An enzymatic preparation method for the intermediates of luimazopazole, with the method of asymmetric reduction reaction (S) in the presence of ketone reductase, cofactor and hydrogen donor in the presence of ketone reductase, cofactor and hydrogen donor in the presence of 2 chlorophenol (2,4 two chlorophenyl) acetone, including the following steps: preparation of (1) ketone reductase The recombinant Escherichia coli containing the ketone reductase gene was inoculated into the liquid LB medium containing kanamycin resistance, at 37 C for overnight culture, and the activated culture was inoculated into the liquid LB medium containing kanamycin, 37 C was cultured to 0.6 OD600 0.8, and the final concentration was 0.1M IPTG, and 25 8 10h was induced and cultured at centigrade; the recombinant ketone reductase was obtained after centrifugation, and the preparation of recombinant ketone reductase was obtained after ultrasonic wall breaking. (2) the preparation of (S) 2 chloride 1 (2,4 two chlorophenyl) ethanol was added into the buffer solution and added a certain amount of cofactor NAD (P) and isopropanol into the buffer solution and added to the recombinant ketone. The original enzyme was biocatalytic at 30 C, and after the reaction was finished, ethyl acetate was added to the extraction, that is, the product (S) was obtained by the 2 chlorine (two chlorophenyl) ethanol.
【技术实现步骤摘要】
一种卢立康唑中间体的酶法制备方法
本专利技术涉及一种卢立康唑中间体的酶法制备方法,尤其涉及一种利用重组酮还原酶不对称催化生产卢立康唑中间体(S)-2-氯-1-(2,4-二氯苯基)乙醇的方法,属于生物制药和生物化工
技术介绍
卢立康唑是日本农药株式会社开发的咪唑类抗真菌药物,其通过抑制羊毛甾醇脱甲基酶从而抑制麦角固醇的合成,减少构成真菌麦角固醇和相对的羊毛甾醇积累,该药适用于真菌治疗,包括体癣、念珠菌病和花斑癣等。与以往抗真菌外用药相比,卢立康唑最大的优势是皮肤贮留率高,用药周期短(为一般药物的一半),疗效好且不易复发,故具有很大的竞争力。卢立康唑的化学结构式如下:(S)-2-氯-1-(2,4-二氯苯基)乙醇是卢立康唑化学合成过程中一个重要的中间体。但目前主要以化学合成为主,但该方法需要使用价格昂贵的手性配体(S,S)-TsDPEN和金属试剂[Ru(p-cymene)Cl2]2,产品收率低,手性ee值不高,化学生产成本大且对环境不优化,不适于规模化生产(CN103044192A)。(S)-2-氯-1-(2,4-二氯苯基)乙醇的大规模生物制备方法目前尚未见报道。但(R)-2-氯-1-(2,4-二氯苯基)乙醇的生物制备,目前已见报道,手性ee值大于99,转化效率大于99(J.Org.Chem.2011,76,2115–2122)。在该文献中亦可见(S)-2-氯-1-(2,4-二氯苯基)乙醇的生物转化,但手性ee值不高(ee值90),因此不能满足工业化生产的需要。(S)-2-氯-1-(2,4-二氯苯基)乙醇生物催化的反应路线为:
技术实现思路
针对现有化学法 ...
【技术保护点】
1.一种卢立康唑中间体的酶法制备方法,所述方法以2‑氯‑1‑(2,4‑二氯苯基)乙酮为底物,在酮还原酶、辅因子及氢供体的存在下,发生不对称还原反应生成(S)‑2‑氯‑1‑(2,4‑二氯苯基)乙醇,其特征在于包括如下步骤:(1)酮还原酶的制备:将含有酮还原酶基因的重组大肠杆菌单菌落接种到含有卡那霉素抗性的液体LB培养基中,37℃过夜培养;将活化后得到的培养物接种到含有卡那霉素的液体LB培养基中,37℃振荡培养至OD600为0.6‑0.8,加入终浓度为0.1M的IPTG,于25℃下诱导培养8‑10h;离心,收集菌体,超声破壁后即获得重组酮还原酶。(2)(S)‑2‑氯‑1‑(2,4‑二氯苯基)乙醇的制备:将底物2‑氯‑1‑(2,4‑二氯苯基)乙酮加入到缓冲液中并加入一定量的辅因子NAD(P)及异丙醇,加入重组酮还原酶于30℃进行生物催化反应,待反应结束后,加入乙酸乙酯进行萃取,即获得产物(S)‑2‑氯‑1‑(2,4‑二氯苯基)乙醇。
【技术特征摘要】
1.一种卢立康唑中间体的酶法制备方法,所述方法以2-氯-1-(2,4-二氯苯基)乙酮为底物,在酮还原酶、辅因子及氢供体的存在下,发生不对称还原反应生成(S)-2-氯-1-(2,4-二氯苯基)乙醇,其特征在于包括如下步骤:(1)酮还原酶的制备:将含有酮还原酶基因的重组大肠杆菌单菌落接种到含有卡那霉素抗性的液体LB培养基中,37℃过夜培养;将活化后得到的培养物接种到含有卡那霉素的液体LB培养基中,37℃振荡培养至OD600为0.6-0.8,加入终浓度为0.1M的IPTG,于25℃下诱导培养8-10h;离心,收集菌体,超声破壁后即获得重组酮还原酶。(2)(S)-2-氯-1-(2,4-二氯苯基)乙醇的制备:将底物2-氯-1-(2,4-二氯苯基)乙酮加入到缓冲液中并加入一定量的辅因子NAD(P)及异丙醇,加入重组酮还原酶于30℃进行生物催化反应,待反应结束后,加入乙酸乙酯进行萃取,即获得...
【专利技术属性】
技术研发人员:唐云平,郑佳文,余芳苗,杨最素,丁国芳,
申请(专利权)人:浙江海洋大学,
类型:发明
国别省市:浙江,33
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