The enantiomers of (4R/S)-4-hydroxy-N, N-diphenyl-2-pentynamide are key chiral synthons for the synthesis of thrombin receptor antagonists such as vorapaxar. In this paper, we report the enzymatic preparation of enantiomerically enriched (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide using lipase A from Burkholderia cepacia ATCC 25416 as the catalyst. First, the lipase gene (lipA) and its chaperone gene (lipB) was cloned and expressed in Escherichia coli system. After purification, lipase A activation was performed with the assistance of foldase lipase B. Enzyme assay revealed that the activated lipase A showed the optimal catalytic activity at 60 ºC and pH 7. The effects of various metals on the activity were investigated and results demonstrated that most of the metals inhibited the activity. To further improve the catalytic outcome, two-phase reaction was studied, and n-hexane proved to be a good organic solvent for the combination system. Using the optimize conditions, (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide with 94.5% ee value and 48.93% conversion ratio was achieved. Our investigation on this lipase reveals lipase A as a promising biocatalyst for producing chiral propargyl alcohol for preparation of novel himbacine analogs.

中文翻译：

---

通过洋葱伯克霍尔德菌脂肪酶 A 对映选择性合成喜巴辛类似物的手性中间体

(4R/S)-4-羟基-N，N-二苯基-2-戊酰胺的对映异构体是合成凝血酶受体拮抗剂（如沃拉帕沙）的关键手性合成子。在本文中，我们报告了使用来自洋葱伯克霍尔德菌 ATCC 25416 的脂肪酶 A 作为催化剂的对映体富集的 (4R)-4-羟基-N，N-二苯基-2-戊酰胺的酶促制备。首先，在大肠杆菌系统中克隆并表达了脂肪酶基因（lipA）及其分子伴侣基因（lipB）。纯化后，脂肪酶 A 在折叠酶脂肪酶 B 的协助下进行活化。酶分析表明，活化的脂肪酶 A 在 60 ºC 和 pH 7 下表现出最佳催化活性。研究了各种金属对活性的影响，结果表明：大多数金属抑制了活性。为了进一步提高催化效果，研究了两相反应，证明正己烷是组合体系的良好有机溶剂。使用优化条件，(4R)-4-羟基-N，N-二苯基-2-戊酰胺获得了94.5%的ee值和48.93%的转化率。我们对这种脂肪酶的研究表明，脂肪酶 A 是一种有前途的生物催化剂，可用于生产手性炔丙醇以制备新型喜巴辛类似物。