TURUN YLIOPISTON JULKAISUJA ANNALES UNIVERSITATIS TURKUENSIS SARJA - SER. D OSA - TOM. 977 MEDICA - ODONTOLOGICA TURUN YLIOPISTO UNIVERSITY OF TURKU Turku 2011 IMMOBILIZATION OF BURKHOLDERIA CEPACIA LIPASE: KINETIC RESOLUTION IN ORGANIC SOLVENTS, IONIC LIQUIDS AND IN THEIR MIXTURES by Piia Hara Department of Pharmacology, Drug Development and Therapeutics, Division of Synthetic Chemistry Institute of Biomedicine, Faculty of Medicine University of Turku Turku, Finland Supervisor and custos: Professor Liisa T. Kanerva, Ph.D. Laboratory of Synthetic Drug Chemistry University of Turku Turku, Finland Reviewers: Research Professor Kristiina Kruus, D. Sc. (Tech.) VTT Espoo, Finland and Professor László Poppe, Ph.D. Budapest University of Technology and Economics Budapest, Hungary Opponent: Docent Ossi Turunen, Ph.D. Aalto University, School of Chemical Technology Espoo, Finland ISBN 978-951-29-4720-1 (PRINT) ISBN 978-951-29-4721-8 (PDF) ISSN 0355-9483 Painosalama Oy – Turku, Finland 2011 Abstract 3 AbStRAct Piia Hara Immobilization of Burkholderia cepacia Lipase: Kinetic Resolution in Organic Solvents, Ionic Liquids and in their Mixtures Department of Pharmacology, Drug Development and Therapeutics/Laboratory of Synthetic Drug Chemistry, University of Turku Annales Universitatis Turkuensis, Painosalama Oy, Turku, Finland, 2011. Biocatalysis opens the door to green and sustainable processes in synthetic chemistry allowing the preparation of single enantiomers, since the enzymes are chiral and accordingly able to catalyze chemical reactions under mild conditions. Immobilization of enzymes enhances process robustness, often stabilizes and activates the enzyme, and enables reuse of the same enzyme preparation in multiple cycles. Although hundreds of variations of immobilization methods exist, there is no universal method to yield the highly active, selective and stable enzyme catalysts. Therefore, new methods need to be developed to obtain suitable catalysts for different substrates and reaction environments. Lipases are the most widely used enzymes in synthetic organic chemistry. The literature part together with the experimental part of this thesis discusses of the effects of immobilization methods mostly used to enhance lipase activity, stability and enantioselectivity. Moreover, the use of lipases in the kinetic resolution of secondary alcohols in organic solvents and in ionic liquids is discussed. The experimental work consists of the studies of immobilization of Burkholderia cepacia lipase (lipase PS) using three different methods: encapsulation in sol-gels, cross-linked enzyme aggregates (CLEAs) and supported ionic liquids enzyme catalysts (SILEs). In addition, adsorption of lipase PS on celite was studied to compare the results obtained with sol-gels, CLEAs and SILEs. The effects of immobilization on enzyme activity, enantioselectivity and hydrolysis side reactions were studied in kinetic resolution of three secondary alcohols in organic solvents, in ionic liquids (ILs), and in their mixtures. Lipase PS sol-gels were shown to be active and stable catalysts in organic solvents and solvent:IL mixtures. CLEAs and SILEs were highly active and enantioselective in organic solvents. Sol-gels and SILEs were reusable in several cycles. Hydrolysis side reaction was suppressed in the presence of sol-gels and CLEAs. Keywords: CLEA, immobilization, ionic liquid, kinetic resolution, lipase, organic solvent, secondary alcohol, SILE, sol-gel. 4 Tiivistelmä tIIvISteLMä Piia Hara Burkholderia cepacian lipaasin immobilisointi ja kineettinen resoluutio orgaanisissa liuottimissa, ionisissa nesteissä ja näiden seoksissa Farmakologia, lääkekehitys ja lääkehoito/ Synteettisen lääkekemian laboratorio, Turun Yliopisto Annales Universitatis Turkuensis, Painosalama Oy, Turku, Finland, 2011. Biokatalyysin käyttö synteettisessä kemiassa on ympäristöystävällinen vaihtoehto puh- taiden enantiomeerien valmistamiseksi, sillä entsyymit eivät ole toksisia ja ne katalysoi- vat monivaiheisia kemiallisia reaktioita suoraviivaisesti, usein miedoissa olosuhteissa. Katalyytteinä käytettävien entsyymien immobilisointi lisää usein prosessien vakautta, stabiloi entsyymiä eri reaktio-olosuhteissa, aktivoi entsyymiä sekä mahdollistaa saman entsyymin uudelleenkäytön useissa reaktioissa. Vaikka lukemattomia sovelluksia erilai- sista immobilisointimenetelmistä on kehitetty, ei ole yhtä yleispätevää menetelmää erit- täin aktiivisten, selektiivisten ja stabiilien entsyymikatalyyttien valmistamiseksi. Uusia menetelmiä entsyymien immobilisointiin tarvitaan, jotta saadaan erilaisille yhdisteille ja reaktioympäristöille sopivia tehokkaita katalyyttejä. Lipaasit ovat yleisimmin käytettyjä entsyymejä synteettisessä orgaanisessa kemiassa. Väitöskirjassa tarkastellaan yleisimmin lipaasien immobilisoinnissa käytettyjä menetel- miä, sekä niiden vaikutusta lipaasien aktiivisuuteen, stabiilisuuteen ja enantioselektiivi- syyteen. Lisäksi tarkastellaan lipaasin katalysoimaa kineettistä resoluutiota orgaanisissa liuottimissa ja ionisissa nesteissä. Väitöskirjan kokeellisessa osassa Burkholderia cepacian lipaasi (lipaasi PS) immobi- lisoitiin käyttäen sooli-geeli-, ristiinsidottu entsyymiaggregaatti- (CLEA) ja kiinteän kantajan päälle immobilisoitua nestekatalyytti- (SILE) menetelmiä. Lisäksi lipaasi PS adsorboitiin celiteen tulosten vertailemiseksi. Immobilisoinnin vaikutusta entsyymin aktiivisuuteen, enantioselektiivisyyteen ja hydrolyysisivureaktioon tutkittiin kolmen sekundäärisen alkoholin kineettisellä resoluutiolla orgaanisissa liuottimissa, ionisissa nesteissä (IL) ja näiden seoksissa. Lipaasi PS sooli-geelit olivat aktiivisia ja enantiose- lektiivisiä katalyyttejä orgaanisissa liuottimissa sekä liuottimen ja ILn seoksissa. CLEAt ja SILEt olivat aktiivisia ja enantioselektiivisiä orgaanisissa liuottimissa. Sooli-geelit ja SILEt olivat uudelleenkäytettäviä useissa reaktioissa. Sooli-geelien ja CLEAn käyttö vähensi hydrolyysisivureaktiota. Avainsanat: CLEA, immobilisointi, ioninen neste, kineettinen resoluutio, lipaasi, or- gaaninen liuotin, sekundäärinen alkoholi, SILE, sooli-geeli. Table of Contents 5 tAbLe OF cONteNtS AbStRAct ....................................................................................................................3 tIIvISteLMä ..............................................................................................................4 tAbLe OF cONteNtS ...............................................................................................5 AbbRevIAtIONS ........................................................................................................7 LISt OF ORIGINAL PAPeRS ....................................................................................9 DeFINItIONS .............................................................................................................10 1. INtRODuctION ..................................................................................................11 2. RevIew OF LIteRAtuRe ................................................................................12 2.1. Lipases .........................................................................................................................12 2.1.1. Classification and general properties of lipases .....................................12 2.1.2. Mechanism of the lipase catalysis ..........................................................13 2.1.3. Enantioselective applications of lipases ...............................................14 2.2. Solvent effects in lipase catalysis................................................................................17 2.2.1. Lipase catalysis in organic solvents .......................................................17 2.2.2. Lipase catalysis in ionic liquids .............................................................19 2.2.3. Effect of water in lipase-catalyzed transesterification reactions in non- aqueous media .......................................................................................21 2.3. Immobilization of lipases ...........................................................................................22 2.3.1. General aspects .......................................................................................22 2.3.2. Support binding ......................................................................................25 2.3.2.1 Support materials for adsorption and covalent attachment ........26 2.3.2.2. Adsorption .................................................................................28 2.3.2.3. Covalent attachment ..................................................................30 2.3.2.4. Supported ionic liquids ..............................................................32 2.3.3. Entrapment .............................................................................................34 2.3.3.1. Sol-gel entrapment .....................................................................35 2.3.4. Cross-linking of enzymes .......................................................................40 2.3.4.1. CLECs ........................................................................................41 2.3.4.2. CLEAs ......................................................................................41 2.3.5. Coating ..................................................................................................44 3. AIM OF tHe StuDy ............................................................................................46 4. MAteRIALS AND MetHODS ............................................................................47 6 Table of Contents 4.1. Materials ......................................................................................................................47 4.2. Enzymes .......................................................................................................................47 4.3. Analytical methods ......................................................................................................47 4.4. Mathematical equations ..............................................................................................49 4.5. Preparation of the lipase PS sol-gels, CLEAs and SILEs .........................................49 4.5.1. Lipase PS sol-gels (Paper I and II) .........................................................50 4.5.2. Lipase PS CLEAs (Paper I) ....................................................................50 4.5.3. Lipase PS SILEs (Paper III) ...................................................................50 4.6. Enzymatic acylation ....................................................................................................51 5. ReSuLtS AND DIScuSSION ..............................................................................52 5.1. Activity of lipase PS preparations (Papers I-III) ........................................................53 5.2. Kinetic resolution of 6-8 (Papers I-III) .......................................................................56 5.3. Reuse of the lipase PS sol-gels, CLEAs and SILEs (Papers I and III) .....................59 5.4. The effect of immobilization on hydrolysis side reaction (Papers I and II) .............61 6. SuMMARy .............................................................................................................64 7. AcKNOwLeDGeMeNtS ...................................................................................66 8. ReFeReNceS .......................................................................................................67 ORIGINAL PAPeRS ...................................................................................................73 Abbreviations 7 AbbRevIAtIONS AC active carbon AnL Aspergillus niger lipase Asp aspartic acid aw water activity [BMIM][BF4] 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][NTf2] 1-butyl-3-methyl bis(trifluoromethylsulfonyl)imide [BMIM][PF6] 1-butyl-3-methyl hexafluorophosphate BTMS butyl trimethoxysilane c conversion CAL A Candida antarctica lipase A CAL B Candida antarctica lipase B CLE cross-linked dissolved enzyme CLEA cross-linked enzyme aggregate CLEC cross-linked enzyme crystal CrL Candida rugosa lipase CSDE cross-linked spray-dried enzyme DES deep euthetic solvent DIPE diisopropyl ether DKR dynamic kinetic resolution DMAP 4,4-dimethylaminopyridine DME dimethoxyethane E enantiomeric ratio ee enantiomeric excess ENT Reichardt´s normalized polarity scale [EMIM][BF4] 1-ethyl-3-methylimidazolium tetrafluoroborate [EMIM][NTf2] 1-ethyl-3-methyl bis(trifluoromethylsulfonyl)imide EC Enzyme Commission FDA U.S. Food & Drug Administration FFA free fatty acid GC gas chromatography 8 Abbreviations Gln glutamine His histidine IL ionic liquid IPA isopropylalcohol iBTMS isobutyl trimethoxysilane k rate constant Km Michaelis constant KR kinetic resolution Leu leucine Lipase PS Burkholderia cepacia lipase log P logarithm for the partition coefficient of a solvent between 1-octanol and water MmL Mucor miehei lipase MTMS methyl trimethoxysilane PEI polyethyleneimine PfL Pseudomonas fluorescens lipase PpL pig pancreatic lipase PrL Penicillium roqueforti lipase PTMS propyl trimethoxysilane PVA polyvinylalcohol RmL Rhizomucor miehei lipase Ser serine SDS sodium dodecyl sulfate SILE supported ionic liquids enzyme SILP supported ionic liquid phases TBME tert-butyl methylether TEOS tetraethoxysilane TlL Thermomyces lanuginosus lipase TMOS tetramethoxysilane UV ultraviolet Vmax maximum reaction rate List of Original Papers 9 LISt OF ORIGINAL PAPeRS This thesis is based on the following papers referred by Roman numerals (I - III) in the text. I. Hara, P.; Hanefeld, U; Kanerva, L.T. Sol-gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents. J. Mol. Catal. B: Enzym., 2008, 50, 80-86. II. Hara, P; Hanefeld, U.; Kanerva, L.T. Immobilized Burkholderia cepacia lipase in dry organic solvents and ionic liquids: A comparison. Green Chem., 2009, 11, 250-256. III. Hara, P.; Mikkola, J.-P.; Murzin, D. Yu.; Kanerva, L.T. Supported ionic liquids in Burkholderia cepacia lipase-catalyzed asymmetric acylation. J. Mol. Catal. B: Enzym., 2010, 67, 129-134. The original papers have been reproduced with the permission of the copyright holders. 10 Definitions DeFINItIONS C B A D C BA D E Biocatalysis Chemical conversion of a substance into desired product with the aid of a free or immobilized enzyme or enzymes inside whole cells.1 Chiral A molecule having the property of chirality.2 Chirality Geometric property of a rigid object (or spatial arrangement of points or atoms) of being nonsuperimposable on its mirror image; such an object has no symmetry elements of the second kind (a mirror plane, centre of inversion, a rotation-reflection axis). If the object is superimposable on its mirror image the object is described as being achiral.2 Conformation Spatial arrangement of the atoms affording distinction between stereoisomers which can be interconverted by rotations about formally single bonds.2 Enantiomer One pair of molecular entities which are mirror images of each other and non-superimposable.2 Enantiopure A sample all of whose molecules having (within limits of detection) the same chirality sense.2 Enantioselectivity Preferential formation in a chemical reaction of one enantiomer over another.2 Racemate An equimolar mixture of a pair of enantiomers.2 Stereochemistry The area of chemistry that deals with spatial arrangements of atoms in molecules and the effects of these arrangements on the chemical and physical properties of substances. Transesterification A reaction where the alcohol part of an ester is replaced by another alcohol, leading to a formation of the new ester.3 nantiomers Introduction 11 1. INtRODuctION Chirality is one of the most important factors in drug discovery and development. During the last decade there has been a growing trend to focus on single enantiomers of chiral drugs. For the first time in 2004, all the approved chiral synthetic drugs went to market as single enantiomers,4 and in 2006, 80 % of small-molecule drugs approved by FDA, U.S. Food & Drug Administration, were chiral and 75 % were single enantiomers.5 The synthesis of enantiopure compounds is a challenging task. Single enantiomers can be produced by chemical, chemo-enzymatic or purely biocatalytic synthesis. Biocatalysis is one of oldest chemical transformation methods known to humans. Biocatalysis offers advantages over chemical synthesis as the enzymes display high enantio-, chemo- and regioselectivity under mild conditions. Because of their excellent functional properties (activity, specificity and selectivity) enzymes are able to catalyze fast modifications of an individual functional group with a high degree of substrate specificity. Moreover, biocatalysis is a clean and ecological way to perform chemical processes. In synthetic chemistry, enzymes are used in different reaction media like aqueous buffers, organic solvents, ionic liquids, supercritical fluids, and even in solvent free systems. Also enzymes need to have large substrate tolerance for natural and unnatural substrates. For synthetic purposes, enzymes are usually stabilized. The stabilization of enzymes towards reaction conditions and substrates is often achieved by immobilizing enzymes on different solid supports, encapsulating or cross-linking. Immobilization often improves activity, specificity and selectivity, and reduces the inhibition caused by substrates, products or a reaction medium. The immobilization of enzymes also makes the handling of the catalyst more convenient, since it enables filtration of the catalysts from reaction mixtures and makes the use of reactors easier. In industrial scale of enzyme catalysis, the immobilization of enzymes is desirable since, in addition to increased stability for reactions, immobilization allows the reuse of the catalyst and the use of continuous processes, which is beneficial from economical and environmental aspects. Literature review in this thesis is focused on lipase catalysis in organic media and in ionic liquids, and on the methods and benefits of lipase immobilization. In the experimental section, the immobilization of Burkholderia cepacia lipase (lipase PS) as sol-gels, CLEAs and supported ionic liquid catalysts, together with the application of the obtained immobilized lipase preparations in enzymatic kinetic resolution of secondary alcohols in organic solvents, ionic liquids and in their mixture is discussed. The results are compared to those obtained with lipase PS on celite, method widely used for lipase immobilization in the Laboratory of Synthetic Drug Chemistry since 1993.6 Moreover, lipase PS on celite is a commercial product as lipase PS-D from Amano Enzyme Inc. Lipase PS sol-gels, CLEAs and supported ionic liquids catalysts are shown to be stable and highly active and selective catalyst for preparing enantiopure secondary alcohols. 12 Review of the Literature 2. RevIew OF LIteRAtuRe This Review of literature focuses on two main subjects. Firstly, lipases and lipase- catalyzed kinetic resolution and the effects of organic solvents and ionic liquids on enzymatic kinetic resolution are discussed. Secondly, the modification of crude lipase preparations by immobilization is discussed. The examples given primarily employ Burkholderia cepacia lipase, lipase PS, which is the catalyst used also in experimental part of the thesis. In addition, some other generally used lipases are discussed. 2.1. Lipases 2.1.1. Classification and general properties of lipases Enzymes are proteins which have a specific 3D structure for catalysis, and they catalyze most of the biological reactions in nature. In official enzyme nomenclature enzymes are numerically classified (EC numbers) to six classes according to the reactions they catalyze (Table 1).7 The naming system is recommended by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology.7 The main industrial applications for enzymes such as proteases and lipases are detergent and food industries. Enzymes, especially lipases, can be produced in high yields from microbial organisms like fungi, yeast and bacteria. Enzymes can also be isolated from slaughter waste or cheap mammalian organs such as kidney or liver. table 1. Classification of enzymes.7 enzyme class Reaction type example of the enzyme 1. Oxidoreductases Oxidation/reduction of C-H, C-C or C=C bonds. Dehydrogenase, oxidase 2. Transferases Tranfers methyl, aldehyde, ketone, acyl, sugar, alkyl, aryl, phosphoryl, nitrogenous, sulphur or selenium-containing groups. Transaminase, kinase 3. Hydrolases Hydrolysis/formation of esters, amides, lactones, lactams, epoxides, nitriles, anhydrides and glycosides. Lipase, amylase, protease 4. Lyases Addition/elimination of small molecules on C=C, C=N and C=O bonds. Decarboxylase 5. Isomerases Isomerizations including racemization and epimerization. Glucose-isomerase, mutase 6. Ligases Formation/cleavage of C-O, C-S, C-N, C-C bonds with concomitant triphosphate cleavage. Synthetase Lipases are most widely used enzymes in organic chemistry. There are hundreds of references of the use of lipases in organic solvents, and they have been reviewed in many books and articles.3,9-11 Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are hydrolytic enzymes that catalyze hydrolysis of triglyserides into fatty acids and glycerol. In non-aqueous solvents Review of the Literature 13 reactions may be shifted towards synthetic direction enabling the formation of esters from acyl donors and alcohols. Lipases exhibit wide substrate specificity, being therefore usable catalysts in organic synthesis. Various lipases are nowadays commercially available as free and immobilized forms. Lipases are used in various industrial applications in preparation of detergents, food, flavours, pharmaceuticals, esters and amino acid derivatives, fine chemicals, agrochemicals, cosmetics and perfumery as well as biosensors.12 2.1.2. Mechanism of the lipase catalysis Based on the structure of the active site, lipases belong to serine hydrolases and they catalyze reactions by the same mechanism as serine proteases do (Scheme 1).13 The active site consists of the catalytic triad and oxyanion hole. The catalytic triad is formed by the nucleophilic serine and an aspartate or glutamate that is hydrogen bonded to the histidine residue acting as a general acid-base catalyst. The oxyanion hole is located next to the catalytic triad, so that one of the backbone NHs of the oxyanion hole is that of the residue next to the catalytic Ser. Oxyanion hole stabilizes the negative charge of the tetrahedral intermediates by hydrogen bonding. Reactions follow the ping-pong bi-bi mechanism (Scheme 1). RCONu1 + E RCO-E Nu2H Nu1H RCONu2 + E a) N NHis286 H O O Asp264 H O Ser87 N H O - O N H N N H His286 + H O O Asp264 - O Ser87 N H R O O Nu1 O H N O -RCONu1 N NHis286 H O O Asp264 - O Ser87 N H O H N O O R Nu1OH N N H His286 + H O O Asp264 - O Ser87 N H O O Nu2 O H N O - Nu2OH RCONu2 Catalytic triad E Oxyanion hole First tetrahedral intermediate (T1) Acyl-enzyme intermediate Second tetrahedral intermediate (T2) b) Scheme 1. Ping-pong bi-bi mechanism of lipases. a) General equation of lipase-catalyzed reaction; b) Catalytic cycle for lipase PS-catalyzed transesterification reaction. E=enzyme 14 Review of the Literature Burkholderia cepacia lipase (lipase PS, previously Pseudomonas cepacia) is one of the most popular lipases used in organic synthesis. Lipase PS has a broad substrate tolerance and has widely been used for regio- and enantioselective hydrolysis and transesterification reactions. Lipase PS is a protein of 320 amino acids and 33 kDa molecular weight. The crystal structure of lipase PS has been determined as an open conformation.14,15 The catalytic triad of lipase PS is formed by the residues Ser87, His286 and Asp264 (Scheme 1 b), and the oxyanion hole by the residues Gln88 and Leu17. Lipase PS contains an essential Ca2+ -site which stabilizes the b-hairpin in residues 214-228.16 The active site is covered by a lid which undergoes conformational rearrangements and switches the enzyme between inactive (closed lid) and active (open lid) states. Lipase PS has high activity over a wide range of pH (3.5-11). The optimum pH is 7.0-8.0. Lipase PS is commercially available as its free and immobilized forms from e.g. Amano Enzyme Inc. (lipase PS “Amano” SD powder and lipase PS “Amano” IM on diatomaceus earth), Sigma-Aldrich (Amano products lipase PS “Amano” SD, lipase PS “Amano” C-I and lipase PS “Amano” C-II, on ceramic, lipase PS “Amano” IM, and lipase PS powder (Sigma)), Sprin Technologies (lipase PS covalently on epoxy acrylic resin and adsorbed on polystyrene resin), and Iris Biotech (adsorbed on DVB cross-linked polystyrene). 2.1.3. enantioselective applications of lipases Three main lipase-catalyzed methods to get enantiopure compounds are kinetic resolution (KR), dynamic kinetic resolution (DKR) and desymmetrization (Scheme 2). S(R) S(S) A P(R) P(S) P(R) P(S) S(R) S(S) P(R) P(S) k(R) krac k(S) k(R) k(S) k(R) k(S) a) Kinetic resolution b) Dynamic kinetic resolution c) Desymmetrization k(R)>k(S) k(R)>k(S), krac>>k(S) k(R)>>k(S) + Scheme 2. Lipase-catalyzed methods to yield enantiopure compounds. S(S), S(R) substrate enantiomers, P(S), P(R) product enantiomers, A prochiral substrate. In kinetic resolution, the substrate enantiomers (S(R) and S(S)) react with different rates to product enantiomers (P(R) and P(S)) (Scheme 2a). When the rate difference between the enantiomers is high enough, both the unreacted substrate enantiomer S(S) and the product enantiomer P(R) are obtained in enantiopure forms at 50 % conversion, expecting that the reaction to product proceeds irreversibly.8 Kinetic resolution provides only 50% theoretical Review of the Literature 15 yield of the enantiopure compounds, which might be a limitation if the other enantiomer is unwanted. In lipase-catalyzed reactions, ester RCOONu1 (acyl donor) reacts with the enzyme to form an acyl-enzyme intermediate which is attacked by nucleophile (Nu2H) to form the product (Scheme 1a). In kinetic resolution, either an acyl donor or a nucleophile can be chiral, and usually the other one is achiral. In lipase-catalyzed kinetic resolution focused on this thesis, acyl donor is achiral and substrate secondary alcohol, Nu2H, chiral. Lipases catalyze several types of reactions in organic media (Scheme 3), which makes them highly potential catalysts in the preparation of chiral compounds, such as pharmaceuticals and fine chemicals, in enantiopure forms. The reactions like hydrolysis, alcoholysis, interesterification, acidolysis, aminolysis, perhydrolysis, ammonolysis and thiolysis can take place. Alcoholysis, thiolysis and interesterification are in fact transesterification reactions where the alcohol part of the acyl donor is replaced by another nucleophile (Nu2H) leading to the formation of another ester. R Nu1 O R E O H2OHydrolysis R2OH Alcoholysis R2COOH Acidolysis R2COOR3 Interesterification R2NH2 Aminolysis H2O2 Perhydrolysis R2SH Thiolysis NH3 Ammonolysis "Acyl-enzyme" E Nu1H R2NHNH2 R NHNHR2 O Hydrazinolysis R NH2 O R NHR2 O R OOH O R OH O R OR2 O R OH O R2 Nu1 O R SR2 O R OR3 O R2 Nu1 O Scheme 3. Lipase-catalyzed reactions in organic media.3,9,11 Enantiomeric ratio E expresses the enantioselectivity of a kinetic resolution reaction where no side reactions occur. E is determined kinetically E=(kcat/Km)A/(kcat/Km)B where kcat/Km is the apparent second-order rate constant for the reaction of the enzyme and the substrate at infinitely low substrate concentrations to give product(s).17 kcat and Km denote catalytic and Michaelis constants, respectively, and A and B are substrate enantiomers. For synthetic purposes E value can be calculated more conveniently from equation E=ln[(1-c)(1-eeS)]/ln[(1-c)(1+eeS)] based on substrate ee and E=ln[1-c(1+eeP)]/ln[1-c(1- 16 Review of the Literature eeP)] based on product ee. 18,19,158 E can be regarded as moderate (15-30), good (30-100) or excellent (>200). The linkage of E to ee and conversion at E-values E=5, E=30 and E=200 is given in Fig. 1. It can be seen that when E=5, only the starting material can be isolated enantiopure at very high conversion (low yield). When E=30, the starting material can be separated enantiopure after 60% conversion and when E=200, both substrate and product enantiomers can be separated enantiopure at 50% conversion. 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 ee (% ) Conversion (%) E=5 E=30 E=200 Product Substrate Figure 1. Dependence of ee on conversion. E=enantiomeric ratio. Dynamic kinetic resolution (Scheme 2b) overcomes the problem of 50 % yield in kinetic resolution, but another enzyme is needed when both enantiomers are required. The unwanted S(S) enantiomer is racemized and recycled in situ giving a theoretical yield of 100 % for the product P(R). The rate constant of racemization (krac) should be much higher than k(S) for achieving very high selectivity. Racemization can be performed by a chemocatalyst, a biocatalyst or it can occur spontaneously. The method has been extensively reviewed.20-23 As another effective method, desymmetrization (Scheme 2c) of prochiral or meso-componds offers a route to enantiopure compounds with 100% theoretical yield. The method has been widely used for the preparation of enantiopure cyanohydrins, diols and diesters. Secondary alcohols as important organic building blocks are by far the most frequently used racemic targets in lipase-catalyzed transesterification reactions. Lipases usually show much higher enantioselectivity in the resolution of secondary than primary or tertiary alcohols. Vinyl acetate is the most common acyl donor for the acylation of secondary alcohols in organic solvents. The use of vinyl esters as acyl donors leads to irreversible reactions. In the case of primary alcohols, the enantioselectivity is frequently from low to moderate and can be increased by optimizing the reaction conditions. Tertiary alcohols are often unreactive towards lipases. There are only few examples of lipase-catalyzed resolution of Review of the Literature 17 tertiary alcohols.24-26 In the experimental part of this thesis, racemic secondary alcohols were used as nucleophiles and vinyl acetate as achiral acyl donor in transesterification. 2.2. Solvent effects in lipase catalysis 2.2.1. Lipase catalysis in organic solvents The choice of reaction medium is an important issue for enzymatic reactions. Moreover, tightening environmental legislations and FDA guidelines set strict demands for the nature of the solvent. For instance, many chlorinated hydrocarbon solvents have already been or are likely to be banned in the near future. In the context of green chemistry, the solvent should be relatively non-toxic, safe and low volatile. The current trend leads away from hydrocarbons and chlorinated hydrocarbons towards lower alcohols, esters and, in some cases, also ethers. Inexpensive natural solvents, such as ethanol, have also the advantage of being biodegradable.27 The nature of the solvent may have clear effect on enzymatic activity, stability and selectivities, and finding the best solvent usually needs solvent screenings and optimizations. Ethers like tert-butyl methyl ether, diisopropyl ether and tetrahydrofuran, esters like ethyl acetate and vinyl acetate, and aliphatic and aromatic hydrocarbons like hexane and toluene have been widely used in lipase-catalyzed transesterifications.3 The catalytic activity of lipases is often lower in neat organic solvents than in aqueous solutions.28 The main reason for this is the rigidity of enzymes in organic solvents. An enzyme needs an essential water layer which maintains the flexibility of the protein molecule necessary to catalysis, and can not be replaced without denaturation of the protein.29 Generally, in hydrophobic solvents the conformation of the active site stays stable and, accordingly, the enzyme maintains its activity and thermostability, whereas in hydrophilic solvents the solvent molecules reach into the active site and strip the essential water from the enzyme.30-32 Some lipases remain active with very small amount of residual water, whereas many others seem to require more bound water.33 In organic solvents the enzymes have “pH memory” meaning that the catalytic activity and conformation of enzymes reflect to the last aqueous solution they have been dissolved.30 Thus, the enzymatic activity can be enhanced by lyophilizing enzymes from aqueous solutions of optimal pH before use in organic solvents. Generally, the partition coefficient of a solvent between water and n-octanol, log P, is used as an indicator of solvent polarity and suitability for enzyme catalysis, however, no clear correlation between log P and enantioselectivity has been observed.34-36 Polar organic solvents having log P<2 are often thought to be unsuitable for biocatalysis due to inactivation or denaturation of enzymes by affecting the essential water layer. Midpolar solvents having log P between 2 and 4 weakly dissolve the water, and their effect on activity and selectivity is unpredictable. Nonpolar solvents having log P>4 do not dissolve the water coat thereby leaving the biocatalyst in an active state. Log P values of commonly used solvents in 18 Review of the Literature biocatalysis are presented in Table 2. Instability of an enzyme in polar solvents can, however, often be partially overcome e.g. by immobilizing the enzyme. Many reactions with free and immobilized lipases have been performed even in THF and acetonitrile.37 table 2. Log P values of commonly used organic solvents.34 Solvent log P acetonitrile -0.33 ethanol -0.24 acetone -0.23 tetrahydrofuran 0.49 ethyl acetate 0.68 isopropanol 0.8 tert-butyl methyl ether (TBME) 1.35 diisopropyl ether (DIPE) 1.9 toluene 2.5 cyclohexane 3.2 hexane 3.5 Table 3 presents the results of solvent screening for the acylation of (±)-menthol with lipase PS. The results indicate that highest E was obtained in CCl4 (E=139, log P 3.0) and the lowest in acetone (E=33, log P -0.23). The results also show that other solvent properties than polarity, e.g. the chain length of alkanes (hexane E=73, decane E=63) and branching of the chain (hexane E=73, cyclohexane E=82), may affect enantioselectivity. table 3. Acylation of (±)-menthol with vinyl acetate in organic solvents in the presence of lipase PS.38 OH (+)-menthol OH OH OCOCH3 (-)-menthol vinyl acetate lipase PS + + Solvent log P34 E hexane 3.5 73 cyclohexane 3.2 82 CCl4 3.0 139 toluene 2.5 53 benzene 2.0 45 NEt3 1.6 38 THF 0.49 40 acetone -0.23 33 dodecane 6.6 65 decane 5.6 63 Review of the Literature 19 2.2.2. Lipase catalysis in ionic liquids Room temperature ionic liquids (RT)ILs have recently received increasing attention as environmentally preferable alternative to organic solvents. They are considered green solvents since they are non-volatile (produce no atmospheric pollution), non-flammable and non-explosive. Additionally, they have high chemical and thermal stability and low melting points (<100°C). Although ionic liquids are considered as green solvents, mostly due to their negligible vapour pressure, the preparation and purification of 1-alkyl-3- methylimidazolium halide ionic liquids are not green according to the twelve principles of green chemistry.39 Also ILs used in biocatalysis have not been designed biocompatible nor biodegradable, and many of them have been observed to be (eco)toxic.40 They also have high solubility in water through which ILs might be run into the environment.40 ILs consist of an organic cation and an inorganic anion. In second generation ILs, mostly used in biocatalysis, hydrophobic anions such as trifluoromethanesulfonate [CF3SO3] - (triflate [OTf]-), bis((trifluoromethyl)sulfonyl)imide [N(CF3)SO2)2] - (bistriflamide, [NTf2] -) and tris((trifluoromethyl)sulfonyl)methanide [C(CF3SO2)3] - ([CTf3] -) are popular due to their low reactivity with water and their large electrochemical windows.41,42 Most common cations are dialkylimidazolium or pyridinium cations. (Scheme 4, Table 4). ILs are beneficial as designer solvents, meaning that their physical and chemical properties like viscosity, density, solubility, polarity and hydrophilicity/hydrophobicity can be tuned optimal for a certain reaction by changing the cation or anion.27,41 ILs can be immiscible with many organic solvents such as hexane and ether, and their water-miscibility varies unpredictably depending more on the anion part.43-46 As an attractive feature, ILs improve water solubility of polar substrates of low solubility like carbohydrates, making them more accessible for the reactions. N NR Me N R + 1-alkyl-3-methyl- imidazolium N-alkyl- pyridinium + NR4+ tetraalkyl- ammonium water-immiscible water-miscible [PF6]- [NTf2]- [BF4]- [OTf]- [CH3CO2]- [CF3CO2]- Br-, Cl-, I- Anions: Cations: Scheme 4. Typically used cations and anions in ILs.41,42 20 Review of the Literature table 4. Properties of ionic liquids commonly used in biocatalysis. Ionic liquid Melting point7 [°c] Density47 [g cm-3] viscosity48 [cP] eNt 49,a water miscibility50 [EMIM][BF4] 15 1.34 43 0.710 yes [EMIM][NTf2] -15 1.53 28 0.676 no [BMIM][BF4] -71 1.20 219 0.673 yes [BMIM][NTf2] 2 1.44 69 0.642 no [BMIM][PF6] 12 1.38 450 0.667 no a ENT : Reichardt´s normalized polarity scale. In 2002, BASF published the first publicly-announced industrial use of ILs, the BASILTM process for the production of the generic photoinitiator precursors alcoxyphenylphosphines.41 Since then several industial methods utilizing ILs have been introduced by BASF, Eastman Chemical Company, IFP, Degussa, BP and other companies. The first biocatalysis in ILs was reported in 2000, when Z-aspartame was synthesized in thermolysin-catalyzed reaction in [BMIM][PF6]. 51 The first lipase-catalyzed reaction in ILs was published in the same year.52 Since then several applications of lipases in ILs have been performed, and the use of lipases have been extensively reviewed by several authors.43-46,50,53-55 The use of ionic liquids or their mixtures with organic solvents as reaction media has improved stability, activity, regio- and enantioselectivity of various enzymes. Frequently used lipases CAL-B and lipase PS have been found to be catalytically active in 1-alkyl-3-methylimidazolium and 1-alkylpyridinium ILs in combination of anions [BF4], [PF6], [OTf] and [NTf2] that are most widely used ILs for biocatalysis (Table 4).46 ILs seem to affect enzymes in much the same way as conventional organic solvents do.44 The factors affecting the activity and stability of lipases in ILs are e.g. ion kosmotropicity and chaotropicity, polarity, ability to form hydrogen bonds, viscosity and hydrophobicity, but no clear correlation between IL property and enzyme activity/ stability has been found.43,54,56 Generally, lipase stability and activity has been higher in hydrophobic ILs.54,57,58 Lipases also have the pH memory in ILs like in organic solvents. Thus, they maintain the pH of the last aqueous environment.59 ILs have high polarity, e.g. [BMIM][PF6], often used in biocatalysis, has log P of -2.39 60, that is much lower than that of suitable organic solvents for biocatalysis (Table 2 vs. Table 4). Although polar organic solvents have been found to denaturate enzymes, ILs with similar polarity do not.42,43 Therefore, log P does not seem to be a useful parameter for indicating enzyme activity in ILs.60 More useful magnitude of polarity is Reichardt´s normalized polarity scale, ENT, (Table 4). [EMIM] and [BMIM] ILs commonly used in lipase catalysis has ENT between 0.6 and 0.8 whereas water has E N T 1 and cyclohexane 0.006. This indicates high polarity of ILs which is not in line with polarity of organic solvents in determination of effects of ILs on enzymes. That is why enzyme activity may Review of the Literature 21 be related more on viscosity and less to the polarity of ILs.43 ILs have high viscosity (Table 4), which can thus cause mass-transfer limitations in enzymatic reactions. Physical properties of ILs depend much on their purity, and users of ILs should be aware of impurities in ILs. Common impurities in ILs are water, halides and organic salts.61-63 Water is a common contaminant in ILs either due to ineffective drying after preparation or due to absorption from atmosphere due to hygroscopicity of an IL. [BMIM][NTf2] saturates with about 1.4% (w/w) of water, and for more hydrophilic ILs, water uptake from air can be even much greater.64 Most hydrophobic ILs can dissolve up to 1% of water. The presence of water (or other cosolvents) reduces the viscosity63, and it can also cause partial hydrolysis of [BF4] and [PF6] with formation of HF which denatures enzymes.46 The common impurity in 3-alkyl-1-methylimidazolium ILs is 3-alkyl-1- methylimidazolium halide (generally chloride) from incomplete metathesis reaction.62 Chloride contamination increases viscosity and inactivates lipases.62,63 ILs should be carefully purified and dried before use in biocatalysis. Third generation ionic liquids (advanced ILs) retain the moderate polarity, stability and distributed negative charge of second generation ILs.42 These third generation ILs use biodegradable, readily available cations or anions of lower toxicity. Cation may be e.g. choline, amino acid or alkylimidazolium, and the anion sugars/sugar analog, amino acid, organic acid, alkyl phosphate or alkyl sulfate. One example of third generation ILs is choline citrate. Third generation ILs tend to be more hydrophilic than second generation ILs and they are often water-miscible. Advanced ionic liquids also contain deep euthetic solvents, DES´s, which are mixtures of salts, such as choline chloride, and uncharged hydrogen bond donors, such as urea, oxalic acid or glycerol.42,65 Their properties are similar to ionic liquids. These advanced ionic liquids are new, and only few examples of biocatalysis have been published in those media. In transesterification of ethyl valerate with 1-butanol, lipase PS, CAL B and CAL A showed good activity in deep euthetic solvents, and in choline chloride:glycine the activity was similar to activity in toluene with all three lipases.65 In the experimental part of this thesis, second generation ILs [EMIM][NTf2], [EMIM][BF4] and [BMIM][PF6] have been prepared and used in kinetic resolution of secondary alcohols. 2.2.3. Effect of water in lipase-catalyzed transesterification reactions in non- aqueous media Thermodynamic water activity (aw) determines the mass action effects of water on hydrolytic equilibrium. It also describes the distribution of water between the various phases that can compete in binding of water.66 It is commonly accepted that water requirements should be discussed in terms of aw. In lipase-catalyzed transesterification reactions, water may act as a competing nucleophile with an alcohol (substrate), causing hydrolytic side reactions of ester substrates and products to liberate free acids (Fig. 2). This water originates by the adsorption from the atmosphere, incomplete drying of the reagents, seemingly dry support materials 22 Review of the Literature or side reactions producing water. This side reaction proceeds until aw is reduced to the level where hydrolysis is no longer favourable. In this way water also influences the equilibrium concentrations. Water will tend to equilibrate between the immediate environment of the biocatalyst and the bulk phase, such that they reach the same aw. 66 Also other type of side reactions like racemization, polymerization or decomposing of the reagents may take place due to water in the reaction system. Water can also change solvent properties, like viscosity of ILs. Elevated water activity increases the activity, Vmax and Km of lipase PS 67,68, and with many lipases, e.g. with CAL B, increasing aw leads to decreased enantioselectivity.69 The components of the system able to compete with the water are bulk phase, reactants and the support. Most support materials like celite does not have effect on activity-aw profile with enzymes. 66 R1CO2R2 ROH R1CO2R R2OH H2O H2O R1CO2H R1CO2H E Transesterification equilibria Hydrolysis equilibr a Figure 2. Equilibria caused by water in transesterification. The enzyme activity can be greatly influenced by water activity. In lipase PS -catalyzed acylation of 1-phenylethanol with lauric acid in isooctane, enantioselectivity was not affected by initial water content ranging from 0 to 0.5% (v/v). However, the activity of lipase PS had decreasing trend when initial water content increased from 0.1 to 0.5% (v/v). As the water content increased, the amount of water in the bulk phase increased, and the reaction became favourable toward hydrolysis. Specific activity decreased from 4.17 to 0.65 mmol/min/g at 2% (v/v) initial water concentration.70 It was explained that since the bulk phase is saturated with water, bound water is more difficult to expel from the enzyme to the bulk phase for the reaction to take place.70 This decrease in activity may also be due to competing nucleophile of 1-phenylethanol with water. Therefore, attention should be paid for proper drying of the components and choice of the reagents. While acylation with acids produces water, the acylation with vinyl esters produces acetaldehyde. Still, in lipase PS –catalyzed acylation of 1-phenylethanol with vinyl acetate in benzene-d6 the main reaction was hydrolysis of vinyl acetate (lipase catalyzed), not the acylation of the substrate even in dry conditions. Product ester hydrolysis was not observed.71 2.3. Immobilization of lipases 2.3.1. General aspects Immobilization of lipases has been thoroughly reviewed.72-77 Immobilization often increases stability, activity, selectivity and offers possibilities to use higher substrate nzyme i Review of the Literature 23 concentrations and different solvent systems, e.g. organic solvents, ionic liquids and supercritical fluids. In hydrophobic media immobilization optimizes enzyme dispersion and helps to avoid aggregation. In addition, immobilization provides more convenient handling, facile separation and efficient re-use of catalyst. Moreover, protein contaminations in the product can be minimized or eliminated. For an industrial scale, immobilization is considered favourable in many applications. Many lipases also undergo interfacial activation, and they need to face a hydrophobic interface to adopt the open/active conformation. This can take place when using hydrophobic supports. Immobilization protocols and supports are strongly dependent on the reaction medium, and several parameters must be taken into account to reach the highest possible stability and activity (Table 5).8,72,73,75-77 Native enzymes that are commercially available as crude preparations contain various additives, such as polyols and sugars, which are added as stabilizers. As a result, the actual protein content may be very low (typically 1-30%). Also the contaminations from the fermentation broth like inactive proteins, medium components, nutrients, buffer salts or carbohydrates can be present. It should be kept in mind that crude preparations are often more stable than purified enzymes. Maximal specific activity is often obtained when enzyme forms a monolayer on the surface of the support.78 Low enzyme loading can be harmful because of the strong interactions with matrix. On the contrary, high loading leads to multilayers of enzyme molecules which decreases the accessibility of the substrate to the active site of the enzyme. table 5. Enzyme and immobilization parameters affecting to efficient immobilization.75,76 Size of an enzyme Stability of an enzyme under immobilization conditions Conformational flexibility Isoelectric point Surface functional groups Glycosylation Additives in enzyme preparation Immobilization time Immobilization pH - optimal to enzyme Immobilization temperature Immobilization buffers Nature and properties of carrier Reaction medium Immobilization techniques applied to lipases include 1) support binding (adsorption and deposition, ionic binding, covalent attachment), 2) entrapment in a polymeric gel, membrane or capsule and 3) cross-linking with polyfunctional agent. The comparison of those techniques is given in Table 6. The first two techniques involve the use of solid support or entrapment of the enzyme. The last method entails covalent linking of enzyme molecules with each other without additional support. There are hundreds of 24 Review of the Literature variations based on combinations of these methods. Covalent coupling, entrapment and cross-linking are irreversible methods, and adsorption, ionic binding, affinity binding, chelation, metal binding and the formation of disulfide bonds are reversible methods where the support can be regenerated and re-loaded when enzyme activity decays. In organic media, non-covalent immobilization methods are often used because enzymes are generally insoluble to that media. table 6. Comparison of enzyme immobilization techniques.61,73,75,79 Method Advantages Disadvantages Adsorption Simple Weak binding (leaking) No chemical modification of an enzyme Little or no stabilization Reversible Non-specific binding Possible to recycle supports May limit mass-transfer Possible to use crude enzyme preparations Often inexpensive Covalent Tight binding, no leakage Chemical modification of enzyme attachment Wide variety of supports and linkers Support not recyclable available Often expensive Rational control of enzyme loading, Activity diluted by carrier distribution and microenvironment May limit mass-transfer Can be used in any medium Too high rigidity Irreversible Sol-gel No chemical modification of enzyme Little or no stabilization entrapment Can be simple Environmental changes can disrupt Inexpensive network and cause leakage Mild May limit mass-transfer Usually very stable Can be brittle Open for chemical modification of matrix Not all entrapped enzymes are by changing components catalytically active Retain high activity Easily recyclable High thermal and mechanical resistance Cross-linking High volumetric activity Chemical modification of enzyme Compatible with elevated temperature Little control of particle properties and organic solvents CLEC requires crystallization of No carrier required enzymes Tight binding May limit mass-transfer Simple Irreversible Possible to use crude enzyme preparations Weak resistance of mechanical No leaching stresses such as stirring Contains high proportion of active enzyme Improved storage and operational stability Easy to recover and recycle Possible to coimmobilize two or more enzymes Review of the Literature 25 In recent years several immobilization methods have been utilized with lipases for different substrates, reaction conditions and reactors, but there is still no universal method for obtaining highly active and stable immobilized enzyme for each conditions. Below the most common lipase immobilization methods are presented. Attention is paid especially to supported ionic liquids, sol-gel entrapment and cross-linking, the methods which are used in the experimental work of this thesis. More detailed procedures of immobilization of lipase PS via sol-gel entrapment, CLEA and supported ionic liquids are presented in the experimental part of this thesis. 2.3.2. Support binding Most commonly used support binding methods are adsorption and covalent attachment. Those and supported ionic liquids, a newer technique utilizing ionic liquids to protect or improve the properties of an enzyme, as well as support materials are discussed below. In Figure 3 different immobilization methods on solid supports are presented. Ionic binding is a simple and reversible method for enzyme binding, however, it is difficult to find conditions under which the enzyme remains strongly bound and fully active.72,75 Any ion exchange resin can act as a carrier, and the charge of an ion exchanger (positively/negatively charged) depends on the charge of the enzyme. The affinity of ionic binding depends on pH and salt concentrations during immobilization, but also during application. During ionic binding, enzyme properties like pH optimum or pH stability may change, and there may be problems with charged substrates and products. The benefits in immobilization of lipases on porous supports encompass fully dispersion of enzyme molecules, which prevents aggregation, autolysis or proteolysis. Also the enzyme will not be in contact with external hydrophobic interface.76 Immobilization of enzymes on solid supports can also increase the interfacial surface area between protein and solvent thereby increasing reaction rate.61 In an ideal situation the immobilized enzyme molecules are uniformly spread over the available surface, and monolayer of enzyme can be formed on the wall of the pores.73 26 Review of the Literature S S S S OH OH OH OH OH OH + - + - + - + - Lipase Adsorption Ionic binding Disulfide bonds Covalent bonding without rigidification Covalent bonding with enzyme rigidification Lipase Lipase Lipase Lipase Lipase Lipase Lipase Lipase Figure 3. Approaches to immobilization of lipases on supports. 2.3.2.1 Support materials for adsorption and covalent attachment Numerous support materials are available for immobilization of enzymes (Table 7.). Physical and chemical properties of the support material strongly affect an enzyme. The support needs to be chemically and mechanically stable. Hydrophilicity/hydrophobicity of the material is one of the most important characters of a support, in addition to mean particle diameter, swelling, mechanical strength, compression behaviour and surface properties.72,73 It is essential to have large surface area, either by small particle size or highly porous materials. In particular pore parameters and particle size determine the total surface area and thus critically affect the capacity for binding enzymes. Porous supports are preferred, since high surface area allows higher enzyme loading and porous materials provides greater protection for the enzyme. The supports are classified as inorganic and organic supports (Table 7). table 7. Classification of support materials for immobilization of enzymes.61,72 Organic supports Inorganic supports Natural polymers Synthetic polymers Natural minerals Processed minerals Polysaccharides e.g. Polystyrene Bentonite Glass (non-porous and cellulose, dextran, Other polymers like Silica controlled pore) agar, agarose, alginate, polyacrylate Mesoporous silicas Metals starch polymethacrylates, Alumina Controlled pore metal Proteins e.g. collagen, polyacrylamide, Zeolites Oxides albumin polyamides, vinyl and Diatomaceous earth Sol-gel Carbon allyl polymers Clays Carrageenan Nylon Review of the Literature 27 The activity of immobilized enzymes depends on the pore size of the support relative to enzyme and particle sizes. Microporous carriers have pores in the range of 0.1-10 mm. Mesoporous pore size is 3-10 nm which is of the same size with enzymes, and macroporous pores with specific areas of 25-100 m2/g have diameter of 8-1000 nm.73 In adsorption of lipases, retention of activity depends on the pore size in the range below 100 nm.73 Above this value, activity is independent of the pore size. Activity of enzymes immobilized on carriers with pore size below 100 nm (e.g. Eupergit C, pore size 10- 20 nm) is lower and strongly related to enzyme loading and pore size, whereas pore size >100 nm results increased accessibility of pores. Thus, when adsorbing lipase PS on mesoporous silicates SBA-15-55Å (55 Å pore diameter) and SBA-15-240 Å (pore diameter 240 Å), the SBA-15-55Å-lipase PS exhibited reduced activity due to limited accessibility of a substrate to enzyme active site.80 Also loadings were lower, suggesting that significant portion of internal pore volume is not available for lipase adsorption. SBA-15-240 Å-lipase PS had sufficient space to allow lipase penetrate deeply into pores, and thus showed similar catalytic activity to free lipase PS. Substrate sorption into support material can cause problems in kinetics. In the esterification of 4-methyloctanoic acid with Novozym 435, Candida antarctica lipase B adsorbed on hydrophobic macroporous polymer, sorption of substrate into pores of hydrophobic beads caused problems in determining E of the esterification.81 Similar problems did not occur in hydrolysis or transesterification reactions. The volume of the beads increased 175-250% by sorption of substrate which correlated 7.2 mmol of substrate per gram of lipase. This amount is not taking part in esterification, and calculation of E must be corrected. Lipase PS and CAL B have been successfully immobilized on several types of supports, such as porous ceramic support Toyonite 200-M82 and Tungsten(VI)oxide coated metal oxides83, supramagnetic nanoparticles based on magnetite Fe3O4 84,85 and zirconia nanoparticles86, and macroporous polystryrene microspheres87 and mesoporous silicates80,88,89,90. Typical hydrophobic and hydrophilic supports used in immobilization of lipases are presented in Table 8. Hydrophobic nature of the support facilitates the opening of the hydrophobic lid and thus activation of the enzyme.77 Acrylic resins are widely used in immobilization of enzymes. Eupergit® C is macroporous copolymer of N,N´-methylene- bi-(methacrylamide), glycidyl methacrylate, allyl glycidyl ether and methacrylamide.77 Reactive epoxy group has made Eupergit C and Eupergit C 250 L popular. Eupergit C binds to proteins via the reaction of its oxirane moieties with free amino groups of an enzyme to form covalent bonds at neutral or alkaline pH.75 Due to high density of oxirane groups on the surface of the beads, enzymes are immobilized at various sites of their structure (=multipoint attachment). Sepabeads® are methacrylic carriers that can be fuctionalized either by epoxy or amino groups and covalently attached to enzymes.75 28 Review of the Literature table 8. Examples of hydrophilic and hydrophobic supports used in enzyme immobilization. Hydrophilic supports Hydrophobic supports Cellulose EP-100 polypropylene Lignin Accurel MP 100 polypropylene Avicel (microcrystalline cellulose) Octyl-silica Celite Octyl-agarose Porous glass XAD-7 (acrylic resin) Silica gel PVA (polyvinyl alcohol) Agarose Celite (diatomaceous earth, diatomite) is one of the most popular carriers in immobilization of lipases, and it can be utilized both in adsorption and covalent attacment. Celite is hydrophilic, and its structure and properties can vary significantly as a function of production process. Several celite types are commercially available. The pore size of celite can vary from mm to several mm, and the shape of the particles can be rods or beads. The shape and porosity greatly affects the adsorption of enzymes as well as the ability to retain water inside the pores. Many commercial lipase preparations are based on celite powder, e.g. Amano lipase PS-D from Burkholderia cepacia. The support material and immobilization method can also induce traces of different compounds to the reaction mixture. It was noticed that number of compounds migrated from Novozym 435 (adsorbed on polyacrylate beads Lewatit® VP OC 1600, poly(methyl methacrylate) cross-linked with divinylbenzene) to organic solvents and ILs.91 Five major components were glycerol, benzoic acid, 2-hydroxyethyl benzoate, 2-hydroxyethyl sorbate and sorbic acid. The presence of these compounds may not have considerable impact on reactions with high substrate concentrations and when an enzymatic reaction is fast, but their presence is good to keep in mind especially in chromatographic analysis. 2.3.2.2. Adsorption Adsorption onto a water-insoluble macroscopic carrier (Fig. 3) is the easiest and oldest method of immobilization.8,72,73 In a most simple case, a lipase in a buffer solution is mixed with a support. The advantages and disadvantages of adsorption method are presented in Table 6. Adsorption forces are relatively weak.8,72,73,75 Basically any type of carrier in Tables 7 and 8 can be used to adsorb an enzyme via physical adsorption. Enzymes with large hydrophobic surface area will interact well with hydrophobic carriers, whereas enzymes with large hydrophilic areas interact with hydrophilic carriers. An enzyme is attached to the matrix through hydrogen bonding, van der Waals forces, or hydrophobic interactions. Although the adsorption of a lipase may be quite strong, it can be reversed by changing the conditions that influence the strength of interaction (e.g. pH and ionic strength in aqueous environment, temperature, polarity of solvent) to allow the recovery and reuse of the support. Deposition method can be used when enzymes are not adsorbed efficiently Review of the Literature 29 enough.72 The enzyme is dissolved in a minimal volume of an aqueous buffer, which is then mixed with the support followed by drying of the complete mixture. Everything present in the solution is deposited on support. Deposition is useful for wide range of enzymes and supports. Celite is typical support used in a deposition method. Most lipases show interfacial activation (Fig. 4.), where the conformational change from lid closed to lid open form.72,75 Adsorption of lipases on hydrophobic supports at low ionic strength is thought to mimic the interfacial activation. It has been suggested that lipases are immobilized in their active conformation (lid open). The final immobilized preparation may be even more active than the native enzyme. Lipase Lipase Lipase Hydrophobic support Lid closed Lid open Adsorbed on hydrophobic support Figure 4. Interfacial activation of lipases on hydrophobic supports. When a lipase is immobilized on a hydrophobic support the only interactions between the support and the enzyme are van der Waals forces.75 For efficient immobilization both the support and the enzyme need to have large lipophilic surface. Too high hydrophobicity of the support prevents the access of the enzyme to the pores and thus leads to decreased enzyme loadings.89 The presence of ethanol has been used to decrease the hydrophobicity enabling better accessibility of the enzyme to pore channels. In many enzymes, hydrophilic amino acid residues, which can be also glycosylated, prevail on the surfaces. Therefore, they can easily form hydrogen bonds and be immobilized on hydrophilic supports like cellulose, lignine, celite, porous glass and silica gel. Particularly popular hydrophilic support in lipase adsorption is celite (diatomaceous earth). Lipases immobilized on celite have been extensively used in organic solvents without any significant leaching. Lipase PS has been adsorbed on celite to enhance the activity and stability of the enzyme directly 92,93 or in the presence of sucrose6,94-97 in several applications. Also commercial preparations of lipase PS on celite has been available, e.g. Amano lipase PS-D and lipase PS “Amano” IM. However, adsorption forces are not strong enough to keep lipase PS adsorbed in polar ILs.98 Celite supports can also be used to control the water activity in the reaction systems. Celite powder adsorbs only minimum amounts of water, but porous celite rods adsorb and release water such way that water content is maintained constant in the reaction system.99 When celite rods R-640 were ground and reduced to fine powder, celite lost most of its ability of adsorbing water. 30 Review of the Literature 2.3.2.3. Covalent attachment Covalent bonding with or without enzyme rigidification (Fig. 3) is widely used because of the stable nature of the bonds between an enzyme and a matrix. This method, usable in any medium, is employed when there are strict requirements for the absence of enzyme in the product.72,75 The advantages and disadvantages of covalent bonding were presented in Table 6. Covalently immobilized enzyme preparation usually contains the support, spacer, linker and enzyme. Covalent bonding of an enzyme to a carrier is based on a chemical reaction between the active amino acid residues located on the enzyme surface and active functionalities that are attached to the carrier surface.73 The most commonly used reactive groups in covalent binding are presented in Fig. 5.72,73 Attention must be paid to amino acid residues essential for catalytic activity since they can not be involved in the covalent linkage to the support. Coupling methods can be divided in 1) activation of the matrix by addition of a reactive functionality to a polymer and 2) modification of polymer backbone to produce activated group.72 HN H2N NH2 OH OH OH O OH O HO O HO S HS HN NH2 HN Enzyme (Lys, ++) (N.terminal, +) (Carbohydrate, +/-) (Asp, +) (Glu, +) (C-terminal, +) (Trp, -) (Tyr, -) (Ser, +/-) (Met, -) (Cys, -) (Arg, -) OH Figure 5. Reactive amino acid residues of an enzyme. ++: very frequently used; +: frequently used; +/-: not frequently used; -: not used in enzyme immobilization.73 In covalent immobilization, both hydrophilic and hydrophobic supports can be used. The supports are often activated before use for binding enzymes. Reactive groups on the support can be attached via short or long spacers to the enzyme. Lipases are known to have remarkable conformational changes during catalysis, and longer spacers may be advantageous since they are expected to allow a wider conformational flexibility.75 Multipoint covalent attachment of enzymes on supports via short spacers involve Review of the Literature 31 many residues on the enzyme surface, which promotes rigidification of the enzyme.72,76 There are numerous reactive groups and linkers used in covalent immobilization.73 Supports functionalized with reactive epoxy, amino or glyoxyl groups are often used in covalent immobilization of lipases. Some examples of covalent attachment of enzymes on supports are presented in Scheme 5. Often used linkers are glutaraldehyde and carbodiimides. O H2N H N OH H O H2N NH2 H H O O H2N N N N Support Support Support Support Carrier + enzyme Carrier + linker + enzyme Support Support Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Scheme 5. Examples of activation methods of the carriers in covalent immobilization. Lipase PS was covalently immobilized on niobium oxide (Nb2O5) and polysiloxane- polyvinyl alcohol (SiO2-PVA) (Scheme 6). 100 SiO2-PVA was activated with epichlorohydrin (epoxy support) (Scheme 6a) and Nb2O5 was pretreated with nitric acid, silanized with g-aminopropyltriethoxysilane (g-APTS) and activated with glutaraldehyde (Scheme 6b). SiO2-PVA has larger pore volume and specific surface area than Nb2O5, and lipase PS on SiO2-PVA was 2 times more stable than lipase PS on Nb2O5 and 17 times more stable than free lipase. Transesterification yield was also higher with lipase PS on SiO2-PVA (89% vs. 40%). When Burkholderia sp. C20 lipase was immobilized covalently on celite carriers by cross-linking with glutaraldehyde, the stability and thermal tolerance was increased.101 32 Review of the Literature Enzyme OH O OH O OH OH Si SiO OR OR OR O Cl OH O OH O OH O Si SiO OR OR OR O OH O OH O OH O Si SiO OR OR OR OH NH a) b) Nb2O5 1. HNO3 2. g-APTS Nb O Nb O O O O Si H2N OHC(CH2)3CHO Nb O Nb O O O O Si CH2CH2CH2N=CHCH2CH2CH2CHO Nb O Nb O O O O Si CH2CH2CH2N=CHCH2CH2CH2CH=N Enzyme E H N2 EH2N Scheme 6. Covalent immobilization of lipase PS on SiO2-PVA (a) and Nb2O5 (b).100,102,103 As an example of environmentally friendly covalent immobilization of lipases, fully biodegradable catalyst was prepared by cross-linking lipase from Aspergillus niger by glutaraldehyde on silk fibers.104 The silk-fiber lipase was successfully used for hydrolysis of sunflower oil for fatty acids (biodiesel production). 2.3.2.4. Supported ionic liquids Supported IL enzyme (SILE) catalysis is a relatively new technique combining the advantages of ionic liquids with those of heterogenous support materials. Immobilization and supporting of ionic liquids and enzymes can be carried out in many ways, such as with simple impregnation, covalent linking, sol-gel method, or using ionic liquid as a vector between a support and an enzyme (Fig. 6).105,106 In SILEs, the ionic liquids are used to protect or improve the properties of the catalyst.107,108 Ionic liquids provide adequate microenvironment for an enzyme allowing high selectivities. The benefits of SILEs are the easier handling of heterogenous catalyst, and use of lower volumes of costly ILs compared to reactions in ILs without loosing the benefits of ILs.106 The diffusion of nzyme nzyme Review of the Literature 33 substrates through IL phase may cause mass-transfer limitations during catalysis. Since SILE method is new, only few examples of the method applied to enzymes are available. The work described in Paper III was the first example of lipase PS SILE. Support material Bulk phase Enzyme I Figure 6. Supported ionic liquid enzymes where IL and enzyme are deposited on a support material. On the other hand, CAL B SILEs were described already in 2005. ILs were used as a vector between ceramic supports and CAL B.105 CAL B was supported to a-alumina macroporous tubular supports with [BMIM][PF6], [BMIM][NTf2] and [EMIM][NTf2]. The activities of the SILEs were tested in the esterification of lauric acid with butyl acetate in hexane. The SILEs were active and stable. The activities were in the order no IL>[BMIM][NTf2]>[EMIM][NTf2]>[BMIM][PF6]. CAL B was also immobilized on a-alumina macroporous tubular supports first by cross-linking CAL B with the support and then coating the active membranes with six different ILs.109,110 The most suitable IL was [OMIM][PF6]. The activities of SILEs were tested in the acylation of 1-butanol with vinyl propionate. Although the activity was lower when ILs were used (compared to absence of IL), the selectivity increased reaching >99 %. In covalently supported ionic liquids phases (SILP), the SILP is first prepared and the enzyme is adsorbed. The technique has been applied to CAL B which was adsorbed on macroporous monolith-supported ionic liquid phase M-SILP-8 and M-SILP-12 (Scheme 7) with high IL/enzyme ratio to ensure full interaction between the protein and IL phases. The catalyst obtained was applied to the synthesis of citronellyl propionate from citronellol and vinyl propionate in ScCO2. 111 The M-SILP-8-CAL B and M-SILP- 12-CAL B were found to be very active and selective, the productivity of M-SILP-8- CAL B being 4.5 times higher (yield 93%) than that obtained for M-SILP-12-CAL B. The product hydrolysis was negligible. No enzyme leaching was observed. L phase 34 Review of the Literature N N+ Cl- M-SILP-8 N N+ Cl- O OH O M-SILP-12 Fe3O4 SiO2 Si(CH2)3 O O OEt N N CnH2n+1 X- + Fe3O4 IL nanoparticles Scheme 7. Covalently supported ionic liquids.111,112 Candida rugosa lipase (CrL) was adsorbed on magnetic nanoparticles supported ionic liquids (Fe3O4-IL) with cation length C1, C4 and C8 and anions Cl -, BF4 - and PF6 - (Scheme 7).112 Magnetic nanoparticles supported ILs were obtained by covalent bonding of IL- silane on magnetic Fe3O4 nanoparticles before adsorbing the enzyme. The activities of the prepared SILP-CrL were analyzed in the esterification of oleic acid with 1-butanol without a solvent. All lipase preparations showed higher activity than the native enzyme (Table 9). Also the operational stability of [C1C(S)Im]Cl-CrL was 92% after 5 cycles, whereas its native counterpart retained 35 % of the initial activity. table 9. Lipase activity with magnetic nanoparticles supported ionic liquids CrL.112 catalyst Activity [μmol min-1 g-1] [%] Native CrL 106.5 100 [C1C(S)Im]Cl-CrL 119.3 112 [C1C(S)Im]BF4-CrL 125.1 118 [C1C(S)Im]PF6-CrL 126.9 119 [C4C(S)Im]PF6-CrL 130.7 123 [C8C(S)Im]PF6-CrL 132.3 124 2.3.3. entrapment Enzymes can be entrapped in polymeric network that allows substrates and products to pass through but retains the enzyme. Entrapment matrix is generally formed during the immobilization process. Enzyme molecules can be physically embedded or covalently linked to the matrix. Several methods for entrapment have been developed, sol-gel method being the most prominent and widely used. Other methods for entrapment of enzymes comprise, for example, fiber entrapping, micro-encapsulation, double entrapment, covalent entrapment, adsorption entrapment, cross-linking entrapment, attachment entrapment and entrapment-coating.73 Entrapment is a mild method and can be useful for enzymes which are easily deactivated e.g. in covalent immobilization. More detailed procedure of sol-gel immobilization of lipase PS is presented in the experimental section and the original paper I. Review of the Literature 35 2.3.3.1. Sol-gel entrapment Conventional sol-gel entrapment refers to a process where enzyme is mixed with sol-gel solution, followed by gelation process under the influence of pH and aging process. Sol- gel entrapment of enzymes is thoroughly reviewed.72,113-116 In sol-gel method, an inert gel network is built by chemical condensation around each enzyme macromolecule. In nano- cages, the enzyme must have room enough to change its conformation required for catalytic cycles, and substrates and products must remain free to diffuse in and out. It is assumed that the enzyme is captured in the matrix in a “lid-opened” (active) conformation.72,113 Lipases entrapped in sol-gel are usually more stable than native enzymes.73 Sol-gels are porous inorganic matrices that have controllable surface area. After aging, the pore sizes of the gel formed are usually in the range of 2-20 nm (mesoporous).73 Materials concerned in sol-gel processes are mostly oxides: silica, alumina, aluminosilicates, titanium dioxide, zirconium dioxide.113 Most favoured precursors are silicon alkoxides Si(OR)4 or alkoxysilanes (XSi(OR)3 or XX´Si(OR)2 where X and X´are organic groups). In alkoxides, R is often methyl (tetramethoxysilane, TMOS) or ethyl (tetraethoxysilane, TEOS). TMOS and TEOS can liberate significant amount of alcohol during hydrolysis.115 Since methanol is less harmful than ethanol, TMOS is often used instead of TEOS.116 Previously the problem of liberating alcohols was solved by changing the precursor to sodium silicate and silicic acid or by using glycerated silanes.115 However, these materials showed large degree of shrinkage. In this thesis, evaporation of alcohol before enzyme addition, the method used for hydroxynitrile lyase,117 was used first time in immobilization of lipase PS. By adding trialkoxysilanes (e.g. methyltrimethoxysilane, MTMS), sol-gels with hydrophobic surface can be obtained. Hydrophobic surfaces can have positive influence on the reactivity of lipases, since they might induce interfacial activation, meaning that the lipase is in its active conformation. They also reduce the leaching of the enzyme (Fig. 7). O NH3 + H Si O H Si Si -O M Enzyme Figure 7. Non-covalent interactions between gel matrix and the enzyme.114 The synthesis of sol-gels includes 1) hydrolysis of precursors, 2) condensation, 3) gelation and aging and 4) drying (Scheme 8).72,73,75 The enzyme and possible additives can be added prior or after hydrolysis and condensation. As the hydrolysis and condensation of atrix 36 Review of the Literature silicon alkoxides are slow, they need to be catalyzed either by acids (H+), bases (O-) or strong Lewis bases, such as F- ions.113 This sol mixture consists of partially hydrolyzed and partially condensed monomers. The change in pH along with the presence of a catalyst and additives promotes a large scale polymerization reaction over the period of minutes to hours, resulting in gelation of the sol and thus, entrapment of enzyme.115 Aging promotes further condensation and strengthens the network. After aging, the gels are dried with an appropriate method (Table 10, Fig. 8) Most gels studied for encapsulation have been dried by evaporation of solvents to form xerogels (Fig. 8b).113 Ambigels (Fig. 8c) are obtained when the proportion of alkoxysilane in a precursor mixture is sufficiently high, and the proportion of hydrophobic groups in the pore surface will be sufficient to give hydrophobic character to gels. Dry ambigels have the same size as the wet gels. The stability of the sol-gel entrapped enzyme was found to be highly dependent on enzyme concentration, pH, ionic strength and other immobilization conditions.73 Si OMe OMeMeO MeO TMOS Si Me OMe OMeMeO MTMS H2O, H + Si OH OHMeO HO Si Me OMe OH HO Condensation and hydrolysis Si OH OMeO HO Si Me OMe OH Sol (HO)2Si O Si O Si O Si O Si OH OHMe O OH O OH Me OO Si Si OMe HO HO O O O G Poly- condensation Gelation (with enzyme and additives) Aging Scheme 8. Synthesis of sol-gels.75,115 table 10. Drying methods and gel types of sol-gels (Fig. 8.).113 Gel type Drying method Gel size Aquagel (a) Pores of the gel filled with water and alcohol Actual size Xerogel (b) Evaporation Shrinking of the gel due capillary forces Ambigel (c) Hydrophobization+evaporation Same size than wet gels Aerogel (d) Supercritical CO2 drying with or without acetone dialysis Size and structure of gel remains el Review of the Literature 37 Enzyme Gel network Liquid matrix a) wet gel Supercritical drying Hydrophobisation+ d H Dr ing by evaporation b) xerogel d) ae c) a rying by evaporation rogel mbigel ydrophobic coating y Figure 8. Drying of sol-gels.113 Reetz et al. showed that immobilization of lipase PS in TMOS gels has relative activities only less than 5%, whereas gels containing more hydrophobic silanes RSi(OCH3)3 (ambigels) has significantly higher activities.118 The relative activity increased from 30% with 50% MTMS up to 1300% for pure MTMS. The relative activity increased with increasing chain length (CH3100, and the enantioselectivity of coated CrL from E=8 to E=21 at pH 5. -ProMe 46 Aims of the Study 3. AIM OF tHe StuDy New methods for immobilization of enzymes are needed for developing more efficient enzyme catalysis. The aim of this study was to develop immobilization methods for lipase PS used in the synthesis of enantiopure intermediates and to study the effects of immobilization on activity, selectivity and possible side reactions in enzymatic acylation. The results are compared to those obtained by using lipase PS on celite (P. Hara, unpublished results). Water content in support materials used in immobilization varies notably and must be taken into account to minimize hydrolytic side reactions in transesterification reactions. The use of enzyme catalysis presumes that the ester product is not hydrolyzed due to side reaction. Understanding and elimination of side reactions offer chiral compounds with increased enantiopurity and yield. Thus, side reactions are studied in this work. The main targets in this thesis were 1. To immobilize lipase PS from Burkholderia cepacia as sol-gels (xerogels), CLEAs and support/IL/lipase PS (SILE) catalysts (Papers I-III). 2. To study the catalytic properties of the immobilized lipase PS preparations using transesterification between 6-8 and vinyl acetate in organic solvents and in ionic liquids as the model reactions (papers I-III) 3. To study the effect of immobilization on hydrolysis as a side reaction in the acylation reaction (Papers I and II). 4. To develop analytical methods needed for developing immobilization methods, following the acylation reactions and activity of the enzyme preparations. The acylation of alcohols 6-8 with vinyl acetate (Scheme 11) was chosen as a model reaction to study the activity and selectivity of the immobilized lipase PS preparations. The secondary alcohols used as substrates are common structures in drugs and fine chemicals. OH O OH H N Pr O OH O O OAc O OAc H N Pr O OAc lipase PS catalysts 6 7 8 (R)-9 (R)-10 (S)-11 + + + (S)-6 (S)-7 (R)-8 OH O OH H NPr O OH Scheme 11. Kinetic resolution of 6-8 catalyzed by lipase PS preparations. Materials and Methods 47 4. MAteRIALS AND MetHODS 4.1. Materials Substrates 6 and 7 were commercially available from Aldrich and Fluka, respectively. Amide 8 was prepared by the reaction of 2-amino-1-phenylethanol with butanoic anhydride (0.9 eqv.). Ionic liquids [EMIM][NTf2], [EMIM][BF4] and [BMIM][PF6] were prepared following the methods in literature62,98,150, methyl trioctylammonium trifluoroacetate ([MTOA] [TFA]), 1-butyl-4-methylpyridinium tetrafluoroborate ([4MBPy][BF4]) and 1-butyl-3- methylimidazolium trifluoromethanesulfonate [BMIM][TfO] were obtained from Merck KGaA and used as received. Silane precursor methyltrimethoxysilane (MTMS) was from Aldrich and tetramethoxysilane (TMOS) from Fluka. The supports applied in this study were active carbon (AC), activated carbon cloth (ACC 507-15, 1500 m2/g) and activated carbon paper (STV 505, 700 m2/g) from Nippon Kynol, Japan, and alumina (Versal VGL-25, 63-100 mm). 4.2. enzymes Burkholderia cepacia lipase (lipase PS) was studied in immobilization experiments, and the enzyme preparations were applied to the kinetic resolution of 6-8 (Scheme 11). Lipase PS was purchased from Amano Enzyme Inc. (Nagoya, Japan). Two forms of free lipase PS powder were used: Lipase PS “Amano”, diluted with diatomaceous earth containing 10% protein according to bicinchoninic acid assay, was used in original paper III. Lipase PS “Amano” SD, diluted with dextrin, containing 3 % protein (bicinchoninic acid assay), was used in original papers I and II as Amano changed the production of the lipase PS. Lipase PS was also adsorbed on celite (20 w/w%) in the presence of sucrose.6 Both Lipase PS “Amano” and lipase PS “Amano” SD gave similar activity when adsorbed on Celite. 4.3. Analytical methods Enzymatic acylations were followed by chiral GC chromatography by the separation of the enantiomers and quantification of the enantiomeric excesses. GC analysis was performed with Agilent 6890 Series II Chromatograph equipped with flame ionization detector (FID) and automatic injection. The acylation of 6 and 7 was analyzed with Varian Chrompack CP-Chirasil-DEX CB column (25 m x 0.25 mm) and the acylation of 8 with Varian Chrompack CP-Chirasil-L-valine column (25 m x 0.25 mm). The samples 48 Materials and Methods from the resolution reactions were derivatized with propionic anhydride in the presence of 4,4-dimethylaminopyridine (DMAP) to achieve good baseline separation for the enantiomers of unreacted 6-8 and product enantiomers 9-11 (Scheme 11). As an example of the chromatographic analysis, the chromatograms of rac-6 derivatized with propionic anhydride (a), rac-6-acetate (b) and the reaction sample from the kinetic resolution of rac-6 with vinyl acetate in the presence of lipase PS (c) is presented in Fig. 10. a) b) c) Figure 10. GC chromatograms of a) rac-6 derivatized as the propionate, b) rac-6 derivatized as the acetate and c) sample taken from the kinetic resolution of rac-6 at 50 % conversion. tr/min. Materials and Methods 49 In the development of CLEAs, the efficiency of the precipitation and cross-linking steps were monitored by following activity yield in the hydrolysis of p-nitrophenyl acetate (0.1 M, at pH 4.5) by UV-spectroscopy at l= 400 nm at 25 °C. The UV measurements were carried out on Perkin Elmer Lambda 650 UV/VIS spectrophotometer. The activity of the enzyme after precipitation and/or the cross-linking step was obtained as enzyme activity (As-1, A=absorbance) and the activity is expressed as v0 (mMmin -1) calculated based on the calibration curve A vs. concentration. The protein content of lipase PS powder was determined with UV-spectroscopy using bicinchoninic acid assay and bovine serum albumin as the standard protein at l=562 nm, 25 °C.151,152 4.4. Mathematical equations For the reaction where no side reactions occur, c is conversion of the reaction at a certain time, and enantiomeric ratio E the enantioselectivity of an irreversible enzymatic kinetic resolution reaction. Enantiomeric excess (ee) is the absolute difference between the mole fractions of each enantiomer. Enantiomeric excess of the substrate and the product (eeS and eeP, respectively) were obtained from the GC chromatograms of the reaction samples (Chapter 4.3., Fig. 10). The correlation between c, E, eeS and eeP is shown by eqs. (1) and (2). 18,19,158 (1) c=eeS/(eeS+eeP) (2) E=ln[(1-c)(1-eeS)]/ln[(1-c)(1+eeS)] In the present work the determination of E has been based on equation (2) using linear regression (E as the slope of the line ln[(1-c)(1-eeS)] versus ln[(1-c)(1+eeS)). When E values are 200 or higher, even minor variation in ee caused by experimental errors leads to significant changes in the value of E and, therefore, E>200 is given rather than more exact values. The catalytic efficiency of the enzymes is measured by determining specific activity. Specific activity of the reaction corresponds to the rate at the beginning of the reaction. The specific activity is calculated over the first smallest possible time interval, and is linear over a short period after the start of the reaction. To measure the specific activity, enzyme assays are typically carried out while the reaction has progressed only a few percent towards total completion. In this study specific activity (mmol min-1g-1) was determined using linear regression c.V.conversion vs. t.m(enzyme), where m(enzyme) is the mass of the enzyme preparation in conversions less than 10%. 4.5. Preparation of the lipase PS sol-gels, cLeAs and SILes Lipase PS sol-gels, CLEAs and SILEs were prepared by straightforward methods without any special equipment and additives. The procedure of immobilization is described in detail in the Experimental parts of the original papers I-III. Despite high 50 Materials and Methods amount of stabilizers in crude lipase PS preparations from Amano (10 % protein in lipase PS “Amano” and 3 % protein in lipase PS “Amano” SD), the crude enzymes were used without further purification. Lipase PS “Amano” solutions in buffer were centrifuged prior to use to remove insoluble material (Paper III). 4.5.1. Lipase PS sol-gels (Paper I and II) Lipase PS sol-gels were prepared without any additives by following the low-methanol method described in literature 117. The sol precursor was prepared by using the mixture of methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS) (1:4), and the condensation reaction was catalyzed by acidic water (HCl, pH 2.85). Since methanol is known to be harmful for enzymes, it was removed from the sol by evaporation prior adding the enzyme. Lipase PS powder was dissolved in phosphate buffer at pH 7 as the optimum pH of the enzyme is between 7 and 8. After addition of the sol, gelation took place immediately and the obtained sol-gel was aged to finish the condensation and dried. Drying in the air either at room temperature or at 40 °C was not efficient enough for acylation reactions in dry organic solvents. Therefore, the gel was lyophilized to xerogel. The optimum lyophilization time was 5 h after which the activity of the sol-gels decreased. 4.5.2. Lipase PS cLeAs (Paper I) Lipase PS CLEAs were prepared according to the literature method known for lipases.135 Since the CLEA method combines the purification and immobilization, lipase PS “Amano” SD was used without further purification in spite of the large amount of dextrin stabilizer (3% protein). Common precipitants, saturated ammonium sulphate, 1,2-dimethoxyethane, acetone and ethanol, were tested to reach the optimal precipitation degree and activity. Dextrin stabilizer caused problems in precipitation when using organic solvents as precipitant, since it formed sticky substance together with the enzyme. As a result, the activity after precipitation and cross-linking was poor. On the contrary, saturated ammonium sulphate gave 100% precipitation and increased activity also after cross-linking. Also dextrin stayed soluble and caused no problems during the procedure. Glutaraldehyde is a common reagent to cross-link proteins via free amino groups in the protein. Optimization of the amount of glutaraldehyde is essential to avoid leaching or excess cross-linking leading to loss of flexibility of the enzyme. Glutaraldehyde amount (100 mM) and cross-linking time (5 h) were optimized to reach the highest activity. 4.5.3. Lipase PS SILes (Paper III) Lipase PS SILEs were prepared by modifying the methods in literature.105,107 In the method used, IL is used as a vector between lipase PS and support material. SILEs were prepared by using lipase PS ”Amano”, containing 10% PS in celite as a crude powder. Celite stabilizer was centrifuged from the enzyme -buffer mixture (phosphate buffer, pH 7.8) prior to immobilization. pH was in lipase PS optimum range 7-8. Ionic Materials and Methods 51 liquids used ([EMIM][BF4] and [EMIM][NTf2]) were chosen as they were known to be suitable for lipase PS.98 Also few other ILs were tested with alumina as a support, but the reactivities in the acylation of 6 with vinyl acetate in toluene were negligible. Support materials used in the study were active carbon, alumina Versal VGL-25 63-100 mm, KynolTM ACC 507-15 active carbon cloth, 1500 m2/g (measured specific surface area 1680 m2/g, pore volume 0.60 cm3/g) and KynolTM STV 505 active carbon paper, 700 m2/g (measured specific surface area 1223 m2/g, pore volume 0.43 cm3/g). Supports were pre-dried and wetted with a solution containing lipase PS in phosphate buffer and dilutant. The SILEs obtained were dried in a rotary evaporator to have seemingly dry enzyme preparations. Dilutant was used to dissolve IL with enzyme mixture. Also the enzyme preparation without any ionic liquid was prepared from ACC 507-15 with the same method to analyze the effects of ILs. 4.6. enzymatic acylation Immobilized enzymes were tested in the enzymatic acylation of racemic alcohols 6-8 with vinyl acetate (Scheme 11) to specify the activity and stability of each enzyme preparation. From each acylation reaction, specific activity as mmol min-1g-1 was determined. The reactions were further followed 24-48 h to reach 50% or higher conversions when possible. For enzymatic acylation in the presence of lipase PS sol-gels and CLEAs, as well as AC and alumina powders, a typical reaction was performed by mixing an organic solvent and vinyl acetate (0.2 M) with a lipase preparation. The racemic alcohol (0.1 M) was added to start the reaction. The reactions were shaken at room temperature or at 48 °C. The progress of the reactions was followed by taking samples (50 mL) at intervals and analyzing the ees and conversions by GC. The samples were derivatized with propionic anhydride in the presence of DMAP (1% in pyridine) to achieve a good baseline separation. For enzymatic acylation in the presence of SILEs, vinyl acetate (0.2 M) was dissolved in an organic solvent and the mixture was stirred (300 rpm) with tailor-made stirrer shaft where the matt-structured SILE cloth was attached as suitable, rectangular pieces. The addition of the substrate (0.1 M) initiated the reaction. Before addition of the substrate, the system was preheated to the desired reaction temperature 25 – 60 °C. The reuse of sol- gels and CLEAs was studied using the enzymatic acylation of 7 and the reuse of SILEs using the enzymatic acylation of 6 as the model reactions. More detailed procedures are described in the experimental parts of the original papers I-III. 52 Results and Discussion 5. ReSuLtS AND DIScuSSION This section summarizes the results obtained experimentally. More detailed discussion is presented in the original papers I-III. As stated before, this study aims at immobilization of lipase PS as sol-gels, CLEAs and SILEs, and at evaluation of their activity and stability using the enzymatic kinetic resolution of secondary alcohols 6-8 in organic solvents and in ionic liquids as model reactions (Scheme 11). The results were compared to those obtained with lipase PS adsorbed on celite (lipase PS-celite). For immobilization of lipase PS, widely used adsorption on celite, sol-gel and CLEA methods as well as modern SILE method were chosen. The activity of immobilized enzymes, kinetic resolution of 6-8, the effect of solvents and immobilization method, possibilities to catalyst reuse as well as the effect on hydrolysis side reaction are discussed. The enantiopreference of the acylation reactions of 6-8 is known yielding (R)-esters of 6 62 and 7 153, and (S)-ester of 8 98. In lipase-catalyzed reaction, condition engineering (choice of solvent, reaction temperature and acyl donor) is important for obtaining high enantioselectivity. In kinetic resolution of 6-8, the solvents, reaction temperature and acyl donor were selected on the basis of screening reactions with the lipase PS powder and lipase PS-celite in the acylation of 6-8 (P.Hara, unpublished results). The demands for enzyme preparations used in organic synthesis are high enantioselectivity, activity and stability. Activity, stability and selectivity of lipase PS xerogels and CLEAs were studied in the acylation of 6-8 with vinyl acetate as an acyl donor (Papers I and II). Organic solvents toluene, DIPE and TBME were used according to solvent screening, and ionic liquids [EMIM][NTf2], [EMIM][BF4] and [BMIM][PF6] were chosen according to the previous results in the laboratory 98. The results were compared to those obtained with the same amount of lipase PS “Amano” SD powder (100 mg/mL vs. xerogel based on 100 mg/mL and 50 mg/mL vs. CLEA based on 50 mg/mL) to observe the effects of the immobilization. The reuse of xerogels and CLEAs is discussed in chapter 5.3. Peracetylated b-cyclodextrin stabilizes and activates lipase PS sol-gels 128 as well as activates lipase PS when co-lyophilized with the enzyme for the acylation of 7 154. In lipase PS “Amano” SD preparation used for sol-gel immobilization, the protein content is only 3% according to bicinchoninic acid assay, diluted to dextrin. In preparing sol-gels, this dextrin used as dilutant may be useful for maintaining the activity during gelation and lyophilization, and it may also have stabilizing effects in acylation. ACC 507-15 is made from phenolic resins and its surface has acidic and basic sites.155 These acidic and basic residues can form hydrogen bonds with IL and enzyme thus stabilizing the immobilized enzyme. Activity, stability and selectivity of lipase PS SILEs were studied with ACC 507-15 SILEs (Paper III), since the reactivity of SILEs prepared using active carbon and alumina as supports was negligible (conversions less than 14 % after 24 h) in kinetic resolution of 6 with vinyl acetate in toluene, and the reactivity of STV 505 supports was lower than that of ACC 507-15 SILEs (See below). ACC 507- 15 SILE preparations are convenient in use, since they can be cut into suitable pieces Results and Discussion 53 and used as such when shaking the reaction mixture, or they can be attached to stirring blade. In this study, ACC 507-15 SILEs were attached to stirring blade and recycled without further washes between the cycles. The reuse of ACC 507-15 SILEs in different temperatures is discussed in chapter 5.3. Lipase PS preparations on active carbon cloth ACC 507-15 had high activity with both [EMIM][NTf2] and [EMIM][BF4] ionic liquids. As the average pore diameter in ACC 507-15 is 1.9 nm 155, and lipase PS is a globular protein with approximate dimensions 30 Å x 40 Å x 50 Å (3 nm x 4 nm x 5 nm) 80, it can be assumed that part of the protein is immobilized on the surface of ACC 507-15, when the stabilization of acidic and basic residues is more important. Alumina and AC were not sufficient supports for lipase PS. Alumina is not adequate support either for immobilization of CAL B (similar size to PS) due to hindered effect of pore diameter (63-200 mm, average pore diameter 60 Å). Accordingly, the enzyme was located in the external surface of alumina and poor enzyme loading was observed.156 In this study the alumina was even smaller fraction 63-100 mm, indicating that lipase PS stayed on the surface of the support, which caused the low activities. This can be also the reason for low activities when active carbon was used as supports. The wettability of STV 505 was poor causing low loadings and thus low activities. 5.1. Activity of lipase PS preparations (Papers I-III) The specific activity of the enzyme preparations is expressed in mmol min-1 g-1, where g-1 refers to rate per gram of lipase PS preparation. Specific activity is the rate of formation of the first few percents of the products such that the product(s) concentrations have not risen to a level to significantly affect the rate. To study the activity of lipase PS preparations, specific activities for the acylation of 6-8 with vinyl acetate in organic solvents, ionic liquids and IL:solvent mixtures were determined and compared to specific activities of free lipase PS powder (Tables 14 and 15). In organic solvents, the specific activities of lipase PS xerogels in the acylation of 6-8 were generally lower than with the lipase PS powder (Table 15, entries 1-4, 11,12,19, Paper I). This can be due to diffusional limitations caused by xerogel. Lyophilization of xerogels also caused shrinking, and the capillary stress during drying can partially crush the enzyme and cause loss of activity.121 Specific activities in ionic liquids and in IL:solvent mixtures (Paper II) were lower than in organic solvents with all the substrates studied, since the high viscosity of ILs increase diffusional limitations and mass-transfer limitations. Thus, the activity was increased in mixtures of IL:organic solvent of low viscosity. In [EMIM] [NTf2]:solvent and [BMIM][PF6]:solvent mixtures the specific activities of xerogels were higher than those of lipase PS powder (entries 6,10,14,18,21,25), whereas in [EMIM] [BF4]:solvent mixtures they were lower (entries 8,23) indicating that hydrophobic ILs are more suitable than hydrophilic [EMIM][BF4]. This trend can also be seen in literature, where the activity of lipase PS has followed the order [BF4]<[PF6]<[NTf2]. 37,54,57,58 Also the possibility of reduced calcium binding in lipase PS due to strong hydrogen bonding ability of BF4 - has been proposed to lower the activities in BF4 ionic liquids. 57 54 Results and Discussion Lipase PS CLEA was significantly more active than lipase PS powder in organic solvents (Table 15, Paper I). Also the specific activity in the acylation of 6-8 with vinyl acetate was higher when CLEA was used in ILs and IL:solvent mixtures rather than lipase PS powder (Paper II). The activities in IL:solvent mixtures were higher than in pure ILs, but considerably lower than in organic solvents. Specific activity of CLEA in the acylation of 6 was higher in hydrophilic TBME than in toluene (Table 15, entries 1-4), whereas xerogel and lipase PS-celite had lower activities in TBME than in toluene. (Table 14, entries 2 and 3, Table 15, entries 1-4). These results show that CLEA is a suitable catalyst also in hydrophilic media, such as hydrophilic organic solvents and ionic liquids. Previously BSA additive yielded high activity CLEAs from lipase PS.140 In that procedure, the activity was extremely low when BSA was not used. In this case, the activity of lipase PS CLEA was decreased when BSA additive was used (specific activity=7.0 mmol min-1 g-1 vs. specific activity=3.9 mmol min-1 g-1 in the acylation of 6 with vinyl acetate in toluene). The differences in activities of lipase PS CLEAs is probably due to different lipase PS preparations used to immobilization, which leads to choice of different precipitant (saturated ammonium sulphate instead of acetone). The immobilization of lipase PS as ACC 507-15 SILEs with or without ionic liquids clearly improved activity (Table 14, entries 1,4-6, Paper III) in the acylation of 6 with vinyl acetate in toluene compared to free enzyme powder (entry 1). The use of [EMIM] [NTf2] in SILE preparation increased activity (entries 4,5,7,8,10,11), and the use of [EMIM][BF4] decreased activity (entry 6). This also indicates that the hydrophobic ILs are more suitable for lipase PS than hydrophilic ones as stated before. The most active lipase PS preparation in the acylation of 6 and 8 was lipase PS-celite (Table 14, entries 2,3,9), however, activity of lipase PS-celite was not studied in ionic liquids, since earlier studies with lipase PS-celite showed very low activity in ILs 98. Specific activity is expressed by gram of lipase PS preparate. The enzyme loading in ACC 507-15 SILEs was 4%, which leads to lower activities compared to lipase PS-celite (enzyme loading 20% (w/w)). ACC 507-15 SILEs were significantly more active than xerogels and CLEAs. CLEAs had higher activity than xerogels. The activity of lipase PS was increased when immobilized as CLEAs, SILEs and adsorbed on celite. Although specific activity of lipase PS xerogels is lower than with free lipase PS powder, the immobilization had stabilizing effect seen in the kinetic resolution of 6-8 (See Chapter 5.2). With lipase PS powder, specific activity was higher with 50 mg/mL lipase PS powder than with 100 mg/mL in most of the acylation reactions of 6-8 (Table 15). This is due to hydrolysis side reaction, which is faster when more lipase PS powder is used. Results and Discussion 55 table 14. Specific activities for the acylation of 6-8 with vinyl acetate in organic solvents in the presence of lipase PS powder, lipase PS-celite and ACC 507-15 SILEs at room temperature. entry compound Solvent Lipase preparation Specific activity [μmol min-1g-1] 1 6 toluene lipase PS ”Amano” 160 mg/mla 8.8 ± 1.0 2 6 toluene lipase PS-celite 20% (w/w)b 103.0 ± 1.0 3 6 TBME lipase PS-celite 20% (w/w) 77.7 ± 1.1 4 6 toluene ACC 507-15/lipase PS 15.7 ± 0.6 5 6 toluene ACC 507-15/[EMIM][NTf2]/lipase PS 19.5 ± 0.9 6 6 toluene ACC 507-15/[EMIM][BF4]/lipase PS 12.0 ± 0.6 7 7 DIPE ACC 507-15/lipase PS 27.1 ± 0.01 8 7 DIPE ACC 507-15/[EMIM][NTf2]/lipase PS 40.0 ± 0.01 9 8c TBME lipase PS-celite 20% (w/w) 87.6 ± 6.5 10 8c TBME ACC 507-15/lipase PS 23.5 ± 1.5 11 8c TBME ACC 507-15/[EMIM][NTf2]/lipase PS 35.1 ± 2.1 a Mechanical stirring instead of shaking used in this experiment. Lipase PS powder amount related to protein amount in ACC 507-15 SILEs. b Lipase PS “Amano” and lipase PS “Amano” SD gave similar activitites on celite c reaction temperature 48 °C table 15. Specific activities (mmol min-1g-1) for the acylation of 6-8 with vinyl acetate in organic solvents and ionic liquids in the presence of lipase PS “Amano” SD powder, xerogels and CLEAs. entry comp. Solventa Powderb Xerogelc Powderd cLeAe 1 6 Toluene 2.7 ± 0.3 1.42 ± 0.02 3.4 ± 0.2 7.0 ± 0.9 2 6 TBME 2.5 ± 0.2 1.27 ± 0.04 3.2 ± 0.3 8.5 ± 0.3 3 6f Toluene 3.8 ± 0.2 2.05 ± 0.06 5.9 ± 0.2 32.3 ± 0.1 4 6f TBME 4.4 ± 0.1 1.69 ± 0.02 5.7 ± 0.1 19.7 ± 0.9 5 6 [EMIM][NTf2] 0.31 ± 0.01 0.32 ± 0.01 6 6 [EMIM][NTf2]:toluene 0.08 ± 0.01 0.98 ±0.08 0.13 ± 0.01 1.57 ± 0.47 7 6 [EMIM][BF4] 0.19 ± 0.01 1.65 ± 0.07 8 6 [EMIM][BF4]:toluene 0.57 ± 0.01 0.35 ± 0.03 1.15 ± 0.01 6.60 ± 0.71 9 6 [BMIM][PF6] 0.41 ± 0.01 1.1 ± 0.1 10 6 [BMIM][PF6]:toluene 0.46 ± 0.01 0.72 ± 0.05 0.79 ± 0.01 1.90 ± 0.41 11 7 DIPE 3.5 ± 0.2 1.47 ± 0.02 3.8 ± 0.2 30.1 ± 0.4 12 7 TBME 3.9 ± 0.1 0.94 ± 0.03 3.6 ± 0.1 18.6 ± 0.2 13 7 [EMIM][NTf2] 0.90 ± 0.01 0.17 ± 0.01 14 7 [EMIM][NTf2]:DIPE 0.21 ± 0.01 0.71 ± 0.04 0.26 ± 0.01 1.80 ± 0.18 15 7 [EMIM][BF4] 0.15 ± 0.02 2.28 ± 0.25 16 7 [EMIM][BF4]:DIPE 1.54 ± 0.01 2.19 ± 0.09 1.27 ± 0.01 4.82 ± 0.13 17 7 [BMIM][PF6] 0.27 ± 0.04 1.34 ± 0.38 18 7 [BMIM][PF6]:DIPE 0.43 ± 0.01 0.68 ± 0.04 0.79 ± 0.01 2.57 ± 0.42 19 8f TBME 1.7 ± 0.1 1.54 ±0.03 2.38 ± 0.03 6.7 ± 0.3 20 8f [EMIM][NTf2] 0.03 ± 0.01 0.06 ± 0.01 21 8f [EMIM][NTf2]:TBME 0.12 ± 0.01 0.31 ± 0.01 0.16 ± 0.01 0.35 ± 0.04 22 8f [EMIM][BF4] 0.04 ± 0.01 1.91 ± 0.01 23 8f [EMIM][BF4]:TBME 0.41 ± 0.01 0.30 ± 0.02 0.84 ± 0.01 1.69 ± 0.40 24 8f [BMIM][PF6] 0.08 ± 0.01 0.67 ± 0.07 25 8f [BMIM][PF6]:TBME 0.07 ± 0.01 1.06 ± 0.11 0.79 ± 0.01 3.29 ± 0.38 a IL:solvent mixtures 1:2 b lipase PS “Amano” SD 100 mg/mL c xerogel based on 100 mg/mL lipase PS “Amano” SD d lipase PS “Amano” SD 50 mg/ml e CLEA based on 50mg/ml lipase PS “Amano” SD f reaction temperature 48 °C 56 Results and Discussion 5.2. Kinetic resolution of 6-8 (Papers I-III) Kinetic resolution catalyzed by lipase PS powder, lipase PS-celite, xerogels, CLEAs and ACC 507-15 SILEs was studied in the acylation of 6-8 with vinyl acetate in organic solvents and ionic liquids (Scheme 11) by following the reactions 24-48 h to reach 50 % or higher conversions when possible. The acylation of lipase PS-celite in ionic liquids was not studied since lipase PS-celite was inactive in the earlier study, probably because the adsorption forces are not strong enough to keep lipase PS immobilized in polar ILs.98 The acylation of 6 with vinyl acetate was studied in toluene, TBME, ionic liquids and IL:toluene mixtures in the presence of lipase PS preparations (Tables 16 and 17). In toluene, lipase PS-celite, xerogel, CLEA, ACC 507-15/lipase PS and ACC 507-15/ [EMIM][NTf2]/lipase PS were highly active and enantioselective (E>200, c=50% in 24 h, in 2 h with lipase PS-celite) (Table 16, entries 2,4-6, Table 17, entries 1,3). In toluene, lipase PS powder had also high activity and selectivity, except when mechanical stirring was used instead of shaking (Table 16, entry 1, Table 17 entry 1). The acylation of 6 in the presence of ACC 507-15/[EMIM][BF4]/lipase PS had lower enantioselectivity (E=152, Table 16 entry 6). This is probably due to hydrophilic nature of [EMIM][BF4] which can strip the essential water layer from the enzyme. In TBME, the acylation of 6 proceeded smoothly and the enantioselectivity was high (>200) in the presence of lipase PS-celite, xerogel and CLEA (Table 16, entry 3; Table 17, entry 2,4). Additionally, the conversion and enantioselectivity were clearly higher than in the presence of lipase PS powder (Table 17, entry 2,4). This indicates the stabilization and activation of lipase PS in hydrophilic solvents like TBME when immobilized as lipase PS-celite, xerogels and CLEAs. The acylation of 6 with vinyl acetate in ionic liquids and IL:toluene mixtures was studied in the presence of xerogels, CLEAs and lipase PS powder (Table 17, entries 5-10, Paper II). In pure ILs (Table 17, entries 5,7,9), the acylation was slow but the enantioselectivity remained high (E>200). The conversion was higher with xerogels than with CLEAs. In IL:toluene mixtures (Table 17, entries 6,8,10), the enantioselectivity was high and the acylation in the presence of xerogel was faster, reaching 45% and 46% conversion in 24 h in [EMIM][NTf2]:toluene and in [BMIM][PF6]:toluene, respectively. The acylation of 6 in IL:toluene mixtures in the presence of CLEAs led to high enantioselectivities in each IL:toluene mixture. The conversion was highest in [EMIM][BF4]:toluene (c=41 %, 24 h, entry 8). Xerogel was more stable in ionic liquids and IL:toluene mixtures than CLEA, hydrophobic [EMIM][NTf2] and [BMIM][PF6] being most appropriate ILs in the acylation of 6, which is in line with literature.54,58 Xerogels were also shown to be stable against storage, since the acylation of 6 with vinyl acetate in toluene proceeded similarly with fresh xerogel and the one stored for 1.5 months at 4°C (unpublished results). The acylation of 7 with vinyl acetate was studied in DIPE, TBME, ionic liquids and IL:DIPE mixtures in the presence of lipase PS preparations (Tables 16 and 17). Immobilization of lipase PS as xerogel improved the enantioselectivity in DIPE and in TBME compared to lipase PS powder while the conversions were similar (Table 17, entries 11,12). CLEAs had similar conversions to the lipase PS powder in DIPE and Results and Discussion 57 in TBME, but the enantioselectivity was decreased. In the presence of ACC 507-15 SILEs, the acylation of 7 was fast (Table 16, entries 7,8), but enantioselectivity was low. Enantioselectivity was slightly higher when ACC 507/[EMIM][NTf2]/lipase PS was used instead of ACC 507-15/lipase PS. In ILs and IL:DIPE mixtures, the enantioselectivity in the acylation of 7 in the presence of xerogels was only moderate and lower than in DIPE, although the acylation reached 50% conversion (Table 17, entries 13-18). The enantioselectivities were also lower than those obtained with lipase PS powder. The conversions and enantioselectivities were moderate when the acylation of 7 in the presence of CLEAs was performed in IL:DIPE mixtures (Table 17, entries 13-18). However, in [EMIM][BF4]:DIPE mixture (Entry 16) the enantioselectivity was higher than in DIPE (E=79 vs. E=58), and the acylation reached 40% conversion. Interestingly, CLEAs had higher conversions and enantioselectivities in the acylation of 6 and 7 in hydrophilic [EMIM][BF4]:IL mixtures than in hydrophobic IL:solvent mixtures. In contrast xerogels performed better in hydrophobic IL:solvent mixtures, although generally hydrophobic ILs are considered more suitable for lipase catalysis.54,58 The acylation of 8 with vinyl acetate was studied in TBME, ionic liquids and IL:TBME mixtures in the presence of lipase PS preparations (Tables 16 and 17). In TBME, the acylation of 8 in the presence of lipase PS-celite, xerogels and CLEAs reached 50 % conversion after 6 h, 30 h and 48 h reaction, respectively, showing that lipase PS-celite was the most active preparation (Table 16, entry 9, Table 17 entry 19). Enantioselectivity was high in each case. The diminishing of the hydrolysis side reaction could be clearly seen when lipase PS-celite, xerogels and CLEAs were used instead of lipase PS powder (reaction stopped at 25 -33% conversion). When the acylation of 8 in TBME was performed in the presence of ACC 505-15 SILEs (mechanical stirring instead of shaking), the reaction stopped at 32% and 36% conversion (Table 16, entries 10,11) due to low solubility of 8, which led to crystallization of substrate and product on SILE catalyst during the reaction. In ILs and IL:TBME mixtures, the acylation of 8 in the presence of xerogels and CLEAs was slow. In [BMIM][PF6]:TBME mixture, both xerogel and CLEA reached 41% conversion, and the enantioselectivity was 102 and 105, respectively. In the earlier study of the acylation of 8 in IL in the presence of lipase PS-celite, the conversions were negligible showing that lipase PS-celite is not suitable catalyst in ILs.98 These results indicate the stabilization of lipase PS when immobilized as xerogels and CLEAs also in ionic liquids. The acylation in the presence of lipase PS powder was faster in many reactions when 50 mg/mL lipase PS powder was used than when 100 mg/mL lipase PS powder was used (Table 17, entries 3-4, 12,14,18,19,25). This reveals the effect of the hydrolysis side reaction, which is stronger when more lipase PS powder is used, and depends on the solvent and temperature. Since all acylation reactions performed in the presence of ionic liquid or ionic liquid:solvent mixture were slower and the selectivity did not increase, ionic liquid is not beneficial for the acylation of 6-8 in the presence of these lipase PS preparations. 58 Results and Discussion table 16. Acylation of 6-8 with vinyl acetate in organic solvents in the presence of lipase PS powder, lipase PS-celite and SILEs at room temperature. entry comp. Solvent Preparation t [h] c [%] E 1 6 toluene lipase PS ”Amano” 160 mg/mla 24 30 112 2 6 toluene lipase PS-celite 20% (w/w)b 2 50 >200 3 6 TBME lipase PS-celite 20% (w/w) 6 50 >200 4 6 toluene ACC 507-15/lipase PS 24 44 >200 5 6 toluene ACC 507-15/[EMIM][NTf2]/lipase PS 24 50 >200 6 6 toluene ACC 507-15/[EMIM][BF4]/lipase PS 24 48 152 7 7 DIPE ACC 507-15/lipase PS 6 46 25 8 7 DIPE ACC 507-15/[EMIM][NTf2]/lipase PS 6 51 33 9 8c TBME lipase PS-celite 20% (w/w) 6 51 >200 10 8c TBME ACC 507-15/lipase PS 24 32 >200 11 8c TBME ACC 507-15/[EMIM][NTf2]/lipase PS 24 36 >200 a Mechanical stirring instead of shaking used in this experiment. Lipase PS powder amount related to protein amount in ACC 507-15 SILEs. b Lipase PS “Amano” and lipase PS “Amano”SD gave similar activitites on celite c reaction temperature 48 °C table 17. Acylation of 6-8 with vinyl acetate in organic solvents and ionic liquids in the presence of lipase PS “Amano” SD powder, xerogels and CLEAs. entry comp. Solventa Powderb Xerogelc Powderd cLeAe t [h] c [%]/E t [h] c [%]/E t [h] c [%]/E t [h] c [%]/E 1 6 Toluene 24 50/>200 24 50/>200 24 49/>200 24 50/>200 2 6 TBME 27 36/56 24 47/>200 27 36/53 24 51/>200 3 6f Toluene 24 49/>200 24 51/>200 24 51/198 24 50/>200 4 6f TBME 24 40/77 24 40/174 24 46/112 30 47/>200 5 6 [EMIM][NTf2] 24 30/>200 24 5/>200 6 6 [EMIM][NTf2]:toluene 48 17/51 24 45/>200 48 17/>200 24 10/>200 7 6 [EMIM][BF4] 24 23/>200 24 24/>200 8 6 [EMIM][BF4]:toluene 24 50/>200 24 39/>200 24 47/>200 24 41/>200 9 6 [BMIM][PF6] 24 34/>200 24 18/>200 10 6 [BMIM][PF6]:toluene 48 50/>200 24 46/>200 48 49/>200 24 20/>200 11 7 DIPE 24 50/115 24 51/139 24 51/131 24 53/58 12 7 TBME 27 42/50 30 40/78 27 46/30 24 56/35 13 7 [EMIM][NTf2] 24 28/38 24 3/- 14 7 [EMIM][NTf2]:DIPE 48 45/88 24 51/66 48 30/51 24 19/49 15 7 [EMIM][BF4] 24 23/46 24 34/40 16 7 [EMIM][BF4]:DIPE 6 50/115 24 54/61 24 51/134 24 40/79 17 7 [BMIM][PF6] 24 41/78 24 21/49 18 7 [BMIM][PF6]:DIPE 24 34/60 24 34/60 24 50/77 24 33/78 19 8f TBME 24 25/30 30 49/>200 48 33/14 48 49/>200 20 8f [EMIM][NTf2] 24 5/>200 24 1/>200 21 8f [EMIM][NTf2]:TBME 48 30/30 24 26/>126 48 24/>200 48 7/- 22 8f [EMIM][BF4] 24 5/>200 24 17/- 23 8f [EMIM][BF4]:TBME 24 33/50 24 28/89 48 23/50 48 33/55 24 8f [BMIM][PF6] 24 13/>200 24 10/>200 25 8f [BMIM][PF6]:TBME 48 16/15 24 41/102 48 48/70 48 41/105 a IL:solvent mixtures 1:2 b lipase PS SD 100 mg/mL c xerogel based on 100 mg/mL lipase PS d lipase PS SD 50 mg/ml e CLEA based on 50mg/ml lipase PS f reaction temperature 48 °C Results and Discussion 59 5.3. Reuse of the lipase PS sol-gels, cLeAs and SILes (Papers I and III) Stability of enzyme preparations allowing reuse of the catalyst in repeating reaction batches is important when used in larger scale and especially when the method is used on industrial application. For this reason, the reuse of lipase PS xerogel and CLEAs was studied in the model reactions: acylation of 7 in DIPE (Paper I), 6 in toluene and 8 in TBME (P. Hara, unpublished results). The reuse of ACC 507-15 SILEs was studied in the acylation of 6 by reusing the same SILE at eight different temperatures between 25-60 °C by increasing the temperature 5 °C after each cycle, and then applying the SILE again at 25 °C (Paper III). Xerogels and ACC 507-15 SILEs were proven to be recyclable several times without significant loss of activity or selectivity. Lipase PS xerogel was used 8 times in the acylation of 7 with vinyl acetate in DIPE (activity loss after 8 cycles 6%, Paper I, Fig. 4) and 5 times in the acylation of 6 with vinyl acetate in toluene (activity loss after 5 cycles 13%, Fig. 11, P. Hara, unpublished results) at room temperature without significant loss of activity or selectivity. This indicates the stabilization of lipase PS during immobilization. In the acylation of 6 with vinyl acetate in toluene at 48 °C, the activity of xerogel started to decrease during third run (24 h, c=34 %, activity loss 33%) although E was >200 in each experiment. In the acylation of 8 with vinyl acetate in TBME at 48 °C, the activity decreased after two cycles (14%). Higher temperature (48 °C vs. room temperature) led to faster inactivation of xerogel during cycles. 0 5 10 15 20 25 0 10 20 30 40 50 Co nv er sio n [% ] Time [h] Figure 11. Recycling of the lipase PS xerogel in the acylation of 6 with vinyl acetate in toluene. Cycle 1 (■), cycle 2 (●), cycle 3 (▲), cycle 4 (▼), cycle 5 (♦). E>200 in each cycle. 60 Results and Discussion Lipase PS CLEA was highly active, and the acylation of 6-8 smoothly proceeded to 50% conversion with a fresh catalyst in organic solvents. It was not, however, stable enough to be recycled. The reuse did not have effects on enantioselectivity (7, Paper I, Fig. 5, 6 and 8 unpublished results), but the activity decreased markedly (12-49%) during the second cycle in each case. The reuse of lipase PS SILEs was studied using the acylation of 6 with vinyl acetate in toluene as a model reaction (Paper III). The stability towards recycling and changes of temperature were studied by subjecting the same catalyst at temperatures between 25 - 60 °C by increasing the temperature 5 °C after each cycle, and finally performing the acylation again at 25 °C (Table 18). ACC 507-15/lipase PS (no IL) maintained the activity and selectivity through temperature range, the conversion loss being 4% after 9 cycles. However, when applied again at 25 °C the specific activity was clearly lower. The activity and enantioselectivity of ACC 507-15/ [EMIM][NTf2]/lipase PS was high after the temperature range and also when applied again at 25 °C, the conversion decreased only 3% and specific activity 0.7 mmol min-1 g-1 after 9 cycles, indicating that [EMIM][NTf2] stabilized the enzyme during these cycles. In case of ACC 507-15/[EMIM][BF4], the activity started to decrease after 50 °C, and when applied again at 25 °C, the enantioselectivity also decreased. This shows slight destabilization of lipase PS by hydrophilic [EMIM][BF4]. This is in line with the results that hydrophobic ILs like [EMIM][NTf2] activate and stabilize enzymes, whereas hydrophilic ILs like [EMIM][BF4] can even cause decrease of activity by stripping off the essential water.54,57,129 Also, as water is a common contaminat in organic solvents, especially in higher temperatures, water can cause a partial hydrolysis of [BF4] anion with formation of HF, which is known to inactivate enzymes.42,46 This stabilization of [EMIM][NTf2] and destabilization of [EMIM][BF4] could be seen when comparing both specific activities and conversions to those of ACC 507-15/lipase PS preparation. As a result, each SILE studied was recyclable several times at varying temperatures, the most active and stable preparation being ACC 507-15/[EMIM][NTf2]/lipase PS. Since the ACC 507- 15 SILEs had high stability and the activity and enantioselectivity remained high after several cycles in various temperatures, and the structure of the immobilized enzyme preparation is convenient and mechanically stable in use, it is possible to use them also in larger scale in batch and continuous flow reactors after optimization. Also other, less harmful ionic liquids e.g. halogen-free Ammoeng and Ecoeng ionic should be tested instead on [EMIM][NTf2], which is known to be toxic. 40 Results and Discussion 61 table 18. Reuse of 6 with vinyl acetate in toluene in the presence of KynolTM ACC 507-15-based SILEs at 25 - 60 °C. s.a.=specific activity. Acc 507-15/lipase PS Acc 507-15/ [eMIM] [Ntf2]/lipase PS Acc 507-15/ [eMIM] [bF4]/lipase PS Number of use T (°C) s.a. [µmol min-1 g-1] Conversion [%]a / E s.a. [µmol min-1 g-1] Conversion [%]a / E s.a. [µmol min-1 g-1] Conversion [%]a / E 1 25 15.7 ± 0.6 44 / >200 19.5 ± 0.9 50 / >200 12.0 ± 0.6 48 / 152 2 30 24.9 ± 0.2 50 / >200 36.5 ± 1.6 51 / >200 13.6 ± 0.9 50 / 136 3 35 20.8 ± 0.9 49 / >200 45.5 ± 3.5 51 / >200 24.5 ± 1.0 50 / >200 4 40 31.3 ± 2.9 51 / >200 42.1 ± 1.4 51 / >200 23.0 ± 0.7 50 / >200 5 45 32.3 ± 0.2 51 / >200 47.6 ± 1.9 51 / >200 24.8 ± 0.6 50 / >200 6 50 36.9 ± 1.2 51 / >200 33.9 ± 0.7 51 / >200 21.8 ± 0.2 50 / >200 7 55 31.0 ± 0.8 51 / >200 32.9 ± 1.0 51 / >200 13.9 ± 0.3 45/ >200 8 60 23.8 ± 0.7 50 / >200 36.7 ± 0.9 51 / >200 9.2 ± 0.4 38 / >200 9 25 6.4 ± 0.3 40 / >200 18.8 ± 1.3 47 / >200 3.2 ± 0.2 15 / 117 a Conversion after 24 h. 5.4. the effect of immobilization on hydrolysis side reaction (Papers I and II) As already stated, the acylation of 8 in TBME in the presence of lipase PS powder started to retard after a certain concentration was reached or the reaction stopped at an early stage. For this reason, attention was paid to the possibility of hydrolysis as a side reaction. Residual water from enzyme preparations, substrates, solvent or atmosphere can lead to the hydrolysis of acyl donors and ester products in dry reaction mixtures. In Scheme 12, possible hydrolysis side reaction in the esterification of 6 is presented as an example. Due to this hydrolysis, the reactions can stop before reaching 50% conversion 157 or the enantioselectivity decreases. The enzyme preparation used plays significant role in hydrolysis in dry conditions. E.g. celite support can be used to decrease undesirable hydrolysis.99 Xerogels, CLEAs and lipase PS-celite reduce hydrolysis in kinetic resolution, since the acylation of 8 proceeds smoothly to 50% conversion (Table 16, entry 9; Table 17, entry 19), contrary to lipase PS powder. The hydrolysis was studied more detailed in the presence of xerogels. H2O H2O OH O O OH OAc OH OH O OH O Lipase PS Esterification Hydrolysis 6 (S)-6 (R)-9 Scheme 12. Hydrolysis as a side reaction in a lipase-catalyzed esterification of 6. 62 Results and Discussion The rate of hydrolysis side reaction depends on the reaction conditions, substrate structure and solvent hydrophilicity. In the case of 6-8, the hydrolysis of 8 took place most rapidly (Table 19, Fig. 12). Also the hydrolysis was faster in more hydrophilic TBME (more water available) than in toluene (entries 1 and 2). Hydrolysis of the product esters 9-11 occurred in all solvents tested with lipase PS powder, conversion being 4-86% after 24 h reaction. The hydrolysis in ILs was tested in [EMIM][BF4]:solvent mixture, since [EMIM][BF4] is the most hydrophilic IL used in this study. Hydrolysis is faster in more hydrophilic IL mixture than in organic solvent with 9 and 10 but, interestingly, slower in case of 11. Xerogel hydrolyzed significantly less of the esters than free lipase PS powder. The suppression of hydrolysis is most evident in the acylation of 8. When the acylation of 8 in the presence of free lipase PS powder stopped around 30% conversion, in the presence of xerogel the acylation proceeded to 49% conversion (Fig. 12). The same effect could be seen in the acylation of 6 in the presence of free lipase PS in TBME (18% hydrolysis), where conversion was 36% after 27 h, whereas in toluene (4% hydrolysis) the conversion was 50% after the same time. When xerogel was used, the conversions in TBME and toluene were 47% and 50%, respectively. Similar effect can also be seen with CLEA, although there were no more detailed studies of hydrolysis. When the acylation of 8 stopped at 33% conversion with free lipase PS, CLEA acylated 8 smoothly (c=49 %, 48 h, Fig. 12.). The same effect was observed with 6 in TBME. The suppression of hydrolysis effected on enantioselectivity, since E of the acylation of 8 improved from 30 to >200 when xerogel was used, and from 14 to >200 in case of CLEA. This improvement occurred also in the acylation of 6 in TBME where E increased from 56 to >200 in case of xerogel and from 53 to >200 in case of CLEA. As a conclusion, both xerogel and CLEA clearly suppress the unwanted hydrolytic side reactions, especially in hydrophilic organic solvents like TBME. Xerogel is also catalyst of choice in IL:solvent mixtures. table 19. Conversion of the side reaction (%) after 24 h in the hydrolysis of enantiopure esters 9-11 (0.05 M) by the residual water in the enzyme preparation and the solvent system at room temperature. entry Substrate Solvent log P c [%] by lipase PSa c [%] b y Xerogelb 1 (R)-9 Toluene 2.8 4 1 2 (R)-9 TBME 1.35 18 3 3 (R)-9 [EMIM][BF4]:Toluene 1:2 27 2 4 (R)-10 DIPE 1.9 20 10 5 (R)-10 TBME 1.35 12 2 6 (R)-10 [EMIM][BF4]:DIPE 1:2 22 26 7 (S)-11c TBME 1.35 86 16 8 (S)-11c [EMIM][BF4]:TBME 1:2 35 45 a 100 mg mL-1 of lipase PS powder b Xerogel based on 100 mg mL-1 of lipase PS powder. c Butanoate instead of acetate; reaction temperature 48 ºC Results and Discussion 63 0 5 10 15 20 25 30 35 40 45 50 0 10 20 30 40 50 Co nv er sio n [% ] Time [h] Figure 12. Acylation of 8 (0.1 M) with vinyl acetate (0.2 M) in TBME at 48 ºC in the presence of lipase PS powder (100 mg mL-1; -▲-), (50 mg mL-1; -▼-), xerogel (100 mg of lipase PS; -■-) and CLEA (50 mg lipase PS; -●-). 64 Summary 6. SuMMARy In this thesis immobilization methods of lipases, mainly Burkholderia cepacia lipase, lipase PS, and the effects of immobilization on activity and kinetic resolution in organic solvents and in ionic liquids is described. Lipase PS “Amano” or lipase PS “Amano” SD was immobilized using three different methods. The methods were sol-gel, cross-linked enzyme aggregates (CLEA) and supported ionic liquids enzymes (SILE) methods. These immobilized lipase preparations were used in enzymatic acylation of racemic alcohols 6-8 to study the the effect of immobilization on activity, enantioselectivity and hydrolysis side reactions in organic solvents, ionic liquids, and in their mixtures. The results were compared to those obtained with lipase PS immobilized on celite, the method widely used for lipase immobilization. Moreover, reuse possibilities of these immobilized enzymes were studied. I have shown that lipase PS xerogels had low specific activity in organic solvents, ILs and in IL:solvent mixtures. However, the kinetic resolution of 6-8 proceeded smoothly in organic solvents. In ILs and IL:solvent mixtures lipase PS xerogel was more stable than free lipase PS powder and lipase PS-celite, and the proceeding of the acylation depends on substrate structure. Xerogel was also reusable 5 times in the resolution of 6 in toluene and 8 times in the resolution of 7 in DIPE. In these reaction conditions, the conversion loss in the acylation of 6 was only 13 % and in the acylation of 7 6 % after the cycles. Hydrolysis of the product esters was suppressed. In this thesis lipase PS CLEAs showed higher activity than free lipase PS powder in all solvent systems used. Kinetic resolution proceeded well in organic solvents. However, CLEA preparation rapidly lost its activity in systems containing IL. CLEA seems to be a good choice in hydrophilic solvents, since it maintained the activity and selectivity in kinetic resolution in TBME, although xerogel and lipase PS-celite were slower in TBME than in toluene. CLEA performed well also in hydrophilic [EMIM][BF4] and in [EMIM][BF4]:solvent mixtures, although xerogel showed higher activity in hydrophobic solvents. CLEA also suppressed the hydrolysis side reaction. Recyclability of CLEAs was limited. I have shown that lipase PS ACC 507-15 SILEs, immobilized on active carbon cloth ACC 507-15, were highly active and enantioselective, the most active being ACC 507- 15/[EMIM][NTf2]/lipase PS. This preparation was reusable 9 times in temperatures 25 - 60°C. [EMIM][NTf2] clearly stabilized lipase PS against inactivation at high temperatures. ACC 507-15 SILEs had also good mechanical resistance in used stirring system. Lipase PS-celite is highly active and enantioselective catalyst in organic solvents. However, it is not a suitable catalyst in polar solvents like ionic liquids, since adsorption forces are not enough to keep the enzyme attached on the support in polar environments. Summary 65 When the results are compared to those of lipase PS-celite, lipase PS-celite and ACC 507-15/[EMIM][NTf2]/lipase PS SILE gave the highest activity. With these catalysts the acylation proceeded smoothly in organic solvents. Xerogel is the choice in ionic liquids, when the product ester is an activated ester and when hydrolysis is possible as a side reaction. CLEA performed well in organic solvents, including hydrophilic TBME. The use of ionic liquids was not beneficial in the acylations of 6-8 in these conditions. SILEs were shown to be suitable especially in larger scale, due to their stability and activity when reused, mechanical resistance and convenient handling. I have shown that there is no “number one” method among the immobilization methods studied, rather the choice of lipase PS preparation depends on the structure of the substrate and the reaction conditions, especially on the solvent used. 66 Acknowledgements 7. Acknowledgements The experimental work of this thesis was carried out at the Laboratory of Synthetic Drug Chemistry, Department of Pharmacology, Drug Development and Therapeutics, Faculty of Medicine, University of Turku. The Academy of Finland is gratefully acknowledged for funding this work and COST D25 for funding short term scientific mission to Delft University of Technology. I want to express my sincere gratitude to my supervisor, Professor Liisa Kanerva for introducing me the world of enzymes and giving me the possibility to do this work. I also want to thank you for the guidance and interesting discussions during the years. Special thanks to Associate Professor Ulf Hanefeld for giving me the possibility to visit Delft University of Technology and for introducing me sol-gel and CLEA methods. I also want to thank Professor Dmitry Yu. Murzin and Professor Jyri-Pekka Mikkola for the possibility to visit the Laboratory of Industrial Chemistry and Reaction Engineering, Åbo Akademi and to learn the supported ionic liquids catalysts technique. Professor Kristiina Kruus and Professor László Poppe are acknowledged for reviewing this thesis. Dr. Arto Liljeblad is acknowledged for all the help during the years and for critical reading of the manuscript of this thesis. I want to thank my colleagues (in alphabetical order) from the Laboratory of Synthetic Drug Chemistry for the nice working environment and for sharing your knowledge with me. Thank you Päivi Alanko, Jürgen Brem, Monica Fitz, Ari Hietanen, Annukka Kallinen, Anu Kiviniemi, Toni Kurki, Niko Laaksovirta, Hanna Launela, Outi Lehtovirta, Dr. Xiang-Guo Li, Tarja Limnell, Dr. Katri Lundell, Otto Långvik, Harri Mäenpää, Tihamér Paál, Päivi Perkiö, Maria Puustinen, Mari Päiviö, Maria Rantapaju, Tiina Saloranta, Elina Siirola, Riku Sundell, Dr. Mihaela Turcu and Marita Vainio. It was a privilege to work with you all. Sincere thanks to my parents Maire and Timo for their love and encourangement for studying. I also want to thank my friends for joy you bring to my life. I would like to express my warmest thanks to my husband Mika and my children Iina and Aleksi for their love and support, and directing my thoughts away from chemistry. Lieto 2011, Piia Hara References 67 8. ReFeReNceS 1. Leresche, J.E.; Meyer, H.-P. Chemocatalysis and biocatalysis (biotransformation): Some thoughts of a chemist and of a biotechnologist. Org. Proc. Res. Dev., 2006, 10, 572-580. 2. 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