Chiral motifs of 2,3-dihydro-1,4 benzodioxane are extensively utilized in diverse medicinal substances and bioactive natural compounds, exhibiting significant biological activities. Notable examples of such therapeutic agents include prosympal, dibozane, piperoxan, and doxazosin. In this work, using 1,4-benzodioxane-2-carboxylic acid methyl ester as the substrate, after screening 38 CALB covariant residues, we found that mutants A225F and A225F/T103A can catalyze the kinetic resolution of the substrate. The effect of temperature, cosolvent, and cosolvent concentration on kinetic resolution was investigated, revealing that the best results were achieved at 30 °C with 20% n-butanol as a cosolvent, resulting in an optimal resolution (e.e._s 97%, E = 278) at 50 mM substrate concentration. Structure analysis showed that mutation sites 225 and 103 are not among the sites that interact directly with the substrate, which means that covariant amino acids that interact remotely with the substrate also regulate enzyme catalysis. This research may provide us with a new strategy for enzyme evolution.

中文翻译：

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使用工程南极假丝酵母脂肪酶 B 高效酶法合成手性 2,3-二氢-1,4-苯并二恶烷基序

2,3-二氢-1,4苯并二恶烷的手性基序广泛应用于多种药用物质和生物活性天然化合物中，表现出显着的生物活性。此类治疗剂的著名实例包括prosympal、二博赞、哌罗生和多沙唑嗪。本工作以1,4-苯并二恶烷-2-甲酸甲酯为底物，筛选38个CALB共变残基后，发现突变体A225F和A225F/T103A可以催化底物的动力学拆分。研究了温度、助溶剂和助溶剂浓度对动力学分辨率的影响，结果表明，在 30 °C、使用 20%正丁醇作为助溶剂时可获得最佳结果，从而获得最佳分辨率（ee _s 97%，E= 278) 在 50 mM 底物浓度下。结构分析表明，突变位点225和103不属于与底物直接相互作用的位点，这意味着与底物远程相互作用的共变氨基酸也调节酶催化。这项研究可能为我们提供酶进化的新策略。