Two-Step Enzymatic Asymmetric Synthesis Of Tert-butyl (3R,5S)-6-chloro-3,5-dihydroxhexanoate

(3R,5S)-te/t-Butyl-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CHOH) is a valuable chiral synthon, which is used for the synthesis of the cholesterol-lowering drugs——statins. To date, the most potential synthetic route of (3R, 5S)-CHOH is via a two-step asymmetric reduction process using tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) as the starting material. In this thesis, a systematic research was done to improve the efficiency of these two reduction reactions. The main contents of this work were as follows:(1) A tetrad mutant of an alcohol dehydrogenase from Lactobacillus kefir, A94T/F147L/L199H/A202L(LkTADH), was screened to be the most efficient biocatalyst in the asymmetric reduction of CDOH to (S)-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate ((S)-CHOH), with a specific activity of 1.37 U/mg and an e.e. value of 99.5%. The best reaction pH and temperature were found to be 5.5 and 20 ℃, respectively, based on the results of enzymatic properties study of purified LkTADH and the stability study of CDOH. Due to the existence of serious substrate inhibitation for LkTADH and the unstability of CDOH in aqueous phase, a fed-batch strategy of substrate was successfully developed to address these issues. The catalytic process was further optimized using LkTADH whole cells as biocatalyst. A final CDOH concentration of 100 g/L was achieved, giving 401.5 mM of (S)-CHOH after a reaction time of 38 h, with only 6.1 mol% furanone (the main byproduct) remaining in the final product. The space-time yield and turnover number of NADP+ in this process were 10.6 mmol/L/h and 16,060 mol/mol, respectively.(2) To study the structure-function relationship of LkTADH, homology modelling study and docking study of LkADH or LkTADH with CDOH were both performed, as well as the back mutation study of LkTADH. A similar partial noncompetitive inhibition was proven to be existing in all of the mutants of LkTADH via the reaction kinetics study. The results of kinetics study also suggest that the mutation of Phe-147 to Leu-147 not only dramatically improves the catalytic efficiency and affinity of enzyme to CDOH, but also partially relieves the substrate inhibition. The conformation of Asn-157 was changed by this mutation, resulting with the formation of an extra hydrogen bond between the amino group of Asn-157 and carboxyl group of Glu-145. It is known that Glu-145 functions as the most important part of the small hydrophobic pocket near the active site. The formation of this new bond was the main reason for the improved catalytic efficiency, as it not only stabilized the active site, but also enlarged the hydrophobic pocket through pushing up the side chain of Glu-145 nearby. As for the mutation of Ala to Leu at 202-site, which is a composition of the large substrate hydrophobic pocket to hold the tert-butyl part of CDOH, the enhancement of hydrophobic interaction helps the improvements of affinity of enzyme to CDOH. Moreover, a remarkable relief of substrate inhibition was also observed with this mutation. However, the other two mutation, A94T and L199H, which are both using huge residues instead of small ones, were proven to be harmful for the affinity of enzyme to CDOH due to the smaller substrate binding pocket.The best mutant obtained during the back mutation was M147-202(F147L/A202L), with a specific activity of 2.61 U/mg and an e.e. value of 99.5%. A final CDOH concentration of 100 g/L (Fed-batch) gave 418.3 mM (S)-CHOH after a reaction time of 21 h with M147-202 whole cells, with only 1.0 mol% furanone remaining in the final product and a TTN number of 16,732 mol/mol. The space-time yield in this process was 19.9 mmol/L/h, which was almost 2-fold of that with LkTADH in the same conditions. This process efficiency was the highest reported for this reduction.(3) The carbonyl reductase CR from Candida magnoliae CGMCC 2.1919 was screened to be the most efficient biocatalyst in the asymmetric reduction of (S)-CHOH to (3R,5S)-CDHH, with a specific activity of 6.98 U/mg and a d.e. value of 99.0%. Addtionally, a mutant of glucose dehydrogenase (GDH-Q) with improved heat stability was obtained through directed mutation. The following coexpressed study of CR and GDH-Q suggested that the coexpressed strains of E.coli BL21(DE3)/pET30-CR+pACYCDuet-GDH-Q was the best one with a matched enzyme activity. Using this coexpressed strain as biocatalyst, this asymmetric reduction process was fully optimized. The results indicated that under the condition of 30 ℃, pH 6.5, mass ratio of glucose to substrate at 1:1(w/w),5 gDCW/L of cell concentration,0.1 mM NADP+,400 g/L of substrate was bio-reducted to (3R, 5S)-CDHH within 24 h with a conversion of 99.5% and a TTN number of 16,796 mol/mol. The purity of product after extraction was 99% with a d.e. valule of 99.6%.