CN108441523A - (3r,5s)-6-氯-3,5,-二羟基己酸叔丁酯的制备方法 - Google Patents
(3r,5s)-6-氯-3,5,-二羟基己酸叔丁酯的制备方法 Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- FIKPWJZUGTVXCO-SFYZADRCSA-N tert-butyl (3r,5s)-6-chloro-3,5-dihydroxyhexanoate Chemical compound CC(C)(C)OC(=O)C[C@H](O)C[C@H](O)CCl FIKPWJZUGTVXCO-SFYZADRCSA-N 0.000 title abstract 2
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- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 4
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- BZDIAFGKSAYYFC-UHFFFAOYSA-N manganese;hydrate Chemical compound O.[Mn] BZDIAFGKSAYYFC-UHFFFAOYSA-N 0.000 claims 1
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Abstract
本发明涉及一种(3R,5S)‑6‑ 氯 ‑3,5‑ 二羟基己酸叔丁酯的制备方法,本发明使用开菲尔乳杆菌(Lactobacillus kefir)作为全细胞生物催化剂,从(S)‑CHOH开始不对称合成,能够实现反应的一步合成,并在发酵液不浓缩的情况下获得较高的转化率。
Description
技术领域
本发明属于制药工业生物技术领域,具体涉及一种(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯的制备方法。
背景技术
具有光学活性的β,δ-二羟基酯已经在天然产品、多羟基化合物以及手性药物的合成中得到了广泛的运用。(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯是一种重要的药物中间体,可用于合成HMG-CoA还原酶的抑制剂(他汀类药物),这类药物可以有效的防止动脉粥样硬化和冠心病的发生。
目前合成(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯的方法主要有化学方法和生物方法。WolbergM等将(S)-6-氯-5-羟基-3-羰基己酸叔丁酯不对称加氢还原成(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯需要低温催化合成,对反应器的要求较高,而且催化剂的价格昂贵,成本较高,因此运用生物法进行催化合成具有良好的前景。
发明内容
本发明的目的在于提供一种(3R,5S)-6-氯-3,5,-二羟基己酸叔丁酯的制备方法,使用开菲尔乳杆菌(Lactobacilluskefir)作为全细胞生物催化剂,从(S)-CHOH开始进行不对称合成。
为实现上述技术目的,本发明采用如下技术方案:
一种(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯的制备方法,包括如下步骤:
(1)发酵培养L.kefirATCC 35411全细胞菌株;
(2)在发酵液中添加底物(S)-CHOH、葡萄糖和MgCl2,进行催化转化;
(3)产物分离提取。
本发明的方法,所述步骤(1)中,富集培养的培养基成分为酵母粉25g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,柠檬酸氢二铵2g/L,七水合硫酸镁0.2g/L,一水合硫酸镁0.05g/L,吐温801g/L;发酵培养基成分为酵母粉8g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.2g/L,一水合硫酸锰0.05g/L,吐温801g/L,发酵pH6.0,温度30℃。
所述步骤(2)中,在发酵液中添加氯化镁,葡萄糖,(S)-CHOH底物进行催化转化;转化过程用氢氧化钠调节pH至6.0~7.0,27℃,转化过程中通入氮气,通气速度为0.1~0.2VVM以维持微正压。
或将发酵液浓缩至OD660=100,后在发酵液中添加氯化镁,葡萄糖,(S)-CHOH底物进行催化转化;转化过程用NaOH调节pH至6.0~7.0,27℃,转化过程中通入氮气,通气速度为0.1~0.2VVM以维持微正压。
所述步骤(3)中,取出转化液,加入1:1乙酸乙酯,混匀,8000rpm离心10min分离菌体细胞,取上清用乙酸乙酯:水=1:1萃取三次,取出产物(3R,5S)-6-氯-3,5,-二羟基己酸叔丁酯后,去除水层,产物留在乙酸乙酯层,加入30g/L无水硫酸钠干燥1-2h,干燥后将溶剂进行旋蒸,45℃,压力从大气压降至150mPa,直至将溶剂旋干,旋蒸后将产物放入真空干燥箱进一步干燥,获取所需产物。
本发明的方法使用L.kefirATCC 35411作为全细胞生物催化剂,从(S)-CHOH开始进行不对称合成,反应产物转化率高,且在发酵液不浓缩的情况下可获取较高的转化率。
附图说明
图1为本发明技术路线图;
图2为不同pH对转化率影响的曲线图;
图3为(3R,5S)-CDHH标准样品的液相色谱图;
图4为本发明制备方法产物的液相色谱图。
具体实施方式
本发明开菲尔乳杆菌(Lactobacillus kefir)菌株购自ATCC菌种保藏中心,菌株型号为ATCC 35411。
实施例1
发酵培养全细胞菌株
(1)将冷冻的L.kefir细胞(-20℃)在室温下解冻,并用磷酸钾缓冲液(200mM,pH6.5)洗涤。以4500rpm离心10分钟,除去上清液。然后将细胞沉淀物悬浮于培养基(酵母粉25g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,柠檬酸氢二铵2g/L,七水合硫酸镁0.2g/L,一水合硫酸镁0.05g/L,吐温801g/L)中富集培养,装液量:200ml/500mL,接种量:2%(v/v),摇床150rpm,温度30℃,培养24h。(培养指标:OD660>1.5)。
(2)在5L的发酵罐中进行发酵,装液量:1.8L/5L,接种量10%,接入发酵培养基(酵母粉8g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.2g/L,一水合硫酸镁0.05g/L,吐温801g/L,pH 6.0)温度30℃,发酵pH 6.0,通空气,通气速度0.6VVM,转速100-200rpm。当葡萄糖浓度<5g/L时,开始补料,补料速率:5mL/L/h补料12h后,加大速率至9mL/L/h,同时进行葡萄糖浓度的实时监控,补料培养基(酵母粉160g/L,葡萄糖400g/L,无水乙酸钠5g/L,三水合磷酸氢二钾5g/L,七水合硫酸镁0.5g/L,一水合硫酸镁0.125g/L),培养72h。(培养指标:最后OD660≈20)。
实施例2
全细胞催化
将发酵液分不浓缩和浓缩两种方式进行全细胞催化;
(1)发酵液不浓缩,直接进行催化转化。按底物浓度20mM、40mM、60mM、100mM进行四组实验,反应体系为3L发酵罐,转化条件分别为:
实验组1:添加2mM氯化镁,80mM葡萄糖,20mM(S)-CHOH底物,转化过程用氢氧化钠调节pH至6.5,27℃,转化过程中通N2,通气速度为0.1~0.2VVM以维持微正压,反应12h后,每间隔5h液相检测转化率及e.e值。
实验组2:添加2mM氯化镁,80mM葡萄糖,40mM(S)-CHOH底物,转化过程用氢氧化钠调节pH至6.5,27℃,转化过程中通N2,通气速度为0.1~0.2VVM以维持微正压,反应12h后,每间隔5h液相检测转化率及e.e值。
实验组3:添加2mM氯化镁,80mM葡萄糖,60mM(S)-CHOH底物,转化过程用氢氧化钠调节pH至6.5,27℃,转化过程中通N2,通气速度为0.1~0.2VVM以维持微正压,反应12h后,每间隔5h液相检测转化率及e.e值。
实验组4:添加2mM氯化镁,80mM葡萄糖,100mM(S)-CHOH底物,转化过程用氢氧化钠调节pH至6.5,27℃,转化过程中通N2,通气速度为0.1~0.2VVM以维持微正压,反应12h后,每间隔5h液相检测转化率及e.e值。
(2)发酵液浓缩至OD660=100,将发酵液在4100rpm离心15min,去上清后用200mM,pH6.5的磷酸钾缓冲液将菌泥重悬,进行催化转化。反应体系为0.6L发酵罐,转化条件如下:
实验组5:10mM氯化镁,400Mm葡萄糖,100mM(S)-CHOH底物,转化过程用氢氧化钠调节pH至6.5,27℃,转化过程中通N2,通气速度为0.1~0.2VVM以维持微正压,反应12h后,每间隔5h液相检测转化率及e.e值。
实施例3
本实施例基于摇瓶培养,说明不同的pH对转化效率的影响
100mL摇瓶培养,添加2mM氯化镁,80mM葡萄糖,20mM(S)-CHOH底物,分别配制pH为5、5.5、6.0、6.5、7.0、7.5、8.0的磷酸盐缓冲液,27℃进行磁力搅拌转化。反应结果如图2所示,pH6.0~7.0时转化率较高,在pH6.5时转化率最高。
实施例4
本实施例说明产物分离提取和检验的方法。
(1)取出转化液,加入1:1乙酸乙酯,混匀,8000rpm离心10min(分离菌体细胞),取上清,用乙酸乙酯:水=1:1萃取三次,取出产物(3R,5S)-6-氯-3,5,-二羟基己酸叔丁酯后,去除水层,产物留在乙酸乙酯层,加入30g/L无水硫酸钠干燥1-2h,干燥后将溶剂进行旋蒸(45℃,压力从大气压降至150mPa,直至将溶剂旋干),旋蒸后将产物放入真空干燥箱进一步干燥,即得所需产物,放于零度以下保存,(乙酸乙酯分相后蒸发回收,55℃,-0.1Mpa)。
(2)转化率及e.e.计算
利用液相进行计算,检测波长220nm,柱温40℃,流动相A(纯水):B(乙腈)=75:25,产物出峰时间37min,流速:0.5ml/min。
供试品溶液的配制:取(S)-CHOH 160mg,精密称定,加4mL乙腈适量超声使溶解,制成每1ml含(S)-CHOH约40.0mg的溶液,作为供试品溶液。
精密量取供试品溶液20μL,注入液相色谱仪,按面积归一化法计算,产物(3R,5S构型)保留时间:约37分钟,异构体(3S,5S构型)保留时间:约44分钟。产物液相色谱图如图4所示。5个实验组不同实验条件下产物转化率如表1所示。
表1反应底物浓度和产物转化率
底物浓度 | 转化率 | |
实验组1 | 20mM | 79%(21h) |
实验组2 | 40mM | 80%(42h) |
实验组3 | 60mM | 40%(51h) |
实验组4 | 100mM | 22%(67h) |
实验组5 | 100mM | 83%(17h) |
Claims (10)
1.一种(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯的制备方法,其特征在于,包括如下步骤:
(1)发酵培养L.kefir全细胞菌株;
(2)在发酵液中添加底物(S)-CHOH、葡萄糖和MgCl2,进行催化转化;
(3)产物分离提取。
2.根据权利要求1所述的方法,其特征在于,所述步骤(1)中,菌株选用L.kefirATCC35411。
3.根据权利要求1所述的方法,其特征在于,所述步骤(1)中,富集培养的培养基成分为酵母粉25g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,柠檬酸氢二铵2g/L,七水合硫酸镁0.2g/L,一水合硫酸锰0.05g/L,吐温801g/L。
4.根据权利要求1所述的方法,其特征在于,所述步骤(1)中,发酵培养基成分为酵母粉8g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.2g/L,一水合硫酸锰0.05g/L,吐温801g/L,发酵pH6.0,温度30℃。
5.根据权利要求1所述的方法,其特征在于,所述步骤(2)中,在发酵液中添加氯化镁,葡萄糖,(S)-CHOH底物进行催化转化;转化过程用氢氧化钠调节pH至6.0~7.0,27℃,转化过程中通入N2,通气速度为0.1~0.2VVM以维持微正压。
6.根据权利要求1所述的方法,其特征在于,所述步骤(2)中,将发酵液浓缩至OD660=100,后在发酵液中添加氯化镁,葡萄糖,(S)-CHOH底物进行催化转化;转化过程用氢氧化钠调节pH至6.0~7.0,27℃,转化过程中通入N2,通气速度为0.1~0.2VVM以维持微正压。
7.根据权利要求5或6所述的方法,其特征在于,转化过程用氢氧化钠调节pH至6.5。
8.根据权利要求1所述的方法,其特征在于,所述步骤(3)中,取出转化液,加入1:1乙酸乙酯,混匀,8000rpm离心10min分离菌体细胞,取上清用乙酸乙酯:水=1:1萃取三次,取出产物(3R,5S)-6-氯-3,5,-二羟基己酸叔丁酯后,去除水层,产物留在乙酸乙酯层,加入30g/L无水硫酸钠干燥1-2h,干燥后将溶剂进行旋蒸,45℃,压力从大气压降至150mPa,直至将溶剂旋干,旋蒸后将产物放入真空干燥箱进一步干燥,获取所需产物。
9.根据权利要求1所述的方法,其特征在于,包括如下步骤:
(1)将-20℃冷冻的L.kefir细胞在室温下解冻,磷酸钾缓冲液200mM,pH6.5洗涤;以4500rpm离心10分钟,除去上清液;将细胞沉淀物悬浮于培养基中富集培养,装液量:200ml/500mL,接种量:2%(v/v),摇床150rpm,温度30℃,培养24h;富集培养基成分为酵母粉25g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,柠檬酸氢二铵2g/L,七水合硫酸镁0.2g/L,一水合硫酸锰0.05g/L,吐温801g/L;
在5L的发酵罐中进行发酵,装液量:1.8L/5L,接种量10%,接入发酵培养基,成分为酵母粉8g/L,葡萄糖20g/L,无水乙酸钠5g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.2g/L,一水合硫酸锰0.05g/L,吐温801g/L,pH 6.0;温度30℃,发酵pH 6.0,通空气,通气速度0.6VVM,转速100-200rpm;当葡萄糖浓度<5g/L时,开始补料,补料速率:5mL/L/h补料12h后,加大速率至9mL/L/h,同时进行葡萄糖浓度的实时监控,补料培养基,成分为酵母粉160g/L,葡萄糖400g/L,无水乙酸钠5g/L,三水合磷酸氢二钾5g/L,七水合硫酸镁0.5g/L,一水合硫酸锰0.125g/L,培养72h;
(2)添加2mM氯化镁,80mM葡萄糖,20mM(S)-CHOH底物,转化过程用氢氧化钠调节pH至6.5,27℃,转化过程中通氮气,通气速度为0.1~0.2VVM以维持微正压;
(3)取出转化液,加入1:1乙酸乙酯,混匀,8000rpm离心10min分离菌体细胞,取上清,用乙酸乙酯:水=1:1萃取三次,取出产物(3R,5S)-6-氯-3,5,-二羟基己酸叔丁酯后,去除水层,产物留在乙酸乙酯层,加入30g/L无水硫酸钠干燥1-2h,干燥后将溶剂进行旋蒸,45℃,压力从大气压降至150mPa,直至将溶剂旋干,旋蒸后将产物放入真空干燥箱进一步干燥,获取产物。
10.根据权利要求9所述的方法,其特征在于,所述步骤(2)中,将发酵液浓缩至OD660=100,发酵液在4100rpm离心15min,去上清后用200mM,pH6.5的磷酸钾缓冲液将菌泥重悬,10mM氯化镁,400Mm葡萄糖,100mM(S)-CHOH底物进行催化转化;转化过程用氢氧化钠调节pH至6.5,27℃,转化过程中通氮气,通气速度为0.1~0.2VVM以维持微正压。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111748527A (zh) * | 2020-05-15 | 2020-10-09 | 上海多宁生物科技有限公司 | 化学成分限定高效补料培养基及其制备方法和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1152054A1 (en) * | 1999-12-03 | 2001-11-07 | Kaneka Corporation | Novel carbonyl reductase, gene thereof and method of using the same |
CN104844457A (zh) * | 2015-03-23 | 2015-08-19 | 爱斯特(成都)生物制药有限公司 | 一种(3r,5s)-6-氯二羟基己酸叔丁酯的制备方法 |
CN105087684A (zh) * | 2015-09-16 | 2015-11-25 | 连云港宏业化工有限公司 | 一种(3r,5s)-6-氯-3,5-二羟基己酸叔丁酯的制备方法 |
-
2018
- 2018-03-22 CN CN201810239859.6A patent/CN108441523A/zh not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1152054A1 (en) * | 1999-12-03 | 2001-11-07 | Kaneka Corporation | Novel carbonyl reductase, gene thereof and method of using the same |
CN104844457A (zh) * | 2015-03-23 | 2015-08-19 | 爱斯特(成都)生物制药有限公司 | 一种(3r,5s)-6-氯二羟基己酸叔丁酯的制备方法 |
CN105087684A (zh) * | 2015-09-16 | 2015-11-25 | 连云港宏业化工有限公司 | 一种(3r,5s)-6-氯-3,5-二羟基己酸叔丁酯的制备方法 |
Non-Patent Citations (3)
Title |
---|
AMIDJOJO M 等: "Asymmetric synthesis of tert-butyl (3, 5) 6-chloro-dihydroxyhexanoate with Lactobacillus kefir", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
SUN XINGYUAN等: "Bioconversion process for synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using liquid-core immobilized Saccharomyces cerevisiae CGMCC No 2233", 《KOREAN JOURNAL OF CHEMICAL ENGINEERING》 * |
何秀娟: "两步酶法不对称合成(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯的研究", 《中国优秀硕士学位论文全文数据库(电子期刊)工程科技Ⅰ辑》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111748527A (zh) * | 2020-05-15 | 2020-10-09 | 上海多宁生物科技有限公司 | 化学成分限定高效补料培养基及其制备方法和应用 |
CN111748527B (zh) * | 2020-05-15 | 2021-01-29 | 上海多宁生物科技有限公司 | 化学成分限定高效补料培养基及其制备方法和应用 |
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