Stereospecific production of double chiral compounds by novel enzymatic processes

手性化合物是医药、精细化工等领域的重要中间体，由于手性化合物具有高度的立体专一性和环境友好性，生物催化合成手性化合物受到了广泛的关注。另一方面，分子中含有两个不对称碳原子的双手性化合物在许多有用的化合物如药物和农药的合成中也是重要的。酶法将双手性中心引入有机化合物的方法有多种，本文通过两步酶反应将双手性中心引入前手性化合物酮基异佛尔酮，形成（4 R，6 R）-放线醇。研究了利用老黄酶和左旋二酮还原酶合成双手性化合物（4 R，6 R）-放线醇的方法。放线醇是合成天然光学活性化合物玉米黄质和黄嘌呤的重要手性中间体。将共表达老黄酶或左旋二酮还原酶基因与辅因子再生酶基因的大肠杆菌E.coli E.coli细胞用作催化剂。100 g/l的酮基异佛尔酮经两步反应几乎按化学计量和立体专一地转化为（4 R，6 R）-放线醇。麻黄碱也是有用的双手性化合物之一，其d-位异构体具有减充血剂或平喘化合物的药理作用。我们发现红串红球菌产生一种新的NADP^+依赖性氨基醇脱氢酶（AADH），该酶催化相应氨基酮MAK的外消旋混合物中的l-异构体不对称还原为d-麻黄碱。以共表达AADH和辅因子再生酶基因的大肠杆菌E.coli细胞为载体，采用全细胞反应技术，催化外消旋MAK转化为d-麻黄碱。与此同时，在24 h内生成178 mM的d-麻黄碱，摩尔产率为89%.

Chiral compounds are useful intermediates for the production of pharmaceuticals, fine chemicals, and so on. Because of high stereospecificity and environmental aspects, biocatalytic production of chiral compounds has been attracting considerable attention. On the other hand, double chiral compounds, which have two asymmetric carbons in their molecules, are also important for the synthesis of many useful compounds, such as pharmaceuticals and agrochemicals. There are several ways to enzymatically introduce two chiral centers into organic compounds.Two-step introduction of double chiral centers into the prochiral compound, ketoisophorone, forming (4R,6R)-actinol through two separate enzyme reactions were performed. By combination of old yellow enzyme and levodione reductase, production of doubly chiral compound, (4R,6R)-actinol, which is a useful chiral intermediate for the synthesis of naturally-occurring optically active compounds, such as zeaxanthin and xanthoxin, was investigated. E.coli transformant cells co-expressing old yellow enzyme or levodione reductase gene with cofactor regeneration enzyme gene were used as the catalysts. 100 g/l of ketoisophorone was almost stoichiometrically and stereospecifically converted to (4R,6R)-actinol by two-step reaction Ephedrine is also one of the useful double chiral compounds, and d-ψ-isomer has pharmacological effects as a decongestant or antiasthmatic compound. We found that Rhodococcus erythropolis produces a novel NADP^+-dependent amino alcohol dehydrogenase (AADH), which catalyze the asymmetric reduction of l-isomer in the racemic mixture of the corresponding amino ketone, MAK, to d-ψ-ephedrine. Using E.coli transformant cells coexpressing the genes of AADH and cofactor regeneration enzyme, biocatalytic conversion of racemic MAK to d-ψ-ephedrine by whole cell reaction was performed. Cosequently, 178 mM of d-ψ-ephedrine was formed in 24 h reaction with a molar yield of 89%.