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Understanding and Engineering the Enantioselectivity of Candida Antarctica Lipase B Towards Sec-Alcohols

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Understanding and Engineering the Enantioselectivity of Candida Antarctica Lipase B Towards Sec-Alcohols Understanding and Engineering the Enantioselectivity of Candida antarctica Lipase B towards sec-Alcohols Didier Rotticci Royal Institute of Technology Department of Chemistry Stockholm 2000 Organic Chemistry ISBN: 91-7170- 550- 3 Cover: The green contour represents an energetically favorable binding site for an organic bromine atom (-6 kcal mol-1) in the active site of the Candida antarctica lipase B mutant Trp104His. The catalytically important Ser105 and His224 are also displayed. Understanding and Engineering the Enantioselectivity of Candida antarctica Lipase B towards sec-Alcohols Didier Rotticci ISRN KTH/IOK/FR--00/60--SE ISSN 110-7974 TRITA-IOK Forskningsrapport 2000:60 Royal Institute of Technology Department of Chemistry Organic Chemistry Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av teknologie doktorsexamen i organisk kemi fredag den 9:e juni, 2000, kl. 10.00 i Kollegiesalen, Administrationsbyggnaden, KTH, Valhallavägen 79, Stockholm. Avhandlingen försvaras på engelska. ISBN: 91-7170-550-3 List of abbreviations and symbols A adenosine RNA ribonucleic acid aw water activity R.t. reaction time BSA bovine serum albumin [S] substrate concentration C cytosine [S0] bulk substrate concentration c extent of conversion SD standard deviation CALB Candida antarctica lipase B T thymine CHES 2-(N-cyclohexylamino) T temperature ethanesulfonic acid TBS t-butyldimethylsilyl d.e. diastereomeric excess TS transition state Deff effective diffusivity U uracil DNA deoxyribonucleic acid Vm maximum reaction rate in the E enantiomeric ratio Michaelis-Menten equation # Eeff effective enantioselectivity ∆G Gibbs free energy of activation # e.e. enantiomeric excess ∆∆G difference in Gibbs free energy e.e.s enantiomeric excess of the of activation for the enantiomers # substrate ∆∆H difference in activation enthalpy e.e.p enantiomeric excess of the for the enantiomers # product ∆∆S difference in activation entropy ES Michaelis-Menten complex for the enantiomers F.r. fast-reacting enantiomer [α] specific optical rotation G guanine β dimensionless substrate GC gas chromatography concentration HPLC high performance liquid ν reaction rate chromatography φ Thiele modulus kb kilobase R η effectiveness factor k turnover number cat K Michaelis-Menten constant M l length of the cell MD molecular dynamics MES 2-(N-morpholino)ethanesulfonic acid MOPS 3-(N- morpholino)propanesulfonic acid MS mass spectrometry NMR nuclear magnetic resonance PCR polymerase chain reaction r radius of the support R gas constant (8.31 J mol-1 K-1) iii Abstract The kinetic resolution of sec-alcohols catalyzed by Candida antarctica lipase B (CALB) was investigated. High enantioselectivity was achieved with some aliphatic, allylic, propargylic and halogenated sec-alcohols. Steric and non-steric interactions were found to be important in the chiral recognition. Empirical rules for qualitative prediction of the enantioselectivity of CALB towards sec-alcohols were defined. High reaction rate and enantioselectivity can be expected for substrates with the following requirements at the stereocenter: (i) large substituent equal to or larger than a propyl group or containing a halogen atom (ii) medium substituent smaller than a propyl group and without one halogen atom. Parameters influencing the enantioselectivity in organic solvents were investigated and reviewed. The enantioselectivity was affected by many parameters, such as temperature, solvent, acyl-donor and enzyme preparation. Particular attention was given to the large differences in enantioselectivity and in reaction rate among the lipase preparations used. Mass-transport limitations were the main causes of the difference in effectiveness between preparations. Diffusion limitations were detrimental to the reaction rate and the enantioselectivity and, thus, should be avoided. Lipase immobilizations and high substrate concentrations reduced mass-transport problems in organic solvents. E- values varied between 60 and 200 for the resolution of 3-methyl-2-cyclohexen-1- ol in hexane. The molecular basis of the enantioselectivity of CALB towards sec-alcohols was studied by means of molecular modeling and experimental kinetic data. The origin of the chiral recognition was traced to the permutation of the large and the medium substituents of the alcohol enantiomers in the active site i.e. the slow- reacting enantiomer placed its large substituent in a pocket of limited volume, whereas the fast-reacting enantiomer placed its medium substituent in this pocket. Rational protein engineering, based on the above model, allowed the creation of one synthetically useful mutant as well as one mutant with annihilated enantioselectivity towards two target 1-halo-2-octanols. These results supported our model concerning the molecular recognition of sec-alcohol enantiomers by CALB. Key words: kinetic resolution, protein engineering, mass-transport limitations, organic media, halohydrins, chiral recognition, empirical rules, biocatalysis. v List of articles I. Candida antarctica lipase B catalysed kinetic resolutions: substrate structure requirements for the preparation of enantiomerically enriched secondary alcanols C. Orrenius, N. Öhrner, D. Rotticci, A. Mattson, K. Hult, and T. Norin, Tetrahedron: Asymmetry, 1995, 6, 1217. II. Enantiomerically enriched bifunctional sec-alcohols prepared by Candida antarctica lipase B catalysis. Evidence of non-steric interactions D. Rotticci, C. Orrenius, K. Hult, and T. Norin, Tetrahedron: Asymmetry, 1997, 8, 359. III. Chiral recognition of alcohol enantiomers in acyl transfer reactions catalysed by Candida antarctica lipase B C. Orrenius, F. Hæffner, D. Rotticci, N. Öhrner, T. Norin, and K. Hult, Biocatal. Biotransform., 1998, 16, 1. IV. Molecular recognition of sec-alcohol enantiomers by Candida antarctica lipase B D. Rotticci, F. Hæffner, C. Orrenius, T. Norin, and K. Hult, J. Mol. Catal. B: Enzym., 1998, 5, 267. V. Candida antarctica lipase B: a tool for the preparation of optically active alcohols D. Rotticci, J. Ottosson, T. Norin, and K. Hult, in Enzymes in non-aqueous media, (Eds.: P. Halling, E. Vulfson, J. Woodley, B. Holland), Totowa, NJ, Humana Press. In press. VI. Mass-transport limitations reduce the effective stereospecificity in enzyme- catalyzed kinetic resolution D. Rotticci, T. Norin, and K. Hult, Org. Lett., In press. VII. Improving the enantioselectivity of a lipase through rational protein engineering D. Rotticci, J. C. Mulder, S. Denman, T. Norin, and K. Hult, manuscript. vii Table of contents 1- Introduction 1 1.1 Scope and outline of the thesis 1 2- Background 3 2.1 Chirality 3 2.2 Determination of enantiomeric purity 4 2.2.1 Chiroptical method 4 2.2.2 Conversion of enantiomers into diastereoisomers 5 2.2.3 Chromatography on a chiral stationary phase 6 2.3 Preparation of optically active compounds 6 2.3.1 Resolution 7 2.3.2 Kinetic resolution 7 2.3.3 Enzymatic kinetic resolution 8 2.3.4 Dynamic kinetic resolution 11 2.3.5 Improving the enzyme enantioselectivity 12 2.4 Biocatalyst engineering 13 2.4.1 Biocatalysis in non-aqueous media 13 2.4.2 Mass-transport limitations 14 2.5 Protein engineering 16 2.5.1 Chemical modification 17 2.5.2 DNA technology 17 2.6 Molecular modeling 18 2.6.1 Molecular graphics 18 2.6.2 Computational chemistry 19 3- The Candida antarctica lipase B 21 3.1 Structure and origin 21 3.2 Kinetics and reaction mechanism 22 4- CALB-catalyzed preparation of optically active sec-alcohols 25 4.1 Substrate structure requirements 25 4.1.1 Acyclic sec-alcohols 25 4.1.2 Cyclic sec-alcohols 28 4.2 Optimization of CALB enantioselectivity 29 4.2.1 Acyl-donor 30 ix 4.2.2 Temperature 31 4.2.3 Solvent and water activity 32 4.2.4 Biocatalyst engineering 32 5- Molecular basis of the enantioselectivity of CALB towards sec-alcohols 37 6- Improving the enantioselectivity of CALB through protein engineering 41 6.1 Improving the enantioselectivity of CALB towards halohydrins 41 6.1.1 Design of the mutants 41 6.1.2 Site-directed mutagenesis 43 6.1.3 Lipase production and purification 45 6.1.4 Kinetic resolution experiments 45 7- Concluding remarks 47 8- Supplementary material 49 8.1 Kinetic resolution of 3-methyl-2-cyclohexen-1-ol (seudenol) 49 8.2 (SP,RP)-(R)-2-octyl hexylphosphonochloridates 49 8.3 (SP,RP)-(S)-2-octyl hexylphosphonochloridates 50 9- Acknowledgments 51 10- References 53 x 1- Introduction This thesis deals with fundamental and applied biocatalysis. Biocatalysis uses natural catalysts namely enzymes to produce synthetic chemicals. Within this field, the thesis focuses on asymmetric transformations catalyzed by a lipase. The concept of catalysis was introduced by the Swedish chemist J. J. Berzelius in 1836. Catalysis is the process by which a small amount of substance (the catalyst) increases the rate of a chemical reaction without being consumed. Enzymes are catalysts evolved by Nature to sustain and develop life. Nearly all enzymes are proteins, i.e. they are built up from the twenty natural L-amino acids. The outstanding properties of these catalysts in terms of substrate specificity, chemoselectivity, regioselectitivty and, especially, enantioselectivity make them particularly attractive for the production of fine chemicals. The latter property, their enantioselectivity, is the center of the research presented herein. Since the pioneering work of Buchner (1897), it is known that enzymes do not require the environment of the living cell to be active. Moreover, it has been known
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