Appl Microbiol Biotechnol (1985) 21 : 16-19 Applied M wrobiology Biotechnology © Springer-Verlag1985 Enzyme catalyzed hydrolysis of the diesters of cis- and trans-cyclohexanedicarboxylic acids Bioorganic preparation of enantiomerically pure cyclohexanedicarboxylic acids, monoesters and lactones Fredrik Bj6rkling 1, John Boutelje 2, Sten Gatenbeck 2, Karl Hult2, and Torbj6rn Norin 1 1 Department of Organic Chemistry, Royal Institute of Technology, S-10044 Stockholm 2 Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-10044 Stockholm, Sweden Summary. Pig liver esterase (EC 3.1.1.1) catalyzed hydrolysis of the dimethyl ester of meso-cis-l,2-cy- clohexanedicarboxylic acid yielded the optically pure (1S,2R)-monoester. The corresponding diethyl ester yielded racemic monoester. The diethyl ester of racemic trans-l,2-cyclohexa- nedicarboxylic acid was kinetically resolved by partial hydrolysis with subtilisin (EC 3.4.21.14) or pig liver esterase. The (1R,2R)-monoester had an enantio- meric excess of 45% and was obtained in an enantiomerically pure form through recrystallisation. The remaining (1S,2S)-diester exhibited an enan- tiomeric excess of 83%. The nature of the ester function (methyl, ethyl, and propyl esters) had a great influence on the enantiomeric excess obtained and on the kinetic parameters. In~oducfion The specificity and the stereoselectivity of enzyme catalyzed reactions are of great interest in the synthesis of simple chiral compounds which can be used as "building blocks" (synthons) in the asym- metric synthesis of more complex products (White- sides and Wong 1983). The commercially available hydrolytic enzymes such as a-chymotrypsin and pig liver esterase are known to catalyze reactions with high stereoselectivity (Jones et al. 1976). Enantiomerically pure y-lactones have previously been prepared by a horse liver alcohol dehydrogenase catalyzed oxidation of cis-l,2-bis(hydroxymethyl)cy- clohexane (Jakovac et al. 1982). However, for preparative purposes the use of hydrolytic enzymes, which do not require cofactors, is the preferred method. Therefore, we have explored the possibility of using the commercially available hydrolases, pig Offprint requests to: J. Boutelje liver esterase, and subtilisin, to asymmetrically synthesize the monoesters of cis- and trans-l,2-cy- clohexanedicarboxylic acids and their related deriva- tives. Additionally, kinetic studies were conducted to find out how the nature of the ester functions affects the kinetics and stereoselectivity of the enzymatic hydrolysis. Materials and methods Enzymes. Pig liver esterase (EC 3.1.1.1) and subtilisin carlsberg (EC 3.4.21.14) were purchased from Sigma. Analytical methods. NMR-spectroscopy was performed on a Bruker WP 200 (200 MHz) insturment using CDC13 as solvent. Capillary GLC was used for purity control of the diesters (25 m Carbowax 20 M, 70-200° C) and for monitoring the enzymatic reactions (30 m DB-1, 70-220 ° C). Diesters of cyclohexanedicarboxylic acids. Diesters of both c/s- and trans-cyclohexanedicarboxylic acid were prepared in good yield by esterification (Rogan 1962) of the correspondingdicarboxylicacid. The trans-dicarboxylic acid was commercially available and the c/s-dicarboxylic acid was synthesized from the corresponding anhydride (Jenkins and Costello 1946). For the kinetic studies, the pure enantiomers of the trans-diethylester of cyclohexanedicar- boxylicacid were used. These reference compounds were obtained via fractional crystallizationof the raeemic dicarboxylicacid in the presence of either S- or R-phenylethylamine (French Demande 1975) followed by esterification as described above. Enantiomeric excess of monoesters. NMR-spectroscopy of the monoesters in the presence of enantiomerically pure phenylethyl- amine gave rise to diastereotopic protons and the e.e. could thereby be determined. For this purpose, the most significant signals were those arising from the methyl protons of the ester group. Crystallization of the trans-monoester was performed in light petroleum (b.p. 40-60° C). The racemate crystallized and the enriched enantiomer remained in the mother liquor. Enantiomeric excess ofdiesters. NMR-spectroscopy of the diesters in the presence of the chiral shift reagent tris-3-(heptafluoropro- pylhydroxymethylene)-d-camphor ato-europium(III) [Eu(HFC)3] gave the e.e.