Understanding and engineering the enantioselectivity of candida antarctica lipase b towards sec-alcohols

by Rotticci, Didier

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-1ol 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 slowreacting 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 enzymecatalyzed 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