Abstract:

Brivaracetam is a new generation of anti-epileptic drugs with broad market prospects. The nitrilase-catalyzed synthesis of (R)-3-cyanohexanoic acid from 3-cyanocapronitrile, followed by hydrogenation, cyclization and chiral resolution to synthesize brivaracetam is a promising route due to the high atomic economy. However, the poor enantioselectivity of the exploited nitrilases has strongly restricted its application. In this study, the nitrilase PgNIT from Paraburkholderia graminis was exploited. The catalytic pocket and subunit interface domain were modified and a mutant F164W/I202R with significantly improved enantioselectivity was obtained. As a result, the ee_p value of F164W/I202R was increased from 86% to 97%, while the E value rose from 17 to 111 compared to wild type, respectively. Molecular dynamics simulation analyses revealed that the mutation of F164 reduced the steric hindrance on the entry of (R)-3-cyanocapronitrile into the catalytic pocket. On the other hand, the modification of I202 site at the “A” interface increased the association distance between subunits and increased the dynamic coordination of atoms in the catalytic pocket domain, thus significantly improved the enantioselectivity.

Key words: nitrilase, enantioselectivity, molecular modification, molecular simulation, biocatalysis, molecular biology

摘要：

布瓦西坦是新一代抗癫痫药物，具有广阔的市场前景。利用腈水解酶立体选择性催化3-氰基己腈合成关键手性中间体（R）-3-氰基己酸，进一步经加氢、环化、手性拆分等合成布瓦西坦路线，具有原子经济性高等优势，然而目前挖掘的腈水解酶存在立体选择性差等缺陷。以腈水解酶PgNIT为研究对象，通过对其催化口袋和亚基界面改造获得了立体选择性大幅提高的突变体F164W/I202R，产物光学纯度由野生型的86%提升至97%，E值由17提高至111。分子动力学模拟研究表明，F164位点的改造减小了（R）-3-氰基己腈深入催化口袋内部的空间位阻，而“A”界面I202位点的改造增加了亚基之间缔合的距离和催化口袋区域的原子动态协同性，从而显著提升了腈水解酶的立体选择性。

关键词: 腈水解酶, 立体选择性, 分子改造, 分子模拟, 生物催化, 分子生物学