Synthesis of chiral ester compounds by enzymatic resolution and reduction

Abstract

In recent years, the most significant development in the field of synthetic organic chemistry is the application of enzymes to chemical reactions. Reactions catalyzed by enzymes or enzyme-containing microorganisms display greater specificities than conventional organic reactions. Some of these reactions have already been shown to have useful applications in the fields of synthetic organic chemistry and biotechnology, especially for the preparation of pharmaceuticals, fine chemicals, food additives and commodity chemicals. In this thesis, new chemo-enzymatic processes for chiral ester compounds were described. First, a novel and efficient resolution process for methyl (R)-2-chloromandelate, the key intermediate for the synthesis of (S)-clopidogrel ($Plavix^R$), has been developed by CAL-A catalyzed transesterification reaction using vinyl propionate as acyl donor with or without organic solvents. The data showed that a CAL-A-catalyzed resolution of methyl 2-chloromandelate, which has steric effect on enzymes is feasible by performing a transesterification reaction in organic solvents with vinyl propionate. Under optimized condition, methyl (R)-2-chloromandelate of high optical purity (>99% ee) was successively prepared in good yield (42%) and high enantioselectivity (E = 34.7). The immobilized enzyme, CAL-A, was reused thirteen times without loss of enantioselectivity and reactivity. It is economical and easy to scale-up for commercial production of mehyl (R)-2-chloromandelate. Several useful chiral intermediates with high optical purity (>99% ee) could be synthesized using this methyl (R)-2-chloromandelate. Second, enzymatic synthesis of chiral $\beta$ -hydroxy-$\alpha$ -amino acids having two chiral centers and three carbon atoms with different functional groups was attempted. These compounds are used as valuable intermediates for the synthesis of chiral pharmaceuticals such as $\beta$ -penem antibiotics. In this study, twenty-two microbial keto reductases/GDH enzymes were tested for reduction of ethyl 2-acetamido-3-oxobutanoate synthesized from ethyl acetoacetate to ethyl D-threonine. A glucose dehydrogenase-coupling reaction was used to regenerate NAD(P)H to perform the reduction reaction continuously. Four keto reductase showed high enantioselectivity and activity. Candida magnolia keto reductase, Candida parapsiliosis keto reductase, and Rhodococcus erythropolis keto reductase produced ethyl D-threonine and ethyl L-allo-threonine, whereas Devosia riboflavin keto reductase produced ethyl L-threonine and ethyl D-allo-threonine. Among four keto reductases screened, Rhodococcus erythropolis keto reductase was extensively studied because it produced ethyl D-threonine predominantly. This enzyme used $NAD^{+}$ as cofactor. The optimum temperature was $20^\circ C$ and optimum pH was 6.5. The keto reductase activity was increased somewhat by the ionic salts such as $Mg^{2+}$ and $Ca^{2+}$. Large scale production (up to several hundreded grams) of ethyl D-threonine and ethyl L-allo-threonine was carried out using Rhodococcus erythropolis keto reductase with good yield (92% yield).