3rd Symposium on Biotransformations for

Pharmaceutical and Cosmetic Industry

The Conference is dedicated to Prof. Janusz Jurczak on

the occasion of his 75th birthday.

Programme and Abstracts

June, 30th – July, 2nd 2016

Warsaw, Poland

Institute of Organic Chemistry

Polish Academy of Sciences

Warsaw 2016

2

Programme and Abstracts

3rd Symposium on Biotransformations for Pharmaceutical and Cosmetic Industry

Cover design:

Group XX ICHO PAN

Editors:

Anna Brodzka

Dominik Koszelewski

Copyright © by Institute of Organic Chemistry,

Polish Academy of Sciences

ISBN: 978-83-940417-2-4

Warsaw 2016

3

Committees

Organizing Committee

Ryszard Ostaszewski – Head of Organizing Committee

Dominik Koszelewski - Secretary

Filip Borys

Anna Brodzka

Rafał Kopiasz

Arleta Madej

Daniel Paprocki

Małgorzata Zysk

Anna Żądło

Damian Trzepizur

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Sponsors

The Organizing Committee gratefully acknowledges the sponsorship of the Symposium

by following companies and organizations:

5

as Supporting Scientific Organization:

6

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7

Conference venue

The 3rd 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

METRO – Rondo Daszyńskiego Station

BUS STOP – Szpital Wolski

105, 109, 155, 178 - from Rondo Daszyńskiego Metro Station

109 – from Central Railway Station

BUS STOP – Krzyżanowskiego

103 – from West Railway Station

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General Information

Registration Desk, Institute of Organic Chemistry Polish Academy of Sciences

Opening hours:

Thursday, 30rd of June: 10.00am –5.00pm

Friday, 1st of July: 9:00am –3:00pm

Please register upon arrival in order to receive your congress materials and personal name

badge.

Conference Dinner: Friday, 30th of June, 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 1st of July 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.

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Programme

10

11

The Conference is dedicated to Prof. Janusz Jurczak on the occasion of his

75th birthday.

Thursday, June 30th, 2016

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)

14:10 – 14:30 The concert of Young Virtuosos (The Polish Children’s Fund)

14:30 – 15:15 Plenary Lecture Prof. Nicolas J. Turner (University of Manchester, UK) Biocatalytic Cascade Reactions Enabled by Synthetic Biology

15:15 – 15:45 Invited Lecture Prof. Francesco Mutti (University of Amsterdam, Nederlands) Biocatalytic Asymmetric Hydrogen-Borrowing Cascades

15:45 – 16:00 Oral Communication Marta Bauer (Tech-Lab.pl, Gdańsk) LP-Control, an 8-Button Idea to Work in the Laboratory

16:00 – 16:20 Coffee break

16:20 – 17:05 Plenary Lecture Prof. Wolfgang Kroutil (University of Graz, Austria) Redox Enzymes in System Catalysis and for Novel Reactions

17:05 – 17:35

Invited Lecture Dr. Paweł Borowiecki (Warsaw University of Technology, Poland) A Tribute to Professor Ernest Alexander Sym (1893–1950). Does Biocatalysis Has Polish Roots?

17:35 – 19:00 Poster Session

12

Friday, July 1st, 2016

9:00 – 9:45 Plenary Lecture Prof. Kurt Faber (University of Graz, Austria) Biocatalytic Cascades for Organic Synthesis

9:45 – 10:15 Invited Lecture Dr. Betina Nestl (Institut für Technische Biochemie, Germany) Imine Reductases: Biochemistry, Engineering and Synthetic Potential

10:15 – 10:30

Oral Communication Dr. Anna Jarosz - Wilkołazka (Maria Curie-Sklodowska University) Dyeing Properties and Biological Activity of Colour Compounds Synthesised by Fungal Laccase

10:30 – 10:45

Oral Communication Ignacy Janicki (Centre of Molecular and Macromolecular Studies PAS) Asymmetric Bioreduction of α,β-Unsaturated Phosphonates Using Ene-Reductases

10:45 – 11:05 Coffee break

11:05 – 11:50 Invited Lecture Dr. Roland Wohlgemuth (Sigma Aldrich, Buchs, Switzerland) Biocatalytic Synthesis of Metabolites

11:50 – 12:20

Invited Lecture Prof. Andrzej Jarzębski (Silesian University of Technology, Poland) Multichannel Monolithic Enzymatic Microreactors- Paving the Way to New Robust Technologies

12:20 – 12:35

Oral Communication Agata Zdarta (Poznan University of Technology) Modification of Bacterial Cell Surface Properties and Oxygenase Activity During Hydrocarbons Biodegradation

12:35 – 12:50

Oral Communication Wojciech Smułek (Poznan University of Technology) Surfactant-Enhanced Biodegradation of Halogenated Compounds by Activated Sludge Microorganisms

12:50 – 14:00 Lunch

14:00 – 14:45

Plenary Lecture Prof. Toshiyuki Itoh (Tottori University, Japan) Ionic Liquid Mediated Activation of a Lipase for Sustainable Preparation of Chiral Secondary Alcohols

14:45 – 15:15

Invited Lecture Prof. Iván Lavandera García (University of Oviedo, Spain) Promiscuous Hydrodehalogenation Activity Can Expand (Even More) the Applicability of Transaminases

15:15 – 15:30 Oral Communication Anna Taraba (Maria Curie-Skłodowska University in Lublin) The Influence of Solvent on the Properties of Quercetin and Rutin

13

15:30 – 15:50 Coffee break

15:50 – 16:20 Invited Lecture Dr. Manuel Perez (Mestrelab Research, Spain) Reaction Monitoring by NMR: an Overview

16:20 – 16:50

Invited Lecture Prof. Laszlo Poppe (Budapest University of Technology and Economics) Biotransformations with Phenylalanine Ammonia Lyase - Mechanism and Process Intensification

16:50 – 17:05 Oral Communication Mădălina Elena Moisă (Babeş-Bolyai University) Click Reactions for Efficient Enzymatic Kinetic Resolution Processing

17:05 – 17:20 Oral Communication Agata Głąb (Wroclaw University of Science and Technology) Cyanobacteria as Biotransformants in Redox Reactions

17:20 – 18:30 Free time

18:30 – 22:00 Conference dinner

14

Saturday, July 2nd, 2016

9:00 – 9:45

Plenary Lecture Prof. Peter Walde (ETH Zürich, Switzerland) Dendronized Polymer-Enzyme Conjugates.− Preparation and Applications for the Immobilization of Enzymes on Silicate Surfaces

9:45 – 10:15 Invited Communication Prof. Leandro Helgueira Andrade (University of São Paulo, Brazil) Enzymatic Reactions of Heteroatom-containing Compounds

10:15 – 10:30 Oral Communication Dr. Danuta Gilner (Silesian University Of Technology) Chemoenzymatic transformation of lignocellulose

10:30 – 10:45

Oral Communication Dorota Domaradzka (University of Silesia in Katowice) Biotransformation of Naproxen in the Presence of Selected Aromatic Compounds and Organic Solvents by Bacillus sp. B1(2015b)

10:45 – 11:10 Coffee break

11:10 – 11:40

Invited Lecture Prof. Maciej Szaleniec (Institute of Catalysis and Surface Chemistry PAS, Poland) Steroid C25 Dehydrogenase, the Biocatalyst for Production of Calcifediol and 25-Hydroxycholesterol

11:40 – 11:55

Oral Communication Krzysztof Żukowski (Adam Mickiewicz University in Poznan) Peroxidase Activity and Physicochemical Characterization of DNAzymes Created by G-quadruplex with Covalently Attached Hemin

11:55 – 12:10

Oral Communication Paulina Majewska (Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wrocław, Poland) Biotransformations of hydroxyphosphonoacetic acid derivatives by bacteria with lipolytic activity

12:10 – 12:25

Oral Communication Dr. Małgorzata Brzezińska-Rodak (Wroclaw Univeristy of Technology) Biocatalytic Modifications of Resveratrol

12:25 – 12:40 Oral Communication Natalia Kwiatos (Lodz Univeristy of Technology) Biosolubilisation of Brown Coal by Recombinant Laccase

12:40 – 12:55 Oral Communication Beata Szmigiel (Wroclaw Univeristy of Technology) Biocatalyzed Synthesis of Tyrosol Derivatives

12:55 – 13:10 Closing remarks

15

Lectures

16

17

L01

Biocatalytic Cascade Reactions Enabled by Synthetic Biology

Nicholas J. Turner School of Chemistry & MIB, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK

Keywords: Cascade reactions, chiral amines, synthetic biology

This lecture will describe recent work from our laboratory aimed at developing new biocatalysts for enantioselective organic synthesis, with a particular emphasis on cascade processes for generating pharmaceutical building blocks. By applying the principles of ‘biocatalytic retrosynthesis’ we have shown that is now increasingly possible to design new synthetic routes to target molecules in which biocatalysts are used in the key bond forming steps.

1

The integration of several biocatalytic transformations into multi-enzyme cascade systems, both in vitro and in vivo, will be addressed in the lecture. In this context MAO-N has been used in combination with other biocatalysts and chemocatalysts in order to complete a cascade of enzymatic reactions.

2-4 Other engineered biocatalysts that

can be used in the context of cascade reactions include -transaminases,5 phenylalanine ammonia lyases,

6

amine dehydrogenases,7 imine reductases

8 and artificial enzymes.

9 We shall also present some very recent work

aimed at the development of a new biocatalyst for enantioselective reductive amination including via hydrogen borrowing.

Acknowledgements NJT thanks all members of his group for their outstanding contributions.

References

[1] N.J. Turner and E. O’Reilly, Nature Chem. Biol., 2013, 9, 285-288.

[2] D. Ghislieri et al., J. Am. Chem. Soc., 2013, 135, 10863-10869. [3] J.H. Schrittwieser et al,, Angew. Chem. Int. Ed., 2014, 53, 3731-3734. [4] N.J. Turner et al., Angew. Chem. Int. Ed., 2014, 53, 2447-2450. [5] A. Green et al., Angew. Chem. Int. Ed., 2014, 53, 10714-10717; P. Both, H. Busch, P.P. Kelly, F.G. Mutti, N.J.

Turner and S.L. Flitsch, Angew. Chem. Int. Ed., 2016, 55, 1511-1513. [6] S.L. Lovelock et al., Angew. Chem. Int. Ed., 2014, 53, 4652-4656; F. Parmeggiani, S.L. Lovelock, N.J. Weise,

S.T. Ahmed and N.J. Turner, Angew. Chem. Int. Ed., 2015, 54, 4608–4611; N.J. Weise, F. Parmeggiani, S.T. Ahmed and N.J. Turner, J. Am. Chem. Soc., 2015, 137, 12977-12983.

[7] F.G. Mutti, T. Knaus, N.S. Scrutton, M. Breuer and N.J. Turner, Science, 2015, 349, 1525-1529.

[8] R.S. Heath, M. Pontini, S. Hussain and N.J. Turner, ChemCatChem, 2016, 8, 117-120. [9] V. Koehler et al., Nature Chem., 2013, 5, 93-99.

18

L02

Biocatalytic Asymmetric Hydrogen-Borrowing Cascades Francesco G. Mutti

1, Tanja Knaus

1, Nicholas J. Turner

2, Nigel S. Scrutton

2, Michael Breuer

3 and Luke

D. Humphreys4

1 Van’t Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The

Netherlands – f.mutti@uva.nl 2 Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, M1 7DN, Manchester, UK.

3 BASF SE, White Biotechnology Research, GBW/B-A030, 67056 Ludwigshafen, Germany.

4 GSK Medicines Research Centre, Gunnel’s Wood Road, Stevenage, Herts, SG1 2NY, UK

Keywords: alcohol amination, α-substituted chiral carboxylic acids, hydrogen-borrowing cascades.

Multi-step chemical reactions in one pot avoid the need for isolation of intermediates and purification steps. In

this context, hydrogen-borrowing processes possess the most elevated atom economy as the electrons liberated in the oxidative step are quantitatively utilized in the interconnected reductive step. As a consequence, stoichiometric amounts of sacrificial cosubstrates for the regeneration of the nicotinamide coenzymes are not required. We have successfully developed this concept for two different biocatalytic processes. In the first study, we investigated the biocatalytic hydrogen-borrowing amination of primary and secondary alcohols (Scheme 1a) because amines constitute the major targets or intermediates for the production of active pharmaceutical ingredients, fine chemicals, agrochemicals, but also polymers, dyes, plasticizing agents, etc.[1] Our method relies on the tandem combination of alcohol dehydrogenases (ADHs) with amine dehydrogenases (AmDHs) to carry out the amination of structurally diverse aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess (R). Furthermore, primary alcohols were also aminated with up to 99% conversion. This redox self-sufficient cascade requires ammonium as a simple and inexpensive source of nitrogen and generates water as the sole by-product. In the second concomitant study, the biocatalytic hydrogen-borrowing concept was applied to the synthesis of α-chiral substituted carboxylic acids from α,β-unsaturated aldehydes via the combination of ene-reductases (ERs) with aldehyde dehydrogenases (AldDHs), (Scheme 1b).[2] Initially, we focused on the biocatalytic synthesis of chiral α-substituted hydrocinnamic acids and acetylated unnatural amino acids since these compounds are important targets for pharmaceuticals. Later on, the applicability of the process was also validated for the synthesis of α-chiral aliphatic acids and heterocycles. The conversion of the α,β-unsaturated aldehydes to the related α-chiral substituted carboxylic acids proceeded generally with elevated yield as well as chemo- and stereo-selectivity. This strategy has several advantages: i) the starting material is an α-substituted α,β-unsaturated aldehyde, a class of compounds extremely reactive for the biocatalytic C=C double bond reduction, whereas α,β-unsaturated carboxylic acids are completely unreactive; ii) no external hydride source from a sacrificial substrate (e.g. glucose, formate) is required; iii) no additional reagent is required; iv) the only by-product is water.

Fig. 1 Biocatalytic asymmetric hydrogen-borrowing cascades.

Acknowledgements

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020

research and innovation programme (grant agreement No 638271, BioSusAmin). The research leading to these results received funding from the European Union’s Seventh Framework Programme FP7/2007-2013 under the grant agreement no. 266025 (BIONEXGEN). The work was funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC; BB/K0017802/1).

References

[1] F.G. Mutti, T. Knaus, N.S. Scrutton, M. Breuer, N.J. Turner, Science, 2015, 359, 1525-1529. [2] T. Knaus, F.G. Mutti, L.D. Humphreys, N.J. Turner, N.S. Scrutton, Org. Biomol. Chem., 2015, 13, 223-233

19

L03

LP-Control, an 8-Button Idea to Work in the Laboratory

Bauer M., Kamysz W. Tech-Lab.pl, ul. Bysewska 26B, 80-298 Gdansk, tech-lab@lipopharm.pl

Keywords: peristaltic pumps, software, control

Modern laboratory equipment is becoming more sophisticated and thus difficult to use. As the result, new devices as well as software are becoming more complicated and their handling restricts the work productivity in many research laboratories. First of all, a scientist should think about designing an experiment rather than about turning to the unit needed. Sometimes making immediate decisions, such as switching the flow of the liquid, are needed. There is also a need to record the temperature, humidity, and the pH during the course of the process in a straightforward way. Sometimes simultaneous control of several pumps (the dosing, HPLC and syringe ones) is also required. The purpose of the presentation is to demonstrate possible applications of an LP-control software in everyday laboratory work. The program was designed by a scientist with a 20-years experience who aspired to design a relatively simple but effective tool allowing to focus on a reliable experiment rather than on turning on and controlling the testied apparatus. For instance, the LP-control software allows to simultaneous controling different types of pumps and to manage various methods for selection of parameters with dedicated sensors. In the main window of the software there are 8 buttons that control the vital functions. More complex options are positioned within the software in an interesting way, namely the LP-control can additionally be installed on tablet devices allowing to operate by touch screen. Some examples of specific applications of the LP-control software in a typical laboratory work will be demonstrated.

20

L04

Redox Enzymes in System Catalysis and for Novel Reactions Eduardo Busto

1, Stefan E. Payer

1, Robert S. Simon

1, Judith Farnberger

1, Somayyeh Gandomkar

1,

Jörg H. Schrittwieser1, Wolfgang Kroutil

1

1 University of Graz, Institute of Chemistry, NAWI Graz, BioTechMed, Graz, Austria – Wolfgang.Kroutil@uni-graz.at

Keywords: cascades, enantioselective N-dealkylation, laccases

Enzyme cascades, thus the combination of several biocatalytic steps in one pot either performed in a simultaneous or in a stepwise fashion circumvent time and resources consuming work-up procedures for intermediates. Furthermore, by this approach labile intermediates are consumed quickly minimizing their degradation. Designing artificial cascades allows then to perform in one pot reactions which are otherwise not feasible by a single enzyme. For instance, the para-vinylation of phenols is only possible by an enzymatic cascade [1]. In a similar fashion phenols were coupled to pyruvate to give enantiomerically pure (R)- or (S)-3-(para-hydroxyphenyl) lactic acids (Fig. 1) [2].

Enzymes are known not only to catalyze their natural reaction but also to catalyze related or different reactions; this is also referred to as reaction promiscuity beside the general substrate promiscuity [3]. In the talk various promiscuous activities will be discussed.

For instance the flavin dependent berberine bridge enzyme (BBE) is known in literature to form a C-C bond between an N-CH3 group and phenol at the expense of molecular oxygen [4]. However, for selected substrates bearing an N-ethyl group instead of the N-methyl group, dealkylation was found. This observation allowed exploiting the BBE for the first enatioselective dealkylation applied for organic synthesis [5].

Another example deals with laccases, which are very well known enzymes applied for instance for di- and polymerization of phenols or dye degradation at the expense of molecular oxygen [6]. By designing a cooperating reaction system combining a chemical reaction with the laccase catalyzed radical formation the first biocatalytic trifluoromethylation was achieved. Trifluoromethylation is a broadly investigated reaction in organic synthesis.

Fig. 1 Examples for discussed cascades: (A) Vinylation of phenols and (B) coupling of phenols to pyruvate to give

para-hydroxy lactic acid derivatives.

Acknowledgements

Various funding sources are acknowledged: COST Action CM1303 “Systems Biocatalysis”; Austrian Science

Fund (FWF, project W9, DK Molecular Enzymology). European Commission/Marie Curie Actions-Intra-European Fellowship (IEF) in the project "BIOCASCADE" (FP7-PEOPLE-2011-IEF). Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT through the Austrian FFG-COMET-Funding Program.

References

[1] E. Busto, R. C. Simon, and W. Kroutil, Angew. Chem. Int. Ed. 2015, 54, 10899. [2] E. Busto, R. C. Simon, N. Richter, and W. Kroutil, ACS Catal. 2016, 6, 2393. [3] a) X. Garrabou, T. Beck, and D. M. Hilvert, Angew. Chem. Int. Ed. 2015, 54, 5609; b) K. Hult, and P. Berglund,

Trends Biotechnol. 2007, 25, 231; c) U. T. Bornscheuer, and R. J. Kazlauskas, Angew. Chem. Int. Ed. 2004, 43, 6032.

[4] J. H. Schrittwieser, V. Resch, J. H. Sattler, W. D. Lienhart, K. Durchschein, A. Winkler, K. Gruber, P. Macheroux, and W. Kroutil, Angew. Chem. Int. Ed. 2011, 50, 1068.

[5] S. Gandomkar, E.-M. Fischereder, J. H. Schrittwieser, S. Wallner, Z. Habibi, P. Macheroux, and W. Kroutil, Angew. Chem. Int. Ed. 2015, 54, 15051.

[6] a) S. Riva, Trends Biotechnol. 2006, 24, 219; b) M. Mogharabi, and M. A. Faramarazi, Adv. Synth. Catal. 2014, 356, 897.

21

L05

A Tribute to Professor Ernest Alexander Sym (1893–1950). Does Biocatalysis Has Polish Roots?

Paweł Borowiecki* Warsaw University of Technology, Faculty of Chemistry, Institute of Biotechnology, Koszykowa St. 3, 00-664 Warsaw.

Keywords: Ernest A. Sym, Lipases, Applied Enzymology, Biocatalysis

Hydrolases (i.e. lipases, esterases etc.) are enzymes, which has received great attention these days. They

are the most versatile and practical biocatalysts used in organic chemistry with the greatest importance in asymmetric synthesis mainly due to their selectivity in action, high reaction rates, wide substrate specificity, vast commercial availability in both free and immobilized forms, low price, and easy handling. Especially lipases exhibit strong compatibility with many types of functional groups present in the transformed substrates, and tolerate high levels of stereochemical complexity in chiral molecules. Moreover, lipases do not need expensive external acceptors and cofactors [i.e. NAD(P)H, FAD etc.], and what is even more important, they are able to catalyze reactions under non-physiological conditions in non-aqueous media, including neat organic solvents, neoteric solvents (supercritical fluids, ionic liquids, fluorous solvents, liquid polymers etc.), deep eutectic solvents (DESs) as well as in solvent-free systems. In this context it should be emphasized that it was Polish biochemist-enzymologist Ernest Alexander Sym (1893–1950) who reported for the first time in a series of papers published in 1930–1936 in Biochemistry Journal and Biochemische Zeitschrift the breakthrough discovery that lipases retain almost full catalytic activity even in nearly anhydrous organic media [1].

Unfortunately, it is now largely forgotten that this basic knowledge had undoubtedly the biggest impact on the development of global lipase-mediated biotransformations since it revealed that lipases can catalyse reactions impossible to carried out in water thereby increasing the accessibility of those enzymes toward transformations of water-insoluble hydrophobic compounds and thus enabling their new applications in synthesis. In addition, this discovery was also crucial as it later turned out that enzymes' stability, activity and selectivity in organic non-polar solvents are much higher than in water. In turn, the reactions performed in aqueous solution are rather thermodynamically impractical, often lead to unwanted by-products, degrade common organic reagents, hinder efficient resolution of enantiomers by promoting racemization as well as water itself is difficult to remove and thus hampers product recovery. For unknown reasons, world scientific community have not noticed Sym’s papers, and the discovery of so-called “non-aqueous enzymology” is attributed to Russian chemist Alexander M. Klibanov who fifty years later in 1985 described a lipase-catalyzed reaction carried out in organic solvents as the reaction media [2].

The author of this lecture wish to report on truly tremendous role of Ernest Aleksander Sym achievements in the field of enzyme catalysis, and give an overview of few spectacular examples of lipases’ applications in organic synthesis with a focus on the preparation of enantiomerically pure active pharmaceutical ingredients (APIs) during the last few years.

Acknowledgements

This lecture is dedicated to Prof. Ernest Alexander Sym to acknowledge his contribution to applied

enzymology, and to Prof. Jan Plenkiewicz on the occasion of his 80th

birthday as well as in honor of his scientific achievements within academic career, in particular the use of lipases as catalyst in organic chemistry. Author wish to thank Prof. Ryszard Ostaszewski for courteous invitation to IIIrd Symposium on Biotransformations for pharmaceutical and cosmetic industry.

References

[1] (a) E.A. Sym, Biochem. J. 1936, 30, 609–617; (b) E.A. Sym, Enzymologia 1936, 1, 156–160; (c) E.A. Sym, Biochem. Z. 1933, 258, 304–324; (d) E.A. Sym, Biochem. Z. 1931, 230, 19–50; (e) E.A. Sym, Biochem. J. 1930, 24, 1265–1281. [2] A. Zaks, A.M. Klibanov, Proc. Natl. Acad. Sci. 1985, 82, 3192–3196.

22

L06

Biocatalytic Cascades for Organic Synthesis E. Busto, A. Dennig, M. Fuchs, S. M. Glueck, M. Hall, W. Kroutil, M. Pickl, J. Pitzer, E. Tassano, K. Faber Department of Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria <Kurt.Faber@Uni-Graz.at>.

Keywords: biocatalytic Cannizzaro, alcohol amination, bio-Mitsunobu, non-canonical amino acid.

The unique compatibility of enzymes with each other allows their combination in a single reactor for the

design of cascade-reactions. By this strategy, tedious isolation and purification of intermediates is omitted and decomposition of sensitive compounds is largely reduced. This strength of this methodology is demonstrated by the following examples:

(i) Redox-neutral disproportionation of racemic -substituted aldehydes by alcohol dehydrogenases in presence of cat. NADH yields equimolar amounts of enantioenriched carboxylic acids and prim-alcohols. This process represents a biocatalytic equivalent to the Cannizzaro-reaction [1].

(ii) One-pot amination of alcohols via ADH-catalysed dehydrogenation yields aldehydes, which are immediately subjected to reductive amination using -transaminases to give prim-amines in a redox-neutral hydrogen-borrowing cascade [2].

(iii) Combination of a retaining and an inverting alkyl sulfatase allows the deracemization of sec-alcohols via biocatalytic hydrolysis of their corresponding racemic sulfate esters. This process constitutes an enantioconvergent parallel cascade and resembles a biocatalytic equivalent to the Mitsunobu-inversion [3].

(iv) Simple aromatics, such as toluene, can be converted to L-Tyr-derivatives in a one-pot process which proceeds via mono-oxygenase catalysed regioselective o-hydroxylation followed by asymmetric C-C coupling with ammonium pyruvate using Tyr-ammonia lyase [4].

Fig. 1 Biocatalytic cascades to carboxylic acids, prim- and sec-alcohols, amines and non-canonical amino acids.

Acknowledgements

Funding by the FWF, BMWFW, BMVIT, SFG, Standortagentur Tirol, Government of Lower Austria and ZIT through the Austrian FFG-COMET-Funding Program is gratefully acknowledged.

References

[1] C. Wuensch, H. Lechner, S. M. Glueck, K. Zangger, M. Hall and K. Faber, ChemCatChem, 2013, 5, 1744. [2] (a) J. H. Sattler, M. Fuchs, K. Tauber, F. G. Mutti, K. Faber, J. Pfeffer, T. Haas and W. Kroutil, Angew. Chem. Int. Ed. 2012, 51, 9156; (b) M. Pickl, M. Fuchs, S. M. Glueck and K. Faber, ChemCatChem 2015, 7, 3132. [3] M. Schober, M. Toesch, T. Knaus, G. A. Strohmeier, B. van Loo, M. Fuchs, F. Hollfelder, P. Macheroux and K. Faber, Angew. Chem. Int. Ed., 2013, 52, 3277. [4] A. Dennig, E. Busto, W. Kroutil and K. Faber, ACS Catal. 2015, 5, 7503.

23

L07

Imine Reductases: Biochemistry, Engineering and Synthetic Potential Bettina M. Nestl

1, Maike Lenz

1, Philipp Scheller

1, Leonie Weinmann

1 and Bernhard Hauer

1

1 Institute of Technical Biochemistry, Universitaet Stuttgart, Stuttgart, Germany – bettina.nestl@itb.uni-stuttgart.de

Keywords: imine reductases, reductive amination, synthesis of N-heterocycles

Chiral amines, amino acids and amino alcohols have proven to be powerful building blocks for defining new

pharmaceutical and agrochemicals due to their high density of structural information. Many methods are available for chiral amine synthesis, yet surprinsingly few chiral amine structural categories can be efficiently synthesized with respect to high overall yield and enantiomeric purity. The asymmetric synthesis via chemical reducing reactions is the most frequently applied approach for the generation of chiral amine molecules. In this light, the recently described enzymatic reduction of prochiral C=N double bonds constitutes a potential alternative due to the easy accessibility of imines from their ketone precursors with the asymmetric addition of hydrogen or a hydride as the key stereo-differentiating step. [1, 2]

By taking advantage of the currently available knowledge about the function of imine reductases from

Streptomyces sp. applied in the asymmetric reduction of imines [3], combined with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference have been identified. The reducing capacity of our newly identified imine reductases has been demonstrated by the reduction of a set of endocyclic imines.[4] We have further extended these successes by the reduction of more challenging exocyclic imine substrates using enzyme catalysts. Recent work on the reductive amination reaction that proceeds in water and in the presence of imine reductase (Fig. 1) will be presented [5] and the enzymatic cascade reaction pathway combining a flavoprotein oxidase and imine reductase for the synthesis of N-heterocycles.

Fig. 1 Imine reductase-catalyzed reductive amination

Acknowledgements

We acknowledge financial support from the European Union and the EFPIA companies’ in kind contribution

for the Innovative Medicine Initiative under Grant Agreement No. 115360.

References

[1] G. Grogan, N. J. Turner, Chem. Eur. J. 2016, 22, 1900. [2] J. Schrittwieser, S. Velikogne, W. Kroutil, Adv. Synth. Catal. 2015, 357, 1655. [3] K. Mitsukura, M. Suzuki, K. Tada, T. Yoshida, T. Nagasawa, Org. Biomol. Chem. 2010, 8, 4533. [4] P. N. Scheller, S. Fademrecht, S. Hofelzer, J. Pleiss, F. Leipold, N. J. Turner, B. M. Nestl, B. Hauer,

ChemBioChem 2014, 13, 2201. [5] P. N. Scheller, M. Lenz, S. C. Hammer, B. Hauer, B. M. Nestl, ChemCatChem 2015, 7, 3239.

24

L08

Dyeing Properties and Biological Activity of Colour Compounds Synthesised by Fungal Laccase

Jolanta Polak

1, Kamila Wlizło

1, Agnieszka Szuster-Ciesielska

2, Jadwiga Sójka-Ledakowicz

3, Joanna

Lichawska-Olczyk3, Anna Jarosz-Wilkołazka

1

1Department of Biochemistry, Maria Curie-Sklodowska University, Lublin, Poland – anna.wilkolazka@poczta.umcs.lublin.pl 2Department of Virology and Immunology, Maria Curie-Skłodowska University, Lublin, Poland 3Textile Research Institute, Łódź, Poland

Keywords: laccase, immobilisation, dyes, antimicrobial activity

Today, various fashion brands, retailers, and consumers are considering alternatives available to devise

green manufacturing processes for eco-friendly products. Decisions made at the design stage greatly affect later decisions that are made by the rest of the supply chain and there is the greatest potential for changing the impact of the production process. The use of low impact dyes, chemicals, and catalysts is an alternative for a more eco-efficient production of coloured textiles. The use of enzymes in textile processes has many advantages as far as the environmentally friendlier processes are concerned. These advantages include water and energy savings, the use of lower amounts of chemicals, and milder process conditions [1].

An easy to handle extracellular laccase from fungal strain Cerrena unicolor was used for homomolecular

and heteromolecular transformations of simple aromatic precursors into coloured compounds, which can be used as textile dyes [2]. The process of synthesis was simple to operate and did not require using harmful and toxic components. The transformation of appropriate precursors occurred in buffered conditions in the presence of the enzyme and molecular oxygen, as the purest co-substrate of reaction. Orange, red, or purple products with high intensity were synthesised from the precursors and, despite the presence of a variety of substituents in the formula of the precursors, especially the reactive amino groups, low toxicity of new dyes against human and Vibrio fisheri cells was noted. The new dyes possessed good dyeing activity towards natural and synthetic fibers and textile products dyed with the best new colourants were characterised by their good quality, and the colour fastness parameters were comparable with the parameters of the commercial dye [3].

Due to the environmental impact associated

with the production of textile dyes, the potential application of fungal laccase as the biocatalyst for the synthesis of valuable colourants will be

demonstrated. The high redox potential fungal

laccase represents ideal biocatalyst because (1) can work efficiently and selectively under mild conditions, (2) can be obtained in the stable form, and (3) can effectively mediate the synthesis of the new compounds about both dyeing and new biological properties [4].

Acknowledgements

This work was partially supported by Ministry of Science and Higher Education Iuventus Plus Program (0433/IP1/2011/71) and National Science Centre (NN 302 633040).

References

[1] R.A. Sheldon, I. Arends, U. Hanefeld, Green Chemistry and Catalysis. Weinheim: Wiley-VCH, 2007 pp. 29-34. [2] J. Polak, A. Jarosz-Wilkołazka, Biotechnol. Progr. 2012, 23, 93-102. [3] J. Polak, A. Jarosz-Wilkołazka, A. Szuster-Ciesielska, K. Wlizlo, M. Kopycinska, J. Sojka-Ledakowicz, J.

Lichawska-Olczyk, J. Cleaner Prod. 2015, 112, 4265-4272. [4] J. Polak, A. Jarosz-Wilkolazka, Process Biochem. 2012, 47, 1295-1307.

25

L09

Asymmetric Bioreduction of α,β-Unsaturated Phosphonates Using Ene-Reductases.

Ignacy Janicki

1, Piotr Kiełbasiński

1, Nikolaus Turrini

2, Mélanie Hall

2, and Kurt Faber

2

1 Department of Heteroorganic Chemistry, Polish Academy of Sciences, Łódź, Poland – ijanicki@cbmm.lodz.pl

2 Department of Chemistry, University of Graz, Graz, Austria

Keywords: ene-reductases, hydrogenation, phosphonates

Biocatalytic reduction of carbon-carbon double bonds is a very useful method in asymmetric synthesis allowing for stereoselective formation of up to two chiral centers in one reaction step [1]. Enzymes that can catalyze these reactions are called ene-reductases and belong to Old Yellow Enzyme family. These flavin-dependent enzymes catalyze the stereoselective reduction of alkenes, which are activated by an electron-withdrawing group (EWG), and rely on nicotinamide cofactor as electron source. So far, the activating groups that are predominantly exploited are carbonyl derivatives, such as aldehydes and ketones, as well as esters. In addition, α,β-unsaturated nitriles, nitroalkenes and cyclic imides have also proven to be excellent substrates [2]. In order to extend this useful bioreduction protocol to more diverse starting materials, we decided to test non-classical alkenyl substrates bearing hetero-organic groups, in particular phosphonate substituents (Fig. 1).

Fig. 1 Asymmetric reduction by ene-reductases.

Optically pure phosphonate derivatives are valuable chiral building blocks for further transformations, for

instance for the synthesis of glutamic acid derivatives used for treatment of nervous system diseases, or fosmidomycin derivatives that show very promising anti-malarial activity.

In total, 12 vatiously substituted alkenylphosphonates (mostly E-isomers) were tested as potential substrates with a set of diverse ene-reductases. Phosphonate derivatives were shown to be accepted and cases of exquisite stereoselectivity along with excellent conversion levels were obtained. Overall, it appears that phosphonate groups are not sufficient to activate/bind alkene substrates alone but are excellent complementary activating groups. Stereocomplementary systems were developed and preparative scale synthesis allowed isolation of products in enantiopure form.

Acknowledgements

The authors would like to thank for support from COST Action CM1303 (SysBiocat). The research leading to

these results has received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. This research is part of project CHEM21 (https://www.chem21.eu/).

References

[1] K. Faber; Biotransformations in Organic Chemistry, 2011, Springer, 166-172. [2] C. K. Winkler, G. Tasnadi, D. Clay, M. Hall, K. Faber, J. Biotechnol. 2012, 162, 381-389.

26

L10

Biocatalytic Synthesis of Metabolites

Roland Wohlgemuth1

1 Sigma-Aldrich, Member of Merck Group, CH-9470 Buchs, Switzerland

Keywords: Asymmetric Synthesis, Chiral Analysis, Metabolites

Biological and chemical syntheses of metabolites, which occur in healthy or diseased biological cells, as well

as metabolite-like compounds attract increased interest in research and development and are relevant for a number of industries. The synthetic routes along metabolic pathways in biological cells, catalyzed by inherently chiral enzymes, provide a blueprint for resource efficiency, selectivity and robustness, which are as well goals for sustainable manufacturing routes and chemical processes. The replacement of stoichiometric by catalytic methods is of key importance and leads to mass and energy savings, selectivity improvements, safety, health and environment benefits in the synthesis of metabolites. As biocatalytic methods have become well established and have even become first choice for certain reaction classes in the route selection [1-2], applying biocatalysis to the synthesis of metabolites is a preferred methodology [3]. Phosphorus is an important element for life on our planet and occurs in living cells as part of many key large and small biomolecules, where the introduction, utilization and removal of the element phosphorus plays major roles. Among the various reaction classes, O-phosphorylations and N-phosphorylations are of major importance in natural biological processes. The large number of known enzymes catalyzing the transfer of phospho-groups from one compound to another is therefore also of much interest in synthetic production processes in industry [4]. Kinases have been produced and applied in a number of biocatalytic asymmetric phosphorylations of small molecules like mevalonate [5] or glyceraldehyde [6] using the phosphoenolpyruvate-pyruvatekinase-system for ATP cofactor regeneration. As phosphorylated metabolites play key roles in biological cells, they are also accessible through other biocatalytic routes than phosphorylations, e.g. using synthases, phosphatases and lyases [7]. Selective defunctionalisation of highly oxidized raw materials instead of introducing functional groups into highly reduced raw materials, e.g. the selective enzymatic elimination of water from sugar acids in the one-step biocatalytic route to 2-keto-3-deoxy-D-gluconate from readily available D-gluconate offers step economy, safety, health and environment benefits, because it avoids the application or production of hazardous chemicals and byproducts, respectively [8]. Selective biocatalytic reductions have become an indispensable synthetic methodology as cofactor regeneration is now a routine task [9-10]. Epoxidehydrolase-catalyzed resolutions of easily available cis-/trans-mixtures of limonene epoxides have enabled the synthesis of all four limonene epoxide enantiomers as well as chiral limonene diols [11-12]. With the progress in chiral analysis, asymmetric synthesis of chiral metabolites using biocatalysts represents a preferred methodology and enables realization of a wealth of opportunities.

References

[1] H.P. Meyer, E. Eichhorn, S. Hanlon, S. Lütz, M. Schürmann, R. Wohlgemuth, R. Coppolecchia, Catal. Sci.

Technol. 2013, 3, 29-40. [2] R. Wohlgemuth, Curr. Opin. Biotechnol. 2010 , 21, 713-724. [3] R. Wohlgemuth, Biotechnol. J. 2009, 4, 1-14. [4] D. Gauss, B Schoenenberger, G.S. Molla, B.M. Kinfu, J. Chow, A. Liese, W. Streit, in: Applied Biocatalysis – From Fundamental Science to Industrial Applications (Eds. A.Liese, L.Hilterhaus, U. Kettling, G.Antranikian)

2016, ISBN: 978-3-527-33669-2, Wiley-VCH, Weinheim, Germany [5] R. Matsumi, C. Hellriegel, B. Schoenenberger, T. Milesi, J. van der Oost, R. Wohlgemuth, RSC Advances

2014, 4, 12989-12994. [6] D. Gauss, B. Schoenenberger, R. Wohlgemuth, Carbohydrate Research 2014, 389, 18-24. [7] G.S. Molla, R. Wohlgemuth, A. Liese, J. Mol. Catal. B: Enzymatic 2016, 124 , 77–82. [8] K. Matsubara, R. Köhling, B. Schoenenberger, T. Kouril, D. Esser, C. Bräsen, B. Siebers, R. Wohlgemuth, J.

Biotechnol. 2014, 191, 69-77. [9] R. Wohlgemuth, in Synthetic Methods for Biologically Active Molecules: Exploring the Potential of Bioreductions, (Ed. E.Brenna) 2014, Wiley-VCH, Weinheim, Germany [10]M.A.K. Vogel, H. Burger, N. Schläger, R. Meier, B. Schönenberger, T. Bisschops, R. Wohlgemuth, Reaction

Chemistry & Engineering, 2016, 1, 156-160. [11]E.E. Ferrandi, C. Sayer, M.N. Isupov, C. Annovazzi, C. Marchesi, G. Iacobone, X. Peng, E. Bonch-

Osmolovskaya, R. Wohlgemuth, J.A. Littlechild, D. Monti, FEBS J. 2015, 282, 2879-2894. [12]E.E. Ferrandi, C. Marchesi, C. Annovazzi, S. Riva, D. Monti, R. Wohlgemuth, ChemCatChem 2015, 7, 3171-

317

27

L11

Multichannel Monolithic Enzymatic Microreactors- Paving the Way to New Robust Technologies

Katarzyna Szymańska

1, Jolanta Bryjak

2 and Andrzej Jarzębski

1,3

1 Department of Chemical Engineering and Process Design, Silesian University of Technology, 44-100 Gliwice, Ks. M.

Strzody 7, Poland - Andrzej.Jarzebski@polsl.pl 2 Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, 50-373 Wrocław, Norwida 4/6,

Poland 3 Institute of Chemical Engineering, Polish Academy of Sciences, 44-100 Gliwice, Bałtycka 5, Poland

Keywords: enzymatic microreactors, flow synthesis, in flow enzymatic catalysis

The siliceous monoliths that feature a very open bi-continuous structure of interconnected meandering flow-through channels of micrometric sizes and of silica skeleton hosting large mesopores can be applied as the reactive cores of continuous-flow microreactors (MRs) [1-3]. Moreover, the preparation enables the size of larger channels and smaller pores (hierarchically connected) to be tuned to specific needs [2]. In effect, the MRs demonstrate huge potentials as the very effective devices operating at low backpressures (1-2 bars) and quite considerable flowrates quantified in mL/min and not μL/min as in most typical MRs. Their large (300-400 m2/g) and easily accessible surface can be functionalised with various groups or metals (Co, Ni) to attach the enzymes either non-specifically or by a tailored adsorption using His6-tagged proteins.

The performance of MR with various enzymes will be reported: e.g. of acyltransferas [1] (tailored adsorption and covalent immobilization), trypsin [2], invertase [3], penicillin acylase (covalently attached), lipases (adsorption). In all the cases examined, the rate of the enzyme catalysed reaction appeared to be very exceedingly high, and this enabled very large productivities to be obtained. Moreover, the MRs appeared to be stable in the continuous flow synthesis with aqueous or organic solvents even for several weeks.

Fig. 1 Schematic representation of the continuous tryptic proteolysis in the monolithic microreactor

Acknowledgements

The support of the National Science Centre (NCN, Poland) for this work under grant No. UMO-2013/09/B/ST8/02420 is gratefully acknowledged.

References

[1] K. Szymańska et al., Catal. Sci. Technol., DOI: 10.1039/c5cy02067k. [2] K. Szymańska et al., Chem. Eng. J., 2016, 287, 148–154. [3] K. Szymańska et al., Micro. Meso. Mat., 2013, 170, 75–82.

28

L12

Modification of Bacterial Cell Surface Properties and Oxygenase Activity During Hydrocarbons Biodegradation

Agata Zdarta, Wojciech Smułek and Ewa Kaczorek Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland – agata.zdarta@doctorate.put.poznan.pl

Keywords: bacterial cell surface, monooxygenase, hydrocarbons

Microbes have demonstrated the ability to degrade a wide range of hydrocarbons, both those occurring naturally as well as environmental pollutants [1]. However there is still much to discover in mechanism of bacterial biodegradation of harmful pollutions in the environment One of the issue is hydrocarbons low bioavailability for bacteria. To defeat this inconvenience, microbes have to adapt their cells parameters to environmental conditions, but not all of them can do it [2, 3]. Several papers have shown that cell surface properties and enzymatic activity of the microbes can be good indicator of bacterial abilities to degrade hydrocarbons [3-4].

The aim of this study was to identify and characterize modification of surface properties and hexadecane monoxygenase activity of Pseudomonas stutzeri K19 during biotransformation of buthylbenzene, tert-butylbenzene, heptane, octane and diesel oil. The strain was isolated form soil in Northern Poland. Liquid cultures were conducted for 7 days as it was described by Smułek et al. 2015 [4] with buthylbenzene, tert-butylbenzene, heptane, octane and diesel oil as a single carbon sources. After that cell surface hydrphobicity, zeta potential, extracellular polymeric substances production and hexadecane monoxygenase activity were measured.

Summing up the results of the research it can be concluded that the type of used carbon source has a significant influence on the surface properties and enzymatic activity of Pseudomonas stutzeri KI9 . Analysis of the concentration of extracellular polymeric substances (EPS), secreted by P. stutzeri strain KI9 revealed that the type of the carbon source in the bacterial culture could stimulate the secretion of the EPS. Conducted analysis did not show effect of the type of carbon source in the culture on the surface hydrophobicity of gram negative P. stutzeri KI9 strain. However, the zeta potential modifications were observed. Furthermore, the analysis of enzymatic activity of hexadecane monooxygenase revealed changes with respect to the control sample on glucose. This results proved that analysis of surface properties and enzymatic activity of microbes could be useful tools for bacterial hydrocarbons biodegradation assessment.

Acknowledgements

This study was supported by The National Science Centre awarded by decisions number DEC-

2012/07/B/NZ9/00950

References

[1] M. Chen, P. Xu, G. Zeng, C. Yang, D. Huang and J. Zhang Bioremediation of soils contaminated with

polycyclic aromatic hydrocarbons, petroleum, pesticides, chlorophenols and heavy metals by composting: Applications, microbes and future research needs Biotechnology Advances 2015, 33, 745–755.

[2] J. Jesus, D. Frascari, T. Pozdniakova and A. S. Danko, Kinetics of aerobic cometabolic biodegradation of chlorinated and brominated aliphatic hydrocarbons: A review Journal of Hazardous Materials 2016, 309, 37–52.

[3] H. Gong, M. Bao, G. Pi, Y. Li, A. Wang and Z. Wang Dodecanol-Modified Petroleum Hydrocarbon Degrading Bacteria for Oil Spill Remediation: Double Effect on Dispersion and Degradation ACS Sustainable Chemistry & Engineering 2016, 4, 169–176.

[4] W. Smułek, A. Zdarta, U. Guzik, B. Dudzińska-Bajorek, E. Kaczorek, Rahnella sp. strain EK12: Cell surface properties and diesel oil biodegradation after long-term contact with natural surfactants and diesel oil Microbiological Research 2015, 176, 38-47.

29

L13

Surfactant-Enhanced Biodegradation of Halogenated Compounds by Activated Sludge Microorganisms

Wojciech Smułek, Agata Zdarta and Ewa Kaczorek Institute of Chemical Technology and Engineering, Poznan University of Technology, Poland – wojciech.smulek@doctorate.put.poznan.pl

Keywords: surfactants, biodegradation, activated sludge

Halogenated organic compounds, which sources are various industrial products, such as: herbicides, dyes or wood preservatives [1], are resistant to biological degradation by naturally existing microbes and therefore remain in the environment. These compounds are not only highly toxic but they also are characterized by mutagenicity and cancerogenicity properties [2].

There are some microorganisms, mainly bacteria and fungi are capable to degrade halogenated organic compounds, but it is slow process of limited effectiveness. Bioremediation of organic compounds can be enhanced by the addition of surfactants to polluted soils. On the one hand surfactants can increase the contact area between hydrocarbons and microorganisms [3]. On the other hand the surface active agents cause the modification of cell surface properties and permeability of biological membrane [4].

The aim of this work was to investigate the three halogenated aromatic compounds biodegradation by activated sludge bacterial strains isolated from genera Kluyvera and Pseudomonas. The degradation of p-bromophenol, p-chlorophenol and p-fluorophenol was determined by HPLC-MS/MS analysis. The results from samples without and with two extracts of plant origin containing saponins were compared. Moreover, to evaluate mechanisms of halophenols uptake by bacterial cells, cell surface hydrophobicity and membrane permeability investigations of tested strains were conducted to explain.

The obtained results indicated that the halophenols can be biodegraded by selected strains without any cometabolites. However, the addition of saponins allowed to short the total degradation time of p-flourophenol as well as p-chlorophenol. In the case of Pseudomonas sp. in cultures with Qullaja saponins p-fluorophenol was totally degradaed after 12 days in comparison with 25 days in Pseudomonas sp. cultures without surfactants. Simultaneously, the changes in biodegradation efficiency caused by natural surfactants addition were accompanied by the significant modifications in the cell membrane permeability.

Acknowledgements

This study was supported by The National Center for Science awarded by decision number

DEC-2012/07/B/NZ9/00950

References

[1] W.-J. Sim, S.-H. Lee, I.-S. Lee et al., Chemosphere 2009, 77, 552-558. [2] M. Pera-Titus, V. Garcia-Molina, M.A. Banos et al., Appl. Catal. B-Environ. 2004, 47, 219-256. [3] G.M. Zeng, Z.F. Liu, H. Zhomg et al. Appl. Microbiol. Biotechnol. 2011, 90, 1155-1161. [4] Y. Shoji, T. Igarashi, H. Nomura et al., Anal. Sci. 2012, 28, 339-343.

30

L14

Ionic Liquid Mediated Activation of a Lipase for Sustainable Preparation of Chiral Secondary Alcohols

Shiho Kadotani, Takashi Nishihara, Toshiki Nokami, and Toshiyuki Itoh

*

Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University Koyama-minami, Tottori 680-8552, JAPAN, E-mail: titoh@chem.tottori-u.ac.jp. Keywords:Lipase, Transesterification acceleration, Ionic Liquid

Lipases are among the most widely used enzymes and are applicable for various substrates in the preparation of chiral ester or alcohols; however, slow reactions or poor enantioselective reactions are sometimes obtained [1].

We have been investigating a method that solves this problem by activation of the lipase using an ionic liquid engineering: coating of lipase PS using imidazolium cetyl(PEG)10 sulfate (IL1) enhanced in both enantioselectivity and reaction rate [2]. We further found that a synergetic activation of a lipase by an amino acid with IL1 was accomplished [3]. For the aim to develop more powerful ionic liquid type activating agents for lipase, we next developed tributyl((2-methoxy)ethoxymethyl) phosphonium (PL1) [4]: PL1-PS displayed excellent enantioselectivity in the reaction of 2-chloro-1-phenylethanol with E>200, though insufficient E values were recorded for IL1-PS (E= 123) for this alcohol [4].

Herein, we report the results of synthesis of two novel IL type activating agents for lipase and their activation properties. 1-Butyl-3-methyl- triazolium (TAZ1) and triaminocyclopropenium (TAC) cetyl(PEG)10 sulfate ionic liquids have been synthesized and used as coating materials of Burkholderia cepacia lipase (Lipase PS) (Fig. 1). These enzymes displayed high reactivity in transesterification of broad types of secondary alcohols using vinyl acetate as an acylating reagent with perfect enantioselectivity (E>200) in an IL solvent system. But they showed different substrate preference with those IL1-PS and PL1-PS. In particular, the activation property of TAC cetyl(PEG)10 sulfates are strongly dependent on the substituents on the amino moiety of the TAC cation and TAC-PS showed a very unique substrate preference. We further succeeded in demonstrating the recyclable use of TZ1-PS ten times in N,N-diethyl,N-methyl,N-(methoxyethoxymethyl)ammnonium bis(trifluoromethylsulfonyl)amide ([N221MEM][Tf2N]) as a solvent.

Since these IL-coated lipases are easily applicable to 10-20 gram-scaled reactions, it is expected that they might be useful for practical preparation of a wide variety of chiral secondary alcohols.

Fig. 1. Enantioselective transesterification catalyzed by IL-coated lipase in an IL solvent

References [1] K. Faber, Biotransformations in Organic Chemistry, 6

th revised and corrected ed.; Springer-Verlag Berlin:

Heidelberg, Germany, 2011. [2] (a) T. Itoh, Y. Matsushita, Y. Abe, S-H. Han, S. Wada, S. Hayase, M. Kawatsura, S. Takai, M. Morimoto, Y.

Hirose, Chem. Eur. J. 2006, 12, 9228-9237. (b) Y. Abe, Y. Yagi, S. Hayase, M. Kawatsura, T. Itoh, Ind. Eng. Chem. Res. 2012, 51, 9952-9958.

[3] K. Yoshiyama, Y. Abe, S. Hayase, T. Nokami, T. Itoh, Chem. Lett. 2013, 42, 663-665.

[4] Y. Matsubara, S. Kadotani, T. Nishihara, Y. Hikino, Y. Fukaya, T. Nokami, T. Itoh, Biotechnol. J. 2015,

10,1944-1951.

31

L15

Promiscuous Hydrodehalogenation Activity Can Expand (Even More) the Applicability of Transaminases

Iván Lavandera

1, Marina García-Ramos

1, Aníbal Cuetos

2, Eva-Maria Fischereder

3, Alba Díaz-

Rodríguez1, Vicente Gotor

1, Gideon Grogan

2 and Wolfgang Kroutil

3

1 Organic and Inorganic Chemistry Department, University of Oviedo, Oviedo, Spain – lavanderaivan@uniovi.es

2 York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK

3 Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Graz, Austria

Keywords: transaminases, catalytic promiscuity, hydrodehalogenation

Among biocatalysts that have demonstrated broad applicability in the last years, transaminases (TAs) can be highlighted, as they perform the amination of an amine acceptor (ketone or aldehyde) using an amine donor (e.g., alanine or isopropylamine), mediated by the cofactor pyridoxal 5’-phosphate (PLP) [1], [2]. Interestingly, TAs can also catalyze the reverse reaction, achieving the kinetic resolution of racemic amines via amination of an amine acceptor (e.g., pyruvate or acetone). This process is hampered by the disadvantage of a maximum of 50% yield, but it is thermodynamically favored in comparison to the amination route.

On the other hand, many enzymes have shown to efficiently catalyze transformations that appear far removed from their natural activity. This catalytic promiscuity can be useful for synthetic purposes, broadening the applicability of biocatalysts. While many examples have recently been described for hydrolases, the application of these unconventional processes for other enzyme classes has not been fully developed [3], [4].

Herein we will show that a protitmiscuous hydrodehalogenation reactivity of transaminases can be applied to obtain a series of dehalogenated ketones starting from the corresponding α-halo ketones, or a series of enantiopure β-fluoroamines by unprecedented formal tandem hydrodefluorination-deamination kinetic resolution starting from the racemic β-fluoroamines [5], in the absence of an amine acceptor, under simple and mild conditions in aqueous medium (Fig. 1). Further experiments also demonstrated that these transformations may occur in hand with enzymatic deactivation. A plausible dual mechanism that could explain these results was also proposed based on different experimental and crystallographic data.

+

NH3 + HF

NH2

F

O

Transaminase / BufferNH2

F

(R)- or (S)up to >99% ee

*

R R R

H

H2O

O

X

O

Transaminase / Buffer

R R

H

X= F, Cl, Br

Fig. 1 Promiscuous hydrodehalogenation activities found with transaminases

Acknowledgements

Financial support from MICINN (Project CTQ2013-44153-P) and the Principado de Asturias (Project FC-15-

GRUPIN14-002) are gratefully acknowledged.

References

[1] M. Fuchs, J. E. Farnberger, and W. Kroutil, Eur. J. Org. Chem. 2015, 6965. [2] W. Kroutil, E.-M. Fischereder, C. S. Fuchs, H. Lechner, F. G. Mutti, D. Pressnitz, A. Rajagopalan, J. H. Sattler,

R. C. Simon, and E. Siirola, Org. Process Res. Dev. 2013, 17, 751. [3] U. T. Bornscheuer and R. J. Kazlauskas, in Enzyme Catalysis in Organic Synthesis, 3

rd Ed., (Eds.: K. Drauz,

H. Gröger, O. May), Wiley-VCH, Weinheim, 2012, p. 1695. [4] M. S. Humble and P. Berglund, Eur. J. Org. Chem. 2011, 3391. [5] A. Cuetos, M. García-Ramos, E.-M. Fischereder, A. Díaz-Rodríguez, G. Grogan, V. Gotor, W. Kroutil, and I.

Lavandera, Angew. Chem. Int. Ed. 2016, 55, 3144.

32

L16

The Influence of Solvent on the Properties of Quercetin and Rutin

Anna Taraba

1, Magdalena Szaniawska

1 and Katarzyna Szymczyk

1

1 Department of Interfacial Phenomena, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska

Sq. 3, 20-031 Lublin, Poland

Keywords: rutin, quercetin, methanol

Quercetin belongs to a group of flavonoids (3,5,7,3’,4’-pentahydroxyflovonol) and it is one of the widely used pharmaceutical flavonoids compound. It generally occurs in plants and in food such as tea, juices, wine or honey. Quercetin has many properties, which have positive effect on humans health [1].

Rutin (rutoside, quercetin-3-O-rutinoside, sophorin) is the glycoside between the quercetin and the disaccharide rutinose. Rutin possesses anti-inflamatory and anti-spasmodic properties. It prevents cancer and protects the liver. Also rutin helps absorb more vitamin C, which prevents cell oxidation [2].

Quercetin and rutin are barely soluble in water, which in turn can limit its absorption in human body. On the other hand, the solubility of organic compounds, such quercetin and rutin, in different solvents plays an important role in their separation and purification applications. The short-chain alcohols are one of the most important groups of organic additives. At their low concentration they behave as cosurfactants while at higher concentration they should be treated rather as cosolvents [3]. Moreover, the mixed water+alcohol solvents are incredibly important from a practical point of view because they have the antiseptic and disinfecting properties, so they are applied in a large variety of products like cosmetics, cleaning agents and drugs [3,4].

Thus, the aim of the presented studies was to determine the influence of the different concentration of methanol on the solubility of quercetin (Cqe=4x10

-5M) and rutin (Cru=4x10

-5M). The analysis of these solutions was

made on the basis of the UV-Vis (spectrophotometer Helios γ) and fluorescence emission spectra measurements (Hitachi 2700).

a) b)

Fig. 1 The structure of rutin (a) and quercetin (b).

References

[1] S. P. Boyle, V. L. Dobson, S.J. Duthie, D. C. Hinselwood, J. A. M. Kyle, A. R. Collins, European Journal of

Clinical Nutrition 2000, 54, 774-782. [2] M. Atanassova , V. Bagdassarian, Journal of the University of Chemical Technology and Metallurgy 2009, 2,

201-203. [3] R. Zana, Advances in Colloid and Interface Science 1995, 57, 1-64. [4] R. Zana, S. Yiv, C. Strazielle, P. Lianos, Journal of Colloid and Interface Science 1981, 80, 208–223.

33

L17

Reaction Monitoring by NMR: an overview Manuel Perez

1

1 Mestrelab Research SL, Santiago de Compostela, Spain – Manuel@mestrelab.com

Keywords: Reaction Monitoring, NMR, high throughput analysis.

NMR has been traditionally used as a tool to gain insight into both static and dynamic systems [1]. The acquisition of information in dynamic systems has always proven to be challenging for many different factors, amongst other homogeneity of the field, turbulence and many others. The analysis of the generated data has also been a limiting factor, in many occasions it involved navigating through hundreds of datasets and treating this in an individual basis.

In this work we will focus on automation of the analysis, on the algorithms that make this automation possible. How deconvolution of the NMR signals makes it possible to obtain information difficult to get to otherwise.

Examples of the type of systems that can be analyzed as well as the different types of information that can be attained will be presented.

Fig. 1 A mixture of metabolites is analyzed under changing pH conditions [2]

References

[1] Khajeh, M., M. A. Bernstein, and G. A. Morris. "A simple flowcell for reaction monitoring by NMR." Magnetic Resonance in Chemistry 48.7 (2010): 516-522.

[2] Xiao, Chaoni, et al. "An optimized buffer system for NMR-based urinary metabonomics with effective pH

control, chemical shift consistency and dilution minimization." Analyst 134.5 (2009): 916-925.

34

L18

Biotransformations with Phenylalanine Ammonia Lyase - Mechanism and Process Intensification

László Poppe

1, Diána Weiser

1, Gergely Bánóczi

1, Ferenc Ender

2, László C. Bencze

3, Hajnal Bartha-

Vári3, Alina Filip

3, Florin-Dan Irimie

3, Beáta G. Vértessy

4, Csaba Paizs

3

1

Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary – poppe@mail.bme.hu

2 Department of Electron Devices, Budapest University of Technology and Economics, Budapest, Hungary

3 Biocatalysis and Biotransformation Research Group, Babeş-Bolyai University of Cluj-Napoca, Romania

4 Department of Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest

Keywords: phenylalanine ammonia-lyase, enzyme immobilization, enzyme mechanism, microreactor

Phenylalanine ammonia-lyase (PAL) catalyze ammonia elimination from their corresponding amino acid

substrates by the aid of an electrophilic prosthetic group, 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO). [Błąd! Nie zdefiniowano zakładki.] The PAL from parsley, Petrosilenum crispum (PcPAL) was especially useful

biocatalyst for preparation of α-L-arylalanines by addition of ammonia onto the corresponding arylacrylates or α-D-arylalanines by kinetic resolutions from racemic arylalanines.[Błąd! Nie zdefiniowano zakładki.]

Successful immobilizations of PcPAL as CLEAs[2] by bisepoxides as cross-linking agent prompted us to study

the attachment of PcPAL by the epoxide strategy to nanostructured supports such as carbon nanotubes (SwCNT-

PAL) [3] or magnetic nanoparticles (MNP-PAL).[4,5] SwCNT-PAL was applied in the ammonia addition reaction onto (E)-3-(thiophen-2-yl)acrylic acid 2 yielding enantiopure (S)-2-amino-3-(thiophen-2-yl)propanoic acid (S)-1 in

batch mode and in continuous-flow packed-bed microreactor (Fig. 1, left).[3] By using MNP-PAL, fixed in a

microfluidic reactor with an in-line UV detector, we first demonstrated that PAL can catalyze the ammonia

elimination from the acyclic propargylglycine (PG) to yield (E)-pent-2-ene-4-ynoate indicating new opportunities to

extend the MIO-enzyme toolbox towards acyclic substrates (Fig. 1, mid).[4] Biotransformation of L-phenylalanine (L-3a) and five unnatural substrates (rac-3b-f) by MNP-PAL was investigated with in-line UV detection in Magne-

Chip device comprises microliter volume reaction cells filled with MNP-PAL (Fig. 1, right).[5]

Fig. 1 Biotransformations in microfluidic reactors with PAL immobilized on different nanosupports

Acknowledgements

Thanks to projects OTKA NN-103242, CNCS–UEFISCDI PN-II-IDPCE-2011-3-0799 and COST Action

CM1303 (SysBiocat) for financial support.

References [1] L. Poppe, C. Paizs, K. Kovács, F.D. Irimie, B.G. Vértessy, Meth. Mol. Biol. 2012, 794, 3-19. [2] D. Weiser, A. Varga, K. Kovács, F. Nagy, A. Szilágyi, B.G. Vértessy, C. Paizs, L. Poppe, ChemCatChem

2014, 6, 1463-1469. [3] J.H. Bartha-Vári, M.I. Toşa, F.D.Irimie, D. Weiser, Z. Boros, B.G. Vértessy, C. Paizs, L. Poppe,

ChemCatChem 2015, 7, 1122-1128. [4] D. Weiser, L.C. Bencze, G. Bánóczi, F. Ender, E. Kókai, A. Szilágyi, B.G. Vértessy, Ö. Farkas, C. Paizs, L.

Poppe, ChemBioChem 2015, 16, 2283-2288. [5] F. Ender, D. Weiser, B. Nagy, L.C. Bencze, C. Paizs, P. Pálovics, L. Poppe, J. Flow Chem. 2016, 6(1), 43-52.

35

L19

Click Reactions for Efficient Enzymatic Kinetic Resolution Processing Mădălina Elena Moisă

1, Csaba Paizs

1, László Poppe

2, Florin Dan Irimie

1, Monica Ioana Toşa

1

1Department of Chemistry, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca, Romania –

mmoisa@chem.ubbcluj.ro 2Department of Organic Chemistry and Technology, Faculty of Chemical Technology and Biotechnology, Budapest University of

Technology and Economics, Budapest, Hungary

Keywords: Click reaction, enzymatic kinetic resolution, lipase

Click reactions represent reliable and versatile tools for covalently linking molecules, allowing the

construction of various complex molecular structures. The usefulness of such transformations is confirmed by the large number of fields in which they have been successfully applied (biochemistry, drug discovery, medicinal chemistry, nanomaterials science, organic synthesis) [1,2]. Among all click processes the copper-catalysed azide-alkyne cycloaddition (CuAAC) with formation of a triazole unit is by far the most frequently employed strategy due to its simplicity, versatility and high selectivity [3]. Numerous studies have been recently carried out in this direction using oligo- or polysaccharides as starting materials [4,5].

The high demand for optically pure compounds and green manufacturing processes leads to a continuous development in biotechnology. Enzymatic kinetic resolution (EKR) of a racemic substrate represents the easiest and preferred biocatalytic route but large scale EKR implies difficulties regarding products isolation and purification.

The present work focuses on the development of an efficient procedure for EKR product separation with the aid of click reactions. In an effort to prove the utility of this approach, chiral heteroaryl-ethanols were used as model compounds in lipase-mediated EKR processes and β-cyclodextrin and cellulose as azido-carriers. Firstly, the chemical synthesis and characterization of racemic heteroaromatic substrates, propargylic acyl donors and azido-modified supports was performed. Next, the optimal reaction conditions for the studied EKR and click processes have been established. Afterwards, the click-based EKR reaction mixture separation procedure was set-up.

Cu(I)

SR

EKR

lipasepropargylic ester

+

support boundoptically pure

product

untransformedoptically pure

substrate

Clickreaction

optically puresubstrate

racemicsubstrate

SR

PRSS Ss

SSPR

+R1 N N N

C R2HC

+

N

N NR1

R2

+

Click reaction

Fig. 1 Click reaction-based strategy for easy EKR products separation

Acknowledgements

This research was supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN-II-PT-PCCA-2013-4-0734.

References

[1] P. Thirumurugan, D. Matosiuk, and K. Jozwiak, Chem. Rev. 2013, 113, 4905-4979. [2] P.K. Avti, D. Maysinger and A. Kakkar, Molecules 2013, 18, 9531-9549. [3] V.D. Bock, H. Hiemstra, and van J.H. Maarseveen, Eur. J. Org. Chem. 2006, 2006(1), 51-68. [4] T. Liebert, C. Hänsch, and T. Heinze, Macromol. Rapid Commun. 2006, 27, 208-213. [5] P.A. Faugera, B. Boëns, P.H. Elchinger, F. Brouillette, D. Montplaisir, R. Zerrouki, and R. Lucas, Eur. J. Org.

Chem. 2012, 2012(22), 4087-4105.

36

L20

Cyanobacteria as Biotransformants in Redox Reactions Agata Głąb

1, Ewa Żymańczyk Duda

2

1 Department of Bioorganic Chemistry, Wrocław University of Science and Technology, Wrocław, Poland –

agata.glab@pwr.edu.pl 2 Department of Bioorganic Chemistry, Wrocław University of Science and Technology, Wrocław, Poland

Keywords: acetophenone, biocatalysis, cyanobacteria

Cyanobacteria are phototrophic microorganisms able to adapt and grow in almost every aquatic environment. They are producers of organic compounds, among which numerous posses biological activity - antibacterial, antifungal, antiviral, anticancer or immunosuppressive [1]. Another important characteristic of cyanobacteria, which makes them an object of many scientific research, is their ability to perform photosynthesis much more efficiently, and therefore potential of producing third generation biofuels [2]. Cyanobacteria biocatalytical potential have not been thoroughly investigated yet, although there are reports showing their ability to biotransform limonene [3], enones [4] or hydroxyphosphonates [5].

The aim of the research is to evaluate the biocatalytic potential of the cyanobacteria basing on the reduction of acetophenone to the phenylethyl alcohol of defined absolute configuration and on the corresponding enantioselective oxidation of (RS)-1-phenylethanol.

Fig 1. Reduction of acetophenone to the phenylethyl alcohol and corresponding enantioselective oxidation of (RS)-1-phenylethanol.

Among investigated cyanobacteria, Nodularia sphaerocarpa turned out to be the most efficient catalysts able

to convert more than 90% of starting ketone, but with low enantiomeric excess (34%). Enantioselecitve reduction was being performed by Leptolyngbya foveolarum and Arthrospira maxima, with efficiency over 60% and ee over 80%. In all reactions (S)-isomer of 1-phenylethanol was formed preferably.

References

[1] R.M.M. Abed, S. Dobretsov, K. Sudesh, J Appl Microbiol 2008, 106, 1-12 [2] I.M.P. Machado, S. Atsumi, J Biotechnol, 2012, 162, 50 [3] H. Hamada, Y. Kondo, K. Ishihara, N. Nakajima, R. Kurihara, T. Hirata, J Biosci Bioeng, 2003, 96, 581 [4] K. Shimoda, N. Kubota, H. Hamada, M. Kajib, T. Hirata, Tetrahedron Asymmetr, 2004, 15, 1677 [5] M. Górak, E. Żymańczyk-Duda, Green Chem, 2015, 17, 4570.

acetophenone (R)-1-phenylethanol (S)-1-phenylethanol

37

L21

Dendronized Polymer-Enzyme Conjugates.− Preparation and Applications for the Immobilization of Enzymes on Silicate

Surfaces Andreas Küchler, Peter Walde Department of Materials, ETH Zürich, Zürich, Switzerland – peter.walde@mat.ethz.ch

Keywords: dendronized polymer, enzyme, immobilized

Dendronized polymers (denpols) are polymers which carry in each repeating unit a dendritic side chain. The water soluble, second generation denpol de-PG2 [1] (Fig. 1) is used for the immobilization of enzymes on silicate surfaces. De-PG2 carries in each repeating unit four peripheral amino groups which provide water solubility at neutral and acidic pH values and nucleophilicity under slightly alkaline conditions.

The enzyme immobilization is achieved by first preparing a denpol-enzyme conjugate in which several copies of an enzyme are covalently bound to the denpol along the denpol chain via chemically stable bis-aryl hydrazone (BAH) bonds. The enzymes used so far include horseradish peroxidase (HRP) [1-3] and proteinase K (proK) [4], yielding the conjugates de-PG2-BAH-HRP and de-PG2-BAH-proK. The conjugates are then immobilized on silicate surfaces through simple adsorption from aqueous solution. The surfaces tested are microscopy glass cover slips [2,4], glass micropipettes [1,3], mesoporous silicates [4], and glass/polydimethylsiloxane microphips [4]. In all cases, stable adsorption and high operational stability are observed, demonstrating the usefulness of the methodology developed for applications of small enzymatic flow reactor devices. The simultaneous binding of two different types of enzymes along the same denpol and their co-immobilization is also possible [1,2,4].

OO

O O

NHHN

OO

O

O

O

OH3N

NH3

OO

OO

HN NH

O

O

OH3N

CH2 co CH2x

y

O

NH3

O

O

NH3

NH3

HN O

N

HN

NH3

N

NH

O

de-PG2

BAH

proK

Fig. 1 Schematic representation the de-PG2-proK conjugates of the denpol de-PG2 and several copies of

covalently linked proK molecules. The linkage is a bis-aryl hydarzone (BAH) bond.

References

[1] A. Grotzky, T. Nauser, H. Erdogan, A. Dieter Schlüter, and P. Walde, J. Am. Chem. Soc. 2012, 134, 11392. [2] A. Küchler, J. Adamcik, R. Mezzenga, A. D. Schlüter, and P. Walde, RSC Adv. 2015, 5, 44530. [3] H. Gustafsson, A. Küchler, K. Holmberg, and P. Walde, J. Mater. Chem. B 2015, 3, 6174. [4] A. Küchler, J. N. Bleich, B. Sebastian, P. S. Dittrich, and P. Walde, ACS Appl. Mater. Interfaces 2015, 7,

25970.

38

L22

Enzymatic Reactions of Heteroatom-containing Compounds Leandro Helgueira Andrade University of São Paulo, Institute of Chemistry, São Paulo, Brazil. leandroh@iq.usp.br

Keywords: lipases, proteases, mono-oxygenases.

Several enzymatic reactions of heteroatom-containing compounds as unnatural substrates have been explored for different purposes. In this talk, we will discuss some results regarding the use of alcohol dehydrogenases, lipases, transaminases and monooxygenases in the transformation of selenium-, boron- and silicon- compounds. [1-4] Besides using enzymes as catalysts for stereoselective reactions, we will also discuss the application of heteroatom-containing compounds as molecular targets for enzymes with biological relevance in medicinal chemistry. [5-7]

Acknowledgements

The authors would like to thank FAPESP (São Paulo Research Foundation; Grant number: 2013/04540-0 and

2014/22457-5) for financial support.

References

[1] Reis, J. S.; Simon, R. C.; Kroutil, W.; Andrade, L. H. Tetrahedron-Asymmetry, 2013, 24, 1495-1501. [2] Brondani, P. B.; Guilmoto, N. M. A. F.; Dudek, H.; Fraaije, M. W.; Andrade L. H. Tetrahedron, 2012, 23, 703-

708. [3] Brondani, P. B.; Gonzalo, G.; Fraaije, M. W.; Andrade, L. H. Adv. Synth. Catal., 2011, 353, 2169-2173. [4] Andrade, L. H.; Silva, A. V.; Milani, P.; Koszelewski, D.; Kroutil, W. Org. Biomol. Chem., 2011, 8, 2043-2051,

2010. [5] Silva, M. S.; Andrade, L. H. Org. Biomol. Chem., 2015, 13, 5924-5929. [6] Andrade, L. H.; Milani, P.; Demasi, M.; Rezende, L.; Do Amaral, A. T. New J. Chem., 2014, 38, 4859-4871. [7] Piovan, L.; Milani, P.; Silva, M. S.; Moraes, P. G.; Demasi, M.; Andrade, L. H. Eur. J. Med. Chem., 2014, 73,

280-285.

39

L23

Chemoenzymatic Transformation of Lignocellulose Danuta Gillner

1, Marta Musioł

1, Karolina Matuszek

2, Anna Chrobok

2, Katarzyna Szymańska

3, Agata

Bujok1, Daria Kowalczykiewicz

1, Jakub Śmiłowski

1

1 Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University Of Technology, Faculty of

Chemistry,Gliwice, Poland - Danuta.Gillner@polsl.pl; 2Department of Organic Chemical Technology and Petrochemistry; Silesian University Of Technology, ,Gliwice, Poland

3Department of Chemical Engineering and Process Design, Silesian University Of Technology, ,Gliwice, Poland

Keywords: biomass, cellulose, lignin, ionic liquids, enzymes

Lignocellulose has attracted considerable attention as a raw material for the production of fuels and wide

range of fine chemicals. Many different methods of biomass pretreatment and further transformation have been investigated. All of them aimed to elaborate efficient, inexpensive and environmentally friendly technologies. Among many methods described in the literature, those with ionic liquids (ILs), used in pretreatment and degradation processes, play very important role [1].

In this paper we present application of new, inexpensive, acidic ionic liquids, based on sulfuric or acetic acids and tertiary/aromatic amines [2], in the cellulose and lignocellulose processing. Various molar ratios of acids used in the preparation, resulted in obtaining of ILs with different acidity and good thermal stability.

Treatment of cellulose with ILs revealed, that they are able to convert cellulose to valuable chemicals (e.g. levulinic acid or 5-hydroxymethylfurfural) under very mild conditions. We have shown, that type of cation plays important role in the process. Some of ILs (especially those based on sulfuric acid) dissolved cellulose even at room temperature. The influence of type of ionic liquid, temperature and role of water on the yield of levulinic acid and 5-hydroxymethylfurfural were determined (example in Fig.1).

It was shown that levulinic acid can be recovered from the reaction mixture in the form of ester, since studied ionic liquids are very good catalysts of esterification processes.

Preliminary investigation on the pretreatment of woody biomass, and especially biomass from walnut tree (leaves, walnut shells, wood waste) as well as oilseed rape straw were also performed.

It is known from the literature, that certain ionic liquids used in pretreatment process, severely inactivate hydrolytic enzymes (e.g. cellulases). Within our study the investigation on the influence of different amounts of new ILs on the activity of cellulases was carried out.

Fig. 1 The influence of temperature and type of ionic liquid on the yield of levulinic acid after 2 h of cellulose transformation

Acknowledgements

This work was partially supported by the National Science Centre, grant No. DEC-2013/09/D/ST8/04002.

References

[1] A.M. da Costa Lopes, R. Bogel-Łukasik, ChemSusChem, 2015, 8(6), 947. [2] K. Matuszek, A. Chrobok, F. Coleman, K.R. Seddon, M. Swadźba-Kwaśny, Green Chemistry, 2014, 16, 3463.

■ [Hmim]HSO4

■ [Hmim][(HSO4)H

2SO

4]

■ [Hmim][(HSO4)(H

2SO

4)2]

40

L24

Biotransformation of Naproxen in the Presence of Selected Aromatic Compounds and Organic Solvents by Bacillus sp. B1(2015b)

Dorota Domaradzka

1, Urszula Guzik

2 and Danuta Wojcieszyńska

2

1 Department of Biochemistry, University of Silesia in Katowice, Katowice, Poland – ddomaradzka@us.edu.pl

2 Department of Biochemistry, University of Silesia in Katowice, Katowice, Poland

Keywords: NSAIDs, microorganism, co-metabolism

Naproxen is one of the most popular over-the-counter painkillers which belongs to the group of polycyclic non-steroidal anti-inflammatory drugs (NSAIDs). Due to its extensive use, it has been found in surface and drinking water [1]. The widespread occurrence of naproxen in the environment has effect potential bioaccumulation in the food chain, and the chronic toxicity [3,6]. Two ways to eliminate naproxen from the environment are known: physicochemical methods and biological methods. Physicochemical methods often lead to more toxic products than parent compounds [2]. The alternative are biological methods. Until now naproxen has been completely transformed by white-rot fungi and some bacterial strain [4,7].

The present work aim was assess the capability of Bacillus sp. B1(2015b) to naproxen cometabolic biological transformation with glucose as a carbon and energy source and in the presence of aromatic compounds and organic solvents as potential inhibitors. Phenol and 4-chlorophenol were chosen as pollutions which have been found in water [5], while methanol, ethanol and DMSO were used as common organic solvents. Bacillus sp. B1(2015b) was used to in these experiments due to capability to complete biotransformation of 3 mg/l of naproxen in cometabolic conditions with glucose in concentration 500 mg/l within the period of month.

The study showed that phenol does not have influence on biotransformation of naproxen such as methanol and DMSO while 4-chlorophenol slightly decreased transformation process. The biotransformation rate was accelerated by ethanol.

Acknowledgements

This work was financed by the National Science Centre (Poland), granted on the basis of decision

DEC-2013/09/B/NZ9/00244.

References

[1] E. Carmona, V. Andreu, Y. Pico, Science of the total Environment, Occurrence of acidic pharmaceuticals and personal care products in Turia River Basin: From waste to drinking water, 2014, Vol. 484, No. 1, 53-63.

[2] Y.H. Hsu, Y.B. Liou, J.A Lee, C.Y Chen, A.B Wu, Biomedical chromatograph, Assay of naproxen by high-performance liquid chromatography and identification of its photoproducts by LC-ESI MS, 2006, Vol. 20, No. 8, 787-793.

[3] R. Nesbitt, Master of Science in Applied Bioscience, University of Ontario Institute of technology. Oshawa Effects of chronic exposure to ibuprofen and naproxen on Florida flagfish (Jordanella floridae) over one complete life-cycle, 2011, 1-153.

[4] E. Marco-Urrea, M. Pérez-Trujillo, P. Blánquez, T. Vicent, G. Caminal, Bioresource Technology, Biodegradation of the analgesic naproxen by Trametes versicolor and identification of intermediates using HPLC-DAD-MS and NMR, 2010, Vol. 101, No. 7, 2159–2166.

[5] J. Michałowicz, Polish Journal of Environmental Studies. The occurrence of chlorophenols, chlorocatechols and chlorinated metoxyphenols in drinking water of the largest cities in Poland. 2005, Vol. 14, No. 3, 327-333.

[6] J. Parrot, D.T. Bennie, Journal of Toxicology and Environmental Health, Life-cycle exposure of Fathead minnows to a mixture of six common pharmaceuticals and triclosan, 2009, Vol. 72, No. 10, 633-641.

[7] D. Wojcieszyńska, D. Domaradzka, K. Hupert-Kocurek, U. Guzik, Journal of Environmental Management,

Bacterial degradation of naproxen – undisclosed pollutant in the environment, 2014, Vol. 145, 157-161.

L25

Steroid C25 Dehydrogenase, the Bocatalyst for Production of Calcifediol and 25-Hydroxycholesterol

Maciej Szaleniec

1, Agnieszka Rugor

3 and Anna Wójcik

2

1 Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, Kraków, Poland – ncszalen@cyfronet.pl

2 Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry, Biophysics and Biotechnology,

Jagiellonian University

Keywords: steroid dehydrogenase, molybdenum enzyme, calcifediol

Steroid C25 dehydrogenase (S25DH) is a bacterial from Sterolibacterium denitrificans catalyzing regioselective hydroxylation of a wide range of 3-hydroxy and 3-ketosterols at C25 carbon atom of the sterol aliphatic side chain. It belongs to EBDH-like subclass of DMSO reductases that in their active site contain a bis-molybdopterin cofactor (Mo-co) which is responsible for both C-H activation of the hydrocarbon and the oxygen transfer [1,2]. Recently it has been demonstrated that it can also be used as an efficient catalyst in the regioselective hydroxylation of cholecalciferol (vitamin D3) to calcifediol [3] and cholesterol to 25-hydroxycholesterol [4]. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D3 used in the treatment of rickets and other calcium disorders.

S25DH was studied by a combination of experimental and theoretical techniques. The kinetic and reactor studies provided a valuable insight not only to the best conditions in which the enzyme can be efficiently employed in hydroxylation of valuable substrates but also delivered information on the rate limiting steps in the reaction mechanism. The latter was achieved with kinetic tests with isotope-labeled substrates such as deuterated cholesterol or

18OH2.

The mechanism of the enzymatic reaction was also studied with modeling tools. Due to the lack of structural data on S25DH such studies required development of a homology model. 100 models were constructed based on the geometry of ethylbenzene dehydrogenase (PDB 2IVF). The best model was selected by docking of the known S25DH substrates and next it was subjected to MD simulations with various substrates. And the geometry of the relaxed structure with low total energy and catalytically active conformation of the substrate was minimized with both MM and QM:MM methods. The mechanism was studied with two simplified cluster models comprised of 83 or 198 atoms. The QM calculations were conducted within DFT theory using B3LYP functional with IEFPCM solvent correction and D2 correction for vdW effects.

On the other hand the modeling studies showed two types of the substrate binding mode, i.e. presenting sterol ring methyl groups towards or away from the Mo-co. These two binding modes seem to be associated with different probability of reactive conformation of the alkyl side chain. The QM-only modeling showed that the mechanism proceeds along similar pathway as that proposed for EBDH [3]: i.e. i) rate limiting C-H activation by Mo=O ligand followed by formation of intermediate product, ii) OH transfer from Mo-OH to the intermediate and iii) addition of H2O to Mo atom followed by enzyme re-oxidation and ligand deprotonation. However, it turned out that the radical intermediate (and Mo

V Mo-co state) is more probable than carbocation (and Mo

IV). The predicted

intrinsic kinetic isotope effect for cholesterol with deuteron at C25 (7.1) is in a good agreement with the observed experimental value (4.5).

The thorough optimization of reactor conditions showed that synthesis efficiently in fed-batch reactors under anaerobic conditions, with 6–9% of an organic co-solvent, 2-methoxyethanol, and the content of 2-hydroxypropyl--cyclodextrin solubilizer adjusted to the substrate type, using 8–15 mM K3[Fe(CN)6] as an electron acceptor. Reaction conducted in such conditions results with high product concentrations (0.8 g/L for 25-hydroxycholesterol and 1.4 g/L for calcifediol) and even higher concentrations can be reached through the hydroxylation of 3-ketosterols (2.2 g/L).

Acknowledgements

Acknowledgement: Authors acknowledge the financial support of the National Center of Science grant

SONATA UMO-2012/05/D/ST4/00277. References

[1] J. Dermer, G. Fuchs, J. Biol. Chem. 2012, 287, 3690 [2] J. Heider, M. Szaleniec, K. Sünwoldt , M. Boll, J. Mol. Microbiol. Biotechnol. 2016, 26, 45-62 [3] M. Warnke et al. Angew. Chem. Int. Ed. 2016, 55, 1881-4. [4] A. Rugor , A. Bojarski, M. Szaleniec, Annual Report, 2015, 46-48

42

L26

Peroxidase Activity and Physicochemical Characterization of DNAzymes Created by G-quadruplex with Covalently Attached Hemin

Krzysztof Żukowski

1, Joanna Kosman

1 and Bernard Juskowiak

1

1 Laboratory of Bioanalytical Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Poznań, Poland –

krzysztof.zukowski@amu.edu.pl

Keywords: DNAzyme, G-quadruplex, Peroxidase activity

G-quadruplexes (G4-DNA) are the nucleic acids structures formed by oligonucleotides rich in guanines. These

structures are stabilized by a Hoogsteen’s hydrogen bonds between guanines and also by a cation (usually potassium) which is located in the channel between the guanine tetrads. Depending on the direction of the DNA strand it can form G-quadruplex of parallel, antiparallel or hybrid topology [1,2].

G4-DNA is able to interact with planar molecules via end stacking or other more specific interactions. As a result of these interaction G-quadruplex can form a complex with porphyrin molecule of hemin. It is proved, that this complex can catalyze peroxidation reaction. The guanosine’s oxygen atoms interact with iron atom, located in hemin molecule (cofactor of this DNAzyme), thereby increasing the enzymatic activity. Oligonucleotide enzymes are much better than protein enzymes, because they are more stable in high temperature. Other advantage of this compounds is the fact, that enzyme-based nucleic acid, enabling the creation of new analytical strategies utilizing the phenomenon of hybridization between complementary DNA strands. [2]

Fig. 1 Scheme of formation peroxidase mimicking DNAzyme.

The problem of measurements with these complexes is the fact that hemin present in the blind experiment

exhibit peroxidase activity. In result of that this experiment gives an analytical signal reducing the sensitivity of the assays based on these type of enzyme at the same time. To sort out this problem, researchers have launched a study on the modification of DNAzymes by covalently attaching hemin molecule to the oligonucleotide. [3,4] In our research we used amino-modified oligonucleotides and NHS-active esters of hemin in the reaction of conjugation. Such synthesized DNAzymes were characterized spectroscopically. In the next step we measured the most important feature of DNazymes, the peroxidase activity. For this purpose we used the reaction of Amplex Red oxidation which results in high fluorescence signal. The research of our research group prove that DNAzymes with covalently attached hemin have a better perspective on their using as an alternative to horseradish peroxidase commonly used in ELISA or Western blot tests.

Acknowledgements

All the Authors are kindly thanked for supporting by a National Science Center Poland – Project “HARMONIA”

2013/10/M/ST4/00490.

References

[1] D.E. Gomez, R.G. Armando, H.G. Farina, P.L. Menna, C.S.Cerrudo, P.D. Ghiringhelli, D.F. Alonso, Int. J. Oncol. 2012, 1561-1569

[2] P. Travascio, Y. Liu, D. Sen, Chem. Biol., 1998, 505-517 [3] S. Nakayama, J. Wang, H.O. Sintim, Chem. Eur. J. 2011, 5691-5698 [4] A.V. Gribas, S.P. Korolev, T.S. Zatsepin, M.B. Gottikh, I.Y. Sakharov, RSC Adv 2015, 51672-5167

43

L27

Evaluation of Cosmetic Usefulness of Biotechnologically Processed Oilseeds Cake

Anna Ratz-Łyko, Jacek Arct and Katarzyna Pytkowska 1 Department of Cosmetic Chemistry, Academy of Cosmetics and Health Care, Warsaw, Poland

Keywords: seedcakes, enzymatic hydrolysis, polyphenols, antioxidant capacity, anti-inflammatory activity, enzymes inhibition,

hydration state and epidermal barrier function, stripping of stratum corneum

The seedcakes of Oenothera biennis (evening primrose), Borago officinalis (starflower), Nigella sativa (black cumin) are found to be rich sources of bioactive compounds, such as flavonoids, phenolic acids and hydrolysable tannins, triterpenes and others [1,2]. Many of them have antioxidant activity, inhibiting oxidation caused by UV radiation and lipid peroxidation, by scavenging free radicals and the reactive oxygen species and have anti-inflammatory activity and inhibition of metalloproteinase activity [3,4]. However, due to the presence of polyphenols in the form of esters and glycosides, these compounds have a limited bioavailability and a lower dermal biological activity as compared to the aglicones. The seedcakes have not yet been used in cosmetology. The aim of the present study was to evaluate the cosmetic suitability of seedcakes processed by enzymatic hydrolysis. The enzymatic hydrolysis by α-amylase, β-glucosidase, β-glucanase (1:1:1) solution was used to bioconversion of glycosides of polyphenols to free aglycones form. The water-ethanolic extracts (1:1) obtained from biotechnologically processed seedcakes were used in cosmetic formulations. In order to evaluate the biological and cosmetic properties of extracts obtained from seedcakes series of experiments: in vitro (polyphenol content, antioxidant capacity, enzymes inhibition activity, antimicrobial activity), in vivo (hydration, TEWL, anti-inflammatory activity), ex vivo (stripping of stratum corneum) were done. The results of in vitro, in vivo and ex vivo studies indicate that extracts from seedcakes due to the antioxidant and anti-inflammatory activities have potential application in anti-aging, moisturizing, mitigating and protective cosmetics. Furthermore, biotechnological processing of seedcakes by enzymatic hydrolysis allows to increase their biological properties.

References [1] A.K. Kiss, A. Kapłon-Cieślicka, K.J. Filipiak, G. Opolski, M. Naruszewicz, Phytother. Res. 2012, 26, 482–487. [2] A.K. Kiss, M. Naruszewicz, Food Chem. 2012,131, 485–492. [3] M. Wettasinghe, F. Shahidi, Food Chem. 2000, 70,17 – 26. [4] M. Wettasinghe, F. Shahidi, J. Agric. Food Chem. 1999, 47, 1801–1812.

44

L28

Biocatalytic Modifications of Resveratrol. Małgorzata Brzezińska-Rodak

1, Ali Kuru

2, Alicja Zakrzewska

1, Sylwia Siudakiewicz

1, Magdalena

Klimek-Ochab1, Ewa Żymańczyk-Duda

1

1 Department of bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland

2 Chemistry Department, Sakarya University, Sakarya, Turkey

Keywords: resveratrol, biocatalysis, O-methylation

Resveratrol, compound, located in the red wine, became important since it was recognized as the one responsible for the “French phenomenon”, that is the good health of French people regardless the fat diet [1]. Resveratrol belongs to the stilbenoids – hydroxylated, stilbene derivatives of mostly trans configurations and of confirmed antioxidative and anticancer activities [2]. Taking into account its activity and the increasing application in food, pharmaceutical and cosmetics industries, there is a need to find the novel methods of synthesis of its derivatives of better properties. Especially biocatalyzed synthesis of O-methylated derivatives is important because of the further their applications as anticancer agents [3,4]. This depends on the conformations of the compounds (cis/trans), so that, selective biotransformations appeared to be preferable methods. Biocatalysts of different origin: prokaryotic (Streptomyces griseus) and eukaryotic (Aspergillus sp., Phanerochaete chrysosporium) were tested in order to obtain new methylated derivatives of resveratrol. Preliminary results are very promising, especially when Aspergillus parasiticus and Aspergillus sydowii were applied.

Fig. 1 Biocatalytic O-methylation of resveratrol

References

[1] Stervbo U., Vang O., Bonnesen Ch. Food Chem., 2007, 2 (101), 449-457. [2] Roberti M. et al. J Med Chem., 2003, 46, 3546-3554. [3] Weng C.J., Wu C.F. et al. J Agric Food Chem., 2010, 58, 2886- 2894. [4] Mazue F., Colin D. et al. Eur J Med Chem., 2010, 45, 2972-2980.

45

L29

Biosolubilisation of Brown Coal by Recombinant Laccase

Natalia Kwiatos1, Bartosz Strzelecki

1 and Stanisław Bielecki

1

1 Institute of Technical Biochemistry, Faculty of Food Science and Technology, Lodz University of Technology, Lodz, Poland –

natalia.kwiatos@dokt.p.lodz.pl

Keywords: laccase, brown coal, biodegradation

Brown coal is one of the most important sources of energy in Poland next to hard coal and gas. Unfortunately, many factors, such as high emission of NOx and SOx and problems with mining, makes it controversial to use directly for combustion. Therefore, there is a need for development of clean coal technology, which would result in obtaining environmentally-friendly material. Biosolubilization is a biological method that uses microorganisms, for example fungi, Fusarium oxysporum, and their products (biosurfactants, enzymes) to convert rock into cleaner black liquid [1,2].

Here we present an efficient two-step lignite biosolubilization. Firstly, the pretreated brown coal is solubilized by F. oxysporum 1101 which results in considerable decrease in sulfur and mercury content in liquid product. In the second step, the liquefied coal is subjected to action of recombinant laccase from F. oxysporum. The experiment was conducted in presence and absence of synthetic or natural mediatiors in optimal enzyme conditions. The process results in high degree of lignite solubilisation, which is illustrated by 50% increase in fulvic acids release. Moreover, the Fourier transform infrared spectroscopy confirms significant changes in coal structure after treatment with laccase.

References

[1] A. Tajduś, P. Czaja and Z. Kasztelewicz, Gór. Geoninżynieria 2011, 2011, 343-365. [2] R.M. Fakoussa and M. Hofrichter, Appl. Microbiol. Biotechn. 1999, 52, 25-40

46

L30

Biocatalyzed Synthesis of Tyrosol Derivatives Beata Szmigiel

1, Ewa Żymańczyk-Duda

2, Małgorzata Brzezińska-Rodak

3

1

Department of Bioorganic Chemistry, Wroclaw University of Technology, Wroclaw, Poland- beata.szmigiel@pwr.edu.pl 2

Department of Bioorganic Chemistry, Wroclaw University of Technology, Wroclaw, Poland 3

Department of Bioorganic Chemistry, Wroclaw University of Technology, Wroclaw, Poland

Keywords: tyrosol, hydroxytyrosol, antioxidants

Antioxidants are compounds having ability to neutralize free radicals- reactive molecules, which stabilizing their structures via interaction with biological molecules, causing their degradation. Antioxidants provide positive effect in the treatment and prevention of such illnesses, which manifest as: cancer, cardiovascular diseases, diabetes, brain stroke, skin diseases. That knowledge about destructive effect of free radicals induces to constantly seek for new substances able to prevent organism damages [1].

There is an interest in the use of biotechnological methods for obtaining various compounds using environmentally friendly methods. Some of them are already introduced into the pharmaceutical and cosmetic companies, which successfully applied biotransformation methods with whole cells biocatalysts or isolated enzymes for the defined compounds synthesis.

The aim of the study is the use of microorganisms for the synthesis of antioxidants- namely tyrosol derivatives, searching for natural biocatalysts with specific activity. The crucial idea is to elaborate the method of conversion of not expensive starting materials (2-phenylethanol) into desired products (tyrosol and hydroxytyrosol) in as simple as possible manner, ending by obtaining of the expected products.

Preliminary results shows that biohydroxylation of 2-phenylethanol catalyzed by Aspergillus niger and

Rhizopus oryzae leads to the tyrosol [2-(4-hydroxyphenyl)ethanol] and then to hydroxytyrosol [2-(3,4-

dihydroxyphenyl)ethanol]. These compounds are known from their extraordinary antioxidant activities and are

usually derived from the natural sources, such as olive oil via expensive extractive methods [2,3]. That is why the

pursuit of the alternative methods of their preparation is still under consideration. Already performed experiments

confirm that Aspergillus niger and Rhizopus oryzae strains are able to hydroxylate the 2-phenylethanol to desired

antioxidants. Biotransformation method using Aspergillus niger strain is relatively a new method, that uses directly

spores of A. niger to the biotransformation, which are recovered from solid medium in Petri dishes, without

inoculation on liquid medium. The results were evaluated using HPLC method. Further studies are intended to

optimize the biocatalytic protocol, to increase the efficiency and the scale of the process.

References [1] Andreassi M, Andreassi L. Antioxidants in dermocosmetology: from the laboratory to clinical application. Journal of Cosmetic Dermatology 2004; 2: 153–160. [2] Ting Hu, Xiao-Wei He, Jian-Guo Jiang, and Xi-Lin Xu. Hydroxytyrosol and Its Potential Therapeutic Effects. J. Agric. Food Chem 2014; 62: 1449−1455. [3] M Nieves Franco, Teresa Galeano-Díaz, Óscar López. Phenolic compounds and antioxidant capacity of virgin olive oil. Food Chemistry 2014; 163: 289–298.

47

Posters

48

49

P01

Bio-based Redox Systems Applied to Mild and Selective Transformations Iván Lavandera

1, Alba Díaz-Rodríguez

1, María López-Iglesias

1, Juan Mangas-Sánchez

1, Lía Martínez-

Montero1, Daniel-Méndez-Sánchez

1, Ángela Mourelle-Insua

1, Nicolás Ríos-Lombardía

1, Vicente

Gotor-Fernández1 and Vicente Gotor

1

1 Organic and Inorganic Chemistry Department, Universidad de Oviedo, Oviedo, Spain – lavanderaivan@uniovi.es

Keywords: alcohol dehydrogenases, laccases, redox processes

The use of redox enzymes applied to the synthesis of valuable molecules has been envisioned as an appealing tool along the last years. In this sense, the application of biocatalysts that can perform mild oxidative reactions employing oxygen as final electron acceptor, or highly selective reductive transformations under hydrogen transfer conditions, has widened the operational window of enzymes applied to industrial processes [1].

Recently, our efforts have been focused on the development of novel redox systems to obtain valuable derivatives making use of laccases and alcohol dehydrogenases (ADHs). Laccases can be found in many fungi, plants and bacteria and, as multicopper-containing catalysts, which are able to activate oxygen to oxidize phenol compounds or aromatic amines. However, to act towards non-activated alcohols or amines, a chemical mediator is necessary, thus providing a laccase mediator system (LMS) [2]. On the other hand, ADHs have provided very efficient and selective reactions to transform carbonylic compounds into chiral alcohols and vice versa [3]. They need a nicotinamide cofactor but the coupling of efficient recycling techniques makes possible the use of catalytic amounts of these expensive molecules.

Herein we will describe some of our recent results (Fig. 1) regarding the oxidation of diols and amino alcohols into the corresponding lactones or hemiaminals, respectively [4], [5], using a laccase from Trametes versicolor with 2,2,6,6-tetramethylpiperidinoxyl radical (TEMPO) as mediator. This chemoenzymatic system has been successfully applied to the deprotection of the benzyl group on a series of primary amines under very mild conditions in aqueous medium [6]. Also, it has been employed together with horse-liver alcohol dehydrogenase to deracemize a profenol derivative in a one-pot sequential manner under dynamic conditions [7]. A similar concept has been performed to deracemize different secondary alcohols through sequential non-selective oxidation catalyzed by the iodine-TEMPO system, followed by stereoselective reduction of the ketone intermediate with an ADH [8].

laccases and/or

alcohol dehydrogenases

Mild oxidative

processes

Deprotection

protocols

Deracemization

systems

Fig. 1

References

[1] B. M. Nestl, B. A. Nebel, and B. Hauer, Curr. Opin. Chem. Biol. 2011, 15, 187. [2] M. Mogharabi and M. A. Faramarzi, Adv. Synth. Catal. 2014, 356, 897. [3] C. V. Voss, C. C. Gruber, and W. Kroutil, Synlett 2010, 991. [4] A. Díaz-Rodríguez, I. Lavandera, S. Kanbak-Aksu, R. A. Sheldon, V. Gotor, and V. Gotor-Fernández, Adv.

Synth. Catal. 2012, 354, 3405. [5] A. Díaz-Rodríguez, L. Martínez-Montero, I. Lavandera, V. Gotor, and V. Gotor-Fernández, Adv. Synth. Catal.

2014, 356, 2321. [6] L. Martínez-Montero, A. Díaz-Rodríguez, V. Gotor, V. Gotor-Fernández, and I. Lavandera, Green Chem. 2015,

17, 2794. [7] A. Díaz-Rodríguez, N. Ríos-Lombardía, J. H. Sattler, I. Lavandera, V. Gotor-Fernández, W. Kroutil, and V.

Gotor, Catal. Sci. Technol. 2015, 5, 1443. [8] D. Méndez-Sánchez, J. Mangas-Sánchez, I. Lavandera, V. Gotor, and V. Gotor-Fernández, ChemCatChem

2015, 7, 4016.

50

P02

Microbial Biotransformation of Bufalin Olga Andrzejczak

1

1 Department of Biotechnology and Food Sciences, Lodz University of Technology, Lodz, Poland –binoz.p.lodz.pl

Keywords: biotransformation, steroid compounds, bufalin

The steroid compounds are some of the most sold products of the pharmaceutical industry [1]. The aim of this work is characteristic of bufalin.

This steroid was originally isolated from the Chinese toad venom [1]. It is a component found in many chinese traditional medicine and shows potential antitumor effect against various malignancies, eg. hepatocellular [2] and lung carcinoma [3]. Research on bufalin has so far mainly involved leukemia, prostate cancer, gastric cancer and liver cancer, and has been confined to in vitro studies [4]. Simultaneously, this compound exhibits cardiotoxic effects [5]. In such situation, biotransformation has played a role in generating new, safer and more active derivatives.

The microorganisms used for this purpose are, for exzample, the strains of Alternaria alternate [6] and Pseudomonas aeruginosa [7]. Others microorganisms used for this purpose are Nocardia sp [8] and Mucor polymorphosporus [6].

Microbial hydrolysis can achieve very high yield. One strain of Alternaria alternate is able to complete metabolized of cinobufagin and resibufogenin to their 12β-hydroxylated products in greater than 90% yield within 8 h. [6]

Fig. 1 The structure of bufadienolide.

References

[1] A. Kamboj and all, International J. of Pharmacy and Pharmaceutical Sciences 2013, 4, 20 [2] Z.J. Zhang and all, J. of Translational Medicine 2014, 12, 57. [3] S.H. Wu and all, The American journal of Chinese medicine 2014, 42, 719. [4] P. H. Yin and all, Asian Pacacyfic J. of Cancer Prevention 2012, 13, 5339. [5] H. Ma and all, Experiomental and Toxicologic Pathology 2012, 64, 417. [6] M. Ye and all, Tetrahedron 2005, 61, 8947 [7] J. Zhang and all, Enzyme and Microbial Technology 2003, 33, 29 [8] J. Zhang and all, Bioorganic & Medicinal Chemistry 2007, 17, 6062

51

P03

Enzymatic Synthesis of β/α-Peptides in Aqueous Medium Krzysztof Morawski

1, Aneta Białkowska

2 and Beata Kolesińska

3

1 Institute of Technical Biochemistry, Lodz University of Technology, Lodz, Poland – krzysztof.morawski@dokt.p.lodz.pl

2 Institute of Technical Biochemistry, Lodz University of Technology, Lodz, Poland

3 Institute of Organic Chemistry, Lodz University of Technology, Lodz, Poland

Keywords: biotransformations, enzymatic peptide synthesis, β/α-peptides,

Peptides play a range of important functions in organisms – from neurotransmitters, through hormones up to antibiotics. Thanks to their properties, they have been used in numerous industries (including food, cosmetics and pharmaceutical ones) [1].

Recently pharmaceutical companies have become interested in β- and β/α-peptides. The presence of β-amino acids in a peptide chain significantly increases its resistance to proteolysis [2]. The first reports concerning this issue show that peptide therapeutic substances including these particles in their structure demonstrate longer biological activity in the body in comparison with homologs built exclusively of α-amino acids.

β-peptides can be obtained by chemical synthesis. Limitations of chemical methods determine the search of other ways of obtaining these valuable substances. Enzymatic methods seem to be an efficient alternative.

During the enzymatic synthesis of peptide bonds, proteolytic enzymes are used as catalysts. This process can take place under kinetic or thermodynamic control [3]. So far, the literature have described only a few enzymes demonstrating biological activity and having substrate specificity towards β-peptides. Due to their uniqueness and application potential, these biocatalysts are extremely valuable.

In the Institute of Technical Biochemistry of Łódź University of Technology preliminary research was carried out on the possibilities of using the producers of proteolytic enzymes deposited in the collection of the Institute of TB in the synthesis of β/α peptides.

The research covered 5 strains of Bacillus type and three strains of cold adapted microorganisms (two strains of Glaciozyma martini and one strain of Pseudoalteromonas type). The reactions were conducted under kinetic control in aqueous medium. As an acyl group donor, 4 substances were used, each of which had an activated amino group β-alanine at the C-terminal end. Three short peptides were used as a nucleophile. MS analyses of post-reaction liquids proved that in all the variants tested so far the expected synthesis products had been obtained. The results seem to indicate a big potential of the tested preparations in the synthesis of β/α-peptides and can be a starting point for the optimization of the yield.

References

[1] K. Fosgerau, and T. Hoffmann, Drug Discovery Today 2015, Vol 20, 122-128. [2] G. Lelais, and D. Seebach, American Peptide Society 2004, Vol 76, 206-243. [3] F. Guzman, and S. Barberis, Electronic Journal of Biotechnology 2007, Vol 10, 279-315.

52

P04

Broad Substrate-Specifity Aminotransferase from the Antarctic Bacterium Psychrobacter sp. B6 as Unique Tool for Biotransformations of Amino Acids

Klaudia Jadczak

1, Tomasz Florczak

2 and Marianna Turkiewicz

3

1,2,3

Institute of Technical Biochemistry, Lodz University of Technology, Lodz, Poland – klaudia.jadczak@dokt.p.lodz.pl

Keywords: aminotransferase, biotransformations, unnatural amino acids

The increasing demand by the pharmaceutical industry for peptidomimetics is driven by the requirement for

single stereoisomers of pharmaceutical compounds such as unnatural and nonproteinogenic amino acids. Biotransformations have become key technologies to produce chiral substances. One of the several used biocatalysts to apply in industrial production of mentioned above a valuable key intermediates – are aminotransferases [1]. They are useful in synthesis of unnatural substrates, because of their capability to reversible transfer of amino groups from a donor substrate to an acceptor. Moreover they are characterized by broad substrate specificity, high enantioselectivity and regioselectivity, high reaction rate and no requirement of addition of cofactor. The existence of transaminases with broad substrate specificity for the synthesis of either D- or L-amino acids makes these enzymes attractive for this type of application, in which the desired products are structurally diverse [2,3]. Examples of unnatural amino acids being involved in the biosynthesis of some therapeutic targets are given below (Fig.1).

The poster shows properties of the psychrophilic aromatic aminotransferase from the Antarcic soli bacterium Psychrobacter sp. B6. Gene isolation, protein expression and purification were done. Analysis indicated dual funcionallity of the enzyme through the ability to carry out the tranamination reaction with aromatic amino acids and aspartate as substates. Kinetic studies were reported. The role of the aminoacid residues responsible for substrate binding in the active site of the enzyme was explained [4]. These results give a good grounding for further research on the modification of the catalytic center of the protein in order to increase its productivity and selectivity in the production of useful chiral building blocks.

NH2

COOH

NH2

OH

COOHNH

2

COOH

NH2

COOH N

NNH

2

COOH

Fig1. Diversity of potential products obtained by transamination reaction: 1-aromatic amino acids (L-diphenylalanine), 2-aromatic β-amino acids (L-phenylisoserine), 3-aliphatic amino acids (L-2-aminobutyric acid),4- aliphatic β-amino acids (L-3- aminobutyric acid), 5- β-heterocyclic amino acids (L-thienylalanine)

References

[1] D. Mundoz Solano, P. Hoyos, M.J. Hemaiz, A.R. Alcantara, J.M. Sanchez-Montero Bioresource Technol.

2012, 115, 196-207 [2] B.Y. Hwang, B.K .Cho, H.Yun, K. Koteshwar, B.G. Kim J. Mol. Catal. B: Enzym. 2005, 37, 47-55 [3] J. Ward, R. Wohlgemuth Curr. Org. Chem. 2010, 14 [4] A. Bujacz, M. Rutkiewicz-Krotewicz, K. Nowakowska-Sapota, M. Turkiewicz Acta Cryst. 2015, 71: 632-645

1. 2. 3.

4. 5.

53

P05

Monoamine Oxidase from Extremophilic Fungi, as a Tool for Synthesis of

Chiral Amine Building Blocks of Pharmaceutical Drugs.

Iga Jodłowska1, Tomasz Florczak

2 and Marianna Turkiewicz

3

1 Institute of Technical Biochemistry, Lodz University of Technology, Lodz, Poland – jodlowska.iga@gmail.com

2 Institute of Technical Biochemistry, Lodz University of Technology, Lodz, Poland

3 Institute of Technical Biochemistry, Lodz University of Technology, Lodz, Poland

Keywords: monoamine oxidase, amine building blocks, HCV

In vivo monoamine oxidases a flavoenzyme catalyze reaction of oxidative deamination of primary and secondary amines including neurotransmitters such as dopamine and serotonin (polyamines) to imines which further reacts to ketons or aldehydes and ammonia. The reaction of oxidative deamination is highly selective, enzyme is able to successively transform/oxidize only one amine enantiomer [1]. According to its highly specificity, and role in amine metabolism it has a great potential in medical chemistry. As it is well known, amines and their derivatives are common compounds of pharmaceutical drugs. The most essential are cyclic amines and their derivatives like pyrrolidines [2]. Our approach is to generate amine building blocks of HCV NS3/4A protease inhibitors, like boceprevir, telaprevir, asunaprevir, simeprevir [3],[4]. The use of biotransformation could allow faster development of drugs and more economical production of those inhibitors.

Poster will present results of preliminary research, that are focused on screening and isolation of monoamine oxidases from extremophilic fungi.

References

[1] M. Höhne, and U. T. Bornscheuer, ChemCatChem. Biocatalitic Routes to Optically Active Amines 2009, 1, 42-51.

[2] V. Köhler, K. R. Bailey, A. Znabet, J. Raftery, M. Helliwell and N. J. Turner, Angew.Chem. Enantioselective Biocatalytic Oxidative Desymmetrization of Substituted Pyrrolidines 2010, 49, 2182-2184.

[3] A. Znabet, M. M. Polak, E. Janssen, F. J. J. de Kanter, N. J. Turner, R. V. A. Orru and E. Ruijter, Chem.Commun. A highly efficient synthesis of telaprevir by strategic use of biocatalysis and multicomponent reactions 2010, 46, 7918-7920.

[4] T. Li, J. Liang, A. Ambrogelly, T. Brenan, G. Gloor, G. Huisman, J. Lalonde, A. Lekhal, B. Mijts, S. Muley, L. Newman, M. Tobin, G. Wong, A. Zaks and X. Zhang, J.Am.Chem.Soc. Efficient, Chemoenzymatic Process for Manufacture of the Boceprevir Bicyclic [3.1.0]Proline Intermediate Based on Amine Oxidase-Catalyzed Desymmetrization 2012, 134, 6467-6472.

54

P06

Chitin Deacetylases from Mucor circinelloides IBT-83, as a Potential Tool for Biotechnological Chitosan Production

Michał B. Kaczmarek, Katarzyna Struszczyk-Świta and Mirosława Szczęsna-Antczak Institute of Technical Biochemistry, Lodz University of Technology – michal.kaczmarek@dokt.p.lodz.pl

Keywords: chitin, chitosan, chitin deacetylase, enzymatic modifications

Chitin, a linear homopolysaccharide composed of (1→4)-2-acetamido-2-deoxy-β-D-glucopyranose units, is one of the most abundant and renewable natural polymer, which has a number of unique properties. Chitin is an extremely insoluble in water and organic solvents, which significantly limited its industrial use, whereas N-deacetylated derivatives, chitosan and chitooligosacharydes are soluble in acidic solutions, which makes them more applicable [1]. Beneficial properties of chitosan and oligosaccharides include: antifungal, antibacterial [2,3], anti-inflammatory [4], antitumor [5] and neuroprotective activities [6].

In order to produce chitosan from chitin, high alkali concentrations at high temperatures are used that might cause uncontrolled depolymerization and increased amorphism [7]. Alternatively or complementary the deacetylation can be achieved enzymatically, using chitin deacetylase (ChDa EC 3.5.1.41) as a useful tool towards biotechnological chitosan production especially when a controlled, non-degradative, and well defined process is required [8].

In this report we present current state of ours research. ChDa activity was detected in partially purified and concentrated crude extract of protein from the cells of Mucor circinelloides IBT-83. Additionally, two open reading frames (ORF) putatively encoding ChDa were identified and amplified from cDNA of this strain. Each ORF were molecularly cloned and sequenced. Nucleotide sequences were translated in silico and analyzed in order to detect conserved amino acid sequences which are involved in binding acetate groups from chitin and/or chitosan. In the future research an attempt will be made to construct a multi-enzyme complex enabling the one-step and selective modification of chitin and chitosan. Chitin deacetylase (ChD), chitosanase (EC 3.2.1.132) and chitinase (EC 3.2.1.14) potentially will be involved in this complex.

References

[1] M. Rinaudo, Prog. Polym. Sci. 2006, 31, 603-632 [2] J.C. Fernandes, F. K. Tavaria, J.C. Soares, O.S. Ramos, M.J. Monteiro, M. E. Pintado, et al., Food Microbiol.,

2008, 25, 922-928 [3] Y. Wang, P. Zhou, J. Yu, X. Pan, Wang , W. Lan, et al.; Asia Pac J. Clin. Nutr. 2007, 16, 174-177 [4] E.J. Yang, J.G. Kim, J.Y. Kim, S. Kim, N Lee; Cent Eur J Biol, 2010, 5, 95-102 [5] H. Quan, F. Zhu, X. Han, Z. Xu, Y. Zhao, Z. Miao; Med. Hypotheses, 2009, 73, 205-206 [6] R. Pangestuti, S.K. Kim, Marine Drug, 2010, 8, 2117-2128 [7] K. L.Chang, G. Tsai, J. Lee, W.R. Fu, Carbohyd. Res., 1997, 303, 327-332 [8] Y-J. Kim, Z. Yong, K-T. Oh, V-N. Nguyen, R-D. Park, J. Microbiol. Biotechnol, 2008, 18 (4), 759-766

55

P07

Transesterification of Sunflower Oil by Lipases: Influence of Diethylamine and Water on Different Lipases

Jakub Szeląg, Aleksandra Tarasiuk, Mirosława Szczęsna-Antczak and Tadeusz Antczak Department of Biotechnology and Food Science, Technical University of Lodz, Lodz, łódzkie

Keywords: transesterification, lipase, esters

It is well known that water plays a key role in functioning of living beings and therefore influence biomolecules. In enzymatic processes water stabilize spatial structure of proteins, maintaining their function. [1] Moreover, in enzymatic processes associated with hydrolysis, the amount of water has direct influence on reaction direction.

Diethylamine (DEA) is a secondary amine with two ethyl groups. As our previous experiments shown, its addition can increase the efficiency of esters synthesis . A mechanism of these processes have not been described yet however, a possible explanation is that DEA increase reaction efficiency by alkalization of reaction environment or directly influences enzyme structure.

During the research an influence of simultaneous addition of different amount of DEA(0-60mM) and water (0-3%) for reaction with different immobilized lipase (lipase TL IM and Mucorcircinnelloides) was studied. The aim of investigation was to check whether previously observed influences occurred only for Mucorcircinelloides lipase or are universal. It was found that not only esters’ synthesis efficiency was changing for different DEA addition, but also addition of DEA influenced the water content in product. In samples where esters ‘ synthesis was the highest, the lowest change of water content between water in substrate and products was noticed. Phenomena of changing of water content was observed for both lipase preparations.

Fig. 1 Esters yield and water content as a function of diethylamine addition.

References

[1] Adachi D, Koda R, Hama S, Yamada R, Nakashima K, Ogino C, Kondo A. Enzyme Microb. Technol 2013 52 118– 122.

0,1

0,2

0,3

0,4

0,5

65

70

75

80

0 10 20 30 40 50 60

Wat

er c

on

ten

t in

pro

du

cts

[%]

Este

rs y

ield

[%

]

DEA addition [mM]

TL IM esters

MC esters

TL IM water

MC water

56

P08

Wastewater Purification Potential of Bacillus cereus in Alliance with Pistia stratiotes

Muhammad Faisal Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore-54590, Pakistan *Corresponding author (Tel: 92-42-35952811 E-mail: mohdfaysal@yahoo.com)

Keywords: Pistia stratiotes, Bacillus cereus, chromium accumulation, chromate reduction

This study deals with chromate resistant bacterial strain Bacillus cereus which has synergistic impact in

association with Pistia stratiotes to detoxify toxic chromium from wastewaters. At 100 µg ml-1

, P. stratiotes plant

was able to uptake 15 mg Cr g-1

after 48 h while bacterial inoculation caused 12.5 mg Cr g-1

which is 17% less as

comparison to control. Addition of various others metallic salts caused much reduction in the uptake of chromate

at various potassium chromate levels of 100 and 500 μg ml-1

. Chromium accumulation by hydrophyte P. stratiotes

was better at acidic pH. The combination of both plants and bacterial strain showed enhanced uptake or

elimination of chromate from the solution.

57

P09

Functional Gene Pools of Microbial Communities Affected by Arsenic and Chromium

Yasir Rehman*

1, Fariha Rizvi

1, Michael McInerney

2, Shahida Hasnain

3

1 Department of Microbiology & Molecular Genetics, University of the Punjab, Lahore Pakistan - Yasir.mmg@pu.edu.pk

2 Department of Microbiology & Plant Biology, University of Oklahoma, Norman OK, USA.

3 The Women University Multan, Multan, Pakistan.

Keywords: arsenic, chromium, functional gene array, functional genes, GEOCHIP, microbial communities

Environmental parameters affect microbial communities. A large number of industries in Pakistan are

releasing their untreated wastes directly into the environment. Arsenic and chromium are two of the many toxicants being released in the environment by industries. As only ~99% soil bacteria can be cultured using standard cultivation techniques, culture independent approach, also called metagenomics, was sued to get a comprehensive view of how microbial communities are affected from arsenic and chromium exposure. A functional gene array was used to estimate the extent of changes that occur in functional gene pool of microbial communities suffering from arsenic and chromium contamination. Soil samples were collected from Kasur, Kalashahkaku and Sialkot along with the collection of control soil samples. DNA was isolated, amplified and hybridized to FGA following manufacturer’s instructions. Diversity indices showed that functional genes are more diverse in contaminated samples as compared to pristine samples. Changes were observed in the number of genes responsible for various functions. Functional genes of Proteobacteria were more abundant in contaminated samples whereas functional of Actinobacteria were more abundant in pristine soil samples. Functional genes of γ-Proteobacteria were more abundant in Sialkot contaminated sites (having higher concentrations of arsenic) whereas functional genes of α-Proteobacteria were more abundant in Kasur contaminated sites (having higher concentrations of chromium). Results indicate that microbial communities in contaminated sites need more diversity of functions in order to survive. Proteobacteria appear to play some role in soils contaminated from chromium and arsenic contamination.

58

P10

PExpanding the Substrate Range of Phenylalanine Ammonia Lyase from Petroselinum crispum Towards Styryl-alanines

Alina Filip

1, Gergely Bánóczi

2, László Poppe

2, Csaba Paizs

1,Florin-Dan Irimie

1, László-Csaba Bencze

1

1 Biocatalysis and Biotransformation Research Group, Department of Chemistry, Babeş-Bolyai University, Cluj-Napoca,

Romania – cslbencze@chem.ubbcluj.ro

2Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary

Keywords: enzyme catalysis, styryl-alanines, rational design

The aim of our research was to extend the substrate range of phenylalanine ammonia lyase from Petroselinum crispum (PcPAL)[1,2,3] towards styryl alanines.

Scheme 1: The ammonia addition (a) and ammonia elimination (b) reactions catalyzed by wt-PcPAL and mutant PcPAL

In the dehydroamination reactions (Scheme 1b), the kinetic parameters (KM, vmax, kcat) showed that styryl

alanine derivatives rac-2a-d are accepted as substrates by wt-PcPAL, however the large KM values suggested weaker binding, and also the reaction velocities obtained were much lower than for natural substrate, L-Phe. The enantioselectivity of the reaction showed high values only for the styryl alanine rac-2a. Furthermore in the hydroamination reaction (Scheme 1a) product formation was not observed, suggesting that styryl-acrylates 1a-d are not accepted as substrates by the enzyme.

This results obtained with the wt-PcPAL directed us to improve the catalytic properties of the enzyme through site-directed mutagenesis, modifying its hydrophobic substrate binding pocket.[4]

The kinetic parameters of the dehydroamination reaction of styryl alanines, catalyzed by the mutant PcPAL show highly improved catalytic properties of the mutant enzyme and suggests that the substrate promiscuity of PcPAL can be further enhanced through rational design.

Acknowledgements

This work was supported by the Romanian National Authority for Scientific Research and Innovation, CNCS-UEFISCDI, under project number PN-II-RU-TE-2014-4-1668

References

[1] L. Poppe, J. Rétey Angew. Chem. Int. Ed. 2005, 44, 3668 – 3688; [2] C. Paizs, A. Katona, J. Rétey Chem. Eur. J., 2006, 12, 2739-2744; [3] C. Paizs, M. I. Toşa, L. C. Bencze, J. Brem. F. D. Irimie, J. Rétey Heterocycles, 2011, 82, 1217-1227; [4] S. Bartsch, U. T. Bornscheuer, Protein Engineering. Design & Selection, 23 (12), 2010, 929–933.

59

P11

Potential of Some Yeast Strains in the Stereoselective Biosynthesis of Acyloins

Mihai Lăcătuș

1, László-Csaba Bencze

1, Csaba Paizs

1,Florin-Dan Irimie

1

1Babes-Bolyai University, Faculty of Chemistry and Chemical Engineering, Cluj Napoca, Romania, irimie@chem.ubbcluj.ro

Keywords: acyloin, yeast, stereoselective

The biocatalytic synthesis of unsymmetrical chiral acyloins as intermediates in the production of active pharmaceutical ingredients has been an attractive goal since the first stereoselective condensation of benzaldehyde and pyruvate by fermenting brewer’s yeast. The product, R-(-)-phenylacetylcarbinol, is the key chiral precursor for pseudoephedrine production [1].

The use of pyruvate decarboxilase (PDC, E.C.4.1.1.1), a Mg(II) and thiamine diphosphate-dependent enzyme from yeasts, as a biocatalyst, has been extensively studied and is now a well-recognized method for condensing aldehydes and pyruvic acid to their corresponding acyloins(α-hydroxy ketones) [2].

Figure 1: Biocatalytic acyloin synthesis.

In order to overcome the possible problems when whole cells systems are employed, such as by-products

formation (the corresponding alcohols,1,2-diols,etc.) an optimization study on the acyloin condensation of benzaldehyde using commercially available yeast Saccharomyces cerevisiae was performed. The optimized biotransformation variables were: pH, temperature, reaction time, and pyruvate concentration. The optimal, as well as the general conditions [3], were further used in the PDC activity screening of newly isolated yeast strain on various novel (hetero)aryl substrates.

Acknowledgements

This work was supported by a grant of then Romanian National Authority for Scientific Research, UEFISCDI,

project number PN-II-PT-PCCA-2013-4-1006

References

[1]. Brovetto, M., Gamenara, D., Méndez, P. S. & Seoane, G. A. Chem. Rev. 2011, 111, 4346–4403. [2]. Patel, R. N.. Coord. Chem. Rev. 2008, 252, 659–701. [3]. Andreu, C. & Del Olmo, M. L. Appl. Microbiol. Biotechnol. 2014, 98, 5901–5913.

60

P12

Biotransformation of 3-Aryl-5-hydroxy-4,5-dihydroisoxazole Acetates

Joanna Główczyk-Zubek

1, Justyna Kliszewicz

1, Edyta Kosińska

1, Jan Maurin

2, Monika Wielechowska

1

1 Department of Drug Technology and Biotechnology, Warsaw University of Technology, Warsaw, Poland,

jmzubek@ch.pw.edu.pl 2 National Medicines Institute,Warsaw, National Centre for Nuclear Research

, Świerk, Poland

Keywords: dihydroisoxazoles, kinetic resolution, lipase

Optically active derivatives of 4,5-dihydroisoxazole are valuable compounds. The dihydroisoxasole moiety is found in many biologically active derivatives with antifungal, antibacterial, antiviral properties.

Esters of 3-arylo-5-hydroxy-4,5-dihydroisoxazoles were prepared via 1,3-dipolar cycloaddition reaction from aryl nitrile oxides and substituted alkenes. 5-Acetoxy-3-phenyl-4,5-dihydroisoxazole was synthesized in Mukayiama reaction from phenylnitromethane and vinyl acetate [1]. Three other isoxazoles were synthesized using relatively stable compounds: 2’,6’-dicholorophenyl nitrile oxide, 2’,4’,6’-trimethylphenyl nitrile oxide and 2’,4’,6’-trimethoxyphenyl nitrile oxide [2].

Racemic esters were hydrolyzed in biotransformation process. Candida antarctica B lipase, Candida rugosa lipase, Pseudomonas fluorescens lipase and Burkholderia cepacia lipase were tested as catalysts in kinetic resolution reactions (Fig.1). Candida antarctica lipase B (Novozyme 435) was the most appropriate enzyme to obtain optically active esters with high ee.

Fig. 1. Hydrolysis of racemic 5-acetoxy-3-aryl-4,5-dihydroisoxazoles

5-Acetoxy-3-phenyl-4,5-dihydroisoxazole was hydrolyzed in the same manner as 5-acetoksy-3-alkyl-4,5-

dihydroisoxazoles. 5-Hydroxy-3-phenyl-4,5-dihydroisoxazole obtained in kinetic resolution reaction turned to be racemate [3]. In situ alcohol racemization provided possibility to transform racemic substrate into optical active ester in one pot hydrolysis-transesterefication reaction (Fig.2.). Enantiomeric excess of product (from 80 to 95%) were determined by HPLC chromatography.

Fig. 2. Biotransformation of 5-acetoxy-3-phenyl-4,5-dihydroisoxazole

References

[1] T. Mukaiyama, T. Hoshino, J. Am. Chem. Soc.,1960, 82,5339

[2] C. Grundman, J.M. Dean J.Org.Chem., 1965,30,2809 [3] J. Główczyk-Zubek, M. Wielechowska et al., 2

ndSymposium on Biotransformations for Pharmaceutical and

Cosmetic Industry, Warsaw, 2014, p.57

61

P13

Lipase-Catalyzed Enantioseparation of Novel Benzoxazole Derivatives Edyta Łukowska-Chojnacka

1, Anna Kowalkowska

1

1Faculty of Chemistry, Institute of Biotechnology, Warsaw University of Technology, 00-664 Warsaw, Noakowskiego St. 3,

Poland, elukowska@ch.pw.edu.pl

Keywords: benzoxazole, lipase, kinetic resolution

Benzoxazole derivatives constitute an interesting subclass of heterocycles with diverse biological and pharmaceutical activity. This ring system is present in many antibiotics e.g. Calcimycin, Routiennocin and Cezomycin, and inhibitors of enzymes e.g. topoisomerase I and II or cyclooxygenase II [1]. Moreover, there are known benzoxazole derivatives possessing anti-inflammatory, antibacterial, antifungal, anticancer, analgesic, antitubercular, anticonvulsant, antioxidant and herbicidal activity [1-3]. Taking into consideration, that the stereochemistry of compounds often determines their activity, studies on the synthesis of enantiomerically pure benzoxazole derivatives with potential antifungal activity were performed. A series of novel 2-mercaptobenzoxazole derivatives including ketones, alcohols and esters was synthesized. All reactions took place in relatively short times (18-24 h) and with satisfactory yields (75-97%). Racemic mixtures of alcohols were separated in enzyme-catalyzed transesterification using commercially available lipases: Amano AK from Pseudomonas fluorescens, Amano PS from Burkholderia cepacia, Lipozyme IM from Mucor miehei and Novozyme SP 435 from Candia antarctica. Additionally, the influence of a solvent (tert-butyl methyl ether, diethyl ether, diisopropyl ether and toluene) on the enantioselectivity of the reaction was investigated. The best results, enantiomerically pure alcohols (e.e.>90%) and esters (e.e.>90%), were obtained, when reactions were carried out in TBME, in the presence of Novozyme SP 435.

References [1] M.K. Gautam, Sonal, N.K. Sharma, Priyanka, K. K. Jha, Int. J. ChemTech Res. 2012, 4, 640.

[2] A. Kaur, S. Wakode, D. P. Pathak, Int. J. Pharm. Pharm. Sci. 2015, 7, 16. [3] R.V. Satyendra, K.A. Vishnumurthy, H.M. Vagdevi, K.P. Rajesh, H. Manjunatha, A. Shruthi, Eur. J. Med.

Chem. 2011, 46, 3078.

62

P14

Lipase Catalyzed Desymmetrization of Heterocyclic Compounds with Hydroxyl

or Carboalcoxyl Group, as a Building Blocks for Biologically Active

Substances

Zbigniew Ochal, Krzysztof Dobrzelewski, Maria Bretner

Institute of Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland, ochal@ch.pw.edu.pl

Keywords: kinetic resolution, lipase, biologically active compounds

Many heterocyclic compounds as benzimidazole, benzotriazole or indole derivatives demonstrated the potential

for a broad spectrum of therapeutic and agricultural applications and they exhibit wide range of the activity - as

antiviral, antibacterial, anticancer and antifungal agents. They are also important building blocks for the synthesis

of biologically active compounds. Among the optically active substances of great importance for the synthesis is

application of the proper enantiomer. Derivatives of benzimidazole, benzotriazole and indole containing at 1 or 2

position moieties with a hydroxyl or a carboalcoxyl group at the asymmetric carbon atom were synthesized, and

enzymatic kinetic resolution was applied to the separation of racemic products into enantiomers. The

transesterification reactions of secondary alcohols, and hydrolysis of alkyl esters of acids with a secondary

carboalcoxy groups were applied. The catalytic efficacy of commercially available lipases in the kinetic resolution

were investigated. The conditions of the enzyme catalyzed reaction, like catalyst, solvent, acylating agent,

temperature and time of process were optimized. The lipase-mediated transesterification approach appeared to

be superior to the hydrolysis reaction and give high enantiomer excess of products. The enantioselectivity of the

investigated enzymatic reactions were from moderate to excellent.

63

P15

Chemiluminescence Measurements of Biopolymer Degradation by Bacteria Tomáš Grivalský

1, Jozef Rychlý

2, Domenico Pangallo

1

1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia – tomasgrivalsky@gmail.com

2 Polymer Institute. Slovak Academy of Sciences, Bratislava, Slovakia

Keywords: biodegradation, chemiluminescence, bacteria

Biodegradable polymers are used in many eco-friendly applications in different kinds of industrial processes. Several factors such as UV, temperature, time period and presence of microorganisms are responsible for their degradation. Our work is focused on analysis of biopolymer degradation by bacteria by chemiluminescence (CL). In our work CL measurements were observed at different intervals from 3 days to 16 weeks to evaluate biodegradation dynamics and evolution. Formally, the light emission reflects the rate of polymer degradation that may be proportional to the product, but our results showed that CL measurement indicate more complicated interpretation affected mainly by the concentration of more reactive terminal group during hydrolysis and presence of bacteria. During biodegradation of polymers the reduction of carbonyls and hydroperoxides are temporarily increased [1]. By CL method we were able to measure (Fig. 1) the process of decomposition of oxygenated structures (hydroperoxides) (line 1) and at the same time we could see the momentaneous state of polymer matrix (line 2). In addition, hydrolytic agar plate assays with biodegradable polymer as unique carbon source were performed for semi-quantitative analysis of enzymatic activity. We also observed the surface of inoculated polymers by scanning electron microscopy. The comparison of the results showed the complexity of biodegradation phenomena.

Fig. 1 Nonisotermal chemiluminescence measurement of polymer degraded by bacteria. Deconvolution of

experimental line by two gaussian lines (1 and 2).

Acknowledgements

This work was financed by the Slovak VEGA Agency, project number: 2/0103/14 "Protecting our memories: investigation into the biodeterioration of photographic and cinematographic materials".

References

[1] C. Abrucsci et al., Int Biodeterior Biodegradation. Biodegradation of photo-degraded mulching films based on

polyethylenes and stearates of calcium and iron as pro-oxidant additives 2011, 65, 451–59.

64

P16

Isoquercitrin Esters with Aliphatic or Aromatic Acids: Enzymatic Preparation and Properties

Eva Vavříková

1, Alena Křenková

1, Kateřina Valentová

1, Lubica Ježová

1 and Vladimír Křen

1

1 Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Science, Vídeňská 1083, CZ 142 20 Prague,

Czech Republic – vavrikova@biomed.cas.cz

Keywords: isoquercitrin, lipase, antioxidant activity

Isoquercitrin (IQ, quercetin-3-O-β-D-glucopyranoside, [1]) is, together with rutin (quercetin-3-O-β-D-rutinoside),

one of the major glycosidic forms of the natural flavonols quercetin. IQ is attracting the researcher's interest due to the growing array of its biological activity reported. Recently, a robust method of biocatalytic production of pure isoquercitrin was developed. [2] Its health benefits seem to be very attractive for food, cosmetic and pharmaceutical industry.

A large series of isoquercitrin esters with mono- or dicarboxylic acids [3] or aromatic acids was designed in order to modulate hydro- and lipophilicity and biological properties.

Regioselectivity of the substitution of flavonoids is essential for successful preparation of their derivatives. Enzymatic approach overcomes protecting and deprotecting strategies, which are inevitable during chemical synthesis. Here, selective acylation of primary hydroxyl groups was accomplished by Novozym 435

® (CAL-B

immobilized on an acrylic resin). Enzymatic reactions were performed with IQ and mono- or dicarboxylic aliphatic acids directly. In the case of aromatic acids, their activated vinyl esters were prepared for enzymatic esterification with IQ.

We have prepared a panel of IQ derivatives substituted at C-6ʺ OH (Fig. 1; acetate, butyrate, hexanoate, octanoate, dodecanoate, palmitate, glutarate, adipate, dodecandioate, benzoate, phenylacetate, 3-phenylpropionate and cinnamate). The conversion of dicarboxylic acids was limited and strictly depended on the chain length of the respective acid. Shorter dicarboxylic acids such as oxalic (C2), malonic (C3), succinic (C4) and maleic (C4) did not react at all, while the lipase accepted C5- to C12-dicarboxylic acids.

To evaluate pharmacological potential of these novel derivatives their lipophilicity (log P), and their antiradical and anti-lipoperoxidant activities were determined.

Fig. 1 Structures of the isoquercitrin esters prepared with lipase Novozym.

Acknowledgements

This work was supported by Czech Science Foundation grant GP14-14373P and project No. LD15082 from

Ministry of Education of the Czech Republic (COST Action FA1403 POSITIVe).

References

[1] K.Valentová, J. Vrba, M. Bancířová, J. Ulrichová, and V. Křen, Food Chem. Toxicol. 2014, 68, 267-282. [2] D.Gerstorferová, B. Fliedrová, P. Halada, P. Marhol, V. Křen and L. Weignerová, Process Biochem. 2012, 47,

828-835. [3] E.Vavříková, F. Langschwager, L. Ježová, A. Křenková, B. Mikulová, M. Kuzma, V. Křen and K. Valentová,

Int. J. Mol. Sci. 2016, submitted

65

P17

Influence of New Synthetized Heavy Metal Chelating Agents on Activated Sludge Bacterial Consortium

Aleksandra Wojciechowska, Wojciech Smułek, Karolina Wieszczycka and Ewa Kaczorek Institute of Chemical Technology and Engineering, Poznan University of Technology, Poland – wojciech.smulek@doctorate.put.poznan.pl

Keywords: activated sludge, chelating agents, bacteria

The development of metallurgical industry is connected with the search for new effective chelating agents. One of the group of promising extractants with interesting properties are pyridine ketoximes. These compounds contain chelating oxime part as well as solvating group – pyridine ring. The last research indicated that pyridine ketoximes can be successfully used in extraction of copper, iron, zinc and cadmium. Moreover, sequencing extraction and reextraction allow to obtain desired selectivity of the process [1,2].

Possible large-scale application of the pyridine ketoximes involve question about their possible negative influence on the environment. Their presence in wastewater may change the equilibrium between microorganism in the activated sludge. Hence, the research on interactions between these new extractants and activated sludge are necessary.

The aim of this work was to synthetize several pyridine ketoxime chelating agents and to investigate their impact on bacterial consortium. The oximes were synthesized in the two-stage process. In the first stage 2-, 3-

and 4-pyridyl ketones were synthesized by treating pyridylcarbonitrile with alkyl magnesium bromide in diethyl ether as diluents. In the second stage, the synthesized ketone was treated with hydroxylamine hydrochloride in the presence of sodium carbonate (at pH = 7) [3]. Then the compounds at different concentrations were added to liquid cultures inoculated with bacterial consortium isolated from activated sludge of wastewater treatment plant. The measurements of optical density of the culture allow to compare growth kinetics. Moreover, the carbon dioxide concentration in head-space of the cultures was also investigated.

Three pyridine ketoximes were synthetized: 1-(-pyridyl)pentadecan-1-one oxime (2PC14), 1-(3-pyridyl)pentadecan-1-one oxime (3PC14), 1-(4-pyridyl)pentadecan-1-one oxime (4PC14). The observation of growth kinetic indicated that the compounds had not significant toxic influence on tested bacterial consortium. Additionally, the increase of carbon dioxide concentration with the increasing compounds concentration may indicate that the pyridine ketoximes can be used by bacteria as carbon and energy source. The obtained results shown that the tree synthetized chelating agents do not constitute a threat to the activated sludge bacteria.

Acknowledgements

Badania zrealizowano ze środków Ministerstwa Nauki i Szkolnictwa Wyższego na działalność statutową

Wydziału Technologii Chemicznej Politechniki Poznańskiej nr 03/32/DSMK/0620.

References [1] A. Parus, K. Wieszczycka, A. Olszanowski, Separation Science and Technology 2011, 46(1), 87-93. [2] A. Parus, K. Wieszczycka, A. Olszanowski, Hydrometallurgy 2011, 105, 284-289. [3] K. Wieszczycka, M. Kaczerewska, M. Krupa, A. Parus, A. Olszanowski, Separation Science and Technology

2012, 95, 157-164.

66

P18

Peroxidase Activity of DNAzyme with a Covalently Attached Hemin Cofactor Alicja Stanisławska

1, Joanna Kosman

1 and Bernard Juskowiak

1

1 Adam Mickiewicz University, Poznań, Poland – joannakosman@gmail.com

Keywords: G-quadruplex, DNAzyme, hemin

The enzyme horseradish peroxidase (HRP) used extensively in bioanalytics is characterized by the excellent catalytic activity, the 0,25 pM level of detection limit. However, due to the disadvatages that come with the processes of using the protein enzyme itself (temperature requirements, expensive production and purification) the probes based on the DNA exhibiting features similar to ones of the natural enzyme are being designed. DNAzyme with peroxidase activity can catalyze the reaction between the hydrogen peroxide and an organic substrate as the result the analytic signal can be observed [1]. The DNAzymes consist of oligonucleotide rich in the guanosine residues which forms the G-quadruplex structure and interacts with the hemin molecule (end-stacking). The porphyrin’s molecules show a slight oxido-reducing activity, which increase after binding with G-quadruplex. The catalytic activity of the DNAzyme is determined by the sequence of oligonucleotide, the presence of the ions and the strength of hemin interaction with the G-quadruplex. The interaction itself is based on increasing the electron density of an iron atom of porphyrin, which results in the complex characterized by a higher analytic signal. Currently the DNAzymes are widely used to detect metal cations i.e. K+, Na+, NH4+, Ca2+, Ag+, Hg+, Pb. Another application is to detect proteins, nucleic acids and various biomolecules [2]. It brings the hope in a fight with cancer, where the G-quadruplex structures are being created basing on a sequence present in some proto-oncogenes (c-myc, c-kit, HIF-1R, RET, bcl-2) and antigens (increased activity of the protein VEGF).

The created G-quadruplex can forms parallel, antiparallel or mixed topology depending on the sequence and type of present cation. The parallel G-quadruplex is characterized by the highest catalytic activity. A flourishing way for increasing the aforementioned activity is to covalently attachment of hemin molecule to the DNA. Such conjugation results in reduction of noises caused by the hemin itself in blind probes. Creating such DNAzyme was possible thanks to the click chemistry. For that purpose the hemin’s derivative was synthesized with the azide-terminated linker and the oligonucleotide was modified with the cyclooctyne group. Such modifications creates conditions for the click reaction to occur without any additional substrates and catalyst. In the case of the click of hemin and G-quadruplexes the click catalyzed by copper can result in cutting of DNA and creation of aggregated complexes difficult to analyze later. We have proved that the click reactions occurring in the presence of copper are less efficient and the oligonucleotides rich in the guanosine strongly interact with the copper ions. Cu-free click reaction (SPAAC) of G-quadruplex and hemin, however, allows on synthesis of probe with good yield (~60%). The activity of obtained DNAzymy with covalently attached hemin was measured in reaction of oxidation of fluorogenic substrate 4-(N-Methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH). DNAzyme based on PS2.M sequence (5’-GTGGGTAGGGCGGGT-3’) exhibits high activity in comparison to hemin itself. Such DNAzyme can be used in design of new bioanalytical assays.

Fig. 1 A G-quadruplex model with the terminally interacting hemin (end-stacking).

Acknowledgements

This work was supported by National Science Center grant no. 2013/10/M/ST4/00490.

References

[1] P. Travascio, Y. Liu, D. Sen, Chem. Biol., 1998, 505-517. [2] J. Kosman, B. Juskowiak, Anal. Chimica Acta, 2011, 7-17.

67

P19

First Example Of The Dynamic Kinetic Resolution of 3-Aryl-4-Pentenoic Acids Dominik Koszelewski, Anna Brodzka, Anna Żądło, Daniel Paprocki, Damian Trzepizur, Malgorzata Zysk, and Ryszard Ostaszewski 1 Institute of Organic Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw (Poland)

Keywords: dynamic kinetic resolution, unsaturated carboxylic acids, racemization, biotransformations, rhodium catalyst

The synthesis of enantiomerically pure compounds is of great importance for pharmaceutical industry [1]. Large amount of medicines are chiral, non-racemic compounds which biological activity is highly dependent on their absolute configuration of the stereogenic centers. Thus, the demand for methods for the synthesis of chiral non-racemic substances has increased rapidly in response to these commercial considerations.

Among others optically active 3-aryl-4-pentenoic acid is an important substrate for the synthesis of numerous medicaments like Rolipram

®, an anti-inflammatory drug and one of the family of γ-aminobutyric acid (GABA)

derivatives as well as Citrocard®, Lioresal

®, Femoxetine

®, or Paroxetine

® another drugs belonging to the GABA

family. Optically active 3-phenyl-4-pentenoic acid is the key intermediate in the synthesis of LG 121071 a modulator of androgen receptors and Neurokinin NK1/NK2 antagonists. The (S)-3-aryl-4-pentenoic acid derivatives were also used for the synthesis of antiproliferation lactones (HL-60 cells inhibitors) antimicrobial agents, and also antitumor antibiotic methylenolactocin .

The first example of dynamic kinetic resolution (DKR) of the chiral unsaturated carboxylic acids is described. The application of tandem metal-enzyme DKR is a powerful tool for the manufacture of high-value chemical commodities. This new protocol of kinetic resolution based on irreversible enzymatic esterification of 3-aryl-4-pentenoic acids with orthoesters was introduced to obtain optically active unsaturated carboxylic acids [2].

A substantial influence of organic co-solvent, and metal catalyst on conversion and enantioselectivity of the enzymatic dynamic kinetic resolution was noted.

Fig. 1 Dynamic kinetic resolution of acid rac-3-aryl-4-pentenoic acid derivatives catalyzed by Novozym 435

Acknowledgements

This work was supported by the Polish National Science Centre project No. 2013/11/B/ST5/02199.

References

[1] Nunez, M. C.; Garcia-Rubino, M. E.; Conejo-Garcia, A.; Cruz-López, O.; Kimatrai, M.; Gallo, M. A.; Espinosa, A.; Campos, J. M. Curr. Med. Chem. 2009, 16, 2064-2074.

[2] Koszelewski, D.; Brodzka, A.; Żądło, A.; Paprocki, D.; Trzepizur, D.; Zysk, M.; Ostaszewski, R. ACS Catal., 2016, 6, 3287-3292.

68

P20

Enzymatic Kinetic Resolution of α-Substituted Esters of 2-Methylbut-2-enoic Acid.

Filip Borys, Dominik Koszelewski and Ryszard Ostaszewski Institute of Organic Chemistry, Polish Academy of Science, Warsaw, Poland – fborys@icho.edu.pl

Keywords: kinetic resolution, lipases, biotransformations

Chiral, enantiomerically pure compounds are becoming more and more important. This is mainly due to the

increasing interest and need for such compounds in pharmaceutical and agricultural industry. Living systems are inherently chiral because many essential biomolecules exist in homochiral form. Thus, the demand for methods for the production of chiral non-racemic compounds has increased rapidly in response to these commercial considerations. The synthesis of enantiomerically pure drugs is of great importance for both academia and pharmaceutical industry.

Unsaturated carboxylic acids are synthetically useful building blocks [1]. Alkylation of the π extended enolates

of unsaturated carboxylic acids and their derivatives is known to occur generally at the α carbon [2]. α-Substituted β,γ-unsaturated carboxylic acids produced from alkylation reaction of unsaturated acids are racemic mixtures. Enzymatic kinetic resolution of unsaturated carboxylic acids is often difficult due to enzyme inhibition caused by substrates [3]. To overcome this difficulty α-substituted β,γ-unsaturated carboxylic acids were converted into activated esters and subsequently resolve to enantiomers by enzymatically catalyzed hydrolysis reaction. As a model compound 2-methylbut-2-enoic acid has been arbitral selected (Figure 1). Obtained in this way enantiopure or enantiomerically enrich unsaturated carboxylic acids can be further functionalized (e.g. metathesis reaction, Heck arylation, epoxidation, oxidation or reduction etc.)

In the present work, we present studies on synthesis of α-substituted esters of 2-methylbut-2-enoic acid and their subsequent enzymatic kinetic resolution. Chosen substrates possess quaternary chiral carbons atom, such compounds are particularly challenging for synthesis in enatipure form as well as for enzymatic resolution due to congested nature of quaternary sterocenter. Our approach allowed to obtained 2-methylbut-2-enoic acid with good yields and enantiomeric excesses.

OH

O

OH

O

Ph

O

O

Ph

R

OH

O

Ph

LDA, THF

-78oC, PhCH2Cl DMAP, DCM

ROH

enzyme,buffer : cosolvent

R: -CH2CF3, -CH2Cl3, -C6H5Cl, -C6H5NO2

Fig. 1.Chemoenzymatic synthesis of enatiopure 2-methylbut-2-enoic acid.

Acknowledgements

This work was supported by the National Science Centre (Grant 2013/11/B/ST5/02199).

References

[1] G. Rajendra, M. J. Miller, J. Org. Chem. 1987, 52, 4471-477. [2] L. R. Domingo, S. Gil, R. Mestres, M. T. Picher, Tetrahedron 1995, 51, 7207-7214.

[3] P. Gunnarsson, G. Pettersson, Eur. J. Biochem. 1972, 27, 564-571.

69

P21

The New Approach to Synthesis of Non-racemic Esters via Double Kinetic Resolution

Anna Brodzka and Ryszard Ostaszewski Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warsaw, Poland Keywords: Double Kinetic Resolution, enzymes, chiral compounds

Chiral, optically 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 methods of synthesis of carboxylic acids

derivatives, especially these containing two chiral centers, in optically pure form are usually difficult and regarded multistep synthesis.

The development of method of synthesis of non-racemic compounds with two chiral centers is desirable. In our studies we propose to apply two racemic chiral substrates (carboxylic acid and alcohol) into lipase mediated esterification reaction. As model substrates we used racemic 3-phenyl-4-pentenoic acid

3 and 3-buten-2-ol which

were subjected to double kinetic resolution process giving non-racemic ester (Figure 1).

Fig. 1 Double Kinetic Resolution reaction.

Acknowledgements

This work was supported by project Preludium UMO-2013/11/N/ST5/02723 financed by the Polish National Science Centre.

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] D. Koszelewski ,A. Brodzka, D. Paprocki, D. Trzepizur, M. Zysk, R. Ostaszewski, R. ACS Catal., 2016, 6,

3287-3292.

70

P22

The Studies Toward Enzymatic Kinetic Resolution of Mixed Carbonates

Arleta Madej

1, Anna Żądło, Dominik Koszelewski, Ryszard Ostaszewski

1Institute of Organic Chemistry PAS, Polish Academy of Sciences, Warsaw, Poland

Keywords: carbonate, resolution, enzyme

Hydrolytic enzymes, such as lipases and esterases, are very important biocatalyst for industrial applications. They are frequently used because of their stability, activity and ability to accept a wide range of substrates.

[1]

Activitty of enzymes can be determined by many analitical methods. Spectrophotometric assays depend upon a direct change in the absorbance of p-nitrophenol.

[2] The carbonates with nitrophenyl group are shown to be

a new class of compound for spectrophotometric assays, very recommendable for high-throughput screening.[3]

In the present study, we report the preparation and evaluation of mixed carbonates of p-nitrophenol

as enzyme substrates and demontrated their application as probes for enzymes screening. The reaction (Fig. 1) is irreversible, which shifts the equilibrium toward products.

Fig. 1 Enzymatic hydrolysis of carbonates (S)-1, (R)-1, 2

The spectophotometric assay is a good method to exlopre the effect of changing conditions on reaction rates.

Progress of the enzymatic reaction and purification can be readily monitored due to the strong UV absorption of the p-nitrophenyl moiety.

Acknowledgements This work was supported by the Polish National Science Center project project No. 2014/14/M/ST5/00030

References

[1] M. Schmidt, U. T. Bornscheuer, Biomol. Engineering 2005, 22, 51 [2] H.U Bergmeyer, New York: Academic Press. 1974, 4, 2066 [3] P 407948 2014: R. Ostaszewski, A. Żądło, M. Zysk, A. Brodzka, S. Kłossowski

71

P23

Environmental Friendly Approach to α-Acyloxy carboxamides via a Chemoenzymatic Cascade

Daniel Paprocki

1, Dominik Koszelewski

1, Anna Żądło

1, Peter Walde

2 and Ryszard Ostaszewski

1

1 Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland

2 Laboratory of Polymer Chemistry, Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 5, 8093 Zurich, Switzerland

Keywords: Passerini reaction, Laccase, oxidation

α-Acyloxy carboxamides are employed for the synthesis of many important classes of organic compounds, such as γ-lactones [1], 2-furanones [2], peptidomimetics [3]. A convenient way for obtaining α-acyloxy carboxamides, skipping multistep synthesis, is performing the Passerini reaction between a carboxylic acid, a carbonyl compound and an isocyanide. The Passerini reaction is usually performed in aprotic organic solvents, e.g. dichloromethane or toluene, which are toxic and carcinogenic. Pirrung, Das Sarma and others have shown that the P-3CR can be performed efficiently also in water [4]. Moreover, we have recently demonstrated that the application of dioctadecyldimethylammonium bromide (DODAB), a vesicle-forming surfactant, has a beneficial effect on the P-3CR, leading to a substantial increase in reaction yield [5].

Aldehydes are typical starting materials of the Passerini reaction. Unfortunately, the use of aldehydes is problematic since they may be unstable during storage, providing the corresponding alcohols as impurities, or undesired products of aldol condensation reactions, or from corresponding acids formed by autoxidation.

We report about a new chemoenzymatic protocol based on a one-pot oxidation and the Passerini reaction procedure. The oxidation of a primary alcohol to the corresponding aldehyde is conducted enzymatically by oxygen from air, with the enzyme laccase and the mediator TEMPO. After the oxidation, a carboxylic acid and an isocyanide are added, which initiate a Passerini reaction in the same “pot”, providing an α-acyloxy carboxamide. All reactions proceed in an aqueous surfactant system, which excludes the usage of organic solvents (Scheme 1). The studies on development of one pot Laccase/TEMPO oxidation followed by the Passerini reaction will be presented.

R

OHCOOH

OMe

NC

+ +O

O RHN

O

OMeLaccaseTEMPODODABbuffer

Scheme 1. Laccase/TEMPO oxidation followed by the Passerini reaction.

Acknowledgements

We gratefully acknowledge the financial support of the NCN project Harmonia No 2014/14/M/ST5/00030

References

[1] M. Passerini, Gazz. Chim. Ital. 1923, 53, 331. [2] R. Bossio, Synthesis 1993, 783, 1. [3] W. Szymański, M. Zwolińska, R. Ostaszewski, Tetrahedron 2007, 63, 7647. [4] M.C. Pirrung, K. Das Sarma, J. Am. Chem. Soc. 2003, 126, 444. [5] D. Paprocki, D. Koszelewski, P. Walde, R. Ostaszewski, RSC Advences 2015, 5, 102828.

72

P24

Novel Enzymatic Ugi Reaction with Amines and Cyclic Imines Anna Żądło, Szymon Kłossowski, Dominik Koszelewski, Daniel Paprocki, Ryszard Ostaszewski

Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland

Keywords: Ugi reaction, multicomponent reaction, enzymatic

Multicomponent reactions (MCRs) are very convenient tools used by organic chemists to assemble novel complex structures from simple starting materials in a one-step procedure. The classical Ugi four-component reaction (U-4-CR) involves the condensation of primary amine, carbonyl compound, isocyanide and carboxylic acid to α-acylamino amides.[1,2]

Amine and carbonyl components could also be replaced by the corresponding

imine. This approach was adopted by several groups to form pyrrolidine and piperidine derivatives.[3,4] Recently, our group reported a new enzymatically catalyzed Ugi-type reaction. The study exploited the

enzyme promiscuity in the multicomponent reaction, which significantly expanded the synthetic methodology for new peptide scaffolds.[5]

In the present study, we overcame the limitations associated with the structure of imines, which were generated from amines. We report MCR with different cyclic imine and amine structures (Scheme 1), which was impossible to perform previously. This methodology was characterized by mild conditions, a simple preparation and good isolated yields. We have proven that lipases show promiscuous activity and are able to catalyze the Ugi-type 3-CR reaction.

Scheme 1. Ugi-type Reaction with Various Cyclic Imines, Amines and Isocyanides.

Acknowledgements

This work was supported by the Polish National Science Center project Nos. 2013/11/B/ST5/02199,

2014/14/M/ST5/00030 and DEC-2011/03/N/ST5/04353.

References

[1] V. G. Nenajdenko, A. V. Gulevich, E. S. Balenkova, Tetrahedron 2006, 62, 5922–5930. [2] S. Kłossowski, A. Muchowicz, M. Firczuk, M. Świech, A. Redzej, J. Golab, R. Ostaszewski, J. Med. Chem.

2012, 55, 55–67. [3] A. Znabet, E. Ruijter, F. J. J. de Kanter, V. Köhler, M. Helliwell, N. J. Turner, R. V. A. Orru, Angew. Chem.

2010, 122, 5417–5420. [4] R. F. Nutt, M. M. Joullie, J. Am. Chem. Soc. 1982, 104, 5852–5853. [5] S. Kłossowski, B. Wiraszka, S. Berłożecki, R. Ostaszewski, Org. Lett. 2013, 15, 566–569.

73

P25

Enantioselective Enzymatic Desymmetrization of 2-Benzyl-2-vinyl-1,3-propanediol.

Rafał Kopiasz

1,2 and Ryszard Ostaszewski

2

1 Department of Chemistry, Warsaw University of Technology, Warsaw, Poland – kopiaszrafal@gmail.com

2 Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland

Keywords: enzyme, desymmetrization, quaternary stereocenter

Compounds containing all-carbon quaternary stereocenter commonly occur in nature. The properties of organic molecules, especially biological activity, are closely linked to their shape, which is dictated by the three-dimensional orientation of substituents at carbon stereocentre. Due to steric hindrance of all-carbon quaternary stereocenter, it has a significant impact on the shape of the molecule. This structural element remains a great challenge in organic synthesis and their asymmetric construction, especially in a catalytic fashion, is one of the most demanding and dynamic topics in the synthetic area of natural products and chiral drugs [1]. Unfortunately, many chemical methods developed in order to overcome this problem require using expensive chiral catalysts or complexes of toxic metals.

Enzymes are attractive alternatives to chemical methods because of their biodegradability and activity under very mild condition. These biocatalysts are widely used to desymmetrization of prochiral molecules [2].

The aim of this study was to use enzymatic procedure for desymmetrization of 2-benzyl-2-vinyl-1,3-propanediol, in order to prepare building block that could be use in a synthesis of analogues of natural compounds, for example aminoacids. The influence of solvents, acyl donors and enzymes on enantioselectivity of the following reaction (Fig. 1.) was investigated. The results of our study will be presented.

Fig. 1. Enzymatic desymmetrization of 2-benzyl-2-vinyl 1,3-propanediol

Acknowledgements

We gratefully acknowledge the financial support of NCN project Opus No. 2013/11/B/ST5/02199. We would

also like to thank Margarita Jurczak for synthesis of 2-benzyl-2-vinyl-1,3-propanediol.

References [1] K. W. Quasdorf and L. E. Overman, Nature 2014, vol 516, 181-191 [2] Vicente Gotor et. all, Chem. Rev. 2011, 111, 110-180.

74

P26

Bioconversion of Chiral Aminophoshonates via Two Step Opposite Redox Reactions: Oxidation and Reduction Employing Fungi.

Natalia Kmiecik

1, E. Żymańczyk-Duda

1

1Department of Bioorganic Chemistry, Wroclaw University of Technology, Poland– natalia.kmiecik@pwr.edu.pl

Keywords: biotransformation, fungi, aminophosphonates

Organophosphorus compounds are characterized by the presence of a phosphorus atom covalently bound to a carbon atom. Phosphonates are very stable and resistant to biochemical decomposition. Derivatives of phosphonic acids have potential biological activities and great industrial importance [3]. Aminophosphonic acids are gained by replacement of carboxylic group by phosphonic moiety. They exhibit inhibitory activity, act as antibiotics, crop protection agents, herbicides and peptides mimics [1-5].

Penicillium funiculosum (Thom 3), Geotrichum candidum (6593) and Beauveria bassiana (271B) were applied for bioconversion of racemic mixture of 1-amino-2-methylbutylphosphonic acid 1. P.funiculosum (Thom 3) and F.oxysporum (DSM 12646) were transformed 1-amino-2-methylpropylphosphonic acid 4. Both substrates were analyzed in order to study the steps of the conversion. The undertaken efforts confirm allowed finding biocatalysts with dual activity towards aminophosphonic substrate. Racemic mixtures of aminophoshonates was bioconverted via oxidative deamination of the substrate, what was followed by ketone bioreduction. Results were evaluated by NMR, MS and IR

technique and enabled to confirm the path of biocatalytic two steps redox reaction:

oxidation and reduction of α- aminophosphonic acid.

PO3H2H2N PO3H2O PO3H2HO

2 3

*

*

* *

*

1

PO3H2H2N PO3H2O PO3H2HO

*

4 5 6

*

Fig. 1. Chemical structures of phosphonic analogues of isoleucine (1-3): substrate 1-amino-2-methylbutylphosphonic acid 1 and products obtained after biotransformation: 2-methylbutanoylphosphonic acid 2, 1-hydroxybutylphosphonic acid 3. Structures of phosphonic analogues of valine (4-6): substrate 1-amino-2-methylpropylphosphonic acid 4 and products of biotransformation: 2-methylpropanoylphosphonic acid 5, 1-hydroxy-2-methylpropylphosphonic acid 6.

Acknowledgements

This work was financed by the project “Biotransformations for pharmaceutical and cosmetics industry” No.POIG.01.03.01-00-158/09-09, which is partly-financed by the European Union within the European Regional Development Fund for the Innovative Economy”.

References

[1] M Brzezińska-Rodak, M Klimek-Ochab, E Żymańczyk-Duda, P Kafarski, Molecules 2011, 16, 5896-5904B [2] ZH Kudzin, DK Gralak, G Andrijewski, J Drabowicz, J Łuczak, J Chromatogr A 2003, 998, 183-199 [3] Nowack, Water Research 2003, 37, 2533–2546 [4] GS Prasad, JR Krishna, M Monajunath, OVS Reddy, M Krishnaiah, CS Reddy, VG Puranik, ARKIVOC 2007, 13, 133-141 [5] E Rudzińska, P Dżygiel, P Wieczorek, P Kafarski, J Chromatogr A 2002, 979, 115-122

75

P27

Biotransformations of Hydroxyphosphonoacetic Acid Derivatives by Bacteria with Lipolytic Activity

Paulina Majewska 1 Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wrocław, Poland

– paulina.majewska@pwr.edu.pl Keywords: hydroxyphosphonates, biocatalysis, kinetic resolution

Stereoselective biocatalytic processes employing whole cells of microorganisms and isolated enzymes are becoming increasingly widely used in the preparation of chiral compounds. Kinetic resolution of enantiomers using biocatalysis is one of the ways for obtaining pure enantiomers in easy and cost effective way. [1,2]

Hydroxyphosphonoacetic acid derivatives are used in the pharmaceutical industry, because many of them have a biological activity (e.g., some of them have antifungal, antibacterial or antiviral properties). [3] Chiral hydroxyphosphonoacetic acid derivatives are also useful as precursors of the various phosphoroorganic compounds. [4]

Racemic 2-butyryloxy-2-(phosphinyl)acetic acid derivatives were synthesized and hydrolyzed using a few bacterial species as biocatalysts. In all cases the reaction was more or less stereoselective.

PO OH

OO

O

O

OP

OH

OO

OH

(SP,S) and (SP,R)

POH

OO

O

O

(RP,R) and (RP,S)

microorganism + O

Fig. 1 Hydrolysis of 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid.

References

[1] A. Liese, K. Seelbach, C. Wandrey, Industrial Biotransformations, VILEY-VCH, Weinheim, 2006. [2] K. Faber, Biotransformations in Organic Chemistry, 6th ed., Springer-Verlag, Berlin Heidelberg, 2011. [3] M. Collinsova, J. Jiracek, Curr. Med. Chem. 2000, 7, 629-647 [4] O. I. Kolodiazhnyi, Tetrahedron: Asymm. 2005, 16, 3295-3340

76

P28

Microbiological Modification of Resveratrol

Sylwia Siudakiewicz

1, Alicja Zakrzewska

1 and Małgorzata Brzezińska-Rodak

1

1 Department of bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland, email:

sylwiasiudakiewicz@gmail.com

Keywords: resveratrol, biotransformation, methylation

Resveratrol (trans-3,5,4-trihydroxystilbene) is a polyphenolic stilbene classified as flavonoid. It is

produced by a plant in response to fungal infections, oxidative stress and UV radiation; synthesized mainly by

grapes. It exhibits a wide range of biological activity, which includes anti-inflammatory, anticancer and antioxidant

properties. In addition, it reduces the risk of cardiovascular disease by inhibiting the oxidation of polyunsaturated

fatty acids.

The aim of the study is to carry out the biotransformation of resveratrol molecules using the strain of

Streptomyces griseus and Aspergillus parasiticus. Pure resveratrol has a short half-life time, and in the human

body is rapidly metabolized. For this reasons, the goal is to obtain its derivatives. The methyl group is attached to

the stilbene and increases the bioavailability and lipophilic properties of molecules. As the donor of methyl group

was used L-methionine. The yield of biotransformation depends on the activity of methyltransferases of both

strains. Therefore, several attempts was carried out in order to optimize the process.

OH

OH

OH

methyl donor

O

O

OH

CH3

CH3

O

O

O

CH3

CH3

CH3

Fig.1. Microbiological methylation of resveratrol.

References

[1] Aneta Kopeć, Ewa Piątkowska, Teresa Leszczyńska, Renata Bieżanowska-Kopeć. Prozdrowotne właściwości resweratrolu. Żywność. Nauka. Technologia. Jakość, 2011, 5 (78), 5–15.

[2] Das DK, Mukherjee S, Ray D. Resveratrol and red wine, healthy heart and longevity. Heart Fail Rev 2010; 15: 467-77.

[3] Justyna Mikuła Pietrasik, Angelika Kuczmarska, Krzysztof Książek. Biologiczna wielofunkcyjność resweratrolu i jego pochodnych. Postępy biochemii 61 (4) 2015, 337-343.

77

P29

Application of Filamentous Fungi for the Bioconversion of Plant Waste Aleksandra Zielonka

1, Magdalena Klimek- Ochab

2

1 Department of Bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland – aleksandra.zielonka@pwr.edu.pl

2 Department of Bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland

Keywords: filamentous fungi, silica nanoparticles, plant waste

Rice husk (RH), horsetail, nettle and covers of corn cobs are an inexpensive biological materials, which contain a large amount of amorphous hydrated silica- congruous form to that present in most subjects in the biosphere. Silicon is deposited mainly in the form of phytoliths in plants, which are primarily built of amorphous hydrated silica [1].

The growing interest in metallic nanoparticles is caused by their diverse properties in chemical and physical field. Recent results of investigations on nanoparticles have ensured that nanotechnology will be an important aspect used in future scientific endeavors [2]. For example, silica nanoparticles can be used as resins, catalytic supports, construction materials and many others [3]. Various methods of their production were reported, involving mainly chemical or physical techniques. From economic and ecological point of view the use of biocatalytic approach is further more reasonable, especially because of some filamentous fungi of Fusarium genus possess desired enzymatic activity. Several fungal catalysts were screened for their ability to transform amorphous hydrated silica of plant origin, to silica nanostructures in room temperature. Rice husk, horstail waste, nettle waste and covers of corn cobs were used as plant substrates for biotransformation processes. Biocatalyzed reaction were carried out under various process condition.

References

[1] Bansal, V., et al., "Fungus-mediated biotransformation of amorphous silica in rice husk to nanocrystalline silica." 2006, Journal of the American Chemical Society 128(43): 14059-14066.

[2] Mahendra Rai, et al., “Myconanotechnology: a New and Emerging Science”, 2009, CAB International 2009. Applied Mycology, 258-267.

[3] Pineda-Vasquez, T. G., et al., "Biogeneration of Silica Nanoparticles from Rice Husk Ash Using Fusarium oxysporum in Two Different Growth Media.", 2014, Industrial & Engineering Chemistry Research 53(17): 6959-6965.

78

P30

Improvement of Biological Activity of Natural Flavonoids by Biotransformation Sandra Sordon

1, Anna Madej, Jarosław Popłoński, Tomasz Tronina, Agnieszka Bartmańska, Ewa

Huszcza 1 Department of Chemistry, University of Environmental and Life Sciences, Wrocław, Poland- sandra.sordon@up.wroc.pl

Keywords: flavonoids, biotransformation, yeast

Flavonoids comprise a large group of polyphenolic compounds ubiquitously presented in plants and characterized by a benzo-γ-pyrone structure. They are widely distributed secondary metabolites with different metabolic functions in plants. They exert considerable influence on growth and development of plants, protect them from UV radiation and bacterial and fungal infections and provide colour to fruits and flowers [1,2].

Flavonoids present in food have beneficial effects on human health. They have been found to exhibit a diverse spectrum of biological activities such as: antioxidant, antimicrobial, antitumor, anti-inflammatory, antiviral and estrogenic [3]. Health benefits associated with flavonoids resulted in a significant increase of interest in this class of compounds. Most of the beneficial health effects of flavonoids are attributed to their antioxidant abilities. In general, it is considered that a higher number of hydroxyl substituents in a flavonoid results in a higher antioxidant activity [4,5].

Biotransformation is one of the possible methods for the preparation of various derivatives of flavonoids. The use of microorganisms as a biocatalyst is a relevant strategy to obtain natural compounds from cheap and readily available substrates.

In opposition to the classic chemical synthesis, biotransformation processes are environmentally friendly, proceed under mild conditions and allow chemo-, regio- and stereoselective modifications of the substrates [6,7]. Moreover, according to the European Union Law, the products obtained by biotransformation of natural compounds are classified as natural ones (EU Directive 88/388/EEC), which significantly increases the probability of their future use in the pharmaceutical and food industries.

In the course of our studies aiming to generate the bioactive compounds from the natural flavonoids by means of biotransformation, we utilize red yeasts as biocatalyst. As a result of the biotransformation hydroxylation products with a catechol moiety were created. Because catechol moiety determines a high capacity for free

radicals scavenging, we used 2,2-diphenyl-1-picrylhydrazyl (DPPH) system to evaluate this activity. All the resulting transformation products much better scavenge the DPPH radical than the starting compounds.

Acknowledgements

The Project is supported by Wroclaw Centre of Biotechnology, The Leading National Research Centre Programme (KNOW) for years 2014-2018

References

[1] G. Forkmann, S. Martens, Current Opinion in Biotechnology 2001, 12, 155–160. [2] L. H. Yao, Y. M. Jiang, J. Shi, F. A. Tomas-Barberan, N. Datta, R. Singanusong, S. S. Chen, Plant Foods for

Human Nutrition 2004, 59, 113-122. [3] A. Wang, F. Zhang, L. Huang, X. Yin, H. Li, Q. Wang, Z. Zeng, T. Xie, Journal of Medicinal Plants Research

2010, 4, 847-856. [4] C. A. Rice-Evans, N. J. Miller, G. Paganga, Free Radical Biology and Medicine 1996, 20, 933-956. [5] P. G. Pietta, Journal of Natural Products 2000, 63, 1035-1042. [6] H, Cao, X. Chen, A.R. Jassbi, J. Xiao, Biotechnology Advances 2015, 33, 214-223. [7] A. Wang, F. Zhang, L. Huang, X. Yin, H. Li, Q. Wang, Z. Zeng, T. Xie, Journal of Medicinal Plants Research

2010, 4, 847-856.

79

P31

Microbial Glycosylation of Flavonoids Jarosław Popłoński1, Sandra Sordon, Tomasz Tronina, Agnieszka Bartmańska, Ewa Huszcza 1 Department of Chemistry, University of Environmental and Life Sciences, Wrocław, Poland- jarek.poplonski@gmail.com

Keywords: biotransformation,glycosylation, flavonoids

Flavonoids are plants secondary metabolites known for numerous positive effects exhibited in humans. Their wide spectrum of biological activities have been used in herbal medicine for years - to threat microbial infections and as anti-inflammatory agents. Nowadays, scientists over the world begins to explain this biological phenomena and currently the anticancer activity is in the spotlight. A great effort was taken to identify bioactive flavonoids, explain their mechanisms of action and significance in diet. The weak point of flavonoids application in medicine is their very low water solubility and bioavailability, thus reaching in vivo required therapeutic plasma levels after ingestion are limited. Although a vast number of these compounds are found as mono-, di-, tri-glycosides, the most of recognized promising anticancer flavonoids, such as isolated from hops - xanthohumol, are known to be in aglycone form. Recently, there is an interest in determination of key factors influencing and increasing flavonoids bioavailability and glucosylation was found to be very promising [1-3]. Chemical synthesis of flavonoid glucosides is far from trivial, since multi step chemical reactions suffer from low yields and limited selectivity. Therefore biotransformations seams to be a „natural” method of choice. There are examples of flavonoid microbial glucosylations, as a relatively simple and cheap methods [4]. Moreover, new applications of biocatalysts appear with such enzyme groups as: glycoside transferases, transglycosidases, glycoside phosphorylases and glycoside hydrolases [5]. Herein we would like to present the results of filamentous fungi glycosylation of flavonoids with an emphasis on hops prenylflavonoids. All products obtained are flavonoid O-glycosides with strain-depending regioselectivity and yields ranging from 30% up to 89%.

Fig. 1 Microbial glucosylation of xanthohumol by Absidia coerulea.

Acknowledgements

This work was financed by National Science Centre, Grant No. DEC-2015/17/D/NZ9/02060.

References

[1] P.C.H. Hollman, M.N.C.P. Bijsman, Y. van Gameren, E.P.J Cnossen, J.H.M. de Vries and M.B. Katan, Free Radical Research 1999, 31, 569-573.

[2] C. Felgines, O. Texier, C. Morand, C. Manach, A. Scalbert, F. Regerat and C. Remesy, American Journal of Physiology Gastrointestinal and Liver Physiology 2000, 279, G1148-G1154.

[3] S.T. Thilakarathena and H.P V. Rupasinghe, Nutrients 2013, 5, 3367-3387. [4] J. Xiao, T.S. Muzashvili and M.I. Georgiev, Biotechnology advances 2014, 32, 1145-1156. [5] T. Desmet and W. Soetaert, Biocatalysis and Biotransformation 2011, 1, 1-18.

80

P32

Paracetamol Degradation by Microbial Consortium Urszula Guzik

1, Danuta Wojcieszyńska

1, and Katarzyna Hupert-Kocurek

1

1University of Silesia in Katowice, Faculty of Biology and Environmental Protection, Department of Biochemistry, Katowice,

Poland; urszula.guzik@us.edu.pl

Keywords: paracetamol, microbial consortium, biodegradation

The increased use of non-steroidal anti-inflammatory drugs, including paracetamol, has contributed to their presence in the environment. Paracetamol has been found in surface water, in the sewage influents and effluents, and even in drinking water [1]. Although detected concentration of this drug is low (nanogram to microgram per liter), it may accumulate in tissues and induce adverse effects in organisms such as acute and chronic damage, reproductive damage, inhibition of cell proliferation or behavioral changes. For that reason it is necessary to develop innovative and cost-effective biological methods for paracetamol wastewater treatment [2].

The aim of the presented work was to evaluate the ability of microbial consortium isolated from the activated sludge of a sewage treatment plant Klimzowiec in Chorzów, Poland to paracetamol degradation.

The microbial consortium consisted of bacterial strains: Pseudomonas sp. KB3, Pseudomonas sp. KB4, Pseudomonas sp. KB5, Acinetobacter sp. KB3, Acinetobacter sp. KB4, Sphingomonas sp. KB3, and Bacillus sp. KB3. The consortium was routinely cultivated in the nutrient broth at 30ºC and 130 rpm for 24 hours. After this, cells were harvested by centrifugation (5,000 x g at 4ºC for 15 min), washed with a fresh sterile medium and used as inoculum.

Degradation of paracetamol was performed in 500 ml Erlenmeyer flasks containing 250 ml of a mineral salts medium [3] inoculated with cells (in the same proportion) to a final optical density of about 0.5 at λ = 600 nm (OD600). Paracetamol was added to obtain a final concentration of 4 - 25 mg/L, and culture was incubated with shaking at 30ºC. Chromatographic analyses of the culture fluid and measurements of the culture growth were carried out every 4 hours.

The concentration of paracetamol was determined by HPLC using the Merck Hitachi HPLC reversed-phase chromatograph equipped with an Ascentis Express ® C18 HPLC Column (100 x 4.6mm), an Opti-Solw ® EXP pre-column, and a UV/VIS DAD detector. The mobile phase composed of 1% acetic acid and methanol (95:5 v/v) at a flow rate of 1 mL/min was used during the analysis of paracetamol concentration. The detection wavelength was set at 260 nm. Paracetamol in supernatant was identified by comparing the HPLC retention time and UV-visible spectra with that of the external standard.

We have shown that the first dose of paracetamol (4 mg/L) was degraded by microbial consortium during 8 hours. 12 mg/L of paracetamol was removed after 12 hours while 25 mg/L was decomposed during 20 hours. Simultaneously, growth of microbial consortium was observed. It indicates that paracetamol is carbon and energy source for the used microorganisms.

The obtained results indicate that the mixed bacterial consortium may be a useful tool in the biological wastewater treatment.

Acknowledgements

This work was financed by the National Science Centre (Poland), granted on the basis of decision DEC-

2013/09/B/NZ9/00244.

References

[1] A. Marchlewicz, U. Guzik and D. Wojcieszyńska, Water Air Soil Pollution 2015, 226. [2] L. Zhang, J. Hu, R. Zhu, Q. Zhou, and J. Chen, Applied Microbiology and Biotechnology 2013, 97, 3687. [3] I. Greń, D. Wojcieszyńska, U. Guzik, M. Perkosz, and K. Hupert-Kocurek, World Journal of Microbiology and

Biotechnology 2010, 26, 289.

81

P33

Effect of Temperature on Microbiological Degradation of Paracetamol Katarzyna Hupert-Kocurek

1, Urszula Guzik

1 and Danuta Wojcieszyńska

1

1University of Silesia in Katowice, Faculty of Biology and Environmental Protection, Department of Biochemistry, Katowice,

Poland; katarzyna.hupert-kocurek@us.edu.pl

Keywords: paracetamol, Acinetobacter, temperature

Paracetamol (4-acetaminophenol) is one of the commonly sold and consumed non-steroidal anti-inflammatory drugs in the world. Because of the high intake, paracetamol and their metabolites continuously enter and accumulate in the environment [1]. Long-term exposure to paracetamol can cause adverse health effects in organisms. It has been shown that N-acetyl-p-benzoquinone imine, one of the intermediate of paracetamol biotransformation in humans, is hepatotoxic [2]. Therefore, there is a need to isolate microorganisms able to degrade paracetamol and characterize the optimal conditions for its degradation.

The aim of the study was to determine the effect of temperature on the degradation activity of Acinetobacter sp. KB3 able to degrade paracetamol.

Acinetobacter sp. KB3 was routinely cultivated in the nutrient broth at 30ºC and 130 rpm for 24 hours. After this, cells were harvested by centrifugation (5,000 x g at 4ºC for 15 min), washed with a fresh sterile medium and used as inoculum.

Degradation of paracetamol was performed in 500 ml Erlenmeyer flasks containing 250 ml of a mineral salts medium [3] inoculated with bacterial cells to a final optical density of about 0.5 at λ = 600 nm (OD600). Paracetamol was added to the culture to obtain a final concentration of 5 mg/L, and all cultures were incubated with shaking at 4, 25, 30, and 40ºC. Chromatographic analyses of the culture fluid and determination of the cultures growth were carried out every 4 hours. All cultures were grown in triplicates. Additionally, an uninoculated control consisted of the mineral salts medium (abiotic degradation control), and a heat-killed control consisted of bacterial cells destroyed by autoclaving (adsorption onto biomass control) were prepared. The optical density of the heat-killed control was the same as for the examined cultures.

The concentration of paracetamol was determined by HPLC using the Merck Hitachi HPLC reversed-phase chromatograph equipped with an Ascentis Express ® C18 HPLC Column (100 x 4.6mm), an Opti-Solw ® EXP pre-column, and a UV/VIS DAD detector. The mobile phase composed of 1% acetic acid and methanol (95:5 v/v) at a flow rate of 1 mL/min was used during the analysis of paracetamol concentration. The detection wavelength was set at 260 nm. Paracetamol in supernatant was identified by comparing the HPLC retention time and UV-visible spectra with that of the external standard.

The results showed that Acinetobacter sp. KB3 was able to degrade paracetamol with the highest efficiency at 30ºC. At this temperature approximately 88.2% of paracetamol was removed within 11 hours. At 25 and 40ºC, 87% and 85.8% of the added paracetamol was degraded, respectively. At 4ºC there was no loss of paracetamol.

We have shown that the Acinetobacter sp. KB3 strain is a typical mesophilic bacterium and could be potentially useful in the removal of pharmaceutical pollutants from the environment.

Acknowledgements

This work was financed by the National Science Centre (Poland), granted on the basis of decision DEC-

2013/09/B/NZ9/00244.

References

[1] L. Zhang, J. Hu, R. Zhu, Q. Zhou, and J. Chen, Applied Microbiology and Biotechnology 2013, 97, 3687. [2] A. Marchlewicz, U. Guzik and D. Wojcieszyńska, Water Air Soil Pollution 2015, 226. [3] I. Greń, D. Wojcieszyńska, U. Guzik, M. Perkosz, and K. Hupert-Kocurek, World Journal of Microbiology and

Biotechnology 2010, 26, 289.

P34

Effect of pH on Microbiological Degradation of Paracetamol Danuta Wojcieszyńska

1, Urszula Guzik

1 and Katarzyna Hupert-Kocurek

1

1University of Silesia in Katowice, Faculty of Biology and Environmental Protection, Department of Biochemistry, Katowice,

Poland; danuta.wojcieszynska@us.edu.pl

Keywords: paracetamol, Acinetobacter, degradation

Paracetamol is the most popular over-the-counter, analgesic and antipyretic drug. Due to its common use, its presence in the environment is frequently observed [1]. Currently, chemical oxidation methods are mainly applied during paracetamol wastewater treatment. However, because of the harsh reaction conditions, high operational costs, and generation of toxic secondary pollutants there is a need to develop technologies for biological removal of paracetamol from the wastewaters [2].

The aim of the presented work was to evaluate the ability of Pseudomonas sp. KB3 to degrade paracetamol at various pH.

Pseudomonas sp. KB3 strain was routinely cultivated in the nutrient broth at 30ºC and 130 rpm for 24 hours. After this, cells were harvested by centrifugation (5,000 x g at 4ºC for 15 min), washed with a fresh sterile medium and used as inoculum.

Degradation of paracetamol was performed in 500 ml Erlenmeyer flasks containing 250 ml of a mineral salts medium [3] (at pH 4-8.2) inoculated with cells to a final optical density of about 0.5 at λ = 600 nm (OD600). Paracetamol was added to obtain a final concentration of 25 mg/L, and all cultures were incubated with shaking at 30ºC. Chromatographic analyses of the culture fluid and measurements of the cultures growth were carried out every 30 minutes. All cultures were grown in triplicates. Additionally, control cultures (250 mL) were prepared: an uninoculated control consisted of the mineral salts medium only (abiotic degradation control), and a heat-killed control consisted of bacterial cells destroyed by autoclaving (adsorption onto biomass control). The optical density of the heat-killed control was the same as for the examined cultures.

The concentration of paracetamol was determined by the HPLC technique using the Merck Hitachi HPLC reversed-phase chromatograph equipped with an Ascentis Express ® C18 HPLC Column (100 x 4.6mm), the Opti-Solw ® EXP pre-column, and a UV/VIS DAD detector. The mobile phase composed of 1% acetic acid and methanol (95:5 v/v) at a flow rate of 1 mL/min was used during the analysis of paracetamol concentration. The detection wavelength was set at 260 nm. Paracetamol in supernatant was identified by comparing the HPLC retention time and UV-visible spectra with that of the external standard.

We have shown that Pseudomonas sp. KB3 was able to degrade paracetamol with the highest efficiency at pH 7.0. Under these conditions, total degradation of paracetamol was observed after 2 hours. Slightly lower degradation rate was observed at pH 8.0. The weakest degradation of paracetamol was observed at pH 4.0-5.0.

The obtained results indicate that Pseudomonas sp. KB3 strain is able to degrade paracetamol in the wide range of pH and may be potentially useful in the biodegradation of pharmaceutical pollutants.

Acknowledgements

This work was financed by the National Science Centre (Poland), granted on the basis of decision DEC-

2013/09/B/NZ9/00244.

References

[1] B. de Gusseme, L. Vanhaecke, W. Verstraete, and N. Boon, Water Research 2011, 45, 1829. [2] L. Zhang, J. Hu, R. Zhu, Q. Zhou, and J. Chen, Applied Microbiology and Biotechnology 2013, 97, 3687. [3] I. Greń, D. Wojcieszyńska, U. Guzik, M. Perkosz, and K. Hupert-Kocurek, World Journal of Microbiology and

Biotechnology 2010, 26, 289.

83

P35

Compounds About Antioxidant and Antibacterial Activity Synthesised by Fungal Laccase

Jolanta Polak, Kamila Wlizło, Katarzyna Szałapata, Monika Osińska-Jaroszuk, Anna Jarosz-Wilkołazka

Department of Biochemistry, Maria Curie-Sklodowska University, Lublin, Poland – anna.wilkolazka@poczta.umcs.lublin.pl

Keywords: laccase, green chemistry, antioxidants, antimicrobial agents

Currently ongoing search for new bioactive compounds that can replace the chemicals used for decades

in the treatment of many diseases. Apart from natural compounds having antimicrobial activity, the search of effective antimicrobial agents are focusing on the synthesis of new compounds via classical organic methods as well as enzyme-catalysed biotechnological processess, which are a “green alternative” to conventional organic synthesis reactions. Particularly noteworthy is the fungal laccase used as catalysts, which has the ability to oxidise many organic compounds into products with new physical and chemical properties, including dyes, antimicrobial, anticancer and antioxidant agents [1, 2]. The use of laccase in organic synthesis has many advantages include water and energy savings, the use of lower amounts of chemicals, and milder process conditions [2].

Laccase from fungal strain Cerrena unicolor catalysed the homomolecular and heteromolecular transformations of simple aromatic compounds into products about new spectroscopic properties. Precursors were benzene or naphthalene derivatives containing amino, methoxy, carboxy, hydroxy or sulfonic groups providing high reactivity of tested precursors during laccase-mediated oxidation [3]. The obtained compounds were tested under their antioxidant and antimicrobial properties. The ability to inhibit the growth of Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli) bacteria was tested using qualitative methods. The antioxidant properties were evaluated using the chemiluminescence assay with the Fe(II)-H2O2-luminol test and the spectrophotometric free radical-scavenging test with ABTS

.+ as radicals [4]. The new compounds exhibit

the antioxidant capacity as well as antibacterial activity against Staphylococcus aureus. The biotransformation of simple organic chemicals

into new antimicrobial agents as well as antioxidants is possible by using of fungal laccase as the catalyst. The process of precursor’s transformation is very efficient, environmentally-friendly, with contribution of molecular oxygen – the purest co-substrate of biotransformation.

Acknowledgements

This work was partially supported by Ministry of Science and Higher Education Iuventus Plus Program (0433/IP1/2011/71) and National Science Centre (NN 302 633040).

References [5] A. Mikolasch, F. Schauer, Appl. Microbiol. Biotechnol. 2009, 82, 605–624. [6] J. Polak, A. Jarosz-Wilkołazka, Biotechnol. Progr. 2012, 23, 93-102. [7] J. Polak, A. Jarosz-Wilkolazka, Process Biochem. 2012, 47, 1295-1307. [8] J. Polak, A. Jarosz-Wilkołazka, K. Szałapata, M. Graz, M. Osińska-Jaroszuk, New Biotechnol. 2016, 33, 255-

262.

84

P36

A Novel Immobilization Approach for the Ectoine Production from Halophiles John Chi-Wei Lan

1, Po-Wei Chen

1 and Yu-Hong Wei

2

1 Biorefinery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze

University, Chungli, Taiwan – lanchiwei@saturn.yzu.edu.tw 2 Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Chungli, Taiwan

Keywords: immobilization, ectoine, fermentation

A halophilic bacterium isolated from a salt environment in southern Taiwan was identified as a Marinococcus sp. The halophiles could synthesize and accumulate intracellular ectoine as a compatible solute capable to resist osmotic stress in a hyper-osmotic environment [1]. The cell immobilization has been shown to offer many advantages for biomass and metabolite productions compared with free-cell (FC) systems such as: high cell density and very high volumetric productivity, reuse of biocatalysts, high process stability (physical and biological) over long fermentation periods, retention of plasmid-bearing cells, improved resistance to contamination, uncoupling of biomass and metabolite productions, stimulation of production and secretion of secondary metabolites and physical and chemical protection of the cells [2].The approaches for enhancing ectoine production with respect to operational stability, sustainable character and cost evaluation by several of cell immobilization manners were investigated and compared. One of the approach is to immobilize cells on modified biodegradable material, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV). The casting and electrospinning PHBV membranes were surface modified by plasma technology as shown in Fig.1. The further comparison of ectoine expression is discussed.

Fig. 1. Scanning electron micrographs of untreated and plasma treated PHBV ((A)PHBV solvent-casting film (B) PHBV Solvent-casting film by plasma (C) PHBV Electrospinning film (D) PHBV Electrospinning film by plasma)

Acknowledgements

This work was granted by the Ministry of Science and Technology (Taiwan) with project number MOST104-

2632-E-155-001.

References

[1] A. E. Onraedt, B. A. W., Wim, K. Soetaert, and E. J. Vandamme. Biotechnology Progress, Optimization of Ectoine Synthesis through Fed-Batch Fermentation of Brevibacterium epidermis (2005), 21, 1206-1212.

[2] A. Ogan, O. Danis, A. Gozuacik, E. Cakmar, and M. Birbir, Production of cellulase by immobilized whole cells of Haloarcula (2012). Applied Biochemistry and Microbiology 48(5): 440-443.

85

P37

Methanotrophic Cell-mediated Biotransformation of Alkane to Alkanol Eun Yeol LEE Chemical Engineering Department, Kyung Hee University, Yongin-si, South Korea – eunylee@khu.ac.kr

Keywords: methanotroph, methane, methanol, alkane, alkanol, biotransformation

A novel methanotroph was isolated from activated sludge and characterized using various techniques such as phylogenetic analysis, electron microscopy, and chemotaxonomic analysis. The isolate was designated as Methylomonas sp. DH-1. Methylomonas sp. DH-1 can utilize methane, ethane, or propane as a sole carbon and energy source. DH-1 grew well in a methane concentration of 30 % (v/v) with a copper concentration of 10 μM, a pH of 9.0 and an optimal temperature of 30

oC in a batch culture condition. Using the strain DH-1 as a biocatalyst,

C1-C3 alkanes were transformed into their corresponding alcohols in the presence of 0.5 mM EDTA as an inhibitor of methanol dehydrogenase and 40 mM sodium formate.

Fig. 1 Biotransformation of C1-C3 alkane to the corresponding alcohols by Methylomonas sp. DH-1.

Acknowledgements

This research was supported by C1 Gas Refinery Program through the National Research Foundation of

Korea (NRF) funded by the Ministry of Science, ICT & Future Plannig (NRF-2015M3D3A1A01064882).

References

[1] I. Y. Hwang, et al., J. Microbiol. Biotechnol. 2014 24, 1597-1605. [2] D.H. Hur, et al., J Chem Technol Biotechnol. 2016 DOI: 10.1002/jctb.5007.

86

87

Author Index

88

89

A

Andrade Leandro Helgueira

University of São Paulo

leandroh@iq.usp.br L22

Andrzejczak Olga

Lodz University of Technology

binoz.p.lodz P02

Antczak Tadeusz

Lodz University of Technology

tadeusz.antczak@p.lodz.pl P07

Arct Jacek

Academy of Cosmetics and Health Care L27

B

Bartha-Vári Hajnal

Babeş-Bolyai University of Cluj-Napoca L18

Bartmańska Agnieszka

Wrocław University of Environmental and

Life Science P30, P31

Bauer Marta

Tech-Lab.pl

tech-lab@lipopharm.pl L03

Bánóczi Gergely

Budapest University of Technology and

Economics L18, P10

Bencze László C.

Babeş-Bolyai University of Cluj-Napoca

cslbencze@chem.ubbcluj.ro

L18, P10,

P11

Białkowska Aneta

Lodz University of Technology P03

Bielecki Stanisław

Lodz University of Technology L29

Borowiecki Paweł

Warsaw University of Technology

pborowiecki@ch.pw.edu.pl L05

Borys Filip

Institute of Organic Chemistry PAS

fborys@icho.edu.pl P20

Bretner Maria

Warsaw University of Technology

mbretner@ch.pw.edu.pl P14

Breuer Michael

BASF SE L02

Brodzka Anna

Institute of Organic Chemistry PAS

anna.brodzka@icho.edu.pl P19, P21

Brzezińska-Rodak Małgorzata

Wrocław University of Technology

malgorzata.brzezinska-rodak@pwr.edu.pl

L28, L30,

P28

Bryjak Jolanta

Wrocław University of Technology

jolanta.bryjak@pwr.wroc.pl L11

Bujok Agata

Silesian University of Technology L23

Busto Eduardo

University of Graz L04, L06

C

Chen Po-Wei

Yuan Ze University P36

Chrobok Anna

Silesian University of Technology L23

Cuetos Aníbal

University of York L15

D

Dennig Alexander

University of Graz L06

Díaz-Rodríguez Alba

University of Oviedo L15, P01

Dobrzelewski Krzysztof

Warsaw University of Technology P14

Domaradzka Dorota

University of Silesia in Katowice

ddomaradzka@us.edu.pl L24

E

Ender Ferenc

Budapest University of Technology and

Economics L18

F

Faber Kurt

University of Graz

kurt.faber@uni-graz.at L06, L09

Faisal Muhammad

University of the Punjab

mohdfaysal@yahoo.com P08

Farnberger Judith

University of Graz L04

Filip Alina

Babeş-Bolyai University of Cluj-Napoca

filip_alina_m@yahoo.com L18, P10

90

Fischereder Eva-Maria

University of Graz L15

Florczak Tomasz

Lodz University of Technology P04, P05

Fuchs Michael

University of Graz L06

G

Gandomkar Somayyeh

University of Graz L04

García-Ramos Marina

University of Oviedo L15

Gilner Danuta

Silesian University of Technology

danuta.gilner@polsl.pl L23

Glueck Sylvia

University of Graz

si.glueck@uni-graz.at L06

Głąb Agata

Wrocław University of Science and

Technology

agata.glab@pwr.edu.pl

L20

Główczyk – Zubek Joanna

Warsaw University of Technology

jmzubek@ch.pw.edu.pl P12

Gotor Vicente

University of Oviedo L15, P01

Gotor-Fernández Vicente

University of Oviedo P01

Grivalský Tomáš

Slovak Academy of Sciences

tomasgrivalsky@gmail.com P15

Grogan Gideon

University of York L15

Guzik Urszula

University of Silesia in Katowice

urszula.guzik@us.edu.pl

L24, P32,

P33, P34

H

Hall Melanie

University of Graz L06, L09

Hasnain Shahida

The Women University Multan P09

Hauer Bernhard

Universitaet Stuttgart L07

Humphreys Luke D.

GSK Medicines Research Centre L02

Hupert-Kocurek Katarzyna

University of Silesia in Katowice

katarzyna.hupert-kocurek@us.edu.pl

P32, P33,

P34

Huszcza Ewa

Wrocław University of Environmental and

Life Science P30, P31

I

Irimie Florin-Dan

Babeş-Bolyai University of Cluj-Napoca

irimie@chem.ubbcluj.ro

L18, L19,

P10, P11

Itoh Toshiyuki

Tottori University

titoh@chem.tottori-u.ac.jp L14

J

Jadczak Klaudia

Lodz University of Technology

klaudia.jadczak@dokt.p.lodz.pl P04

Janicki Ignacy

Centre of Molecular and Macromolecular

Studies PAS

ijanicki@cbmm.lodz.pl

L09

Jarosz-Wilkołazka Anna

Maria Curie-Sklodowska University

anna.wilkolazka@poczta.umcs.lublin.pl L08, P35

Jarzębski Andrzej

Silesian University of Technology

andrzej.jarzebski@polsl.pl L11

Ježová Lubica

Institute of Microbiology, Czech Academy

of Science P16

Jodłowska Iga

Lodz University of Technology

jodlowska.iga@gmail.com P05

Juskowiak Bernard

Adam Mickiewicz University in Poznan L26, P18

K

Kaczmarek Michał B.

Lodz University of Technology

michal.kaczmarek@dokt.p.lodz.pl P06

Kaczorek Ewa

Poznan University of technology L12, L13,

P17

Kadotani Shiho

Tottori University P14

Kamysz Wojciech

Tech-Lab.pl

tech-lab@lipopharm.pl L03

91

Kiełbasiński Piotr

Centre of Molecular and Macromolecular

Studies PAS

piokiel@cbmm.lodz.pl

L09

Klimek-Ochab Magdalena

Wrocław University of Technology L28, P29

Kliszewicz Justyna

Warsaw University of Technology L12

Kłossowski Szymon

Institute of Organic Chemistry PAS

szymon.klossowski@icho.edu.pl P24

Kmiecik Natalia

Wroclaw University of Technology

natalia.kmiecik@pwr.wroc.pl P26

Knaus Tanja

University of Amsterdam L02

Kolesińska Beata

Lodz University of Technology P03

Kopiasz Rafał

Warsaw University of Technology

Institute of Organic Chemistry PAS

kopiaszrafal@gmail.com

P25

Kosińska Edyta

Warsaw University of Technology P12

Kosman Joanna

Adam Mickiewicz University in Poznan

joannakosman@gmail.com L26, P18

Koszelewski Dominik

Institute of Organic Chemistry PAS

dominik.koszelewski@icho.edu.pl

P19, P20,

P22, P2,

P24

Kowalczykiewicz Daria

Silesian University of Technology L23

Kowalkowska Anna

Warsaw University of Technology P13

Křen Vladimír

Institute of Microbiology, Czech Academy

of Science P16

Křenková Alena

Institute of Microbiology, Czech Academy

of Science P16

Kroutil Wolfgang

University of Graz

Wolfgang.Kroutil@uni-graz.at

L04, L06,

L15

Küchler Andreas

ETH Zürich L21

Kuru Ali

Sakarya University L28

Kwiatos Natalia

Lodz University of Technology

natalia.kwiatos@dokt.p.lodz.pl L29

L

Lăcătuș Mihai

Babes-Bolyai University

lacatusmihai@chem.ubbcluj.ro P11

Lan John Chi-Wei

Yuan Ze University

lanchiwei@saturn.yzu.edu.tw P36

Lavandera Iván

University of Oviedo

lavanderaivan@uniovi.es L15, P01

Lee Eun Yeol

Kyung Hee University

eunylee@khu.ac.kr P37

Lenz Maike

Universitaet Stuttgart L07

Lichawska-Olczyk Joanna

Textile Research Institute L08

López-Iglesias María

Universidad de Oviedo P01

Ł

Łukowska – Chojnacka Edyta

Warsaw University of Technology

elukowska@ch.pw.edu.pl P13

M

Madej Anna

University of Environmental and Life

Sciences P30

Madej Arleta

Institute of Organic Chemistry PAS

arleta.madej@icho.edu.pl P22

Majewska Paulina

Wroclaw University of Technology

paulina.majewska@pwr.wroc.pl P27

Mangas-Sánchez Juan

Universidad de Oviedo P01

Martínez-Montero Lía

Universidad de Oviedo P01

Matuszek Karolina

Silesian University of Technology L23

92

Maurin Jan

Warsaw, National Centre for Nuclear

Research P12

McInerney Michael

University of Oklahoma P09

Méndez-Sánchez Daniel

Universidad de Oviedo P01

Moisă Mădălina Elena

Babeş-Bolyai University of Cluj-Napoca

mmoisa@chem.ubbcluj.ro L19

Morawski Krzysztof

Lodz University of Technology

krzysztof.morawski@dokt.p.lodz.pl P03

Mourelle-Insua Ángela

Universidad de Oviedo P01

Musioł Marta

Silesian University of Technology L23

Mutti Francesco G.

University of Amsterdam

f.mutti@uva.nl L02

N

Nestl Bettina M.

Universitaet Stuttgart

bettina.nestl@itb.uni-stuttgart.de L07

Nishihara Takashi

Tottori University L14

Nokami Toshiki

Tottori University L14

O

Ochal Zbigniew

Warsaw University of Technology

ochal@ch.pw.edu.pl P14

Osińska-Jaroszuk Monika

Maria Curie-Sklodowska University P35

Ostaszewski Ryszard

Institute of Organic Chemistry PAS

ryszard.ostaszewski@icho.edu.pl

P19, P20,

P21, P22,

P23, P24,

P25

P

Paizs Csaba

Babeş-Bolyai University of Cluj-Napoca L18, L19,

P10, P11

Pangallo Domenico

Institute of Molecular Biology, Slovak

Academy of Sciences P15

Paprocki Daniel

Institute of Organic Chemistry PAS

dpaprockizolw@gmail.com

P19, P23,

P24

Payer Stefan E.

University of Graz L04

Perez Manuel

Mestrelab Research SL

Manuel@mestrelab.com L17

Pickl Matthias

University of Graz L06

Pitzer Julia

University of Graz L06

Polak Jolanta

Maria Curie-Sklodowska University

jpolak@poczta.umcs.lublin.pl L08, P35

Popłoński Jarosław

Wrocław University of Environmental and

Life Science

jarek.poplonski@gmail.com

P30, P31

Poppe László

Budapest University of Technology and

Economics

poppe@mail.bme.hu

L18, L19,

P10

Pytkowska Katarzyna

Academy of Cosmetics and Health Care L27

R

Ratz-Łyko Anna

Academy of Cosmetics and Health Care

anna.ratz-lyko@wszkipz.pl L27

Rehman Yasir

University of the Punjab

Yasir.mmg@pu.edu.pk P09

Ríos-Lombardía Nicolás

Universidad de Oviedo P01

Rizvi Fariha

University of the Punjab P09

Rugor Agnieszka

Jerzy Haber Institute of Catalysis and

Surface Chemistry PAS

ncrugor@cyfronet.pl

L25

Rychlý Jozef

Polymer Institute Slovak Academy of

Sciences P15

S

Scheller Philipp

Universitaet Stuttgart L07

93

Schrittwieser Jörg H.

University of Graz L04

Scrutton Nigel S.

University of Manchester L02

Simon Robert S.

University of Graz L04

Siudakiewicz Sylwia

Wrocław University of Technology

sylwiasiudakiewicz@gmail.com L28, P28

Smułek Wojciech

Poznan University of Technology

wojciech.smulek@doctorate.put.poznan.pl

L12, L13,

P17

Sordon Sandra

University of Environmental and Life

Sciences

sandra.sordon@up.wroc.pl

P30, P31

Sójka-Ledakowicz Jadwiga

Textile Research Institute L08

Strzelecki Bartosz

Lodz University of Technology L29

Stanisławska Alicja

Adam Mickiewicz University in Poznan

stanislawska.alicja@gmail.com P18

Struszczyk-Świta Katarzyna

Lodz University of Technology

katarzyna.struszczyk@p.lodz.pl P06

Szaleniec Maciej

Jerzy Haber Institute of Catalysis and

Surface Chemistry PAS

ncszalen@cyfronet.pl

L25

Szałapata Katarzyna

Maria Curie-Sklodowska University P35

Szaniawska Magdalena

Maria Curie-Sklodowska University L16

Szczęsna-Antczak Mirosława

Lodz University of Technology

miroslawa.szczesna-antczak@p.lodz.pl P06, P07

Szeląg Jakub

Technical University of Lodz

jakub.szelag@dokt.p.lodz.pl P07

Szmigiel Beata

Wroclaw University of Technology

beata.szmigiel@pwr.edu.pl L30

Szuster-Ciesielska Agnieszka

Maria Curie-Sklodowska University L08

Szymańska Katarzyna

Silesian University of Technology

Katarzyna.Szymanska@polsl.pl L11, L23

Szymczyk Katarzyna

Maria Curie-Sklodowska University L16

Ś

Śmiłowski Jakub

Silesian University of Technology L23

T

Taraba Anna

Maria Curie-Sklodowska University

annataraba@gmail.com L16

Tarasiuk Aleksandra

Technical University of Lodz P07

Tassano Erika

University of Graz L06

Toşa Monica Ioana

Babeş-Bolyai University of Cluj-Napoca L19

Tronina Tomasz

Wrocław University of Environmental and

Life Science P30, P31

Trzepizur Damian

Warsaw University of Technology P19

Turkiewicz Marianna

Lodz University of Technology P04, P05

Turner Nicolas J.

University of Manchester

nicholas.turner@manchester.ac.uk L01, L02

Turrini Nikolaus

University of Graz L09

V

Valentová Kateřina

Institute of Microbiology, Czech Academy

of Science P16

Vavříková Eva

Institute of Microbiology, Czech Academy

of Science

vavrikova@biomed.cas.cz

P16

Vértessy Beáta G.

Budapest University of Technology and

Economics L18

W

Walde Peter

ETH Zürich

peter.walde@mat.ethz.ch L21, P23

94

Wei Yu-Hong

Yuan Ze University P36

Weinmann Leonie

Universitaet Stuttgart L07

Weiser Diána

Budapest University of Technology and

Economics L18

Wielechowska Monika

Warsaw University of Technology

mwielechowska@ch.pw.edu.pl P12

Wieszczycka Karolina

Poznan University of Technology P17

Wlizło Kamila

Maria Curie-Sklodowska University L08, P35

Wohlgemuth Roland

Sigma-Aldrich

wohlgemuth@catv.rol.ch L10

Wojciechowska Aleksandra

Poznan University of Technology P17

Wojcieszyńska Danuta

University of Silesia in Katowice

danuta.wojcieszynska@us.edu.pl

L24, P32,

P33, P34

Wójcik Anna

Jagiellonian University

L25

Z

Zakrzewska Alicja

Wrocław University of Technology L28, P28

Zdarta Agata

Poznan University of Technology

agata.zdarta@doctorate.put.poznan.pl L12, L13

Zielonka Aleksandra

Wrocław University of Technology

aleksandra.zielonka@pwr.edu.pl P29

Zysk Małgorzata

Institute of Organic Chemistry PAS

malgorzata.cwiklak@icho.edu.pl P19

Ż

Żądło Anna

Institute of Organic Chemistry PAS

anna.zadlo@icho.edu.pl

P19, P22,

P23, P24

Żukowski Krzysztof

Adam Mickiewicz University in Poznan

krzysztof.zukowski@amu.edu.pl L26

Żymańczyk – Duda Ewa

Wrocław University of Technology

ewa.zymanczyk-duda@pwr.wroc.pl

L20, L28,

L30, P26

95

List of Participants

96

97

A

Andrade Leandro Helgueira

University of São Paulo

leandroh@iq.usp.br

Andrzejczak Olga

Lodz University of Technology

binoz.p.lodz

B

Bauer Marta

Tech-Lab.pl

tech-lab@lipopharm.pl

Borowiecki Paweł

Warsaw University of Technology

pborowiecki@ch.pw.edu.pl

Borys Filip

Institute of Organic Chemistry PAS

fborys@icho.edu.pl

Brodzka Anna

Institute of Organic Chemistry PAS

anna.brodzka@icho.edu.pl

Brzezińska-Rodak Małgorzata

Wrocław University of Technology

malgorzata.brzezinska-rodak@pwr.edu.pl

D

Domaradzka Dorota

University of Silesia in Katowice

ddomaradzka@us.edu.pl

Drabowicz Józef

Centre of Molecular and Macromolecular Studies PAS

Jan Dlugosz University in Czestochowa

draj@cbmm.lodz.pl

F

Faber Kurt

Acib GmbH, University of Graz

kurt.faber@uni-graz.at

Faisal Muhammad

University of the Punjab

mohdfaysal@yahoo.com

Falcioni Francesco

Piramal Healthcare Ltd.

Francesco.Falcioni@piramal.com

Fichman Merav

The Hebrew University of Jerusalem.

merav.fichman@mail.huji.ac.il

Filip Alina

Babeş-Bolyai University of Cluj-Napoca

filip_alina_m@yahoo.com

G

Gilner Danuta

Silesian University of Technology

danuta.gilner@polsl.pl

Głąb Agata

Wrocław University of Science and Technology

agata.glab@pwr.edu.pl

Główczyk – Zubek Joanna

Warsaw University of Technology

jmzubek@ch.pw.edu.pl

Grivalský Tomáš

Slovak Academy of Sciences

tomasgrivalsky@gmail.com

Guzik Urszula

University of Silesia in Katowice

urszula.guzik@us.edu.pl

H

Hupert-Kocurek Katarzyna

University of Silesia in Katowice

katarzyna.hupert-kocurek@us.edu.pl

I

Itoh Toshiyuki

Tottori University

titoh@chem.tottori-u.ac.jp

J

Jadczak Klaudia

Lodz University of Technology

klaudia.jadczak@dokt.p.lodz.pl

Janicki Ignacy

Centre of Molecular and Macromolecular Studies PAS

ijanicki@cbmm.lodz.pl

Jarosz-Wilkołazka Anna

Maria Curie-Sklodowska University

anna.wilkolazka@poczta.umcs.lublin.pl

Jarzębski Andrzej

Silesian University of Technology

andrzej.jarzebski@polsl.pl

Jodłowska Iga

Lodz University of Technology

jodlowska.iga@gmail.com

98

Jurczak Janusz

Institute of Organic Chemistry PAS

janusz.jurczak@icho.edu.pl

K

Kaczmarek Michał B.

Lodz University of Technology

michal.kaczmarek@dokt.p.lodz.pl

Kafarski Paweł

Wroclaw University of Technology

pawel.kafarski@pwr.edu.pl

Kiełbasiński Piotr

Centre of Molecular and Macromolecular Studies PAS

piokiel@cbmm.lodz.pl

Kmiecik Natalia

Wroclaw University of Technology

natalia.kmiecik@pwr.wroc.pl

Kopiasz Rafał

Warsaw University of Technology

Institute of Organic Chemistry PAS

kopiaszrafal@gmail.com

Koszelewski Dominik

Institute of Organic Chemistry PAS

dominik.koszelewski@icho.edu.pl

Kroutil Wolfgang

University of Graz

Wolfgang.Kroutil@uni-graz.at

Kwiatos Natalia

Lodz University of Technology

natalia.kwiatos@dokt.p.lodz.pl

L

Lăcătuș Mihai

Babes-Bolyai University

lacatusmihai@chem.ubbcluj.ro

Lan John Chi-Wei

Yuan Ze University

lanchiwei@saturn.yzu.edu.tw

Lavandera Iván

University of Oviedo

lavanderaivan@uniovi.es

Lee Eun Yeol

Kyung Hee University

eunylee@khu.ac.kr

Ł

Łukowska – Chojnacka Edyta

Warsaw University of Technology

elukowska@ch.pw.edu.pl

M

Madej Arleta

Institute of Organic Chemistry PAS

arleta.madej@icho.edu.pl

Majewska Paulina

Wroclaw University of Technology

paulina.majewska@pwr.wroc.pl

Moisă Mădălina Elena

Babeş-Bolyai University of Cluj-Napoca

mmoisa@chem.ubbcluj.ro

Morawski Krzysztof

Lodz University of Technology

krzysztof.morawski@dokt.p.lodz.pl

Mutti Francesco G.

University of Amsterdam

f.mutti@uva.nl

N

Nestl Bettina M.

Universitaet Stuttgart

bettina.nestl@itb.uni-stuttgart.de

O

Ochal Zbigniew

Warsaw University of Technology

ochal@ch.pw.edu.pl

Ostaszewski Ryszard

Institute of Organic Chemistry PAS

ryszard.ostaszewski@icho.edu.pl

P

Paprocki Daniel

Institute of Organic Chemistry PAS

dpaprockizolw@gmail.com

Perez Manuel

Mestrelab Research SL

Manuel@mestrelab.com

Polak Jolanta

Maria Curie-Sklodowska University

jpolak@poczta.umcs.lublin.pl

Popłoński Jarosław

Wrocław University of Environmental and Life

Science

jarek.poplonski@gmail.com

Poppe László

Budapest University of Technology and Economics

poppe@mail.bme.hu

99

R

Ratz-Łyko Anna

Academy of Cosmetics and Health Care

anna.ratz-lyko@wszkipz.pl

Rehman Yasir

University of the Punjab

Yasir.mmg@pu.edu.pk

S

Siudakiewicz Sylwia

Wrocław University of Technology

sylwiasiudakiewicz@gmail.com

Smułek Wojciech

Poznan University of Technology

wojciech.smulek@doctorate.put.poznan.pl

Sordon Sandra

University of Environmental and Life Sciences

sandra.sordon@up.wroc.pl

Stanisławska Alicja

Adam Mickiewicz University in Poznan

stanislawska.alicja@gmail.com

Steib Andreas Bayer AG andreaskarl.steib@bayer.com

Szaleniec Maciej

Jerzy Haber Institute of Catalysis and Surface

Chemistry PAS

ncszalen@cyfronet.pl

Szeląg Jakub

Technical University of Lodz

jakub.szelag@dokt.p.lodz.pl

Szmigiel Beata

Wroclaw University of Technology

beata.szmigiel@pwr.edu.pl

T

Taraba Anna

Maria Curie-Sklodowska University

annataraba@gmail.com

Turner Nicolas J.

University of Manchester

nicholas.turner@manchester.ac.uk

V

Vavříková Eva

Institute of Microbiology, Czech Academy of Science

vavrikova@biomed.cas.cz

W

Walde Peter

ETH Zürich

peter.walde@mat.ethz.ch

Wohlgemuth Roland

Sigma-Aldrich

wohlgemuth@catv.rol.ch

Wojcieszyńska Danuta

University of Silesia in Katowice

danuta.wojcieszynska@us.edu.pl

Z

Zakrzewska Alicja Wrocław University of Technology

Zdarta Agata

Poznan University of Technology

agata.zdarta@doctorate.put.poznan.pl

Zielonka Aleksandra

Wrocław University of Technology

aleksandra.zielonka@pwr.edu.pl

Ż

Żądło Anna

Institute of Organic Chemistry PAS

anna.zadlo@icho.edu.pl

Żukowski Krzysztof

Adam Mickiewicz University in Poznan

krzysztof.zukowski@amu.edu.pl

Żymańczyk – Duda Ewa

Wrocław University of Technology

ewa.zymanczyk-duda@pwr.wroc.pl