specific odours some of them have also considerable value in the flavors and perfumery industry.
One of the tasks of our studies is the chemical and microbial synthesis of isoprenoid derivatives. Here we present
the process of biotransformation as a very useful tool in the synthesis of new isoprenoid compounds with enhanced biological
activity. This technique makes possible for example hydroxylation of inactivated carbon atoms in the structure of isoprenoids
in a single step what is a difficult challenge using chemical methods. On the example of farnesol, nerolidol or nootkatone we
demonstrate that microorganisms can be also employed to study the metabolism of natural compounds. In our experiments
fungal strains display the role of specific models, which imitate the metabolism of isopenoids in mammalians cells and let us
to observe their metabolic pathways. Moreover in the process of biotransformation we produce the compounds, which occur
in the nature but in very small quantity. The practical application of microbial transformation can be illustrated by
chemoenzymatic synthesis of pure enantiomers of lavandulol.
Acknowledgements
This project was financed by European Union from the European Regional Development Found Grant No.
POIG.01.03.01.-00-158/09.
References
[1] B.M. Fraga, Nat. Prod. Rep. 1999, 16, 711-711.
[2] P.B. Hylemon and J. Harder, FEMS Microbiol. Rev. 1999, 22, 475-488.
[3] J.C.G.Galindo, A. Hernandez, F.E. Dayan, M.R. Tellez, F.A. Macias, R.N. Paul, S.O. Duke, Phytochemistry 1999, 52,
805-813.
[4] M. Jimenez-Estrada, R.R. Chilpa, T.R. Apan, F.Ledias, W. Hansberg, D. Arrieta, F.J.A. Aguilar, J. Ethnopharmacol.
2006, 105, 34-38.
[5] A. Roja, A.R. Pizzy, C.M. Marson, N.S.B. Thomas, FEBS Lett. 2000, 467, 291-295.
20
L05
Biotransformation of Terpenoid Lactones with the p-Menthane System
Aleksandra Grudniewska, Radosław Gniłka and, Czesław Wawrzeńczyk
Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland - [email protected]
Keywords: biotransformation, lactones, p-menthane system
Lactones with the p-menthane system constitute a large group of naturally occurring and synthetic compounds.
Many of them them (e.g., mintlactone, isomintlactone and wine lactone) posses interesting odoriferous properties [1].
Synthetic lactones of this group are known for their antifungal and antifeedant activity [2].
The introduction of the hydroxy group into a molecule often leads to changes in its biological activity. Microbial
hydroxylation of non-activated carbon atom is a very valuable method of functionalization of organic molecules [3].
Here we present the results of microbial transformations of saturated (1, 2) and unsaturated (3) racemic lactones
with the p-menthane system (Fig. 1). Several biocatalysts were screened to check their ability to transform compounds
studied. Three of them (Absidia cylindrospora, Absidia glauca and Syncephalastrum racemosum) were found the most
effective biocatalysts. They transformed racemic substrates into the corresponding optically active hydroxy lactones.
Additionally, to confirm the absolute configuration of products obtained from the conversion of racemic lactones,
enantiomerically enriched substrates, with known absolute configuration, were subjected to the biotransformation. The
reaction progress was checked by chiral gas chromatography.
Fig. 1 Structure of lactones 1-3.
Acknowledgements
This work was financed by European Union from the European Regional Development Found Grant No.
POIG.01.03.01-00-158/09.
References
[1] J.M. Gaudin, Tetrahedron 2000, 56, 4769-4776.
[2] A. Grudniewska, K. Dancewicz, A. Białońska, C. Wawrzeńczyk, and B. Gabryś, J. Agric. Food Chem. 2013, 61, 33643372.
[3] H.L. Holland, and H.K. Weber, Curr. Opin. Biotechnol. 2000, 11, 537-542.
21
L06
Microbial Transformations of Halolactones
Witold Gładkowski, Marcelina Mazur , Aleksandra Grudniewska, and Czesław Wawrzeńczyk
Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland - [email protected]
Keywords: halolactones, dehalogenation, fungi
Haloorganic compounds exhibit wide spectrum of biological activity e.g. antimicrobial [1], anticancer [2] or
antifeedant [3]. They found application in many branches of industry i.a. pharmacy, medicine or chemical industry.
Unfortunately, large group of these compounds has a tendency to accumulate in natural environment. Their low
biodegradability and high toxicity are a serious ecological problem. One of the methods of their decomposition is the process
of microbial degradation which starts with removing the chlorine atom from the molecule. Different mechanisms can be
involved during these transformations including hydrolytic dehalogenation, intramolecular nucleophilic substitution
catalyzed by halohydrin dehalogenase, reductive dehalogenation or elimination of the HX molecule [4].
In our research group different halolactones exhibiting antifeedant or anticancer activity have been synthesized
[3,5]. One of the main requirements of future application of these compounds in natural environment is their easy
degradability. In this communication we report the studies over the biotransformations of halolactones with alkylsubstituted
cyclohexane or p-methoxyphenyl ring using fungal cultures Absidia cylindrospora, Absidia glauca or Mortierella isabellina.
The knowledge of the metabolism of these compounds by fungi will be useful in subsequent studies over their
biodegradability. On the other hand, as the products of biotransformations we expected to obtain new lactone oxyderivatives,
especially hydroxylactones, with some new biological activities.
The most common transformations of halolactones were hydrolytic dehalogenation and hydroxylation of nonactivated carbon atom. Formation of double bond followed by epoxidation was also observed. In some cases the action of
fungi led to the mixture of lactone oxyderivatives [4] (Fig. 1).
O
O
OH
O
O
O
O
O
I
O
O
O
O
O
Fig. 1 Metabolism of the iodolactone with trimethyl substituted cyclohexane ring in Absidia cylindrospora culture
In this communication different transformation pathways of halolactones will be presented and the effect of
substrate structure on the course of biotransformation will be discussed.
Acknowledgements
This research was partially financed by the European Union within the European Regional Development Found
(grant no.POIG.01.03.01-00-158/09).
References
[1] H.N. Bhargava and P.A Leonard, Am. J. Infect. Control. 1996, 24, 209.
[2] L.G. León, R.M. Carballo, et.al. Bioorg. Med. Chem. Lett. 2007, 17, 2681.
[3] M. Mazur, W. Gładkowski, M. Podkowik, J. Bania, J. Nawrot, A. Białońska and C. Wawrzeńczyk, Pest Manag. Sc.
2014, 70, 286.
[4] W. Gładkowski, M. Mazur, A. Białońska and C. Wawrzeńczyk, Enzyme Microb. Technol., 2011, 48, 326.
[5] W. Gładkowski, A. Skrobiszewski, M. Mazur, M. Siepka, A. Pawlak, B. Obmińska-Mrukowicz, A. Białońska, D.
Poradowski, A. Drynda and M. Urbaniak, Tetrahedron, 2013, 69, 10414.
22
L07
Systems Biocatalysis: Organizing Multiple Enzymatic Catalysts for Improved Synthetic
Functions
Stefano Servi2, Paola D’Arrigo1,2, Davide Tessaro1,2 and Loredano Pollegioni2,3
1
2
3
Dipartimento CMIC Giulio Natta, Politecnico di Milano, 20131 Milano, Italy
The Protein Factory, Polimi, Uninsubria, ICRM CNR - [email protected]
Dipartimento di Biotecnologie e Scienze della vita, Università dell’Insubria Varese,Italy
Keywords: multi step enzyme catalysed reactions, deracemization, artificial metabolism
Hydrolytic enzymes have dominated the era of modern biocatalysis, an intensive research period where enzymes
have been applied to the preparation of chiral synthons as single enantiomers. The advancement in protein engineering has
given synthetic organic chemists new instruments allowing to project complex synthetic sequences with a combination of
chemical and enzymatic catalysts. Systems Biocatalysis [1] aims at the construction of non natural pathways in what is
defined as an artificial metabolism [2], challenging metabolic engineering.
The Authors will retrace this itinerary remembering the application of phospholipases in reverse hydrolysis
conditions, [3] of amino acid oxidases/ amino transferases system for the production of non-natural amino acids in a
deracemization process, [4] proteases and bases in a dynamic kinetic resolution process [5] up to the construction of an open
artificial metabolic path were six enzymes work toghether building a futile cycle [6].
Acknowledgements
The Author (SS) wish to thank the Organizers for the invitation to the conference and the opportunity to visit their
University, Town, Country
References
[1] Systems Biocatalysis is the COST Action CM1303
[2] W. D. Fessner, C. Walter, Angew. Chem. Int. Ed. 1992, 31, 614-616.
[3] S. Servi, Phospholipases as synthetic catalysts in Topics in Current Chemistry. Biocatalysis: from discovery to
application, 1998, 200, 127-158, W.-D. Fessner editor, Springer Verlag.
[4] D. Tessaro, L. Cerioli, S. Servi, F. Viani, P. D’Arrigo, Adv. Synth. Catal. 2011, 353, 2333-2338
[5] P. D’Arrigo, L. Cerioli, S. Servi, F. Viani and D. Tessaro Catal. Sci. Technol., 2012, 2, 1606 - 1616
[6] D.Tessaro, L.Pollegioni, L. Piubelli, P. D’Arrigo and S.Servi, submitted.
23
L08
The Application of Lipases Promiscuity for Enzymatic Multicomponent Reaction With
Isocyanides
Szymon Kłossowski, Ryszard Ostaszewski
Institute of Organic Chemistry PAS, Kasprzaka 44/52, 01-224 Warszawa – [email protected]
Keywords: peptidomimetics, Ugi reaction, enzyme promiscuity
Enzymes offer the powerful platform for catalyzing the chemical reactions. Apart engineered enzymes which can
catalyze new reactions, the natural enzymes often possess activity toward reactions other than those catalyzed in nature. Such
ability (enzyme promiscuity) gives perspectives in developing new reaction catalyzed by proteins. The significant examples
includes the C-C bond formation in enzymatic reaction as well as multicomponent reaction accelerated by lipases.[1,2]
Here, the studies on the course of isocyanide-based multicomponent reaction catalyzed by enzymes will be
presented.[3] The development of the reaction was based on previously proposed mechanism for catalytic three-component
reaction and the mechanism of action of the lipases. [4] We will present the model studies on the influence of the substrates
and conditions on the reaction course.
Acknowledgements
This work was supported by project ”Biotransformations for pharmaceutical and cosmetics industry”
No. POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund.
References
[1] M. Svedendahl, K. Hult, P. Berglund, J. Am. Chem. Soc. 2005, 127, 17988 - 17989.
[2] K. Li, T. He, C. Li, X. W. Feng, N. Wang, X. Q. Yu, Green Chem. 2009, 11, 777 - 779.
[3] S. Klossowski, B. Wiraszka, S. Berlozecki, R. Ostaszewski, Org. Lett., 2013, 15, 566–569
[4] S. C. Pan, B. List, Angew. Chem. Int. Ed. 2008, 47, 3622 - 3625.
24
L09
Sucrose Phosphorylase – a Potential Biocatalyst for Industrial Glycosylation Reactions
Rama Krishna Gudiminchi1, Christiane Luley1, Andrew Towns2, Subhash Varalwar3 and Bernd Nidetzky4
1
Austrian Center of Industrial Biotechnology, Graz, Austria – [email protected]
Vivimed Labs Europe Ltd, Huddersfield, West Yorkshire, HD1 6BU, England
3
Vivimed Labs Ltd, Veernag Towers, Hubsiguda, Andhra Pradesh, India
4
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria
2
Keywords: glycosylation, active ingredient, SPase
Glycosylation is a process of attaching a glycosyl group to a hydroxy group or other functional groups of acceptor
molecule forming a glycoside. Glycosylation of small molecules (used as drugs, cosmetics, fragrances and food ingredients)
can significantly improve their physicochemical and biological properties. Chemical glycosylation requires several tedious
steps and often less selective. In contrast, enzymatic glycosylation allows single step glycosylation with high regio- and
stereo-selectivity under mild reaction conditions. Enzymes that degrade, modify, or create glycosidic bonds are collectively
known as Carbohydrate-Active Enzymes (CAZymes). The vast majority of glycosylation reactions are performed in nature
by glycosyltransferases (GTs), transglycosidases (TGs), glycoside hydrolases (GHs) and glycoside phosphorylases (GPs).
Often these enzymes also glycosylate wide range of non-natural acceptor substrates in vitro. All these enzymes have their
own advantages and disadvantages in industrial glycosylation reactions.
In the current studies sucrose phosphorylase was identified as a potential biocatalyst for glucosylation of a
commercial active ingredient among the well-known glycosylating enzymes α-glucosidases (α-Gase), cyclodextrin
glucanotransferases (CGTase) and sucrose phosphorylases (SPase). Utilizing sucrose, a cheap and efficient glucosyl donor
substrate, SPase catalyzed highly regio-specific glucosylation of target molecule with fewer by-products.
Fig. 1 Glucosylation mechanisms of various glycosylating enzymes.
Acknowledgements
This work has been supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal
Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur
Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian
Research Promotion Agency FFG.
References
[1] T. Desmet, W. Soetaert, P. Bojarova, V. Kren, L. Dijkhuizen, V. Eastwick-Field, and A. Schiller, Chem. Eur. J. 2012, 18,
10786-10801.
[2] C. Goedl, T. Sawangwan, M. Mueller, A. Schwarz, and B. Nidetzky, Angew. Chem. Int. Ed. 2008, 47, 10086-10089.
25
L10
Diphenolic Compounds Production by Tyrosinase from Agaricus bisporus
Małgorzata Cieńska, Jolanta Bryjak
Department of Bioorganic Chemistry, Wroclaw University of Technology, Wrocław, Poland – [email protected]
Keywords: tyrosinase, immobilization, diphenols
Tyrosinase (polyphenol oxidase, PPO, EC 1.14.18.1) is the copper-containing enzyme which, in the presence of
molecular oxygen, has two catalytic activities: monophenolase and diphenolase. In the monophenolase route, tyrosinase
catalyzes hydroxylation of monophenols to o-diphenols, while in diphenolase cycle o-diphenols are oxidized to
corresponding quinones which can be non-enzymatically converted to polymeric compounds [1].
As tyrosinase is capable to oxidize a large range of phenolic compounds it has a wide potential in practical
applications [1]. For example, tyrosinase can be used for synthesis of diphenolic drugs as 3,4-dihydroxy-L-phenylalanine (LDOPA) [2] or diphenolic antioxidants such as 2-(3,4-dihydroxyphenyl)ethanol (3-HTyr) [3].
Marin-Zamora et al. tested immobilized tyrosinase for production of several diphenolic compounds [4]. Their
system contained: borate ions at pH 9.0 to complex diphenols; ascorbic acid (AA) to avoid further non-enzymatic oxidation;
and hydroxylamine (HA) to accelerate the regeneration of oxy-tyrosinase form, that is necessary to carry out the reaction of
ortho-hydroxylation. Accordingly, in our study L-tyrosine and tyrosol were used for L-DOPA and 3-HTyr production, using
tyrosinase in native form and immobilized on cellulose based carrier [5]. In the experiments different combinations of
reaction mixtures and reaction conditions were tested, with the pH value fixed at 9.0, 8.0 and 7.0. It was stated that the
substrate conversion increased with a decrease of pH and the most promising results were obtained in reaction system with
native and immobilized enzymes at pH 7.0. At these conditions the conversion degrees achieved 100% for both substrates.
However, instability of the immobilized enzyme in successive runs was observed, what caused that no more than 2
consecutive batch processes could be conducted. It seems that 3-HTyr and L-DOPA production with immobilized tyrosinase
has rather low industrial applicability because of so called “suicide inactivation” of tyrosinase, which can be lowered but not
completely overcomed by immobilization. Hence, the most efficient hydroxylation of L-Tyrosine or tyrosol can be achieved
using native tyrosinase, in reaction system contained 0.5 M borate buffer at pH 7.0, 6.7 mM hydroxylamine and double
excess of ascorbic acid, in relation to substrates concentration.
Acknowledgements
This study was supported by the project “Biotransformations for pharmaceutical and cosmetics industry” No.
POIG.01.03.01-00-158/09 partly financed by the European Union within the European Regional Development Fund.
References
[1] G. Faccio, K. Kruus, M. Saloheimo, L. Thony-Meyer, Process Biochem. 2012, 47, 1749-1760.
[2] C. Algieri, L. Donato, P. Bonacci, L. Giorno, Biochem. Eng. J. 2012, 66, 14-19.
[3] J.C. Espin, C. Soler-Rivas, E. Cantos, F.A. Tomas-Barberan, H.J. Wichers, J. Agric. Food Chem. 2001, 49,1187-1193.
[4] M.E. Marin-Zamora, F. Rojas-Melgarejo, F. Garcia-Canovas, P.A. Garcia-Ruiz, J. Biotechnol. 2009, 139, 163-168.
[5] K. Labus, A. Turek, J. Liesiene, J. Bryjak, Biochem. Eng. J. 2011, 56, 232-2
26
L11
Biotransformation of Aminophosphonic Acids Using Fungi
Natalia Kmiecik, Ewa Żymańczyk-Duda
Department of Bioorganic Chemistry, Wroclaw University of Technology, Wrocław, Poland – [email protected]
Keywords: biotransformation, fungi, aminophosphonates
Biotransformation is a process which used biological systems to converse chemicals into certain products.
Nowadays, biotransformation is very important way to get valuable products. This bioprocess is apply as an alternative tool
to pursue new pharmaceuticals or chemical compounds. Bioconversion also allowed to control the stereochemistry of the
process, usually in a simple and cheap manner [1-4].
Microorganisms are the enormous source of enzymes of different activities, that’s why they have been applied in
industry. Enzymes features (stereo-, regio- and enantioselectivity) are crucial for the asymmetric synthesis of optically pure
chiral compounds [5-7]. Fungal strains are used to bioconversion of different compounds to optically pure products. The
appropriate choice of microbial strain should consider suitable cultivation and reaction medium to achieve the highest level
of effectiveness of the process [8].
Aminophosphonic acids and their derivatives are compounds with stable carbon to phosphorus bond. They have
wide range of promising biological activities and variety of current applications in industry, for instance they act as plant
growth regulators, herbicides or peptide mimics [9].
The aim of presented work was to analyze the ability of diverse fungal strains to enantioselective transformation of
aminophosphonic acid. Thus, biocatalytic methods, which allowed obtaining optically pure derivatives of aminophosphonic
acids via stereoselective biooxidation were elaborated. Application of P.funiculosum resulted in obtaining derivatives of
phosphonates (Fig. 1). Discussed procedure is a good starting point for further scaling up the process.
Fig. 1 Bioconversion of aminophosphonic acid.
Acknowledgements
This work was financed by the project “Biotransformations for pharmaceutical and cosmetics industry”
No.POIG.01.03.01-00-158/09, which is partly-financed by the European Union within the European Regional Development
Fund for the Innovative Economy”.
References
[1] R.O. Lopes, J. B. Ribeiro, et. al., Tetrahedron: Asymmetry 2011, 22, 1763–1766
[2] M. Olmo, C. Andreu, G. Asensio, J. Mol. Catal. B: Enzymatic 2011, 72, 90– 94
[3] K. B. Borges, W. S. Borges, et. al., Tetrahedron: Asymmetry 2009, 20, 385–397
[4] E. Żymańczyk-Duda, Phosphorus, Sulfur and Silicon, 2008, 183, 369-382
[5] F. Z. Abas, M. H. Uzir, M. H. M. Zahar, Journal of Applied Science, 2010, 10, 3289- 3294.
[6] K. B. Borges, W. S. Borges, M. T. Pupo, PS Bona, Appl. Microbiol. Biotechnol. 2007, 77, 669– 674
[7] P. M. Albuquerque, M. A. Witt, B. U. Stambuk, M. G. Nascimento, Process Biochemistry, 2007, 42, 141–147
[8] S. E. Milner, A. R. Maguire, ARKIVOC, 2012, 321-382
[9] A. Mucha, P. Kafarski, Ł. Berlicki, J. Med. Chem. 2011, 54, 5955-5980
27
L12
Hydrolytic Enzyme-Based Syntheses of Enantiopure Heteroatom Derivatives as
Precursors to Chiral Catalysts
Piotr Kiełbasiński, Małgorzata Kwiatkowska, Lidia Madalińska
Department of Heteroorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Łódź, Poland [email protected]
Keywords: chiral phosphines, hydrolytic enzymes, enzymatic kinetic resolution, enzymatic desymmetrization
The authors’ investigations of the application of hydrolytic enzymes in the preparation and transformations of
chiral, non-racemic heteroorganic derivatives will be briefly reviewed. A number of recent achievements will be presented,
among them those resulting in the chemoenzymatic syntheses of chiral heteroorganic ligands and catalysts. Thus,
enantiomerically pure hydroxymethyl sulfoxides 1 [1] and phosphine oxides 2, 3 [2] have been obtained by an enzymepromoted desymmetrization of prochiral, or kinetic resolution of racemic substrates and the sulfinyl analogs 1 transformed
into highly efficient chiral catalysts for asymmetric synthesis [3,4].
Due to the unexpected racemization of phosphine oxides 2 and 3 [5], a new approach is under investigation, which
envisages the synthesis of other phosphorus analogs. Hence, the ongoing experiments of the analogous syntheses and
transformations of P-chiral phosphines, phosphine sulfides, phosphine boranes and C-chiral PTA derivatives will be
discussed.
HO
OH
P
R
HO
AcO
OAc
BH3
HO
P Me
Ph
P
, Enzyme
HO
AcO
CAL-B
R
Solvent
ee ca. 90%
OH
OH
N
N
Ph
N
P Me
Ph +
AcO
BH3
P Ph
Me
Solvent
R = Ph, yield 30%, ee = 95%
R = Me, yield 49%, ee > 90%
P
BH3
AcO
Enzyme
Solvent
P
N
N
N
Ph
+
ee ca 90%
OAc
P
N
N
Ph
N
Acknowledgements
The project is partially financed from the Parent Bridge Program of the Foundation for Polish Science
POMOST/2013-8/9 (for M.K.)
References
[1] M. Rachwalski, M; Kwiatkowska, J. Drabowicz, M. Kłos, W. M. Wieczorek, M. Szyrej, L. Sieroń, P. Kiełbasiński,.
Tetrahedron: Asymmetry 2008, 19, 2096-2101.
[2] S. Kaczmarczyk, M. Kwiatkowska, L. Madalińska, A. Barbachowska, M. Rachwalski, J. Błaszczyk, L. Sieroń, P.
Kiełbasiński, Adv. Synth. Catal. 2011, 353, 2446-2454
[3] P. Kiełbasiński, M. Rachwalski, S. Kaczmarczyk, S. Leśniak, Tetrahedron: Asymmetry 2013, 24, 1417-1420 and
references therein.
[4] M. Rachwalski, S. Kaczmarczyk, S. Leśniak, P. Kiełbasiński, ChemCatChem 2014, 6, 873-875 and references therein.
[5] S. Kaczmarczyk, L. Madalińska, P. Kiełbasiński, Phosphorus, Sulfur, Silicon Relat. Elem. 2013, 188, 249-253.
28
L13
Mixed Carbonates as a New Class of Substrates for Fluorogenic Assays
Anna Żądło, Ryszard Ostaszewski
Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warsaw, Poland - [email protected]
Keywords: fluorogenic probes, enzyme catalysis, high-throughput screening
Hydrolytic enzymes, such as lipases and esterases, are very important enzymes for industrial applications. They are
frequently used because of their stability, activity and ability to accept a wide range of substrates.[1] Although many
enzymes are known, the achievable activities and selectivities are often insufficient. For this reason, discovery of new
variants of enzymes by biodiversity mining, mutagenesis or directed evolution are desirable. These approaches require the
ability to examine large number of enzymes in parallel. It could be achieved by screening enzyme libraries in highthroughput format.[2-4] Mixed carbonates of 4-methyl-7-hydroxycoumarin are shown to be a new class of probes for
examination hydrolytic activities. [5] These substrates proved particularly useful due to the low level of non-specific
degradation, in contrast to assays based on simple esters of umbelliferone. These probes represent new class of fluorogenic
compounds, easy to synthesize and highly relevant for screening of lipases and esterases libraries. These advantages make
umbelliferyl carbonates very recommendable substrates for high-throughput screening. Moreover, we report the use of chiral
fluorogenic carbonates as probes for determination of enantioselective hydrolytic activities. Use of two derivatives of
coumarin in one reaction allows recording of reaction rates for both enantiomers simultaneously.
CH3
CH3
O
O
R1
k1
O
O
O
R2
O
+
+ CO2
HO
O
O
OH
O
O
CF3
OH
k2
O
R2
enzyme
PBS pH 7.4
R2
O
R1
O
CF3
R1
OH
R1
O
R2
+ CO2
+
HO
O
O
k1>k2
Fig. 1 Enzymatic hydrolysis of fluorogenic probes.
Acknowledgements
This work was supported by project ”Biotransformations for pharmaceutical and cosmetics industry”
No. POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund.
References
[1] M. Schmidt, U. T. Bornscheuer, Biomol. Engineering 2005, 22, 51-56.
[2] E. Leroy, N. Bensel, J.-L. Reymond, Adv. Synth. Catal. 2003, 345, 859-865.
[3] E. Leroy, N. Bensel, J.-L. Reymond, Bioorg. Med. Chem. Lett. 2003, 13, 2105-2108.
[4] J.-P. Goddard, J.-L. Reymond, Current Opinion in Biotechnology 2004, 15, 314-322.
[5] P 407948 2014: R. Ostaszewski, A. Żądło, M. Zysk, A. Brodzka, S. Kłossowski
29
L14
Stimuli Responsive CPC Gels: Effect of pH on Gel Formation
Illa Ramakanth1,2, Jaroslav Hamrle1 and Jaromir Pištora1
1
2
Nanotechnology Centre, VŠB - Technical University of Ostrava, Ostrava - Porub, Czech Republic –[email protected]
Department of Chemistry, Rajiv Gandhi University of Knowledge Technologies, Hyderabad 500 032, India
Keywords: surfactants, self-assembly, gelation
Self-assembly of molecules to form soft materials have attracted growing interest in areas ranging from chemistry
and biology to materials science [1]. Among various soft materials, supramolecular gels have attracted potential attention
because of their ability to form highly ordered superstructures with specific functional properties and their biodegradability
which is not exhibited by polymer gels [2]. Molecular gels (Organo / Hydrogels) formed as a result of the entrapment and
adhesion of the liquid in the large surface area solid 3D matrix, have attracted potential interest. Assembling organic
molecules into well-defined supramolecular self-assembled gelator aggregates is important owing to their potential
applications in various optoelectronic fields, including enhanced charge transport, fluorescence and sensing abilities [3].
In continuation of our previous work reported on stimuli-responsive cetylpyridinium chloride (CPC) gels formed
from CHCl3:H2O binary solvent mixture at a specific composition ratio [4], we have focused our attention on gelation of
various binary solvent systems like DCM:H2O using CPC. The aim is to investigate the behavior of CPC gel phase with
varying pH of the solvent system and entrapment of guest molecules by stimulating external factors such as pH or
temperature, responsible for the supramolecular network. CPC in binary solvent mixtures showed a turbid gel at a critical
solvent composition of DCM:H2O mixture. The gelation ability was evaluated in various other organic solvents in the
presence of water and the phase evolution was studied. It was observed that the selfassembly of CPC was found to be
dependent on pH of the medium. The absorption and emission characteristics of CPC showed significant response with
strong alkaline medium. CPC’s fluorescence behavior at strongly basic solution has been investigated using UV-visible
absorption, Fluorescence emission and 1H NMR spectroscopy. The microstructure of the CPC gels in various solvents was
proposed based on spectroscopic and microscopic investigations.
Fig. 1 Fluorescence emission spectra of CPC solutions in alkaline medium (pH ~ 11.8) at an excitation λ=396 nm.
References
[1] A.R. Hirst, B. Escuder, J.F. Miravet, and D.K. Smith, Angew. Chem, Int. Ed., 2008, 47, 8002.
[2] S. Malik, and A.K. Nandi, J. Phys. Chem. B., 2004, 108, 597.
[3] Y.J. Seo, S. Bhuniya, and B.H. Kim, Chem. Commun., 2007, 1804.
[4] I. Ramakanth, and A. Patnaik, J. Phys. Chem. B., 2012, 116, 2722.
30
L15
Lipase-Mediated Kinetic Resolution Towards Enantiomerically Enriched α- and βHydroxy Acids Derivatives
Mara Ana Naghi, Monica Ioana Tosa, Csaba Paizs, Florin Dan Irimie
Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, Cluj-Napoca, Romania – [email protected]
Keywords: lipase, kinetic resolution, hydroxy acids
Chiral carboxylic acids are important building blocks for the synthesis of many pharmaceutical drugs[1].
Biocatalysis has emerged as a powerful strategy for the production of high enantiopurity hydroxy acids due to its remarkable
stereoselectivity and high yield.
Our work has been devoted to the synthesis of highly enantiopure α- and β-hydroxy acid derivatives by means of
lipase mediated kinetic resolution. Due to the necessity of the preparation of both enantiomers of a drug and its key
intermediates in order to recognize the physiological properties of these molecules, we developed biocatalytic approaches
toward both enantiomers of 2-heteroaryl-2-hydroxyacetic acids [2], ethyl-3-hydroxy-3-(2-aryl-thiazol-4-yl)propanoates [3],
and 2-hydroxy-2-(5-arylthiophene-2-yl)acetonitriles, schematically presented in Figure 1.
The synthesis of enantiomerically enriched (ee=99%) (R)- and (S)-α-hydroxy acids was accomplished by the CaLA mediated O-acylation of the racemic α-hydroxyesters rac-2a-d, followed by the CaL-B mediated hydrolysis of the
untransformed enantiomer of the substrate and a two step enzymatic hydrolysis. The four new optically pure (R)- and (S)- 3eh β-hydroxy esters and their butanoates 5e-h were obtained by enantioselective O-acylation of the racemic alcohols and by
ethanolysis of the corresponding racemic esters, mediated by CaL-B in organic solvents. Vinyl acetate and CH2Cl2 proved to
be most appropriate for obtaining the (S)-1i-l α-cyanohydrins and the corresponding (R)-α-cyanohydrin O-acetates, by
enantioselective O-acylation mediated by lipase AK from Pseudomonas fluorescens.
Fig. 1 Enzymatic kinetic resolution of rac-1-5, by means of O-acylation, hydrolysis or alcoholysis reactions.
Acknowledgements
M.N. thanks the financial support provided by PN II ID PCE 3-0775/2011 and POSDRU/159/1.5/S/132400.
References
[1] (a) R. Kobayashi, M. Konomi, K. Hasegawa, M. Morozumi, K. Sunakawa, K. Ubukata, Antimicrob. Agents Chemother.
2005, 49, 889–894;(b) A. Zaks, D.R. Dodds, Drugs Discovery Today 1997, 2, 513–531.
[2] M.A. Naghi, L.C. Bencze, J. Brem, C. Paizs, F.D. Irimie, M.I. Tosa, Tetrahedron: Asymmetry 2012, 23, 181–187.
[3] J. Brem, M. Naghi, M.I. Tosa, Z. Boros, L. Poppe, F.D. Irimie, C. Paizs Tetrahedron: Asymmetry 2011, 22, 1672–1679.
31
L16
Screening of Carriers for Lipase Immobilization for Reactions in Water System
Zofia Hrydziuszko1, Katarzyna Szymańska2, Jolanta Liesiene3 and Jolanta Bryjak1
1
Department of Bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland – [email protected]
Department of Chemical Engineering, Silesian University of Technology, Gliwice, Poland
3
Faculty of Organic Technology, Kaunas University of Technology, Kaunas, Lithuania
2
Keywords: lipase, immobilization, operational stability
Lipases are the most widely used class of enzymes in bioprocesses. Production and purification of lipases are very
expensive procedures thus these enzymes are used typically in immobilized forms [1]. Presented studies focused on the
selection of an appropriate enzyme-carrier preparation for hydrolytic reaction in aqueous media with the help of lipases from
Pseudomonas cepacia (PCL), Candida rugosa (CRL), Mucor circinelloides (MCL), or lipase B from Candida antarctica
(CALB). The work focused on four groups of carriers - mesoporous cellular foams, silica (MN Kieselgel 60 or Silicagel),
acrylic copolymer and cellulose-based Granocel – on which lipases were immobilized by simple adsorption and/or covalent
attachment.
Basing on the common procedures of immobilization on the carriers with different functionality, 86 enzyme-carrier
preparations were obtained and their activities were tested in the reaction of p-nitrophenyl palmitate hydrolysis at 37 oC in a
batch mode. All preparations were used in 4-h incubation at 60 oC (70 oC for PCL) to evaluate their thermal stability. In the
case of selected enzyme-carrier preparations, operational stability was determined by running 10 – 23 consecutive batch
processes.
On average, in the selection of a proper carrier for an enzyme immobilization, the highest activity is regarded as the
major factor but in practical application their operational stability under processing condition should be used as the most
important criterion, even at the cost of lower activity. However, running series of 10-20 consecutive batch processes are time
consuming solution (minimum 5-7 days), thus researchers are looking for quick and easy preliminary test that evaluates
higher operational stability on the base of a simple activity measurements. In this work it was assumed that thermal stability
would be such simple criterion. To prove the assumption, 14 immobilized PCL preparations with different activities were
subjected to inactivation at elevated temperature (70 oC for 4 h) and were used in 20 subsequent batch processes. Examined
preparations were characterized by all combinations of low and high activity and thermal stability. Obtained results clearly
showed that the highest activity did not provide sufficient stability under operational conditions. Among all immobilized
PCL preparations, 6 of them were operationally (preserving over 22% of initial activity during last 15 processes) and
thermally (thermal stability over 80%) stable. The rest 8 preparations showed operational stability below 20% and thermal
stability under 50%. Thus it might be claimed that thermal stability can be treated as a very useful indicator of higher
longevity of immobilized preparations under processing conditions. Taking into account above correlation, operational
stability tests with immobilized CRL, CALB, and MCL were carried out for enzyme-carrier preparations with the highest
thermal stability.
Operational stability was the main selection criterion among the lipase-carrier preparations with the highest thermal
stability. It was stated that PCL immobilized on Silicagel with amino groups by adsorption or covalent attachment provided
stable activity level of 23% and 29% of initial value, respectively, during the last 15 batch processes. Taking into account
CRL, the enzyme covalently bound onto NH2-Kieselgel preserved 50% of activity during last 15 batch processes. In the case
of CALB and MCL immobilization the most appropriate carrier was Granocel with hydroxyl groups (68% and 44%,
respectively, in 10 batch processes). Additionally, obtained results evidenced again lack of an universal carrier for enzymes
immobilization, even tested in the same reaction system.
Acknowledgements
This work was supported by project ”Biotransformations for pharmaceutical and cosmetics industry”
No. POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund.
References
[1] A. Liese, K. Seelbach, A. Buchholz and J. Haberland, Processes. In Industrial Biotransformations, 2nd ed. A. Liese, K.
Seelbach, Ch. Wandrey, eds, 2006, 273–320. Wiley-VCH, Weinheim.
32
L17
Decarboxylases as Promising 'Green' Biocatalytic Tools for Synthetic Applications
C. Wuensch,1,2 T. Reiter,1,2 J. Gross,1,2 G. Steinkellner,1,3 A. Hromic,1,3 K. Fauland,1,3 T. Pavkov-Keller,1,3 K.
Gruber,3 K. Faber,2 S. M. Glueck1,2
1
Austrian Centre of Industrial Biotechnology (ACIB) c/o
Department of Chemistry, University of Graz, Graz, Austria, email: [email protected]
3
Institute of Molecular Biosciences, University of Graz, Graz, Austria
2
Keywords: enzymatic carboxylation, asymmetric hydration, decarboxylases
The development of sustainable and eco-friendly synthetic strategies is an important task in modern synthetic
chemistry. The regioselective enzyme-catalyzed carboxylation of aromatics [1] represents a promising and 'green' alternative
to classic chemical methods (Kolbe-Schmitt reaction) which often require harsh reaction conditions [2]. The desired reactions
are catalyzed by (de)carboxylases at the expense of bicarbonate as CO2-source [3].
Depending on the type of enzyme, either the ortho- or para-carboxylation of phenols (catalyzed by benzoic acid
decarboxylases) or the complementary beta-carboxylation of styrenes (catalyzed by phenolic acid decarboxylases) were
achieved in a highly regioselective fashion [4].
Furthermore, an unexpected promiscuous 'hydratase-activity' of phenolic acid decarboxylases was discovered,
catalyzing the stereoselective asymmetric hydration of hydroxystyrenes to yield the corresponding sec-alcohols [5]. By this
means, a new chiral center can be introduced into a prochiral alkene in 100% atom economy.
In summary, these enzymatic transformations represent novel biocatalytic tools, which often lack a counterpart in
traditional chemistry. Various carboxylated products (such as salicylic acid or p-aminosalicylic acid) are highly interesting
building blocks for pharmaceuticals, cosmetics, agro- or fine chemicals.
Acknowledgements
This work has been supported by the Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT through the
Austrian FFG-COMET-Funding Program.
References
[1] S. M. Glueck, S. Gümüs, W. M. F. Fabian, K. Faber, Chem. Soc. Rev. 2010, 39, 313.
[2] A. S. Lindsey, H. Jeskey, Chem. Rev. 1957, 57, 583.
[3] T. Matsui, T. Yoshida, T. Yoshimura, T. Nagasawa, Appl. Microbiol. Biotechnol. 2006, 73, 95.
[4] C. Wuensch, S. M. Glueck, J. Gross, D. Koszelewski, M. Schober, K. Faber, Org. Lett. 2012, 14, 1974; C. Wuensch, J.
Gross, G. Steinkellner, A. Lyskowski, K. Gruber, S. M. Glueck, K. Faber, RSC Adv. 2014, 4, 9673.
[5] C. Wuensch, J. Gross, G. Steinkellner, K. Gruber, S. M. Glueck, K. Faber, Angew. Chem. Int. Ed. 2013, 52, 2293.
33
L18
t-Butyl Acetoacetate as a Substrate for Enzymatic Transesterification
Catalina Wiśniewska and Ryszard Ostaszewski
Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warsaw, Poland – [email protected]
Keywords: enzymes, transesterification, t-butyl esters
The application of esterases or lipases for the kinetic resolution of esters of tertiary alcohols is hampered by the
fact that most of the commercially available enzymes did not accept tertialy alcohols as substrates [1]. Only a limited group
of enzymes possessing a special structure of active site is able to hydrolyse esters of tertially alcohols.
We have found that the enzyme catalysed transeserification of ethyl and t-butyl acetoacetates enables to obtain
various acetoacetates with good yields. The result is very surprising because the mechanism of the hydrolysis of t-butyl ester
cannot be the same as ethyl ester.
In this work the results of enzyme catalysed transeserification of t-butyl acetoacetate and systematical studies on
mechanism of the reaction of enzymatic transesterification of t-butyl acetoacetate will be discussed. Proposed mechanism
that explains the obtained results will be presented.
O
O
O
O
+
O
ROH
enzyme
O
R
+
OH
Fig 1: Enzymatic transesterification of t-butyl acetoacetate
Acknowledgements
This work was partially financed by Polpharma Starogard Gdański and “Biotransformations for pharmaceutical and
cosmetic industry”
References
[1] R. Kourist, U. T. Bornscheuer Appl Microbiol Biotechnol 2011, 91, 505-517
[2] G. Nguyen et. al Journal of Molecular Catalysis B: Enzymatic 2011, 70, 88–94
[3]J. Rehdorf et al. Appl Microbiol Biotechnol 2012, 93, 1119–1126
34
L19
Cyanobacteria as Biocatalyst for the Reduction of β - Oxoalkylphosphonates
Monika Górak, Ewa Żymańczyk-Duda
Department of Bioorganic Chemistry, Wroclaw University of Technology, Wrocław, Poland – [email protected]
Keywords: cyanobacteria, reduction, β- oxophosphonates
Optically active chiral compounds play an important role in the chemical and life science industry. Regulatory
requirements, the prospects of lower toxicity and higher efficacy have increased demand for chiral compounds. In response to
this trend, biocatalytic production method has been rapidly applied to chiral industry. Despite the fact, that the use of the
biocatalytic methods in industrial processes increased, the practicability of technical applications of these methods is often
limited by the lack of suitable biocatalysts. [1]
One of the most efficient ways to produce chiral alcohols of high purity is biocatalyzed reduction of ketones. This
strategy, mostly, based on bioconversion with non-photosynthetic and heterotrophic microorganisms or their purified
enzymes. [2] However, phototrophic prokaryotes such as cyanobacteria have also been identified as a potential bioreductant.
Cyanobacteria have gained a lot of attention in recent years because of their divers, possible applications to bioconversions of
structurally different substrates into desired usable products. [3]
Biocatalysis is an effective and in many cases preferable alternative to the standard synthesis of optically active
isomers of fine chemicals, including phosphonates of define structure and absolute configuration. [4] Hydroxyphosphonates
are a class of organophosphorus compounds with potential biological activity. [5]
Morphologically different strains of cyanobacteria were used as a novel source of reductive activities towards βoxoalkylphosphonates. However, only filamentous strains of Arthrospira maxima CCALA 027, Nodularia sphaerocarpa
CCALA 114 and heterocystous photoheterotrophic cyanobacterium Nostoc cf-muscorum CCALA 129 are efficient
biocatalysts in reduction of chosen substrate to the corresponding β- hydroxyphosphonates of high enantiomeric purity (Fig.
1 R = -CH3; -C2H5; -C6H5). Furthermore, several efforts have been undertaken to optimize the bioconversion conditions to
obtain higher degree of conversion of the substrate. The effect of cultivation medium, light source, light intensity and light
cycle (day/ night) on the effectiveness of the biotransformation process was examined.
O
R
OH O
P OC H
R
2 5
OC2H5
O
P OC H
2 5
OC2H5
Fig 1: Bioreduction of β –oxoalkylphosphonates by cyanobacteria.
Acknowledgements
This work was financed form Project „Biotransformation for pharmaceutical and cosmetics industry” no
POIG.01.03.01–00–158/09 part – financed by the European Union within the European Regional Development Fund for the
Innovative Economy.”
References
[1] A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt, Nature 2001, 409, 258–268.
[2] K. Nakamura, R. Yamanaka, T. Matsuda, T. Harada, Tetrahedron: Asymmetry 2003, 18, 2659-2681.
[3] F. Jiittner, R. Hans, Applied Microbiology and Biotechnology 1986,1, 52-54.
[4] E. Żymańczyk-Duda, M. Klimek-Ochab, Curr. Org. Chem. 2012, 11, 1408-1422.
[5] O.I. Kolodiazhnyi, Russian Chemical Reviews 2006, 3, 227-253.
35
L20
Filamentous Fungi as a Tool for Obtaining Hops Minor Prenylflavonoid Glycosides
Jarosław Popłoński, Sandra Sordon, Tomasz Tronina, Agnieszka Bartmańska, Ewa Huszcza
Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland - [email protected]
Keywords: hops, xanthohumol, glycosylation
Hops are the resinous inflorescences of Humulus lupulus used in industry with brewing industry being the main.
Medicinal use of hops is associated with its sedative and phytoestrogenic properties. There are many interesting compounds
with extraordinary biological activities found in hops, but the flavonoid fraction is currently in the spotlight. Xanthohumol is
the main prenylated chalcone isolated from hops, displaying broad spectrum of biological activity with anticancer activity
being the most important. Beside xanthohumol there are other minor prenylated chalcones, a group of xnathohumols
(xanthohumol B,C, to H). Little is known about the biological potential of these compounds which is the result of their very
limited abundance in the plant material [1]. At this point, worth to mention is that prenylflavonoids isolated form hops are
only in aglycone form, what is quite unusual for plants. Chemical synthesis of prenylflavonoid glycosides would be a multistep process, but even with a fairly good yield, such products lose their “natural product” status. Glycosylation reactions are
common for living organisms and with the use of plant cultures flavonoid glycosides are obtained, however such processes
would be very difficult to transfer to a larger scale. Recently, there are many reports concerning microbial glycosylation of
flavonoids with considerably good yields [2].
Herein we would like to introduce a novel approach to the synthesis of hops minor prenylflavonoid compounds and
show structure-activity assay results of these compounds towards antiproliferative activity against cancer cell lines. Finally,
we would like to present biotransformation studies concerned in increasing bioavailability of this group of compounds with
impact on transformations leading to glycosides. Thereby, presenting filamentous fungi as a competitive tool to obtain
flavonoid glycosides.
Fig 1 Examples of microbiological glycosylation of minor prenylflavonoids using filamentous fungi.
Acknowledgements
This research was supported financially by the European Union within the European Regional Development Fund
as a part of the scholarship program “Grant Plus”.
References
[1] A. Bartmańska, T. Tronina, J. Popłoński and E. Huszcza, Current Drug Metabolism, 2013, 14, 1083-1097.
[2] J. Xiao, T.S. Muzashvili, M.I. Georgiev Biotechnol. Adv. 2014 http://dx.doi.org/10.1016/j.biotechadv.2014.04.006
36
L21
Synthesis of Chiral Alcohols by Ethylbenzene Dehydrogenase – from Modeling to
Application
Maciej Szaleniec1, Mateusz Tataruch1, Tomasz Borowski1, Jolanta Bryjak2, Johann Heider3
1
Jerzy Haber Institute of Catalysis and Surface Chemistry, Niezapominajek 8, 30-239 Kraków, Poland – [email protected]
Department of Bioinorganic Chemistry, Wrocław Technical University, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
Laboratory for Microbial Biochemistry and Synmikro Center for Synthetic Microbiology, Philipps University of Marburg, 35043 Marburg,
Germany
2
3
Keywords: ethylbenzene dehydrogenase, QM:MM, chiral alcohols
Chiral alcohols are important building blocks for the synthesis of fine chemicals and pharmaceuticals. Many of
them can be produced biotechnologically via enantioselective reduction of the respective ketones by various alcohol
dehydrogenases. The available enzymes exhibit a wide range in substrate and coenzyme specificities, reaction rates and
enantiomeric excesses of the products, and economically feasible processes have been implemented with a number of model
enzymes, particularly by coupling the ketone reduction reaction with cost-efficient regeneration techniques for the required
NAD(P)H cofactors. However, the process is limited to ketones contained within the substrate range of the used enzymes and
their compatibility with the NAD(P)H regeneration process. A fundamentally new process for chiral alcohol production may
be derived from the recent discovery that the first enzyme of the anaerobic ethylbenzene metabolic pathway, ethylbenzene
dehydrogenase (EbDH), produces (almost) exclusively (S)-alcohols from ethylbenzene and many alternative substrates [1].
EBDH is monomolybdenum enzyme originating from denitrifying bacteria that is involved in the anaerobic
catabolism of hydrocarbons. The enzyme contains a bisMGD molybdenum cofactor in their active site that is responsible for
an activation and enantiospecific hydroxylation of the substrates.
In order to understand the mechanistic basis of such a high enantioselectivity we have employed QM:MM
modeling (ONIOM approach at B3LYP/AMBER/electronic embedding level of theory) combined with kinetic isotope tests.
The comparison of energy profiles of the reaction pathways leading to either (S)- or (R)-1-phenylethanol showed that the C-H
activation step is both rate limiting and enantio-determining step of the reaction process. On the other hand the OH rebound
process turns out to be non-enantiospecific and is characterized by lower energy barrier [2]. These conclusions are validated
by comparison of the calculated and the experimental kinetic isotopic effects.
EbDH was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic
compounds. The optimization of the synthetic procedure involved use of the enzyme in a crude purification state that saves
significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions,
immobilization of the enzyme on a crystalline cellulose support (Granocel type) and changes in reaction conditions in order
to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization
of ferricyanide as alternative electron acceptor in high concentrations).
We report here on an extension of effective enzyme activity from 4 h to more than 10 days (the change of enzyme
inactivation constants from 0.174±0.005 h-1 to 0.0051± 0.0001 h-1 for homogenous enzyme working in aerobic conditions
and anaerobic immobilized enzyme in anaerobic conditions, respectively). In optimal conditions in 40 ml fed-batch reactor
we were able to obtain product yields of up to 0.4 to 0.5 g/L, which represent a decent starting point for further optimization
[3].
Acknowledgements
The authors acknowledge the financial support of Polish National Science Center under grant N N204 269038, as
well as the financial support of the project ‘Biotransformations for pharmaceutical and cosmetics industry’ No.
POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund.
References
[1] AM. Szaleniec, C. Hagel, M. Menke, P. Nowak, M. Witko, J. Heider, Biochemistry 2007, 46, 7637.
[2] M. Szaleniec, A. Dudzik, B. Kozik, T. Borowski, J. Heider, M. Witko, J. Inorg. Biochem. 2014, 139, 9.
[3] M. Tataruch, J. Heider, J. Bryjak, P. Nowak, D. Knack, A. Czerniak, J. Liesiene, M. Szaleniec, J. Biotechnol. 2014 DOI:
10.1016/j.jbiotec.2014.06.021.
37
L22
High Performance Continuous Microreactors for Enzymatic Biotransformations
Katarzyna Szymańska1, Aurelia Zniszczoł1, Jacek Kocurek1, Wojciech Pudło1, Andrzej Jarzębski1,2
1
2
Faculty of Chemistry, Silesian University of Technology, Gliwice, Poland - [email protected]
Institute of Chemical Engineering, Polish Academy of Sciences, Gliwice, Poland
Keywords: enzymatic microreactors, monolithic silica, continuous biotransformations
Recently much attention has been paid to the development of immobilized enzyme reactors (IMERs), porous
monolith silica demonstrate huge potentials in this area. They are environmentally acceptable, structurally stable and resistant
to microbial attacks [1].
In this work monolithic silica rods 6 x 40 mm (length), exhibiting very open (total pore volume 3.8 cm3/g) and
uniform 3D hierarchical pore structure of 35-50 µm flow-through macropores and ca. 20 nm mesopores (MH-1, Fig 1A),
were synthesized using the sol–gel method combined with pore templating and phase separation. These monoliths were
successfully converted into miniaturized, multichannel continuous-flow reactors which (for a single rod) could operate at
flow rates of up to about 800 [mL/cm2*min] at pressure drop not exceeding 60 [kPa/cm] (Fig. 1B). The lower pressure drop
eliminates the need for application of HPLC pumps.
Monoliths’ surface could easily be covered with various functional groups: amino, epoxy, hydrophobic; Ni and Co
could also be introduced. Different enzymes were immobilized by: adsorption (lipases), tailored adsorption using His6tagged protein (acetyl transferase) and covalent attachment (trypsin (Fig. 1C), invertase, penicillin acylase). Microreactors
thus obtained appeared to be very effective and stable in con-flow enzyme catalysed reactions carried in aqueous or organic
solvents even for several weeks.
Fig. 1. Scheme of microreactor (A), Pressure drop in microreactors with different pore size (B), Myoglobin digestion by
trypsin immobilized on MH-1(C).
Acknowledgements
This work was financed from Project “Biotransformation for pharmaceutical and cosmetics industry”
No.POIG.01.03.01-00-158/09- part-financed by the European Union within the European Regional Development Fund for
the Innovative Economy”.
References
[1] K. Szymańska, W. Pudło, J. Mrowiec-Białoń, A. Czardybon, J. Kocurek, A.B. Jarzębski, Microporous and Mesoporous
Materials 2013,170, 75.
38
L23
First Chemoenzymatic Stereodivergent Total Synthesis of Both Enantiomers of
Promethazine and Ethopropazine
Paweł Borowiecki, Daniel Paprocki, Maciej Dranka and Maria Bretner
Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland. –[email protected]
Keywords: promethazine, ethoproprazine, lipase-catalyzed kinetic resolution, Mosher methodology, stereodivergent synthesis,
enantiodifferentiation, BINOL.
For over four decades it has been considered that both enantiomers of promethazine posses exactly the same
therapeutic activities [1,2], and thus it was administered in clinics as racemate. However, recent findings have shown that the
different physiological and pharmacological effects of promethazine enantiomers is a fact, what makes this drug returned to
the laboratories. For example, it has been determined that (+)-promethazine reduced cytokine IL-6 production to 90% of the
histamine-stimulated cell while the (−)-promethazine induced only 50% reduction in cytokine IL-6 production [3]. Moreover,
Boland and McDonought [4,5] found that preferably the (+)-enantiomer of promethazine is particularly effective in inhibiting
the formation of bone resorbing cells (osteoclasts) thus providing a new class of agents capable for preventing or even
treating bone loss, mainly associated with periodontitis and osteoporosis. Moreover, resolution of the key intermediate 1(10H-phenothiazin-10-yl)propan-2-ol enantiomers may simultaneously provide an access to other valuable compound in
enantioenriched form, that is: ethopropazine (profenamine). In turn, this anti-dyskinetic drug has been widely used in clinical
practice for over 30 years in the treatment of Parkinson's disease, although there is no data available regarding its
pharmacokinetic properties in humans. Only one study on mechanism of stereoselective interaction between
butyrylcholinesterase (BChE) and ethopropazine enantiomers was performed leading to final conclusion as BChE posses
significantly higher affinity for (R)-configurated ethopropazine [6]. In order to find whether ethopropazine displays
stereoselectivity in its pharmacokinetics and exhibit different biological profiles of its stereoisomers, obtaining both
enantiomers in more than analytical quantities is particularly desirable. To the best of our knowledge, heretofore no reports
concerning preparative-scale synthesis of optically active promethazine and ethopropazine have been reported.
Therefore, the aim of this study was to take advantage of the extraordinary properties of enzyme catalysis and
develop new method based on lipase-mediated kinetic resolution of 1-(10H-phenothiazin-10-yl)propan-2-ol enantiomers,
which combined with convenient chemical reactions could provide simple and straightforward approach for the preparation
of optically active promethazine and ethopropazine molecules
Acknowledgements
We gratefully acknowledge the financial support from the project „Biotransformations for Pharmaceutical and
Cosmetics Industry” No. POIG.01.03.01-00-158/09, funded by the European Union within the European Regional
Development Fund. The study was co-financed by Warsaw University of Technology, Faculty of Chemistry.
References
[1] (a) W. T. Nauta, R. F. Rekker, Handbook of Experimental Pharmacology, Rocha e Silva, M., Ed.; New York, SpringerVerlag, 1978, 215.
[2] L. Toldy, et al., Acta. Chim. Acad. Sci. Hung. 1959, 19, 273.
[3] J. McDonough, H. Dixon, J. Niño, Intranasal Drug Delivery Technology for space motion sickness, 01-9303. Southwest
Research Institute, inclusive dates 004/01/2002; http://www.swri.org/3pubs/IRD2003/Synopses/019303.htm
[4] E. J. Boland, J. McDonough, WO 2004/110458 A1 2004.
[5] E. J. Boland, US 2006/0258650 A1 2006.
[6] G. Šinko, Z. Kovarik, E. Reiner, V. Simeon-Rudolf, J. Stojan, Biochimie 2011, 93, 1797-1807.
39
L24
Selective Modifications of Sterols Performed by Enzymes from Sterolibacterium
denitrificans
Agnieszka Rugor1, Tomasz Janeczko2, Agnieszka Dudzik1, Jakub Staroń3, Andrzej Bojarski3, Maciej Szaleniec1
1
2
3
Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, Kraków, Poland – [email protected]
University of Environmental and Life Sciences, Wrocław, Poland
Institute of Pharmacology, PAS, Kraków, Poland
Keywords: steroids, sterols, regioselectivity
Sterolibacterium denitrificans is a denitrifying bacterium that under anaerobic conditions mineralizes cholesterol
[1]. It is a source of new regio- and chemoselective enzymes that can be consider as an interesting biocatalysts for the
industry. The initial degradation step of choresterol, ring A oxidation and isomerisation to cholest-4-en-3-one, is catalyzed by
cholesterol dehydrogenase/isomerase (AcmA, Anaerobic cholesterol metabolism). This product is further oxidized to
cholesta-1,4-dien-3-one by cholest-4-en-3-one-∆1-dehydrogenase (AcmB) [2]. Subsequently, both products are hydroxylated
at tertiary C25 of the alkyl side chain by steroid C25 dehydrogenase (S25DH) using water as an oxygen donor (Fig. 1) [3].
Fig. 1 Initial steps of cholesterol degradation pathway with formation of cholest-1,4-dien-3-one and 25- hydroxylated
steroids.
In our work a purified S25DH and crude protein fractions of AcmB were tested as catalyst in batch or fed-batch
reactors using various sterols and steroids. For S25DH substrates the reaction rate was monitored by HPLC-MS. For crude
AcmB a products of oxidation were extracted using SPE (40-100 ml reactors containing app. 20 mg of a substrate) and
analyzed by HPLC-MS and NMR. The S25DH catalyzed hydroxylation in the range of cholesterol derivatives while AcmB
was active in introduction of a double bond in some compounds of pharmaceutical interest.
Acknowledgements
The authors acknowledge the financial support from the project Interdisciplinary PhD Studies "Molecular sciences
for medicine" (co-financed by the European Social Fund within the Human Capital Operational Programme) and The
National Centre of Research and Development for the grant LIDER/33/147/L- 3/11/NCBR/2012.
References
[1] S. Tarlera, Other., Int. J. Syst. Evol. Microbiol, 2003, 53, 1085–1091.
[2] Y. R. Chiang,Other,, J. Bacteriol, 2008, 190, 905-914.
[3] J. Dermer and G. Fuchs, J. Biol. Chem, 2012, 287, 36905–36916
40
Posters
41
42
P01
Lipase Catalyzed Acidolysis of Dipalmitoyl Phosphatidyl Choline with Alpha Lipoic
Acid to Produce a Novel Phospholipid with Potential Biomedical Applications
Shiva Shanker Kaki, M. Balakrishna and R.B.N. Prasad
Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad-500007, INDIA –
[email protected]
Keywords: phosphatidylcholine, alpha lipoic acid, lipase
Lipoic acid (LA), a well-known biological antioxidant is a medium chain fatty acid with a wide range of biological
activities with both medicinal and cosmetic applications. It is reported to be a clinically safe supplement which exhibits
significant antioxidant and cell regulating properties. Keeping in view of the biological activities of LA, several molecules
were designed and prepared as LA derivatives with interesting properties [1]. However, the stability and the bioavailability of
LA were found to be improved when LA was administered in a formulation that was composed of lipids. Very few reports
are available where lipoic acid and lipids were together employed. In a recent study, LA was incorporated into a triglyceride
and the product was shown to increase the oxidative stability of the membrane where it was incorporated [2]. In the present
study, bioorganic synthesis of a novel lipoconjugate of LA in the form of a phospholipid is described which could be an
interesting product for application as cosmeceutical as phospholipids are considered to be efficient delivery vehicles of
bioactive molecules. Synthesis of 1-lipoyl-2-palmitoyl phosphatidylcholine (LPPC) was carried out employing
dipalmitoylphosphotidylcholine (DPPC) and LA as substrates via enzymatic route. Immobilized lipases were reported to be
effective biocatalysts for these types of biotransformation involving lipidic substrates. In the present study, a high LA
concentration and low water activity were necessary for the enzymatic acidolysis reaction in an organic solvent medium at
ambient temperature. Among the lipases examined, Lipozyme RM IM was found to be the best lipase for the incorporation of
LA at a water activity of 0.11 in toluene medium. The transesterification resulted in formation of the desired product (73 %)
along with lyso PC as the by product (24 %) at a water activity of 0.11 in 96 hours with maximum conversion of DPPC (>
96%). The composition of the product showed that 41.07 mol % of lipoic acid was incorporated into DPPC. The
transesterification was regioselective with nearly 92 % incorporation of lipoic acid in the sn-1 position with negligible
incorporation in the sn-2-position of the phospholipid in the product. The prepared phospholipid derivative could find
potential applications in cosmeceutical related product along with use in drug solubilisation and release studies.
S
S
O
O
O
O
O
O
+
Lipase
HO
solvent, aW
S
S
O
O O
P
O O
O
O
O O
P
O O
N
N
Fig. 1 Enzymatic synthesis of LPPC from DPPC.
Acknowledgements
The authors gratefully acknowledge SERB (DST), New Delhi for the financial assistance under Fast Track Scheme
for Young Scientist.
References
[1] S.A.Kates, R.A. Casale, A. Baguisi, R. Beeuwkes, Bioorg. Med. Chem. 2014, 22, 505–512.
[2] J. A. Laszlo, K. O. Evans, D. L. Compton, M.Appell, Chem. Phys. Lipids. 2012, 165,160-168.
43
P02
Enzymatic Hydrolysis of Type I Collagen as a Potential Source of New Macromolecules
with Amphiphilic Character
Aleksandra Kezwoń, Ilona Chromińska and Kamil Wojciechowski
Department of Microbioanalytics, Warsaw University of Technology, Warsaw, Poland – [email protected]
Keywords: enzymatic hydrolysis, collagen, surface properties
Protein hydrolysis is a process resulting in destruction of peptide bonds at specific locations, e.g., in the vicinity of
specific groups of amino acids. In the first part of the process, peptide chains is untangled resulting in uncovering the amino
acids groups hidden inside of the complex protein structure. The chains are then cut in specific places forming products with
surface properties dependent on the content of polar or non-polar amino acids.
For the purpose of the presented studies enzymatic hydrolysis of collagen type I was employed. The process does not
require extreme reaction conditions because enzymes usually work at room temperatures and pH values close to neutral.
They offer a possibility to control the products and the rate of conversion by using specific amount of enzyme and reaction
conditions. Type I collagen from bovine calf skin was chosen as the most common representative of fibrous proteins from
mammalian organisms [1, 2], and a potential source of new biomaterials with exceptional biocompatibility, biodegradability,
low toxicity and high mechanical strength [3]. The appropriate reaction conditions (i.e., specific enzyme, pH, temperature,
incubation time, etc.) were chosen based on literature information with the aim to achieve the best surface activity of the
hydrolysis products [4, 5].
The effect of the protein hydrolysis on surface activity and surface dilational rheology of collagen was investigated in the
course of this study. The dynamic surface tension after different hydrolysis treatments was measured during 5000 sec by drop
shape analysis method. In the first part of measurement (1 s – 3600 s) the volume drop was kept constant (21 µl). This
provided information about the ability of the collagen hydrolysates to reduce dynamic surface tension. In the second part of
the measurement (3600 s – 5000 s) the harmonic (sinusoidal) modulations of the volume of the drop were applied. This
enabled us to retrieve the surface dilational elastic E’ (storage) and viscous E’’ (loss) moduli of the hydrolysates’ films,
which describe the mechanical properties of the layers and are believed to play crucial role in kinetic stabilization of foams
and emulsions.
Fig. 1 Conventional surfactants vs protein-hydrolysate surfactants
References
[1] T.I. Nikolaeva, E.I. Tiktopulo, R.V. Polozov, Y.A. Rochev, Biophysics 2007, 52, 191 - 195.
[2] M. Fang, E.L. Goldstein, E.K. Matich, B.G. Orr, M.M. Banaszak-Holl, Int. J. Biol. Macromol. 2013, 29, 2330 - 2338.
[3] V.K. Yadavalli, D.V. Svintradze, R.M. Pidaparti, Int. J. Biol. Macromol 2010, 46, 458 - 464
[4] A. Nimptsch, S. Schibur, C. Ihling, A. Sinz, T. Riemer, D. Huster, Cell Tissue Research 2011, 343, 605 - 617 .
[5] H. Lin, D. O. Clegg, R. Lal, Biochemistry 1999, 38, 9956 – 9963
44
P03
Lipase Catalyzed Acidolysis of Phosphatidylcholine from Egg Yolk with Conjugated
Linoleic Acid Preparations Obtained from Sunflower and Safflower Oils
Anna Gliszczyńska, Natalia Niezgoda, Witold Gładkowski, Anna Chojnacka, Grzegorz Kiełbowicz, Czesław
Wawrzeńczyk
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Wrocław, Poland - [email protected]
Keywords: acidolysis, phosphatidylcholine, conjugated linoleic acid
Phosphatidylcholine (PC) is a naturally occurring cell membrane phospholipid in every living organism. PC is usually
produced from egg yolks [1] and soybeans [2]. It is available commercially in pure form and it is used in food industries as
an emulsifier or emulsion stabilizer.
These studies described enzymatic acidolysis of egg-yolk phosphatidylcholine in the sn-1 position. As the acyl donor the
concentrated preparation of conjugated linoleic acid was used. CLA is a mixture of geometrical and positional isomers of
linoleic acid. CLA gained special interest due to its biological properties. Studies have shown that CLA contribute to the
reduction of body fat level [3]. CLA were obtained by alcali isomerization of oils rich in linoleic acid (sunflower and
safflower oils). The enrichment of CLA from the FFA mixture was accomplished by urea complexation method to give the
preparations containing about 90% of CLA with the isomer composition which is presented in Table 1.
Phosphatidylcholine from egg yolk was subjected to enzymatic acidolysis catalyzed by lipase from Mucor miehei
(Lipozyme RM IM). Reaction conditions were as follows: 24 wt.% enzyme load at 45oC for 200 mg of PC and 1:8 (PC/CLA)
molar ratio. Water activity of substrates mixture and enzyme was adjusted to 0.33. After 12 h of the reaction water activity
was changed to 0.11. A yield of ca. 50% of CLA incorporation was achieved in phospholipid fraction (PC and LPC).
Comparison of fatty acid composition in native phosphatidylcholine from egg yolk and structured phospholipid indicates that
the incorporation of CLA occurred mainly in sn-1 position (Table 1)
Structured PC with conjugated linoleic acid may be a new agent used in mesotherapy in treating localized collections of
fat.
Table. 1 Relative fatty acid composition of PL before and after lipase catalyzed acidolysis with CLA concentrates.
CLA concentrates
sunflower oil
safflower oil
16:0
0.3
2.0
16:1 POA
0.2
0.1
18:0 SA
3.9
nd
18:1 OA
1.1
1.4
18:2 LA
nd
2.8
18:2 c9,t11+t8,c10
35.2
33.9
18:2 c11,t13
1.5
1.2
18:2 t10,c12
57.1
58.1
18:2 c9,c11+c10,c12
0.4
0.3
18:2 t,t
0.3
0.2
20:4 ARA
nd
nd
ΣCLA
94.5
93.7
a
Data are presented as mean ± SD of three independent experiments
nd; not detected
Fatty acid
Egg yolk PC
sn-1
sn-2
64.4±0.44a
6.8±1.66
1.9±0.05
0.8±0.34
27.9±0.35
3.18±0.92
4.9±0.33
51.29±4.26
0.8±0.10
36.82±2.66
1.12±0.01
Acknowledgements
This project was financed by National Science Center Project no. 2012/05/B/NZ9/03358
References
[[1] W.Gładkowski, et al., Journal of the American Oil Chemists' Society 2012, 89, 179.
[2] L. Montanari, et al., The Journal of Supercritical Fluids 1999, 14, 87.
[3] H. Blankson, et al., The Journal of Nutrition, 2000, 130, 2943.
45
Structured PL (36 h)
sunflower oil
safflower oil
5.54
5.71
0.36
0.33
2.51
2.89
22.09
21.79
16.75
16.29
17.76
17.66
0.56
0.60
28.95
30.07
0.89
0.55
1.33
1.01
3.25
3.09
49.50
49.90
P04
The Influence of Several Additives on the Phenylalanine Ammonia Lyase Activity
Alexandra Radu1, Diána Weiser2, Monica Ioana Toșa1, Florin-Dan Irimie1, László Poppe2 and Csaba Paizs1
1
2
Department of Chemistry, Babeș-Bolyai University, Cluj-Napoca, Romania – [email protected]
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary
Keywords: phenylalanine ammonia lyase, enzyme activity, enzyme stability
Several organic solvents, inhibitors and a series of mono- and divalent metallic ions were studied through PcPAL –
phenylalanine ammonia lyase from parsley – catalyzed reactions in order to obtain trans-cinnamic acid from Lphenylalanine.
PcPAL catalyzes the reversible cross migration of the amino group and a hydrogen atom present on the vicinal carbon
atom of a substrate. This enzyme is involved into a wide range of industrial processes, such as paper, food and
pharmaceutical production1. In medicine it is used to treat phenylketonuria disorder, and several types of cancer. Another
important feature of PcPAL is its ability to catalyze the stereoselective preparation of unnatural amino acids and also to
obtain the corresponding α, β-unsaturated compounds through the elimination process2.
In order to establish if the presence of the additives mentioned above led to increase the enzyme performance, multiple
experiments were designed. The investigations performed in the presence of polar solvents revealed that PcPAL showed
increasing stability and activity. The enzyme activity was enhanced with approximately 33% and 36% in presence of ethanol
and respectively methanol, while 10% of other solvents as tetrahydrofuran and acetonitrile reduced with almost 50% the
enzyme activity. In what concern the experiments carried out in the presence of metal ions it could be observed that Mn2+ and
Co2+ enhanced considerably the activity of the enzyme, while others like Na+ and K+ caused a negligible increase of the
enzyme activity. Other metal ions like Mg2+, Cu2+, Zn2+ proved to inhibit at small concentration the lyase activity.
The behavior of the mentioned additives may significantly enhance the productivity of the PcPAL catalyzed preparativescale procedures.
Acknowledgements
The present study was conducted with the financial support of the O IPOSDRU MEN under the project POSDRU
number 159/1.5/S/132400.
References
[1] a) K. Hahlbrock and D. Scheel, Ann. Rev. Plant Phys. Plant Mol. Biol. 1989, 40, 347-369; b) C. Appert, E. Logemann, K.
Hahlbrock, J. Schmid and N. Amrhein, Eur. J. Biochem. 1994, 225, 491-499.
[2] C. Paizs, A. Katona and J. Rétey, Chem. Eur. J. 2006, 12, 2739-2744.
46
P05
The Synthesis of 3-Pyrroline-2-on Derivatives from Amino Acids and Their Phosphonic
Acid Analogues
Nezire Saygili and Cemil Aydogan
Department of Basic Pharmaceutical Sciences, Hacettepe University, Ankara, Turkey – [email protected]
Keywords: pyrrolinone, amino acid, acyl phosphonates
3-Pyrrolin-2-ones are important starting materials for the preparation of a variety of biologically active compounds
[1-3]. On the other hand, excitatory amino acids (EAA) are the most common neurotransmitters in the mammalian central
nervous system (CNS) [4]. Thus, EAA receptors offer an opportunity to develop therapeutic compounds for the treatment of
several pathological conditions affecting the brain, such as Parkinson’s and Alzheimer’s diseases [5]. Besides, phosphonic
analogues of the aminodicarboxylic acids are modulators for the N-methyl-D-aspartate (NMDA) receptor site.
NH2
RO2C
(CH2)n
1
MeO
CO2R
O
OMe
%10 HCl, H2O, 25oC
N
RO2C
1) HCl
2) SOCl2
O
(CH2)n
CO2R
N
RO2C
(CH2)n
3) (RO)3P
2
n = 0,1,2
O
C O
RO
3
R = Me-, Et-
RO
P
O
In our previous study we have synthesized N-substituted 3-pyrroline-2-ones [6]. In course of our studies on Nsubstituted pyrrolinone systems, we decided to investigate the condensation reaction of amino dicarboxylic acid esters with
dimethoxydihydrofurane to obtain pyrrolinones (2) which will be used as intermediate products for the further synthesis of
pyrrolinone acyl phosphonates (3). We used one-pot condensation reaction to furnish the corresponding pyrrolinones (2) in
acceptable yields. Pyrrolinone derivatives were formed by using various amino dicarboxylic acid esters and their structures
were identified by using various spectroscopic methods. Synthesis of the phosphonate analogues of pyrrolinones are still
underway.
Acknowledgements
We are kindly thanked for financial support by Hacettepe University, Ankara Turkey.
References
[1] J.P. Dittami, F. Xu, H. Qi and M.W. Martin, Tetrahedron Lett. 1995, 36, 4201-4204.
[2] R. Shiraki, A. Sumino, K. Tadano and S. Ogawa, J. Org. Chem. 1996, 61, 2845-2852.
[3] B. Kenda, A.C. Matagne and P. Talaga, J. Med. Chem. 2004, 47, 530-549.
[4] J.W. Ferkany, J. Willetts, S.A. Borosky, D.B. Clissold, E.W. Karbon and G.S. Hamilton, Bioorg. Med. Chem. Lett. 1993,
3(1), 33-38.
[5] D.D. Schoepp and B.G. Johnson. J. Neurochem. 1989, 53, 1865-1870.
[6] N. Saygili, A. Altunbas and A. Yesilada. Turk. J. Chem. 2006, 30, 125- 130.
47
P06
Optimization of Regeneration and Transformation Conditions for Chamomile
(Matricaria chamomilla L.)
Katarzyna Bandurska, Malgorzata Król
Department of Microbiology and Biotechnology, Jan Dlugosz University, Częstochowa, Poland – [email protected]
Keywords: biopharmaceuticals, medicinal plants, plants transformation
Goal of this research was to test efficiency of regeneration and transformation properties of chamomile plants for
future production of biopharmaceuticals. For this purpose the influence of seedling age, type of explant, addition to medium
of hormones in different concentrations, the density of Agrobacterium tumefaciens culture at OD550, as well the length of
co-cultivation and post-cultivation periods on the regeneration ability of transformed chamomile’s explants were tested. The
highest percentage of transformed regenerants (99,6 %) was observed for hypocotyls. First putative transgenic chamomile
plants that produced roots on kanamycin-containing medium were confirmed by PCR analysis for the presence of transgenes.
Acknowledgements
This work was supported by a grant Homing Plus/2010-1/1 from the Foundation for Polish Science.
48
P07
Optimization of the Aromatization Step in the Synthesis of Evernyl
Jolanta Jaśkowska 1, Zbigniew Majka 2 and Aleksandra Sosin 1
1
2
Institute of Organic Chemistry and Technology, Cracow University of Technology, Kraków, Poland – [email protected]
Adamed Pharmaceuticals, Pieńków, Poland
Keywords: evernyl, aromatization
Evernyl (methyl 2,4-dihydroxy-3,6-dimethylbenzoate, Verymoss, Veramoss, Oakmoss, Everniate), which is associated
with the fragrance described as oakmoss, olibanum resinoid and powdery woody-sweet is often used primarily in perfumery
and cosmetics [1]. Is present, among others, in fragrance compositions such as Chanel 19, Cristalle, Diorella, Cool Water,
Polo, Chamade. Introduces an earthy aroma that very well with floral notes. Depending on the origin, the smell may be
slightly different [2]. In addition to its advantages fragrance, Evernyl has also antioxidant [3], antimicrobial [4] and
antifungal activity [5]
Most often the method described in the literature synthesis of Evernyl is a two-stage process, which relies on the
synthesized methyl 2-hydroxy-3,6-dimethyl-4-oxocyclohex-2-ene-1-carboxylate (3), which in the next step is aromatized to
the expected product.
The aim of the study was to optimize the aromatization step. We evaluated the effect of a type of oxidant, solvent,
temperature and reaction time on yield of the process. In addition to the typical oxidizing agents used in an aromatization
process such as N-bromoderivatives of imides (NBA, NBP, NBSac) the reaction may also be carried out in the presence of an
enterobactin – a substance produced by Escherichia coli. The progress of all reactions was monitored by TLC, and the
structures of the obtained compounds were confirmed on the basis of 1H NMR, IR, UPLC-MS spectra.
O
H3C
CH3
O
OH
1
CH3ONa
+
O
H3C
O
O
OH
O
O
CH3
O
H3C
CH3
enterobactin
CH3
3
H3C
O
O
HO
CH3
CH3
Evernyl
2
Fig. 1 Two-step synthesis of Evernyl.
References
[1] D. Joulain, R. Tabacchi, Flavor Fragr. J. 2009, 24, 49-61.
[2] D. Joulain, R. Tabacchi, Flavor Fragr. J. 2009, 24, 105-116.
[3] V. M. Thadhani, M. I. Choudhary, S. Ali, I. Omar, H. Siddique, V. Karunaratne, Nat. Prod. Res., 2011, 25, 1827 – 1837.
[4] (a) N. Sultana, A. Afolayan, J. Asian Nat. Prod. Res., 2011, 13, 1158 – 1164; (b) FIRMENICH SA
Patent: WO2008/68683 A1, 2008; (c) L. Acevedo, E. Martinez, P. Castaneda, S. Franzblau, B. Timmermann, E. Linares, R.
Bye, R. Mata, Planta Med., 2000, 66, 257 – 261.
[5] (a) I. Ali, D. Khan, F. Ali, H. Bibi, A. Malik, J. Chem. Soc. Pakistan, 2013, 35, 139 – 143; (b) M. Goel, P. Dureja, A.
Rani, P. Uniyal, H. Laatsch, J. Agricult. Food Chem. 2011, 59, 2299 – 2307
49
P08
Synthesis of New Cytotoxic Derivatives of Betulin and Betulinic Acid
Katarzyna Sidoryk1,2, Piotr Cmoch1, Anna Korda1, Jana Oklešťková3, Zbigniew Pakulski1, Lucie Rárová3, and
Miroslav Strnad3
1
Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warszawa, Poland
Pharmaceutical Research Institute, Warszawa, Poland
3
Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký
University & Institute of Experimental Botany ASCR, Olomouc, Czech Republic
2
Keywords: betulin, saponins, SAR study, cytotoxic activity
Betulin (1) and betulinic acid (2) are natural compounds with proven anticancer, anti-bacterial, antimalarial, antiviral and
anti-inflammatory activities [1, 2]. Recent investigations have demonstrated that even a simple modification of the structure
can significantly increase their biological activity. Lupane saponins (derivatives bearing sugar moiety at C-3 or C-28
positions) are also considered as very promising cytotoxic derivatives [3,4].
In this communication, we will report on the synthesis of series of new lupane derivatives as well as novel lupane-type
saponins. All new derivatives were examined for the cytotoxic activity against cancer cell lines of various histopathological
origins, including T-lymphoblastic leukemia (CEM), breast carcinoma (MCF-7), and cervical carcinoma lines (HeLa) as well
as normal fibroblasts (human BJ-H-tert). Our investigations demonstrate that the modifications at C-3 and/or C-28 position
of lupane triterpenes and saponins, significantly changed the cytotoxic activity of the resulting new compounds.
Acknowledgements
The support from the National Science Centre (Grant No. 2012/07/B/ST5/00823) is acknowledged.
References
[1] A. Sami, M.Taru, K. Salme, Y.K. Jari Eur J. Pharm. Sci. 2006, 29, 1–13.
[2] M.G. Moghaddam, B.H. Faujan, A. Samzadeh-Kermani Pharmacol. Pharm.2012, 3, 119–123.
[3] P. Cmoch, A. Korda, L. Rárová, J. Oklešťková, M. Strnad, R. Luboradzki, Z. Pakulski, Tetrahedron 2014, 70, 2717–
2730.
[4] P. Cmoch, A. Korda, L. Rárová, J. Oklešťková, M. Strnad, K. Gwardiak, R. Karczewski, Z. Pakulski, Eur. J. Org. Chem.
2014, 4089–4098.
50
P09
The Studies on Dynamic Kinetic Resolution of Racemic β-Hydroxyacids
Malgorzata Zysk, Anna Brodzka and Ryszard Ostaszewski
Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland
Keywords: DKR, biocatalysis, hydroxyesters
3-Hydroxyacids of structure 1 and their derivatives are versatile substrates in synthesis of biologically active
compounds, e. g. duloxetine[1]. However, efficient synthesis of enantiomerically pure esters of carboxylic acids usually
evokes many difficulties. Commonly used methods are based on kinetic resolution (KR) processes, however these methods
provide products only in 50% yield. To overcome this limitation we have proposed a new approach to synthesis of
enantiomerically pure esters 1[2], based on dynamic kinetic resolution of carboxylic acids (Figure 1).
Fig. 1 DKR of 3-hydroxy-3-(p-NO2-phenyl)-propanoic acid.
Results of dynamic kinetic resolution (DKR) of 3-hydroxy-3-(p-NO2-phenyl)-propanoic acid will be presented.
Acknowledgements
This work was supported by project ”Biotransformations for pharmaceutical and cosmetics industry” No. POIG.01.03.01-00158/09 part-financed by the European Union within the European Regional Development Fund “
References
[1] D. Titu, A. Chadha J. Mol. Cat. B: Enzymatic 2008, 52-53, 168-172.
[2] a) R. Ostaszewski, M. Ćwiklak, A. Wóltańska, Sz. Kłossowski, A. Żądło, Patent Appl: P.394228, 2011; b) R.
Ostaszewski, M. Ćwiklak, A. Wóltańska, Sz. Kłossowski, A. Żądło, Patent Appl: P.394723, 2011; c) R. Ostaszewski, M.
Ćwiklak, A. Wóltańska, Sz. Kłossowski, Patent Appl: P.394722, 2011, d) R. Ostaszewski, A. Brodzka, M.Ćwiklak, Patent
Appl: P.396417, 2011.
51
P10
The Studies on Dynamic Kinetic Resolution of 3-Phenyl-4-pentenoic Acid
Anna Brodzka, Malgorzata Zysk and Ryszard Ostaszewski
Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland – [email protected]
Keywords: DKR, biocatalysis, hydroxyesters
Chiral, enantiomerically pure compounds are of great importance for both academia and pharmaceutical industry.
Among others, enantiomerically pure carboxylic acids and their derivatives are valuable building block for the synthesis of
biologically active compounds.1,2 Unfortunately, efficient synthesis of derivatives of carboxylic acids as a single enantiomer
usually are difficult and regarded multistep synthesis.
We propose to use dynamic kinetic resolution (DKR) to prepare enantiomerically pure derivatives of carboxylic acid.
Theoretically, DKR can provide single enantiomeric products (99% enantiomeric excess or greater) in 100% yield in the case
where efficient racemization catalyst is combined with a highly enantioselective enzyme. The use of DKR seems to be
increasing and it would appear that these strategies become serious alternatives to conventional methods for asymmetric
synthesis. According to our knowledge, this method has never been used to synthesis of carboxylic acids before.
As a model compound for our studies, we choose 3-phenyl-4-pentenoic acids (Figure 1).
O
OH
Toluene, PhC(OEt)3,
Novozym,
cat., 5d, 55oC
rac-1
O
OEt
(S)-2
Fig. 1 DKR of 3-phenyl-4-pentenoic acid.
Acknowledgements
This work was supported by project ”Biotransformations for pharmaceutical and cosmetics industry”
No. POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund “.
References
[1] K. R. Campos, M. Journet, S. Lee, E. J.J. Grabowski, R. D. Tillyer, J. Org. Chem. 2005, 70, 268-274
[2] S. Pichlmair, M. de Lera Ruiz, K. Basu, L. A. Paquette, Tetrahedron 2006, 62, 5178–5194.
[3] R. Ostaszewski, M. Ćwiklak, A. Wóltańska, Sz. Kłossowski, A. Żądło, Patent Appl: P.394228, 2011; b) R. Ostaszewski,
M. Ćwiklak, A. Wóltańska, Sz. Kłossowski, A. Żądło, Patent Appl: P.394723, 2011; c) R. Ostaszewski, M. Ćwiklak, A.
Wóltańska, Sz. Kłossowski, Patent Appl: P.394722, 2011, d) R. Ostaszewski, A. Brodzka, M.Ćwiklak, Patent Appl:
P.396417, 2011.
52
P11
A High-sensitivity Fluorogenic Probes for Hydrolytic Enzymes
Dominik Koszelewski, Anna Żądło and Ryszard Ostaszewski
Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warsaw, Poland – [email protected]
Keywords: enzyme assays; fluorogenic substrates, hydrolases
One of the major activities in biocatalysis consists in developing enzymes for industrial applications, particularly in the
areas of fine chemical synthesis. Molecular imaging of the activity of target enzymes is therefore crucial for fundamental
research. Recognition of enzymatic activity is limited by the ability to establish a sufficient probe concentration on-site. It is
therefore essential to develop probe technologies providing maximum detection sensitivity. off-ON enzyme-responisve
probes, offering zero background signal plus catalytic activation by the target enzyme, are prime candidates to deliver it.
Most current off-ON (fluorogenic) probes are often based on FRET mechanism [1].
Here we would like to present the synthesis and characterization of new type of fluorogenic probes based on phenolic Excited
State Intramolecular Proton Transfer (ESIPT) (Fig. 1) [2].
Fig. 1 Action of hydrolase probes.
Acknowledgements
This work was supported by project ”Biotransformations for pharmaceutical and cosmetics industry” No.
POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund.
References
[1] B. Law, and C.-H. Tung, Bioconjugate Chem. 2009, 20, 1683.
[2] O. Thorn-Seshold, M. Vargas-Sanchez, S. McKeon and J. Hasserodt, Chem. Comm. 2012, 48, 6253.
53
P12
The Studies on Ugi and Passerini Reactions in Liposomes
Daniel Paprocki, Szymon Kłossowski and Ryszard Ostaszewski
Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warsaw, Poland – [email protected]
Keywords: Liposomes, Ugi Reaction, Passerini Reaction
Liposomes are spherical vesicles composed of a lamellar phase lipid bilayer. They can be formed in aqueous
solution of certain amphiphilic molecules. Liposomes have been used in a broad range of pharmaceutical applications e.g.
drug delivery systems, carriers for molecules in cosmetic [1]. Moreover there are known enzyme-catalyzed reactions [2],
even the PCR reaction [3] occurring inside lipid vesicles.
We propose the studies on the influence of the liposomes on the course of multicomponent reactions such as Ugi
and the Passerini reactions (Fig. 1). We compared the results of these reactions in standard conditions (organic solvents) and
where substrates are entrapped in liposomes.
O
R
OH
O
R
+
NH2
R1
+
OH R 1
+
R2
+N
H
R
R1
O
+N
H
R
2
R
1
R3
O
1
H
N
O
R
H
N
N
R3
C
O
R2
O
C
O
R2
O
2
3
R, R , R , R = alkyl or aryl
Fig. 1 The Ugi Reaction (above) and the Passerini Reaction (below).
Acknowledgements
This work was supported by project ”Biotransformations for pharmaceutical and cosmetics industry”
No. POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund“.
References
[1] A. Akbarzadeh, et al., Nanoscale Res. Lett. 2013, 8, 102-110.
[2] P. Walde, S. Ichikawa, Biomolecular Engineering 2001, 18, 143-177.
[3] T. Oberholzer, et al., Chem. Biol. 1995, 2, 677-682.
54
P13
"DetoxPad" Detoxifying Action or Placebo
Marcin Stasko1, Adriana Żyła2
1
2
Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warsaw, Poland - [email protected]
Faculty of Chemistry, University of Opole, Opole, Poland
Keywords: Detox Pads, analysis, placebo, industrial application
Fig. 1 Foot acupuncture http://www.detoxpads.com/images/detox-pads-reflexology.jpg
Too often we hear about heavy metals and toxins polluting our environment and our bodies, which can cause many
ailments in our organisms. Many pharmaceutical companies and similar facilities are coming out to our needs with
introducing many doubtful medicines and prophylactics calling them "golden remedy for everything". The most engaging
products are these which marked with slogans like 'ecological' or 'natural'. It is caused by ourselves thanks to our belief that
these products are not harmful. One of these products are detox foot patches (pads).
Detox food pad patched contain ingredients based on natural ancient recipe and they aim purify the body through the skin
feet. According to the producers the appearance of dark ugly smelling gunk after using was to bear witness to passage of
contaminants to the patch.
The question is: does detox food pad patched to our skin really has ability of cleaning our body and can heal us from
illness caused by toxins and heavy metals? Why does foot pads change their colors, and why suddenly they stop? Do they
really work? We will try to answer that questions, by carrying out scientific tests.
Performed an analysis of the composition of the recipe reported by the producer with respect to properties of components
described in the literature and also was conducted a series of tests those which aim determine which changes the color of the
detox food pad patched and what not cause. Conclusions reached were confronted with the results (online survey) conducted
on the 320 persons, including 100 users.
References
[1] S. Barrett, The detox foot pad scam. Device Watch, Feb 5, 2009
[2] J. Stossel, Ridding yourself of toxins or money? Company says Kinoki Food Pads 'capture toxins from your body' ABC
News, April 11, 2008
[3] S. Varney Japanese foot pad is latest health fad. KQED Aug 18, 2008
[4] FTC charges marketers of Kinoki Foot Pads with deceptive advertising seeks funds for consumer redress. FTC news
release Jan 28, 2000
55
P14
Biosynthesis of Zeaxanthin by Selected Psychrophilic Strain of Flavobacterium sp.
Małgorzata Milner-Krawczyk, Monika Wielechowska, Joanna Główczyk-Zubek, Kamila Petryka, Anna
Sobiepanek and Maria Bretner
Department of Drug Technology and Biotechnology, Warsaw University of Technology, Warsaw, Poland – [email protected]
Keywords: zeaxanthin, Flavobacterium, biosynthesis
Zeaxanthin - (3R,3'R)-beta,beta-carotene-3,3'-diol is a xanthophyl belonging to the carotenoid natural pigments family. It
is one of the most popular carotenoids responsible for the yellow color of many plants, poultry, fish and birds. Zeaxanthin
plays critical role in the prevention of age-related macular degeneration and cancer. It is also used as a colorant in the
cosmetic and food industries. Currently, there are two main methods of zeaxanthin production: chemical synthesis and
extraction from plant tissues. However, these methods are very time consuming and harmful for the environment. Therefore,
the particular interest in microbial sources of zeaxanthin is still growing.
Searching of the new bacterial strains producing zeaxanthin was the subject of our intensive research. From the samples
of antarctic soil we isolated psychrophilic bacterium from Flavobacterium species. Subsequently, we decided to optimize
conditions of 11E3 strain cultivation allowing for efficient pigment production. Initially, the method of the zeaxanthin
extraction was developed allowing the quick assessment of carotenoid production. Subsequently, the effect of various
nitrogen and carbon sources on zeaxanthin production was studied. The greatest impact on the zeaxanthin biosynthesis had
peptone from soymeal as nitrogen source and glucose as the carbon source. The selected mineral ATCC 1687 medium
supplemented with 3% peptone from soymeal and 0,5% glucose led to six-fold improvement in zeaxanthin production with
yield from about 0,9 mg/l to 6,0 mg/l.
56
P15
One-pot Deracemization of 5-hydroxy-4,5-dihydroisoxazole Derivatives
Joanna Główczyk-Zubek, Monika Wielechowska, Edyta Łukowska-Chojnacka, Maciej Dziachan, Agnieszka
Kowalska
Department of Drug Technology and Biotechnology, Warsaw University of Technology, Warsaw, Poland – [email protected]
Keywords: chiral 4,5-dihydroisoxazole, dynamic resolution, lipase
Chiral 4,5-dihydroisoxazole derivatives are useful substrates for synthesis of many chiral compounds, e.g. βhydroxyketones, γ-aminoalcoholes, β-hydroxynitiles. These derivatives have a high biological activity, the best example is
antitumor antibiotic Acivicin. It has also been found that the derivatives of 4,5-dihydroisoxazole show antibacterial,
antifungal and antiviral activity, as well they have potential to be applied as insecticides.
The 5-acetoxy-4,5-dihydroisoxazole derivatives were synthesized via 1,3-dipolar cycloaddition reaction using the
appropriate nitrile oxide (generated in situ) and vinyl acetate as a dipolarophile. Racemic esters were hydrolyzed with several
enzymes e.g. pig liver esterase (PLE), Candida antarctica B lipase (Novozyme 435), Candida rugosa lipase, Pseudomonas
fluorescens lipase. 5-Hydroxy-4,5-dihydroisoxazoles obtained in kinetic resolution reactions turned out to be racemates,
while enantiomeric excesses of unchanged esters were very high (>90%). In situ alcohol racemization provided an
opportunity to use dynamic resolution in one-pot hydrolysis-transestryfication reaction. The replacement of phosphate buffer
in hydrolysis step with buffer saturated TBME resulted in the significant increase in reaction yield. Novozyme 435 and
Candida rugosa lipase were the most appropriate enzymes for this biotransformation and allowed to obtained both
enantiomers of 5-acetoxy-4,5-dihydroisoxazoles derivatives with high enantiomeric excesses (80-93%) and 65-75% overall
yields.
57
P16
Characterization of the Collection of Environmental Microorganisms, Able to Conduct
Lactic Acid Fermentation and Analysis in Terms of Biotechnological Application.
Urszula Kasprowicz, Joanna Cieśla and Anna Kulińska
Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland – [email protected]
Keywords: lactic acid, fermentation, microorganisms
The collection of 17 bacterial strains was isolated from natural sources, in different parts of Poland (Bialystok,
Warsaw) and from different ecological niches (silage, fermented milk, fermented cabbage and pickles). Morphological
characteristic of the isolates showed that they belong to the Lactobacillus genus. Genotypic identification carried out by
partial 16S-23S rDNA gene sequence analysis using PCR technique revealed that strains belong to the Lactobacillus brevis,
Lactobacillus plantarum/ pentosus, Lactobacillus casei/ paracasei, Lactobacillus buchneri and Lactobacillus crustorum
species. Partial metabolic profile of lactic acid fermentation was tested for all 17 strains. Lactic acid concentration was
quantified using enzymatic method and HPLC technique [1]. The analysis showed that lactic acid production during
fermentation varied between isolates and yields from 1 g/l to 32,2 g/l in fermentation medium. The amount of isomeric forms
of a lactic acid produced by isolates, was quantified using enzymatic method. The L−lactic acid content percentage was from
33 to 93%. All strains produced acetic acid in a concentration of 3-8 g/L, which proves that they are heterofermenters.
Antibiotic resistance of isolated strains was tested with a microdilution method [2]. All strains were resistant to vancomycin,
kanamycin and ciprofloxacin, which is consistent with previous reports. In addition, isolates resistant to erythromycin and
ampicillin were detected. In contrast, all isolates were susceptible to rifampicin.
Acknowledgements
Research was supported by Warsaw University of Technology.
References
[1] M. S. Qureshi, S.S. Bhongale, and A.K. Thorave, J. Chrom. A 2011, 1218, 7147-7157.
[2] M. Egervärn, H. Lindmark, S. Roos, G. Huys, and S. Lindgren, Antimicrob. Agents Chemother. 2007, 51, 394-396.
58
P17
A New Recombinant Saccharomyces cerevisiae Strain for Production of 2-phenylethanol
Jolanta Mierzejewska, Aleksandra Mularska and Patrycja Okuniewska
Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland – [email protected]
Keywords: 2-phenylethanol, biotransformation, yeast
2-phenylethanol (2-PE) is an aromatic alcohol with rosy scent which is widely used in the food, fragrance and
cosmetic industries. Because of the costs of production and the quality of product, most of the 2-PE originates from chemical
synthesis and only small amount of 2-PE is being extracted from plants. Since there is an increasing demand on natural
products the alternative method for production of 2-PE needs to be drawn up. The promising sources of natural 2-PE are
microorganisms, especially yeasts which can produce 2-PE either by biosynthesis or biotransformation, however at low level
[1].
We constructed by fusion procedure a new diploid Saccharomyces cerevisiae strain producing 3,8 g l-1 2-PE by
conversion of 5 g l-1 L-phenylalanine in shaking flasks. To our knowledge it is the highest amount of 2-PE per litre achieved
in batch-culture of yeast carried out in shaking flasks.
According to the literature, we obtained a boundary concentration of 2-PE, around 4 g l-1, which is toxic for yeast
and inhibits the production of 2-PE [2]. Thus, to achieve higher efficiency genetic modification of yeast strain or one of the in
situ product removal (ISPR) method are necessary.
References
[1] M.M.W. Etschmann, W. Bluemke, D. Sell, and J. Schrader, App. Microbiol. Biot. 2002, 59, 1.
[2] D. Stark, D. Zala, T. Munch, B. Sonnleitner, I.W. Marison, and U. von Stockar, Enzyme Microb. Technol. 2003,
32,212.
59
P18
2-Hydroxy-2-(ethoxyphenylphosphinyl)acetic Acid as a Chiral Solvating Agent and
Chiral Derivatizing Agent for Alcohols and Amines
Paulina Majewska and Jagoda Szyszkowiak
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland –
[email protected]
Keywords: chiral discriminator, enantiomeric excess, absolute configuration
Preparation of compounds in enantiomerically pure forms is of great importance in the pharmaceutical industry, as the
biological activity of many drugs heavily depends on their absolute configuration. This, alongside with development of
synthetic organic chemistry have caused expanding interest in the synthesis of novel chiral compounds as well as the
intensive development in methods for determination of their three-dimensional structure and enantiomeric purity.[1]
The aim of the presented research was to check whether the 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid is a
versatile chiral phosphonic auxiliary (readily seen in 31P NMR).
The preliminary studies indicate that this compound may be used as chiral derivatizing agents for amines and alcohols,
since the separation of selected examples of diastereomeric alcohols and amines in 31P NMR spectra was found to be
satisfactory. In addition, the self-discrimination have been observed, when using diastereomeric mixture with high
enantiomeric excesses. The preliminary research about using this compound as a CSA are also promising.
Fig. 1 Interactions that occur between the chiral phosphonic auxiliary and the test molecule visible in 31P NMR.
Acknowledgements
This work was financed from the Project “Biotransformations for pharmaceutical and cosmetics industry” No.
POIG.01.03.01-00-158/09 partly-financed by the European Union within the European Regional Development Fund for
Innovative Economy.
References
[1] G.-Q. Lin, Y.-M. Li, A. S. C. Chan; Principles and Applications of Asymmetric Synthesis John Wiley: Chichester, 2001.
60
P19
Synthesis of Novel Fragrant Oxime Ethers from the Main Constituents of Caraway and
Spearmint Essential Oils
Daniel Jan Strub1, Józef Kula2, Stanisław Lochyński1,3
1
2
3
Department of Bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland – [email protected]
Institute of General Food Chemistry, Faculty of Biotechnology and Food Sciences, Technical University of Łódź, Łódź, Poland
Institute of Cosmetology, Wrocław University College of Physiotherapy, Wrocław, Poland
Keywords: oxime ethers, carvone, olfactory evaluation
Monoterpenes are a large class of natural compounds which comprises many fragrant molecules [1]. The
variety of their odoriferous properties results from the structural diversity, volatility, solubility and stability [2].
Monoterpenes are also a vast pool of substrates for the synthesis of novel semisynthetic fragrances [3,4]. From a
structural point of view, most fragrances are ketones, aldehydes, alcohols, esters or ethers. Studies regarding
structure-odoriferous activity relationship of oxime ethers are scarce. Literature data is limited to two patents of
International Flavors and Fragrances Inc. [5,6], where O-methyl ethers of aldehydes commonly used in
perfumery are described; and one patent of Givaudan [7], where fragrance of various non-terpenoid O-methyl
ethers is presented.
We have prepared novel fragrant oxime ethers with p-menthane system from (+)-carvone – constituent of
caraway essential oil and (–)-carvone – from spearmint essential oil. Two-step procedure – oximation of
carbonyl substrates and following O-alkylation was employed. Use of both enantiomers of carvone allowed us to
compare olfactory properties of both isomers of oxime ethers from the same series. Synthetic details and
olfactory evaluation of all novel oxime ethers will be presented. Fragrance diversity is observed between
homologous series of carvone oxime ethers. Their odours range from turpentine like to vegetable nuances.
Acknowledgements
This work was supported by the project “Biotransformations for pharmaceutical and cosmetics industry” No.
POIG.01.03.01-00-158/09, which is partly-financed by the European Union within the European Regional Development
Fund for the Innovative Economy”.
References
[1] C. Sell, A Fragrant Introduction to Terpenoid Chemistry. The Royal Society of Chemistry: Cambridge, 2003.
[2] S. Firestein, Nature 2001, 413, 211-218
[3] R. Kuriata-Adamusiak, D.J. Strub, P. Szatkowski, S. Lochyński, Flavour Frag. J. 2011, 26, 351-355
[4] D.J. Strub, L. Balcerzak, S. Lochyński, Curr. Org. Chem. 2014, 18, 446-458
[5] A.P.S. Narula, R. Mahesh, M. Pawlak, C.D.W. Brooks, U.S. Pat. 6924263 B2, 2005
[6] A.P.S. Narula, R. Mahesh, M. Pawlak, P.M. Merritt, C.D.W. Brooks, U.S. Pat. 7015189 B2, 2006
[7] R. Kaiser, P. Naegeli, C. Nussbaumer, Eur. Pat. 0672746 A1, 1995
61
P20
Hydrogenation of (+)- and (–)-Carvone by Cyanobacteria
Lucyna Balcerzak1, Stanisław Lochyński1 and Jacek Lipok2
1
2
Department of Bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland – [email protected]
Department of Analytical and Ecological Chemistry, Faculty of Chemistry, Opole University, Poland
Keywords: oxime ethers, carvone, olfactory evaluation
Monoterpenes comprise the largest and structurally diverse class of terpenes [1,2] Some monoterpenes are
cheap and readily available, what is of special importance regarding the common use of these compounds in
cosmetic industry. Not without the meaning, concerning low cost of obtaining such substances is the fact that
these compounds are often considered as a by-products, appearing in a processing of plants, in particular herbs
and conifers [3]. These attributes make them ideal substrates for biotransformation.
Cyanobacteria require only water, carbon dioxide, simple inorganic salts and light for their growth and
development [4]. Such limited environmental requirements, cause that blue-green algae are pioneer
microorganisms, which possess a high tolerance towards environmental factors [5]. Considering flexible
metabolism that reflects impressive ability of these microorganisms to ecological adaptations, the information
that only 2% of studies of biotransformations of chemical compounds is connecting to cyanobacteria that are
used as catalysts, seems to be quite surprising.
Therefore in our studies, the abilities of three cyanobacterial species: Chroococcus minutus, Anabanea sp.
and Nodularia moravica to biotransform (+)- and (–)-carvone were examined . Results of conducted experiments
have showed that freshwater species of cyanobacteria can perform hydrogenation of transformed compounds.
The main product of biotransformation was dihydrocarvone, what suggests that at the first step, the reaction may
occur at the C=C double bond of carvone, and it is independent on stereochemistry of added substrate – thus it is
an example of nonselective biotransformation. Additionally, in case of Anabanea sp., carveol was obtained as
the product of biotransformation..
Acknowledgements
This work was supported partially by Polish Ministry of Science and Higher Education in the frame of National
Science Centre grant No. UMO-2011/01/B/NZ9/04722.
References
[1] J. Degenhardt, and T. G. Köllner, Phytochemistry 2009, 70, 1621-1637.
[2] Y. Gounaris, Flavour and Fragrance Journal 2010, 25, 367-386.
[3] A.L. Groussin, and S. Antoniotti, Bioresour Technol 2012, 115, 237-243.
[4] A. Vioque, Adv Exp Med Biol 2007, 616, 12-22.
[5] H.J Humm, and S. R. Wicks, Introduction and guide to the marine bluegreen algae, NewYork 1980, Wiley
[6] C.C.C.R de Carvalho,. and M.M.R. da Fonseca, Biotechnol Adv 2006, 24, 134-142.
62
P21
Effective Continuous Acid Blue 62 Decolorization by Free Laccase Immobilized in a
Membrane Reactor
Marcin Lewańczuk, Jolanta Bryjak
Department of Bioorganic Chemistry, Wroclaw University of Technology, Wrocław, Poland - [email protected]
Keywords: laccase, decolorization, membrane reactor
Research focuses on using an enzyme membrane reactor (EMR) for the effective continuous decolorization of Acid Blue
62 (AB62). The following factors were considered for the effective use of Cerrena unicolor laccase immobilized in the EMR
volume: the enzyme was stable in 20 successive runs in a batch reactor; no aeration was necessary; AB62 and the oxidized
products sorbed onto the membrane but were not rejected; and the enzyme was stable in the EMR system. It is obvious that
any continuous process must be predictable and thus the objective was to verify the process model experimentally. For this
reason, a proper isoenzyme kinetic equation was selected and the parameters were evaluated. The obtained kinetic parameters
were used to plan processes and to verify their applicability to long-term AB62 decolorization, and a very good agreement
between the calculated and the measured data was obtained. In the main designed continuous decolorization process, the
conversion reached 98% and was stable for 6 days. The membrane reactor with C. unicolor laccase appears to be very
promising for AB62 decolorization.
Fig. 1 Scheme of continuous Acid Blue 62 decolorization by free laccase immobilized in a membrane reactor.
Acknowledgements
This study was supported by the project “Biotransformations for pharmaceutical and cosmetics industry” No.
POIG.01.03.01-00-158/09 partly financed by the European Union within the European Regional Development Fund.
63
P22
Influence of Storage Conditions on the Stability of Emulsions Containing Jasmonates
Alicja Kapuścińska and Izabela Nowak
Faculty of Chemistry, Adam Mickiewicz University in Poznan, Poznań, Poland – [email protected]
Keywords: jasmonates, particle size, emulsion stability
Skin aging is a natural and inevitable physiological process, signs of which are wrinkles, dryness and loss of the elasticity
[1]. Cosmetic manufacturers attempt to find a solution that could improve the appearance of the mature skin. In order to give
cosmetic desired properties, a cosmetic base is enhanced by different substances. These substances are called active
ingredients of cosmetics and exhibit certain physical effects on the physiology and / or functions of the skin. A new issue on
the cosmetics market is jasmonic acid and its derivatives, also known as jasmonates. In the plant organisms jasmonates act as
plant hormones that regulate their growth and development [2]. It has been shown that jasmonates have an effect on the
functioning of the human skin and, therefore may be a cosmetic active ingredient. These compounds are in fact capable of
regulating the activity of the sebaceous glands of the skin and stimulate exfoliation and skin renewal [3]. Tetrahydrojasmonic
acid sodium salt has been given the trade name LR2412 and has been used in brand L'Oreal cosmetics.
The influence of storage conditions on the stability of emulsions containing jasmonates was examined. Four types of
cosmetic formulations were prepared: O/W emulsion at elevated temperat ureheat, O/W emulsion at room temperature with
the addition of autoemulsifier, W/O emulsion at elevated temperature with acrylic acid carbomer-based gel base. For each
formulations either jasmonic acid (JA), methyl jasmonate (MeJA) or methyl dihydrojasmonate (MeDiJA) was added. After
100 days the stability of formulations stored at room temperature and in the refrigerator were examined. Examination of the
stability of prepared cosmetic emulsions was based on the backscattering phenomenon, where Turbiscan Lab Expert
Formulaction apparatus was used. The change in the particle size distribution of cosmetic formulations at the time was also
examined. For this study, particle size distribution analyzer, Mastersizer 2000 was used. Measurements of both phenomena
allows detection of the emulsion destabilization due to the aggregation of the dispersed phase particles.
Fig. 1 Differences in the particle size distribution of O/W emulsion prepared cold (a- the day of preparation, b- after 100 days
of storage in the fridge, c- after 100 days of storage at room temperature).
References
[1] M. Noszczyk, Kosmetologia pielęgnacyjna i lekarska 2010, Wyd. Lekarskie PZWL, 92-96.
[2] J. Kopcewicz, Lewak S., Fizjologia roślin 2001, Wydawnictwo Naukowe PWN. 2: 138 – 166.
[3] B. A. Bernard, R. Pereira, Experimental Dermatology 2012. 21, 398-400.
64
P23
Chemical Processes Allowed by COSMOS Organic Certification for Processing of
Vegetable Oils
Aleksandra Zielińska, Izabela Nowak
Faculty of Chemistry, Adam Mickiewicz University in Poznań, Poznań, Poland – [email protected]
Keywords: natural cosmetics, chemical processes, certifying organizations
Natural cosmetics containing vegetable oils in their composition have become a global trend in the recent times.
With the increasing awareness not only about our own health, but also about the environment we live in, there is
growing interest in products of natural origin [1,2]. Before a cosmetic will be recognized as an organic, ecological or
natural product, it has to comply with the conditions defined by certifying organizations [3]. In addition, the
manufacturer is obligated to undergo regular audits, which assures the compliance with all rules of the standard that will
be confirmed by the certifying unit [1,3].
In 2009, COSMOS Standard (Organic and Natural Cosmetics Standard) was created and accepted at the global
market, in order to harmonize designations of all of the most important certifying organizations such as: BIOFORUM
(Belgium), BDIH (Germany), COSMEBIO and ECOCERT (France), AIAB and ICEA (Italy) and SOIL
ASSOCIATION (United Kingdom) [1,3,4]. The main purpose of this integration was to complement labels of the
above-mentioned European certifiers, thereby providing consumers with a certified product quality [1,4]. What is
important, after fulfilling the requirements of a common standard, each of organizations can still use their own criteria
to allocate the certificate [3]. The main tasks of COSMOS Standard include: promoting the use of products from
organic farming, use of natural resources in a responsible manner, certifying respecting the environment, processing
and production of environmentally friendly, integration and development of the concept of Green Chemistry,
certification of natural cosmetics in accordance with the standards of COSMOS [1,4]. To obtain certification, it is
necessary to know that the application of green chemistry principles as well as the nanotechnology use is severely
limited [1]. These very strict European standards involve two different certification levels: Cosmos Organic (for organic
products) and Cosmos Natural (for natural products) [1,3]. Cosmos Organic requires that organic product must contain
at least 95% ingredients of natural origin, that can be extracted with simple physical actions (vegetable oils, fats and
waxes, herbal extracts and the nectar of flowers, natural essential oils) [1,4]. In contrast to that, Cosmos Natural product
should not contain more than 2% of synthetic raw materials [4].
Products that are certified to COSMOS Standard, such as more and more common and popular vegetable oils, can
be obtained by some of allowed chemical processes. There are three groups of processes, which [4]: a) allow the
formation of biodegradable molecules, b) respect natural active substances that are present in ingredients, and c)
encourage good waste management and energy use and take into account ecological balance. These include: alkylation,
amidation, calcination of plants residues, carbonization (resins, fatty organic oils), condensations and addition,
esterification, trans-esterification and inter-esterification, etherification, fermentation (natural / biotechnological),
hydration, hydrogenation, hydrolysis, ionic exchange, neutralization, oxidation and reduction, ozonolyze,
phosphorylation, saponification, sulphation and sulphatation [4,5].
As the market for natural personal care and cosmetics is growing globally, CBI (Centre for the Promotion of
Imports from developing countries) in the Netherlands offers free certification for such countries as e.g. Benin, Burkina
Faso, Cape Verde, Colombia, Ecuador, El Salvador, Indonesia, Mali, Peru, Philippines, Senegal, Uganda. These
countries are offering in the area of vegetable oils exotic varieties, sustainability claims or products with interesting
functionalities [6].
References
[1]
[2]
[3]
[4]
[5]
[6]
B. Mirkowska, Świat Przemysłu Kosmetycznego. 2012, 3(12), 8-13.
H.H. Rech Kosmetyki naturalne. 2006.
Organizacje certyfikujące. Retrieved July 28, 2014 from http://www.zdrowe-kosmetyki.pl/certyfikaty.php.
COSMOS-standard, Cosmetics organic and natural standard. Version 2.0. Appendix II. 2013, 21-22.
N. Karak, Processing and Applications. 2012, Woodhead Publishing Limited, 120-130.
Natural ingredients for cosmetics. Retrieved July 28, 2014 from
http://www.cbi.nl/About%20CBI/sectors/Natural%20ingredients%20for%20cosmetics/1858
65
P24
A Comparative Study of Transmembrane Diffusion of Low Molecular Peptides
Across Synthetic Membranes and Their Interaction with a Model Lipid Bilayer
Anna Olejnik, Izabela Nowak
Faculty of Chemistry, Adam Mickiewicz University in Poznan, Poznań, Poland – [email protected]
Keywords: tetrapeptides, release studies, dipalmitoyl phosphatidylcholine
Recently there has been a growth of interest in the novel skincare formulations containing active ingredients such
as low molecular peptides. These compounds have recently revolutionized the cosmetics and pharmaceutical industries
and have become one of a main bioactive component in the anti-age formulations [1]. Peptides are involved in several
natural processes with relevance to skincare, such as the cell migration, modulation of cell proliferation, melanogenesis,
angiogenesis and protein synthesis and their regulation [1,2]. Some researchers state that there is no significant
evidence that peptides work better than moisturizers, though other investigators claim that they represent a group of
compound with a future potential [3]. In fact peptide can be found in a large number of cosmetics [2] including antiage, anti-wrinkle and skin moisturizing products, therefore it is important to analyze the formulations containing these
compounds and to study their interactions with bilayers.
The aim of the present work was to assess the diffusion rate of the selected tetrapeptide from various semisolids
such as hydrogels, oil-in-water emulsions and water-in-oil emulsion. The impact of different synthetic membranes on
the tetrapeptide release kinetics was also investigated. The additional usage of the atomic force microscopy (AFM)
enabled to characterize the applied membranes that was helpful to assess their suitability for the release experiment.
The obtained results proved that the diffusion rate of tetrapeptide from semisolid dosage form reflected the effect of
both physical and chemical parameters of the product, including its rheological properties. The formulations analyzed in
this study had different properties however they had the same amount of tetapeptide. The results reported herein
indicated that the higher viscosity of the semisolid the slower permeation through the membrane.
The interaction of the low molecular peptides with a model lipid bilayer (DPPC - dipalmitoyl phosphatidylcholine)
was studied by using molecular dynamics. The simulations were conducted when the peptide was inserted in three
positions, initially in an aqueous medium, then was partly or completely inserted into the lipid bilayer. These theoretical
studies enabled to understand and visualize the potential behavior of tetrapeptides in the biological environment.
Acknowledgements
The authors would like to thank JKP Instruments for their professional support and technical collaboration in
analyzing the synthetic membranes.
References
[1] K. Fields and T.J. Falla, Other, J. Cosmet. Dermatol. 2009, 8, 8.
[2] L. Zhang, T.J. Falla, Clin. Dermatol. 2009, 27, 485.
[3] J. P. Bowler, Clin. Interv. Aging. 2009, 4, 81.
66
P25
Determination of Antioxidant Activity of Commercial Creams Containing
Carotenoids
Joanna Igielska-Kalwat and Izabela Nowak
Faculty of Chemistry, Adam Mickiewicz University in Poznan, Poznań, Poland –[email protected]
Keywords: carotenoids, antioxidant, 1,1-diphenyl-2-picrylhydrazyl
Free radicals are species that have unpaired valence electron or an open electron shell necessary for stable
molecular structure. Notable examples are singlet oxygen or superoxide are examples of free radicals that are
characterized by a high reactivity. To compensate for the lack of electrons, they take electrons from other molecules.
The human body has many defense mechanisms that neutralize the harmful effects of reactive oxygen species [1]. An
important role in reducing oxidative damage in the human body plays antioxidants. The strongest antioxidants include
carotenoids. Carotenoids are substances, which impart color from yellow to red in both plants and animals. They consist
of 11 conjugated double bonds, so that they can be counted among the polyprenoids..
We can divide them into carotenes and xanthophylls Food carotenoids positive effect on inhibiting the development of
many diseases today. The group characterized by the most powerful carotenoid antioxidant properties include lycopene,
lutein, astaxanthin and β-carotene [2].
Antioxidant activity was determined by the modified method of Brand-Williams and workers using synthetic
DPPH (1,1-diphenyl-2-picrylhydrazyl). Absorbance of the solution was measured at λ= 517nm. The results are shown
as a percent of quenching of free radicals according to the formula:
% Inhibition = 100 (Ao-A1) / A0 ,
where:
A0 - DPPH absorbance of the solution after 30 minutes without the addition of antioxidant,
A1 - DPPH absorbance of the solution after 30 minutes in the presence of antioxidant.
The antioxidant properties of the commercial creams containing carotenoids were measured by the method of free
radical DPPH. These studies confirm effectiveness of carotenoids as natural antioxidants. The figure 1 shows that free
radical DPPH is dimmed by creams containing carotenoids only after 30 minutes.
Fig. 1 DPPH capacity measurements for selected commercial creams containing carotenoids
References
[1] M. Guerin, M.E. Huntle, M. Olaizola, Trends in Biotechnol. 2003, 21, 210-216.
[2] F.A. Juola, K. McGraw, D.C. Dearborn D.C, Biochem. Physiol. 2008,149, 370-7.
67
P26
Amine-Functionalized Mesoporous Silica as Catalysts for Knoevenagel
Condensation
Agata Wawrzynczak, Magdalena Gorzynska and Izabela Nowak
Faculty of Chemistry, Adam Mickiewicz University in Poznan, Poznań, Poland – [email protected]
Keywords: hybrid mesoporous materials, amine-functionalized silica, Knoevenegel condensation
Hybrid amine-functionalized mesoporous silica, may have a wide range of applications, among others in
adsorption and catalysis [1]. For example, Knoevenagel condensation between benzaldehyde and ethyl acetoacetate
may be catalyzed by active centers with basic nature, generated by amino groups. This reaction yields, among others, in
ethyl 2-benzylidene-3-oxobutanoate, which is a valuable compound for pharmaceutical and cosmetic industry. Further
transformations of this compound enable obtaining derivatives of coumarin, nifedipine or nitrendipine. The first
compound is a popular fragrant, whereas the latter two serve in medicine as calcium channel blockers [2].
Two different synthetic approaches may be utilized in order to incorporate amino groups into the matrix of
hybrid mesoporous materials. One-pot technique involves co-condensation step between functional group precursor and
inorganic silica source, whereas in the post-functionalization route the modification occurs when mesostructurization
step is already completed.
During our studies hybrid organic-inorganic mesoporous silica materials of SBA-15 and SBA-16 type,
bearing amino groups, were synthesized, using one-pot and post-functionalization techniques. Samples were obtained
both in the bulky form and as free standing silica films. Tetraethylortosilicate (TEOS) and triblock copolymers
(Pluronic F-127 and P-123) were used as a silica source and structure directing agents, respectively. Two different
aminosiloxanes were applied a source of amino groups. Various extraction procedures were applied in order to remove
template from silica matrix. Template removal procedure, recognized as the most effective, was based on extraction
with HCl/ethanol solution.
Several techniques were employed to characterize structural, textural and physicochemical properties of the
obtained materials, namely: XRD, SEM/TEM analysis, low-temperature N2 sorption measurements, FT-IR
spectroscopy and elemental analysis. Small-angle XRD measurements and TEM analysis confirmed mesoporosity and
hexagonal (SBA-15) or regular (SBA-16) pore ordering in the obtained materials. Low-temperature N2 sorption
analysis revealed also that the synthesized hybrid materials possess well-developed surface area. FT-IR and elemental
analysis confirmed the presence of amino groups grafted onto silica matrix.
Basic character of the obtained materials predestines them as potential catalysts in the Knoevenagel
condensation, e.g. amine-functionalized mesoporous silica film of SBA-16 type yields in ethyl 2-benzylidene-3oxobutanoate with selectivity of 95%. Moreover, all synthesized hybrid materials exhibit high adsorption capacity and
could be used, for example, as adsorbents for waste water purification.
Acknowledgements
The National Science Center is kindly acknowledged for partial financing of presented results (grant nr: DEC2013/10/M/ST5/00652).
References
[1] F. Zhu, D. Yang, F. Zhang and H. Li, J. Mol. Catal. A: Chemical 2012, 363-364, 387.
[2] A. Corma, R.M. Martín-Aranda and F. Sánchez, Stud. Surf. Sci. Catal. 1991, 59, 503.
68
P27
Biocatalytic Reduction of Fluorinated β-Keto Phosphonate Derivatives by Baker’s
Yeast
Magdalena Rapp, Marta Z. Szewczyk and Henryk Koroniak
Faculty of Chemistry, Adam Mickiewicz University, Poznań, Poland - [email protected]
Keywords: bioreduction, yeast, fluorinated β-ketophosphonates
The wide interest in methylenephosphonates is connected with their nature as hydrolytically stable analogues
of naturally occurring phosphate esters. Mono and difluoromethylene phosphonates can serve as the example, how
incorporation of the fluorine atom into an organic molecule can influence chemical [1] and biological activity [2]. As
chiral compounds very often caused desirable effect only in appropriate enantiomeric form, we become interested in
biocatalytic reduction leading to compounds 1 and 2 - interesting alternative of asymmetric synthesis. Between the
enzymatic method, the application of Baker’s yeast (Saccharomyces cerevisiae) in enantioselective reduction was
chosen [3]. The comparison of the biotransformations with and without some additives will be presented.
OH
OH
P(O)(OEt)2
P(O)(OEt)2
R
R
F
F
F
R = Me, Ph
Fig. 1
Additionally, the stereochemistry of these reactions will be discussed.
Acknowledgements
The research was supported by Wroclaw Research Centre EIT+ under the project „Biotechnologies and
advanced medical technologies” – BioMed (POIG.01.01.02-02-003/08) financed from the European Regional
Development Fund (Operational Programme Innovative Economy, 1.1.2)
References
[1] V. P. Kukhar, V.D. Romanenko, In: Amino Acids, Peptides and Proteins; Hughes A. B., Ed.; Wiley-VCH: 2009; 2,
189
[2] a) G. M. Blackburn, Chem. Ind. (London) 1981, 134; b) G. M. Blackburn, D. L. Jakemen, A. J. Ivory, M.P.
Wiliamson Bioorg. Med. Chem. Lett. 1994, 4, 2573; c) C. E. McKenna, P. D. Shen, J. Org. Chem. 1981, 46, 4573; d) S.
Halazy, A. Ehrhard, C. Danzin, J. Am. Chem. Soc., 1991, 113, 315
[3] E. Żymańczyk-Duda, B. Lejczak, P. Kafarski, J. Grimaud, P. Fischer, Tetrahedron 1995, 51, 11809
69
P28
(S)-1-Phenylethanol Dehydrogenase as a Catalyst in Prochiral Compounds
Reduction
A. Dudzik1, M. Szaleniec1, W. Snoch1, J. Opalińska – Piskorz1, M. Witko1, J. Heider2
1
2
Jerzy Haber Institute of Catalysis and Surface Chemistry PAS, Kraków, Poland – [email protected]
Laboratory of Microbial Biochemistry, Philipps-University of Marburg, Germany
Keywords: alcohol dehydrogenase, optically pure alcohols, hydrogen-transfer biocatalysis
Short-chain dehydrogenases/reductases (SDRs) have received an increasing attention due to their broad
substrate specificity, tolerance to high temperatures and organic solvents [1], which are favorable characteristics of
biocatalysts for chiral alcohol synthesis. Enantiopure alcohols are valuable synthons for the fine chemicals production.
A straightforward approach to the synthesis of chiral alcohols is the asymmetric reduction of the corresponding
carbonyl compounds.
In our research we have used (S)-specific phenylethanol dehydrogenase (PEDH) from Aromatoleum
aromaticum capable to reduce more than 50 prochiral ketones and β-keto esters to enantiopure secondary alcohols. The
enzyme overexpressed in E. coli cells was used to convert a range of para substituted acetophenone derivatives in batch
reactor tests simulating industrial conditions (i.e. cofactor recovery system with high content of isopropyl alcohol,
whole-cell biocatalyst, high loading of the reactor – i.e. 50 mM and 300 mM substrate concentration). The obtained
reactions progress curves were used for development of artificial neural network models describing enzyme reactivity.
The resulting models connected reaction conditions as well as thermodynamic and electronic features of the
substrates with concentration of the respective products.
The obtained results allowed identification of parameters influencing the conversion rate, which will help in
planning of the reactions in a bigger scale. Moreover, the analysis of the models provided insight into the influence of
physicochemical parameters on the PEDH reactivity.
Fig. 1 A) Schematic representation of the MLP 3-5-1 neural network B) Scatter plot for experimental versus predicted
product concentrations obtained for neural network model in external validation set: circles - experimental data points –
concentrations of 1-(4-fluorophenyl)ethanol, triangles – neural network predictions.
Acknowledgements
The authors acknowledge the financial support of the project ‘Biotransformations for pharmaceutical and
cosmetics industry’ No. POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional
Development Fund.
References
[1] H.W. Hӧffken, M. Duong, T. Friedrich, M. Breuer, B. Hauer, R. Reinhardt, R. Rabus, J. Heider, Biochemistry 2006,
1, 82-93.
70
P29
Biotechnological fatty acids esters for pharmaceutical and cosmetic applications
Łukasz Stańczyk, Katarzyna Struszczyk-Świta, Mirosława Szczęsna-Antczak and Tadeusz Antczak
Institute of Technical Biochemistry, Lodz University of Technology, Łódź, Poland - [email protected]
Keywords: lipases, biotransformation, Mucor, cosmetics, transesterification
Effects of the growing demand for crude oil and natural gas are increasing prices of these fossil feedstocks
and all their derivatives, which account for over 90% of raw materials for worldwide chemicals production. In some
industries the crude oil and its derivatives are difficult to replace, but in some other - their replacement is not only
possible, but even desired.
Pro-ecological trends and growing awareness of consumers cause that crude oil derivatives in cosmetics and
pharmaceutical products like mineral oil or vaseline have not been accepted anymore. These two components, which
often constitute over 30% of formulation, can be easily replaced by fully natural fatty acids esters obtained in
biotechnological processes. Depending on substrates structure, like carbon chain length and its branching degree, their
physiochemical and sensory parameters can be modified.
Using the generally regarded as safe and unique filamentous fungi Mucor circinelloides and Mucor
racemosus, which were isolated by our group, an effective and “ecofriendly” biotechnological method of synthesis of
the aforementioned esters has been developed. Catalyzed by the fungal lipases biotech esterification is a one-step
process carried out at low temperature and atmospheric pressure oppositely to the chemical method. The process can be
carried out in kilogram scale with yields ranging between 65 and over 93% depending on alcohol substrates. The
immobilized preparations of Mucor lipases with extremely long half-life (over 1 year) can be used in a column system
with and without recirculation.
Using renewable raw materials, like rapeseed oil or wastes from its production and C2 - C18 alcohols,
including branched ones, a wide range of fatty acids esters with different functionalities like: emollients, emulsifiers,
surfactants, rheology modifiers or even bacteriostatic agents can be obtained.
Acknowledgements
Project POIG.01.03.01-00-158/09 partially financed by the European Regional Funds within the scope of
Operation Program Innovative Economy.
71
P30
Enzymatic Transesterification Reactions Using Lipolytic Fungal Biomass in NonAqueous Aystems
Mirosława Szczęsna-Antczak, Łukasz Stańczyk, Jakub Szeląg, Agnieszka Borowska, Katarzyna
Struszczyk-Świta, and Tadeusz Antczak
Institute of Technical Biochemistry, Lodz University of Technology, Łódź, Poland - [email protected]
Keywords: bioconversion, alcoholysis, acidolysis, whole-cell lipase preparation, high operational stability
Bioeconomy has been one of most dynamically developing sectors of EU economy. Its basic goal is the
replacement of fossil fuels with biomass as a renewable source of valuable products and/or feedstocks for industry.
Foundation of the industry on natural resources and bioprocesses is the prerequisite of sustainable development.
Presented results were achieved within the frames of a research project realized by a consortium of several
research institutions in Poland: Lodz University of Technology, Institute of Biopolymers and Chemical Fibers in Lodz,
Centre of Molecular and Macromolecular Studies of Polish Academy of Science, University of Agriculture in Krakow
and Central Mining Institute in Katowice. One of objectives of this project is the development of a chemo-enzymatic
method of oleaginous biomass conversion into biodegradable components of aliphatic-aromatic polymers for
fabrication of agro-textiles.
In the Institute of Technical Biochemistry of LUT has been devised a biocatalyst, which is inexpensive and
highly active in non-aqueous systems. Conditions of its effective usage in processes of oil bioconversion (mainly
rapeseed, sunflower, soybean and waste oils) into esters of aliphatic primary alcohols (mainly branched) or structured
SUS-type triacylglycerols (saturated-unsaturated-saturated acid bound to glycerol) were also optimized. Products of
these enzymatic reactions may be further converted (by physiochemical way) into dimers and macrodiols, which can be
used in polymerization processes.
Immobilized in porous carriers, whole-cell (mycelial) preparations of intracellular lipases produced by
oleaginous and lipolytic fungal strains Mucor from the culture collection at ITB, which are robust and highly active in
non-aqueous systems, have been used to develop semi-continuous transesterification processes, e.g. plant oil
alcoholysis by 2-methylbutan-1-ol (or other medium-chain alcohols) and acidolysis by saturated fatty acids (especially
palmitic and stearic). Operational stability of these biocatalysts in column PBR reactors (working volume of 0.2-0.5L)
either with petroleum ether used as a solvent (or without it), under suitable process (acidolysis and alcoholysis)
parameters reaches about half a year (or more) without any decrease in bioconversion yield. Identification of crucial
parameters deciding of transesterification processes efficiency and high stability of the biocatalyst guarantees the
successful up-scaling of these processes.
Acknowledgements
This work was realized within the frames of the project No POIG 01.01.02-10-123/09 (entitled ”Application
of biomass in production of environmentally friendly polymer materials”), co-financed from the European Regional
Development Fund (the Innovative Economy Operational Programme 2007-2014.
72
P31
Immobilization of Lipase from Pseudomonas fluorescens on Different Carriers
A. Zniszczoł1, K. Szymańska2, S. Boncel1, A. Jarzębski2 and K. Z. Walczak1
1
2
Department of Organic, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, Gliwice, Poland
Department of Chemical Engineering, Silesian University of Technology, Gliwice, Poland – [email protected]
Keywords: lipase, immobilization,transesterification
Enzymes catalyze reactions usually under very mild conditions with a high degree of substrate specificity and
reduced amounts of by-products. Furthermore enzymes including lipases can catalyze reactions in different media [1]
and in various physical states [2]: as individual molecules, in solution or attached to surfaces of different supports. The
purpose of our research was to produce effective and recyclable catalysts for enantioselective transesterification
reaction for the industrial applications. The ideal carrier should exhibit several properties include physical resistance,
hydrophilicity, resistance to microbial attack, biocompatibility and availability at low cost. There are many
commercially available supports for immobilization, unfortunately, the ideal carrier does not exist.
In the study lipase from Pseudomonas fluorescens was covalently immobilized on Santa Barbara Amorphous
(SBA-15) mesoporous silica modified with different groups (epoxy, amino) and on multi-wall carbon nanotubes
(MWCNTs) which contain various functional groups e.g. amino, hydroxyl or carboxyl. The presence of enzyme
attached to the different types of carriers was verified by thermal analysis and FT-IR spectroscopy. All obtained
biocatalysts were also characterized in terms of their surface properties.
These immobilized enzymes were examined in the model reactions, namely transesterification of vinyl esters
with racemic (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (Fig. 1). The activity of the variously immobilized preparations
was confirmed in the series of experiments. The obtained results indicated very high operational stability of
immobilized lipases and the ability of the biocatalysts for multiple use.
O
OH
*
O
O
+
R
O
2
O
O
Lipase
R
1
O
2
R2
O
O
O
R
R2
1
+
OH
2
R =H
O
1
R
+
O
O
R =CH3
OH
O
H
Fig. 1 Esterification of solketal by vinyl esters in the presence of lipases immobilized on different supports
In covalent immobilization of enzymes the different morphology of carrier or chemical functionalization surface are
crucial and may change the physicochemical properties of enzyme.
Acknowledgements
This work was financed from the Project “Biotransformation for pharmaceutical and cosmetics industry”
No.POIG.01.03.01-00-158/09- part-financed by the European Union within the European Regional Development Fund
for the Innovative Economy”
References
[1] L.T. Kanerva, J. Vihanto, M.H. Halme,, J.M. Loponen, E.K. Euranto, Acta Chemica Scandinavica, 1990, 44, 10321035.
[2] H. Tümtürk, N. Karaca, G. Demirel, F. Sahin, International Journal of Biological Macromolecules, 2007, 40, 281285.
73
P32
Kinetic Resolution of 3-Amino-1,2-Propanediol Racemic Mixture in the Presence of
Immobilized Lipase
Danuta Gillner, Katarzyna Szymańska, Mateusz Maciński, Natalia Szymura, Krzysztof Walczak, Andrzej
Jarzębski
Faculty of Chemistry, Silesian University of Technology, Gliwice, Poland – [email protected]
Keywords: aminodiols, immobilized lipase, kinetic resolution
Chiral aminoalcohols are known synthons of many popular pharmaceuticals, such as chloramphenicol, HIV
protease inhibitor [1] and many others. Aminopropanediols can be found in the structure of many β-adrenolytics (e.g.
propranolol). Since the majority of medicines require pure enantiomers, many methods have been developed to obtain
them. That includes the synthetic methods as well as resolution of racemic mixtures.
This report describes research on the kinetic resolution of racemic 3-amino-1,2-propanediol derivatives via
transesterification (Scheme 1) as well as hydrolysis routes. Phthalic anhydride was used to protect the amino group. The
preliminary selection of several bacterial, fungal and mammal lipases revealed that the best resolution was obtained
using pancreatin and lipase from porcine pancreas (PPL) type II. Here we present the potency of immobilized PPL in
the resolution of racemic 3-amino-1,2-propanediol derivatives.
O
N
OH
OH
O
O
O R
lipase
O
N
O
R +
OH
OH
O
O
R/S
O
Fig. 1. Enzymatic acylation of N-(2,3-dihydroxypropyl)phthalimide
Comparison of different functionalized silica carriers revealed better behavior of silica monoliths with
hierarchical pores structure, modified with hexadecylsilane (MH-HDS). The influence of pH of immobilization was
investigated. It showed that lipase immobilized at pH 7-8 gives the best results in the resolution of racemic N-(2,3dihydroxypropyl)phthalimide via transesterification (eeR 75-80%; α 46-48%).
Several acylating agents were also studied in the transesterification process. The best results were obtained when vinyl
butyrate or myristate were used. The mixture of t-butanol/toluene (2:1 v/v) was used as a solvent.
Concerning the hydrolysis route, diesters of N-(2,3-dihydroxypropyl)phthalimide were synthesized and used
as substrates. Taking into account the regioselectivity of PPL it was expected, that the ester group of primary alcohol
will be hydrolyzed much more rapidly. Unfortunately the hydrolysis led to the complex mixture of products, including
monoesters of primary and secondary alcohols as well as dihydroxy derivative. Such phenomenon, described previously
in the literature [2], can be explained by intramolecular acyl group migration. It was observed not only in the case of
acetate or butyrate but also myristate with long carbon chain.
Acknowledgements
The research was supported by the project “Biotransformations for pharmaceutical and cosmetic industry”
No. POIG.01.03.01-00-158/09 co-financed by the European Union from European Regional Development Fund
References
[1] J.C. Barrish, E. Gordon, M. Alam, P.F. Lin, G.S. Bisacchi, P. Chen, P.T. Cheng, A.W. Fritz, J.A. Greytok, and M.A.
Hermsmeier. J.Med.Chem., 1994, 37(12), 1758
[2] M.A. Mbappe and S. Sicsic, Tetrahedron: Asymmetry, 1993, 4(5),1035.
74
P33
Biotransformations of Chosen Chalcones by Photosynthetic Bacteria – Potential
Applications
Beata Żyszka, Jacek Lipok
Department of Analytical and Ecological Chemistry, Opole University, Opole, Poland - [email protected]
Keywords: cyanobacteria, biotransformation, chalcones
Regardless of the way, the obtaining of pure flavonoids, either via separation from plant material, or during
chemical synthesis, such processes are known as difficult, costly, requiring often an extreme conditions of reaction, or
even toxic reagents [1]. Meanwhile, the presence of some flavonoids, as natural components of cyanobacterial (blue
green algae) cells [2], suggests the biosynthesis of this substances and thereby the existence of biochemical pathways
that enable their transformation. Cyanobacteria – one of the largest, phylogenic unique group of Gram–negative,
photosynthetic prokaryotes – seem to be specially predestined for use as effective tool in structure modification of
bioactive compounds, to improve their bioavailability and specific properties [3]. Desired features of these
”charismatic” microorganisms such as: rapid biomass increase, ease and low costs of maintaining culture, ability to
perform different modes of metabolism, formation of many secondary metabolites that possessing biological activity,
were just some of the reasons leading us to study of interactions of cyanobacteria with chalcones. Chalcones (1,3diarylprop-2-en-1-ones), are open-chain flavonoids, which molecule consists of two aromatic rings joined by threecarbon (α,β-unsaturated) carbonyl system. Many studies reveal that compounds of chalcone-based structure possess
interesting antioxidative, cytotoxic, anticancer, antimicrobial, antiprotozoal, antiulcer, antihistaminic and antiinflammatory properties. Therefore several chalcones are used for treatment of viral disorders, cardiovascular diseases,
parasitic infections, pain, gastritis, and stomach cancer, as well as food additives and cosmetic ingredients. However,
full pharmacological potency of chalcones is still not utilized [4].
The available data suggest that only heterotrophic bacteria, fungi and yeasts are able to biotransform
chalcones on the ways described as: cyclizations, hydroxylations, reductions (double bonds and ketones), O-alkylations,
dealkylations and glycosylations [5-7]. In our study, microbial transformations of chalcones were carried out using
halophilic and freshwater species of blue green algae that had not been reported before, as possessed such an ability.
The influence of tested compounds on the growth of cyanobacteria was also determined and it was observed that
chalcones acted on cyanobacteria in species-dependent manner. Chemical analysis of post-culture liquids, which were
performed by using HPLC and GC-MS, revealed the presence of newly formed metabolites, in an amount that
corresponded to disappearance of transformed maternal chalcones.
Acknowledgements
Reported study were partially supported in the frame of Grant No UMO-2011/01/B/NZ9/04722, financed by
National Science Centre (NCN) of Poland. Beata Żyszka was granted scholarships from programme “PhD scholarship –
investment in scientific staff of Opole Voivodeship” co-funded by the European Union within the European Social
Fund.
References
[1] F. Du, F. Zhang, F. Chen, A. Wang, Q. Wang, X. Yin and S. Wang, Afr. J. Microbiol. Res. 2011, 5, 2566-2574
[2] D.P. Singh, R. Prabha, K.K. Meena, L. Sharma and A.K. Sharma, AJPS. 2014, 5, 726-735
[3] A. Wang, F. Zhang, L. Huang, X. Yin, H. Li, Q. Wang, Z. Zeng and T. Xie, J. Med. Plants Res., 2010, 4, 847-856
[4] D.I. Batovska and I.T. Todorova, Curr. Clin. Pharmacol., 2010, 5, 1-29
[5] M. Sanchez-Gonzalez and J.P.N. Rosazza, J. Nat. Prod., 2004, 67, 553-558
[6] S. Das and J.P.N. Rosazza, J. Nat. Prod., 2006, 69, 499-508
[7] T. Janeczko, W. Gładkowski and E. Kostrzewa-Susłow, J. Mol. Catal. B: Enzym., 2013, 98, 55-61
75
P34
Antifungal Activity of Fluorinated Benzoxaboroles
Dorota Wieczorek1, Agnieszka Adamczyk-Woźniak2, Andrzej Sporzyński2, Jacek Lipok1
1
2
Faculty of Chemistry, Opole University, Opole, Poland – [email protected]
Faculty of Chemistry, Warsaw University of Technology , Warsaw, Poland
Keywords: boronic acids, benzoxaboroles, fungicidal activity
Boronic acids and their derivatives are group of substances, which possess wide range of applications [1]. The
possibility to use some of boronates in medicine seems to be especially interesting, since there are known examples of
boron-containing compounds which may act as a anticancer, antibacterial, antifungal or antiviral agents [2, 3].
Benzoxaboroles or internal hemiesters of ortho-hydroxymethylphenylboronic acids are the group of boronic acid
derivatives that have recently drawn much attention mainly because of their biological activity [4]. An example of
benzoxaborole, which is found to be very potent antifungal drug is 5-fluoro-1,3-dihydro-2,1-benzoxaborol-1-ol
(AN2690, Fig. 1) [5]. This compound has just received the FDA approval to be used as drug against onychomycosis.
OH
F
B
O
Fig. 1 The structure of AN2690.
The goal of our experiments was to verify activity of benzoxaboroles – isomers of AN2690 that were variously
substituted with fluorine atom – against filamentous fungi: Aspergillus niger, Aspergillus terreus, Fusarium solani,
Fusarium oxysporum, Penicillium ochrochloron, and yeast Candida tenuis. Our experiments, which have been
performed using agar diffusion method, revealed that these compounds possess antifungal activity that is dependent on
the structure of tested compound.
References
[1] A. Adamczyk-Woźniak, M. K. Cyrański, A. Żubrowska, A. Sporzyński, J. Organomet. Chem. 2009, 694, 35333541.
[2] S. J. Baker, C. Z. Ding, T. Akama, Y.-K. Zhang, V. Hernandez, Y. Xia, Future Med.Chem. 2009, 1, 1275 -1288.
[3] P. C. Trippier, C. McGuigan, J. Balzarini, Antiviral Chem. Chemother. 2010, 20, 249-257.
[4] D. Wieczorek, J. Lipok, K. M. Borys, A. Adamczyk-Woźniak, A. Sporzyński, Appl. Organometal. Chem. 2014, 28,
347-350.
[5] S. J. Baker, Y.-K. Zhang, T. Akama, A. Lau, H. Zhou, V. Hernandez, W. Mao, M. R. K. Alley, V. Sanders, J. J.
Plattner, J. Med. Chem. 2006, 49, 4447-4450.
76
P35
Biotransformation of Glyphosine as a Factor Promoting the Growth of Freshwater
Cyanobacterium Anabaena sp.
Damian Drzyzga, Ewa Kasowska-Żok, Paweł Kafarski and Jacek Lipok
Faculty of Chemistry, Opole University, Opole, Poland – [email protected]
Keywords: glyphosine, cyanobacteria, biotransformation
Glyphosine [N,N-bis(phosphonomethyl) glycine] (Fig.1.) is structural phosphonic derivative of the smallest amino acid
– glycine.
OH
HO
OH
P
O
N
O
HO
P
O
HO
Fig.1 Structure of Glyphosine.
Within the last decade a new implementation of this compound appeared, especially in medicine, where a high affinity
of N,N-bis(phosphonomethyl) glycine to the mineral bone matrix (hydroxylapatite), resulted in the use of this
phosphonate as a ligand for platinum(II). The main advantage of introducing such [(Bis(phosphonomethyl)aminoκN)acetato-κO(2-)]platinum(II) complexes to medical treatment, results from their selectivity towards bone tumor and
high activity against the human ovarian cancer cell [1]. Also the use of glyphosine in the treatment of diabetes type I,
seems to be beneficial, since this compound enhances the presentation of insulin peptide to T cells that upregulates IL10 secretion in splenocytes, and prevents diabetes in NOD mice [2].
Glyphosine belongs to the group of phosphonic compounds that often influenced the metabolism of some water
organisms. It has been proved that the presence of phosphonates could change the composition of community of
freshwater algae, affecting negatively on some phytoplankton strains [3,4]. From the other hand it has been reported
that some aquatic photoautotrophs are able to decompose phosphonates and use such substances as a sources of
nutritive phosphorus [5].
Herein, we present the results, which confirm the ability of examined cyanobacterium to biotransformation of
glyphosine in a self-beneficial manner. Our studies proved that Anabaena sp. may decompose [N,Nbis(phosphonomethyl) glycine] via decarboxylation of its molecule with releasing aminomethylphosphonic acid
(AMPA). Using analytical method basing on high performance liquid chromatography (HPLC), we were able to track
the concentration of releasing product during whole experiment. Moreover, our studies confirm that glyphosine may
serve either as additional, or as the only nutritive source of phosphorus for examined freshwater cyanobacterium.
Acknowledgements
Reported studies were funded by the National Science Centre, from grant No. UMO-2011/01/B/NZ9/04722. Damian
Drzyzga is a recipient of a Ph.D. scholarship under a project funded by the European Social Fund.
References
[1] M. Galanski, et al., J Med Chem 2003, 46(23), 4946-51.
[2] A.W. Michels, et al., J Immunol 2011, 187(11), 5921-30.
[3] S. Pesce, I. Batisson, C. Bardot, C. Fajon, C. Portelli, B. Montuelle, J. Bohatier, Ecotoxicol. Environ. Saf. 2009, 72,
1905–1912.
[4] E. Vendrell, D.G.D.B. Ferraz, C. Sabater, J.M. Carrasco, Bull. Environ. Contam. Toxicol. 2009, 82, 538–542.
[5] V. Ravi, H. Balakumar, J. Sci. Ind. Res. India 1998, 57, 790–794.
77
P36
A New and Improved Chemocatalytic and Enzymatic Approach to Unnatural
Amino Acids
Magdalena Kaik-King, Gary King
AdvaChemLab, Poznań Science and Technology Park, Poznań, Poland- [email protected]
Keywords: chemocatalysis, enzymatic reactions, unnatural amino acids
The research work carried out in the lab of AdvaChemLab has found a modified DiPAMP catalyst for the synthesis of
unnatural amino acids. The new family of analogues gives a much higher asymmetric induction and TOF compared to
the original DiPAMP catalyst. The removal of the N-acetyl amino acid protection using a chemo- approach involves
the use of strong acids at refluxing temperatures and leads to partial racemisation of the amino acid. In comparison,
enzymatic deprotection is far milder and can be performed at only slightly elevated temperatures using innocuous
reagents in water solvent. Also, the enzymatic deprotection also furnishes product of ee > 99%. But, enzymatic
deprotection of racemic substrates only give a maximum theoretical yield of 50%, hence a marrying of chemo- and an
enzymatic methodology gives high yields and high enantiomeric excess unnatural amino acids.
As an example the synthesis of unnatural (R)-3,4-dimethoxyphenyl alanine using this approach was performed on a
100 g scale. The modified DiPAMP catalyst only requires a loading of 100 000:1, compared to 20 000:1 for standard
DiPAMP-Rh catalyst. Standard DiPAMP catalyst gives an enantiomeric excess of only 92% [1]. Whereas the
AdvaChemLab modified catalyst gives an enantiomeric excess of 99%. Attempted chemo- deprotection using strong
refluxing acid leads to erosion of ee and a poor overall yield. But, N-Acetyl deprotection using D-Acylase enzyme in a
standard KH2PO4 buffer in water at 40oC gave the required free amino acid in high yield and >99% ee.
OMe
OMe
MeO
OMe
AcHN
OMe
MeO
MeO
CO2H
100000:1 loading
NaOH
MeOH, 50oC, 10bar H2
Ac2O, NaOAc, reflux
O
MeO
O
N
AcHN
CO2H
AcHN
CO2H
O
Me
modified DiPAMP cat - ee = 99%
standard DiPAMP - ee = 92%
OMe
MeO
D-Acylase
KH2PO4 buffer, pH 8.0
40oC, H2O
H2N
CO2H
ee > 99%
Fig. 1 Unnatural amino acid synthesis using a chemo- and enzymatic approach.
Acknowledgements
Financial support from the National Centre for Research and Development (grant no. HI1/2/157250, INNOTECH
programme) is gratefully acknowledged.
References
[1] W. S. Knowles, Angewandte Chemie International Edition 2002, 41, 1998.
78
P37
BioNanoPark –
Research Potential and Possibility of Cooperation with Science and Business
Partners
Krzysztof Makowski, Aleksandra Włodarczyk
Industrial Biotechnology Laboratory, Lodz Regional Science and Technology Park Ltd., Łódź, Poland –
[email protected]
BioNanoPark is established in 2012 modern research center for science and business. Unique structure of
BioNanoPark laboratories is dedicated to broadly-taken biotechnology but also biophysics, nanotechnology and some
aspects of material and biomedical engineering.
During the conference will be presented the potential of laboratories situated in BioNanoPark and possible
ways of cooperation with universities and private investors, including activities related to biocatalysis and
biotransformations.
79
80
Author Index
81
82
A
Antczak Tadeusz
Lodz University of Technology
[email protected]
G
Gilner Danuta
Silesian University of Technology
[email protected]
P32
Gliszyńska Anna
Wrocław University of Environmental
and Life Science
[email protected]
L04, P03
Glueck Sylvia
University of Graz
[email protected]
L17
Gładkowski Witold
Wrocław University of Environmental
and Life Science
[email protected]
L06, P03
P14, P15
L23, P14
Główczyk – Zubek Joanna
Warsaw University of Technology
[email protected]
Brodzka Anna
Institute of Organic Chemistry PAS
[email protected]
L19
P09, P10
Górak Monika
Wroclaw University of Technology
[email protected]
Bryjak Jolanta
Wrocław University of Technology
[email protected]
L10, L16,
L21, P21
Grudniewska Aleksandra
Wrocław University of Environmental
and Life Science
[email protected]
L05, L06
Gudiminchi Rama Krishna
Acib GmbH
[email protected]
L09
P29, P30
B
Balcerzak Lucyna
Wrocław University of Technology
[email protected]
P20
Bandurska Katarzyna
Jan Dlugosz University in
Częstochowa
[email protected]
P06
Borowiecki Paweł
Warsaw University of Technology
[email protected]
L23
Bretner Maria
Warsaw University of Technology
[email protected]
C
Cieńska Małgorzata
Wrocław University of Technology
[email protected]
Cieśla Joanna
Warsaw University of Technology
[email protected]
L10
H
Hrydziuszko Zofia
Wrocław University of Technology
[email protected]
P16
I
D
Drzyzga Damian
Opole University
[email protected]
Dudzik Agnieszka
Jerzy Haber Institute of Catalysis and
Surface Chemistry PAS
[email protected]
F
Faber Kurt
Acib GmbH
University of Graz
[email protected]
Fryszkowska Anna
Dr. Reddy’s Laboratories Ltd
[email protected]
L16
Igielska – Kalwat Joanna
Adam Mickiewicz University in Poznań
[email protected]
P35
P25
J
L24, P28
L02, L17
Jarzębski Andrzej
Silesian University of Technology
[email protected]
L22, P31, P32
Jaśkowska Jolanta
Cracow University of Technology
[email protected]
P07
K
L03
83
Kafarski Paweł
Wroclaw University of Technology
[email protected]
L01, P35
Kaik-King Magdalena
AdvaChemLab
[email protected]
P36
Kaki Shiva Shanker
Indian Institute of Chemical
Technology
[email protected]
P01
Kapuścińska Alicja
Adam Mickiewicz University in
Poznań,
[email protected]
P22
Kasowska-Żok Ewa
Opole University
[email protected]
P35
Kezwoń Aleksandra
Warsaw University of Technology
[email protected]
P02
N
Naghi Mara Ana
Babes-Bolyai University
[email protected]
L15
O
Kiełbasiński Piotr
Centre of Molecular and
Macromolecular Studies PAS
[email protected]
L12
King Gary
AdvaChemLab
P36
Kłossowski Szymon
Institute of Organic Chemistry PAS
[email protected]
L08, P12
Kmiecik Natalia
Wroclaw University of Technology
[email protected]
L11
Koszelewski Dominik
Institute of Organic Chemistry PAS
[email protected]
P11
Olejnik Anna
Adam Mickiewicz University in Poznań
[email protected]
P24
Ostaszewski Ryszard
Institute of Organic Chemistry PAS
[email protected]
L08, L13,
L18, P09,
P10, P11, P12
P
Pakulski Zbigniew
Institute of Organic Chemistry PAS
[email protected]
P08
Paprocki Daniel
Institute of Organic Chemistry PAS
[email protected]
L23, P12
Popłoński Jarosław
Wrocław University of Environmental
and Life Science
[email protected]
L20
R
L
Radu Alexandra
Babeș-Bolyai University
[email protected]
P04
Ramakanth Illa
Nanotechnology Centre VSB-Technical
University of Ostrava
[email protected]
L14
Lewańczuk Marcin
Wroclaw University of Technology
[email protected]
P21
Rapp Magdalena
Adam Mickiewicz University in Poznań
[email protected]
P27
Lochyński Stanisław
Wroclaw University of Technology
[email protected]
P19, P20
Rugor Agnieszka
Jerzy Haber Institute of Catalysis and
Surface Chemistry PAS
[email protected]
L24
Ł
Łukowska – Chojnacka Edyta
Warsaw University of Technology
[email protected]
S
Saygili Nezire
Hacettepe University, Faculty of
Pharmacy
[email protected]
P15
M
P05
Majewska Paulina
Wroclaw University of Technology
[email protected]
P18
Servi Stefano
The Protein Factory
[email protected]
L07
Makowski Krzysztof
Lodz Regional Science and Technology
Park Ltd.
[email protected]
P37
Sidoryk Kararzyna
Institute of Organic Chemistry PAS
[email protected]
P08
Mierzejewska Jolanta
Warsaw University of Technology
[email protected]
P17
Stańczyk Łukasz
Lodz University of Technology
[email protected]
P29, P30
84
Staśko Marcin
Institute of Organic Chemistry PAS
[email protected]
P13
Wiśniewska Catalina
Institute of Organic Chemistry PAS
[email protected]
Strub Daniel
Wroclaw University of Technology
[email protected]
P19
Włodarczyk Aleksandra
Lodz Regional Science and Technology
Park Ltd.
[email protected]
Struszczyk-Świta Katarzyna
Lodz University of Technology
[email protected]
P29, P30
Z
Szaleniec Maciej
Jerzy Haber Institute of Catalysis and
Surface Chemistry PAS
[email protected]
L21, L24, P28
Szczęsna-Antczak Mirosława
Lodz University of Technology
[email protected]
P29, P30
Szymańska Katarzyna
Silesian University of Technology
[email protected]
L16, L22,
P31, P32
L04, L05,
L06, P03
Wawrzyńczak Agata
Adam Mickiewicz University in Poznań
[email protected]
P26
Wieczorek Dorota
Opole University
[email protected]
P34
Wielechowska Monika
Warsaw University of Technology
[email protected]
P14, P15
P37
Zielińska Aleksandra
Adam Mickiewicz University in Poznań
[email protected]
P23
Zysk Małgorzata
Institute of Organic Chemistry PAS
[email protected]
P09, P10
Ż
W
Wawrzeńczyk Czesław
Wrocław University of Environmental
and Life Science
[email protected]
L18
85
Żądło Anna
Institute of Organic Chemistry PAS
[email protected]
L13, P11
Żyła Adriana
Opole University
[email protected]
P13
Żymańczyk – Duda Ewa
Wrocław University of Technology
[email protected]
L11, L19
Żyszka Beata
Opole University
[email protected]
P33
86
List of Participants
87
88
A
F
Antczak Tadeusz
Lodz University of Technology
[email protected]
Faber Kurt
Acib GmbH, University of Graz
[email protected]
B
Fryszkowska Anna
Dr. Reddy’s Laboratories Ltd
[email protected]
Balcerzak Lucyna
Wrocław University of Technology
[email protected]
G
Bandurska Katarzyna
Jan Dlugosz University in Częstochowa
[email protected]
Gilner Danuta
Silesian University of Technology
[email protected]
Borowiecki Paweł
Warsaw University of Technology
[email protected]
Gliszyńska Anna
Wrocław University of Environmental and Life
Science
[email protected]
Bretner Maria
Warsaw University of Technology
[email protected]
Glueck Sylvia
University of Graz
[email protected]
Brodzka Anna
Institute of Organic Chemistry PAS
[email protected]
Gładkowski Witold
Wrocław University of Environmental and Life
Science
[email protected]
Bryjak Jolanta
Wrocław University of Technology
[email protected]
C
Główczyk – Zubek Joanna
Warsaw University of Technology
[email protected]
Cieńska Małgorzata
Wrocław University of Technology
[email protected]
Górak Monika
Wroclaw University of Technology
[email protected]
Cieśla Joanna
Warsaw University of Technology
[email protected]
Grudniewska Aleksandra
Wrocław University of Environmental and Life
Science
[email protected]
Czajkowska-Wojciechowska Ewa
Adamed Sp. Z o.o.
[email protected]
Gudiminchi Rama Krishna
Acib GmbH
[email protected]
D
H
Drzyzga Damian
Opole University
[email protected]
Hitce Julien
L’Oreal
[email protected]
Dudzik Agnieszka
Jerzy Haber Institute of Catalysis and Surface
Chemistry PAS
[email protected]
Hrydziuszko Zofia
Wrocław University of Technology
[email protected]
89
Koszelewski Dominik
Institute of Organic Chemistry PAS
[email protected]
I
Igielska – Kalwat Joanna
Adam Mickiewicz University in Poznań
[email protected]
L
Lewańczuk Marcin
Wroclaw University of Technology
[email protected]
J
Jarzębski Andrzej
Silesian University of Technology
[email protected]
Lochyński Stanisław
Wroclaw University of Technology, Faculty of
Chemistry
[email protected]
Jaśkowska Jolanta
Cracow University of Technology
[email protected]
Ł
K
Łukowska – Chojnacka Edyta
Warsaw University of Technology
[email protected]
Kafarski Paweł
Wroclaw University of Technology
[email protected]
M
Kaik-King Magdalena
AdvaChemLab, Poznań Science and Technology Park
[email protected]
Majdak Dagmara
Wroclaw University of Technology
[email protected]
Kaki Shiva Shanker
Indian Institute of Chemical Technology
[email protected]
Majewska Paulina
Wroclaw University of Technology
[email protected]
Kapuścińska Alicja
Adam Mickiewicz University in Poznań
[email protected]
Makowski Krzysztof
Lodz Regional Science and Technology Park Ltd.
[email protected]
Kasowska-Żok Ewa
Opole University
[email protected]
Mierzejewska Jolanta
Warsaw University of Technology
[email protected]
Kezwoń Aleksandra
Warsaw University of Technology
[email protected]
N
Naghi Mara Ana
Babes-Bolyai University
[email protected]
Kiełbasiński Piotr
Centre of Molecular and Macromolecular Studies PAS
[email protected]
O
King Gary
AdvaChemLab, Poznań Science and Technology Park
Olejnik Anna
Adam Mickiewicz University in Poznań
[email protected]
Kłossowski Szymon
Institute of Organic Chemistry PAS
[email protected]
Ostaszewski Ryszard
Institute of Organic Chemistry PAS
[email protected]
Kmiecik Natalia
Wroclaw University of Technology
[email protected]
90
Strub Daniel
Wroclaw University of Technology
[email protected]
P
Pakulski Zbigniew
Institute of Organic Chemistry PAS
[email protected]
Struszczyk-Świta Katarzyna
Lodz University of Technology
[email protected]
Paprocki Daniel
Institute of Organic Chemistry PAS
[email protected]
Szaleniec Maciej
Jerzy Haber Institute of Catalysis and Surface
Chemistry PAS
[email protected]
Popłoński Jarosław
Wrocław University of Environmental and Life
Science
[email protected]
Szczęsna-Antczak Mirosława
Lodz University of Technology
[email protected]
R
Szymańska Katarzyna
Silesian University of Technology
[email protected]
Radu Alexandra
Babeș-Bolyai University
[email protected]
U
Ramakanth Illa
Nanotechnology Centre VSB-Technical University of
Ostrava
[email protected]
Ufnal Marcin
The Medical University of Warsaw
[email protected]
W
Rapp Magdalena
Adam Mickiewicz University in Poznań
[email protected]
Wawrzeńczyk Czesław
Wrocław University of Environmental and Life
Science
[email protected]
Rugor Agnieszka
Jerzy Haber Institute of Catalysis and Surface
Chemistry PAS
[email protected]
S
Wawrzyńczak Agata
Adam Mickiewicz University in Poznan
[email protected]
Saygili Nezire
Hacettepe University
[email protected]
Wieczorek Dorota
Opole University
[email protected]
Servi Stefano
The Protein Factory
[email protected]
Wielechowska Monika
Warsaw University of Technology
[email protected]
Sidoryk Kararzyna
Institute of Organic Chemistry PAS
[email protected]
Wiśniewska Catalina
Institute of Organic Chemistry PAS
[email protected]
Stańczyk Łukasz
Lodz University of Technology
[email protected]
Włodarczyk Aleksandra
Lodz Regional Science and Technology Park Ltd.
[email protected]
Staśko Marcin
Institute of Organic Chemistry PAS
[email protected]
91
Żyła Adriana
Opole University
[email protected]
Z
Zielińska Aleksandra
Adam Mickiewicz University in Poznań
[email protected]
Żymańczyk – Duda Ewa
Wrocław University of Technology
[email protected]
Zysk Małgorzata
Institute of Organic Chemistry PAS
[email protected]
Żyszka Beata
Opole University
[email protected]
Ż
Żądło Anna
Institute of Organic Chemistry PAS
[email protected]
92