作为心血管药物地尔硫的关键合成中间体,甲基(2R,3S)-3-(4-甲氧基苯基)缩水甘油酸盐((2R,3S)-MPGM)(1)可通过氯醇(3S)-2-氯-3-羟基-3-(4-甲氧基苯基)丙酸甲酯((3S)-2)的闭环获得。我们报告了使用具有4.5倍提高的比活性的工程酶SSCRM2将2-氯-3-(4-甲氧基苯基)-3-氧代丙酸甲酯(3)有效还原为(3S)-2,通过缓解空间位阻和不希望的相互作用,通过酮还原酶SSCR的结构指导位点饱和诱变获得。结合使用共表达微调策略,重组大肠杆菌(pET28a-RBS-SSCRM2/pACYCDuet-GDH),共表达SSCRM2和葡萄糖脱氢酶,构建并优化蛋白质表达。优化反应条件后,实现了工业相关的300克/升3的全细胞催化完全还原,提供99%ee的(3S)-2和519.1g·L-1·d-1的时空产率,代表迄今为止报道的(3S)-2生物催化合成的最高记录。该生物催化合成的E因子为24.5(包括水)。该原子经济合成中产生的手性醇(3S)-2转化为(2R,3S)-MPGM,产率为95%,ee为99%。本文受版权保护。保留所有权利。
As a key synthetic intermediate of the cardiovascular drug diltiazem, methyl (2R,3S)-3-(4-methoxyphenyl) glycidate ((2R,3S)-MPGM) (1) is accessible via the ring closure of chlorohydrin (3S)-methyl 2-chloro-3-hydroxy-3-(4-methoxyphenyl)propanoate ((3S)-2). We report the efficient reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (3) to (3S)-2 using an engineered enzyme SSCRM2 possessing 4.5-fold improved specific activity, which was obtained through the structure-guided site-saturation mutagenesis of the
ketoreductase SSCR by reliving steric hindrance and undesired interactions. With the combined use of the co-expression fine-tuning strategy, a recombinant E. coli (pET28a-RBS-SSCRM2 /pACYCDuet-GDH), co-expressing SSCRM2 and glucose dehydrogenase, was constructed and optimized for protein expression. After optimizing the reaction conditions, whole-cell-catalyzed complete reduction of industrially relevant 300 g L-1 of 3 was realized, affording (3S)-2 with 99% ee and a space-time yield of 519.1 g∙L-1 ∙d-1 , representing the highest record for the biocatalytic synthesis of (3S)-2 reported to date. The E-factor of this biocatalytic synthesis was 24.5 (including water). Chiral alcohol (3S)-2 generated in this atom-economic synthesis was transformed to (2R,3S)-MPGM in 95% yield with 99% ee.