WO2014205917A1 - 一种罗素伐他汀中间体的制备方法 - Google Patents

一种罗素伐他汀中间体的制备方法 Download PDF

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WO2014205917A1
WO2014205917A1 PCT/CN2013/083294 CN2013083294W WO2014205917A1 WO 2014205917 A1 WO2014205917 A1 WO 2014205917A1 CN 2013083294 W CN2013083294 W CN 2013083294W WO 2014205917 A1 WO2014205917 A1 WO 2014205917A1
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monomethyl
reaction
tert
hydroxyglutarate
substrate
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陶军华
唐苏苏
李斌
刘根
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苏州汉酶生物技术有限公司
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    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/62Carboxylic acid esters

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  • the invention belongs to the field of biopharmaceutical and biochemical technology, and particularly relates to a biological preparation method of (R)-3-hydroxyglutaric acid monomethyl ester.
  • Cardiovascular and cerebrovascular diseases are one of the most common and serious diseases that endanger humans (especially middle-aged and older people).
  • Dyslipidemia is an important risk factor for atherosclerosis, coronary heart disease, and other cardiovascular and cerebrovascular diseases. According to the survey of nutrition and health status of Chinese residents conducted by the Ministry of Health in recent years, the total prevalence rate is 18.6%, the total number of patients in the country reached 160 million.
  • Lipid-lowering drugs can effectively reduce the incidence and mortality of such diseases, and have a positive and far-reaching impact on the prevention and treatment of cardiovascular diseases.
  • Statins 3- Hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are currently the world's best-selling cholesterol-lowering and hypolipidemic drugs due to fewer side effects.
  • AstraZeneca (Astrazeneca) The company's rosuvastatin (Rosuvastatin, English trade name Crestor) was first approved in the Netherlands in November 2002. Approved by the US FDA in August 2003, global sales in 2009 44 Billion dollars, ranked 17th. Rusvavastatin has achieved satisfactory results in terms of efficacy and safety and is known as 'super statin'.
  • Chinese invention patent disclosure CN101805756A A method for producing a statin intermediate by biocatalysis is disclosed, which discloses the production of (R)-3- using racemic 4-cyano-3hydroxybutyrate ethyl ester. Ethyl hydroxyglutarate. Taking this method, although it can overcome the deficiencies of the chemical method, only 50% of the target products obtained after the hydrolysis of the racemic body The theoretical yield means that half of the substrate is wasted, does not meet the principles of green chemistry and atomic economy, and the method has a low substrate feed rate of only 20 mmol/L, which does not have the value of industrial application.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a rosuvastatin intermediate (R)-3-
  • the preparation of monomethyl hydroxyglutarate and hydroxy-protected monomethyl (R)-3-hydroxyglutarate achieves nearly 100% conversion and high reaction substrate concentration.
  • the present invention adopts the following technical solution: a method for preparing (R)-3-hydroxyglutaric acid monomethyl ester, the method (R)-4-Cyano-3-hydroxybutyric acid methyl ester as a substrate, which is reacted under the action of an enzyme to form the (R)-3-hydroxyglutaric acid monomethyl ester, and the reaction is at a pH value. 7.0 ⁇ 9.0 Aqueous buffer and temperature 25 °C It is carried out at ⁇ 35 °C.
  • the enzyme used in the reaction is a recombinant nitril hydrolase prepared by inoculating a single colony of recombinant Escherichia coli containing a nitrilase gene into a liquid LB containing ampicillin resistance. In the medium, activate at 35 ⁇ 40 °C for 8 ⁇ 12 hours, inoculate the culture obtained after activation into liquid LB medium containing ampicillin resistance, shake culture at 35 ⁇ 40 °C, and culture to OD600.
  • the inducer can and preferably is isopropyl-b-D- Thiogalactoside.
  • the aqueous buffer may be phosphate buffer or Tri-HCl buffer.
  • the mass ratio of the recombinant nitrilase to the substrate may be 0.01 to 0.1:1, preferably 0.02 ⁇ 0.05:1 For example 0.03:1.
  • the substrate concentration may be 0.8 to 1.5 mol/L, preferably 1.0 to 1.5 mol/L.
  • the method is embodied as follows: the recombinant nitrilase is added to the aqueous buffer, and then the substrate is added by a constant flow pump, and the substrate is added at a temperature of 25 after the substrate is added. °C The reaction was carried out at ⁇ 35 ° C under stirring, until the substrate conversion rate was 99.0% or more, and the reaction was terminated.
  • the system is adjusted to be acidic with hydrochloric acid (for example, a pH of 2 to 6, preferably a pH of 2 to 3) Then, it is filtered with celite, and the residue is washed with ethyl acetate. The filtrate is extracted with ethyl acetate. The organic phase is combined. The organic phase is dried over anhydrous sodium sulfate and concentrated to give a yellow brown oil (R)-3 - Monomethyl hydroxyglutarate.
  • hydrochloric acid for example, a pH of 2 to 6, preferably a pH of 2 to 3
  • a further technical solution adopted by the present invention is a method for preparing a hydroxyl group-protected monomethyl (R)-3-hydroxyglutarate, which is protected by a hydroxyl group.
  • the (R)-3-hydroxyglutaric acid monomethyl ester is (R)-3-tert-butyldimethylsilyloxyglutaric acid monomethyl ester, which comprises the following steps:
  • the catalyst is 4-dimethylaminopyridine (DMAP), which is combined with (R)-3-
  • DMAP 4-dimethylaminopyridine
  • the organic base acid can be imidazole, N, N- Diisopropylethylamine, pyridine, triethylamine, etc. may be one kind or a combination of various types.
  • a combination of methanol, tetrahydrofuran and water is used as a solvent, the potassium carbonate and 3-tert-butyldimethylsilyloxy group.
  • the molar ratio of methyl glutarate tert-butyldimethylsilylate is 4.5 ⁇ 5:1.
  • the present invention has the following advantages compared with the prior art:
  • the method of the invention solves the problem that the substrate concentration in the existing biological method is too low and the conversion rate is low by adopting a specific recombinant nitrilase.
  • the method has mild reaction conditions, high reaction efficiency and simple operation, and in particular, the concentration of the reaction substrate can be greatly improved, and the preparation is improved.
  • the efficiency of (R)-3-hydroxyglutaric acid monomethyl ester and the cost of the reaction are reduced, and have important industrial application value.
  • the hydroxyl group provided by the present invention is protected (R)-3-
  • the preparation method of monomethyl hydroxyglutarate is simple in operation, mild in conditions, and the yield of the obtained hydroxyl group-protected monomethyl (R)-3-hydroxyglutaric acid is high.
  • Figure 1 is a hydrogen nuclear magnetic spectrum of the product prepared in Example 4.
  • the present invention provides a process for producing (R)-3-methylhydroxyglutarate monomethyl ester using a recombinant nitrilase, and the reaction equation is as follows:
  • a single colony of recombinant E. coli containing the nitrilase gene was inoculated from 4 g of ampicillin-resistant liquid LB medium from a glycerol tube or a transformation plate overnight (37 ° C, 200 rpm). 100 mL of ampicillin-resistant liquid LB medium was transferred from the overnight culture at 1/100 inoculation, and cultured at 37 ° C, 200 rpm to an OD 600 value of 0.6-0.8, and IPTG was added to continue incubation at 30 ° C overnight. The cells were collected by centrifugation and the cells were suspended in 10 mL of phosphate buffer (2 mM, pH 7.0).
  • the cell suspension was ultrasonically disrupted in an ice bath for 10 minutes, centrifuged, and the supernatant was pre-frozen overnight, and lyophilized for 24 h to 48 h to obtain a freeze-dried powdery recombinant nitrilase.
  • the synthetic route is as follows:
  • step (2) Add the crude product obtained in step (1) to a 5 000 ml round bottom flask and add methanol (330 mL). , tetrahydrofuran (330 mL), water (660 mL), add potassium carbonate (850 g, 6.16 mol, 4.93 eq), stir at room temperature, TLC After monitoring for 12 h, the reaction was complete and the reaction was terminated. Add water (1500 mL) and petroleum ether (1200 mL) to the reaction system.
  • the solution is divided into three phases, the intermediate phase is taken out, and the methanol and tetrahydrofuran are removed by concentration; diluted with water and washed with petroleum ether (500 mL ⁇ 3), the aqueous phase is adjusted to pH 1 with hydrochloric acid, and extracted with ethyl acetate (1000) ⁇ / RTI> ⁇ / RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; After filtration, it was concentrated to give 265 g (yield: ⁇ /RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; ⁇ /RTI> ⁇ RTIgt; Product optical purity is greater than 99.6%.

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Abstract

提供了罗素伐他汀中间体(R)-3-羟基戊二酸单甲酯和羟基被保护的(R)-3-羟基戊二酸单甲酯的制备方法,以(R)-4-氰基-3-羟基丁酸甲酯为底物,使其在酶的作用下反应生成所述(R)-3-羟基戊二酸单甲酯,所述的酶为重组腈水解酶,所述的反应在pH为7.0-9.0的水相缓冲液中以及温度25°C~35°C下进行。该方法反应条件温和,反应效率高,操作简便,并且通过采用特定的重组腈水解酶,解决了现有生物法中底物浓度过低,转化率低的问题。

Description

一种罗素伐他汀中间体的制备方法
技术领域
本发明属于生物制药和生物化工技术领域,具体涉及一种 (R)-3- 羟基戊二酸单甲酯的生物制备方法。
背景技术
心脑血管疾病是危害人类(特别是中老年)健康最常见、最严重的疾病之一。血脂异常是动脉粥样硬化、冠心病以及其它心脑血管疾病的重要危险因素。根据近年卫生部在全国范围内进行的中国居民营养与健康状况调查显示,总患病率为 18.6 %,全国患者总人数达 1.6 亿。
调脂药可有效降低此类疾病的发生率和死亡率,对心血管疾病的防治产生积极的作用和深远的影响。他汀类药物( 3- 羟基 3- 甲基戊二酰辅酶 A(HMG-CoA) 还原酶抑制剂)由于副作用较少,是目前世界上最为畅销的降胆固醇和降血脂药物。由阿斯利康 (Astrazeneca) 公司开发的罗素伐他汀 (Rosuvastatin, 英文商品名 Crestor) 于 2002 年 11 月首次在荷兰获批, 2003 年 8 月通过美国 FDA 上市审批, 2009 年在全球销售额 44 亿美元,排名第十七位。罗素伐他汀在药效和安全性方面均达到令人满意的结果,被称为 ' 超级他汀 ' 。
罗素伐他汀的手性侧链合成是其生产的关键步骤。 (R)-3- 羟基戊二酸单甲酯及其后续的羟基被保护的 (R)-3- 羟基戊二酸单甲酯是合成罗素伐他汀侧链的重要手性前体。利用化学方法合成 (R)-3- 羟基戊二酸单甲酯的方法,由于条件极端且水解无选择性,极易破坏手性基团和其它活性基团,生成副产物,因此并没有得到广泛的应用。
中国发明专利公开 CN101805756A 公开了一种生物催化法生产他汀类药物中间体的方法,其中公开了利用消旋的 4- 氰基 -3 羟基丁酸乙酯生产 (R)-3- 羟基戊二酸乙酯。采取该方法,虽然能够克服化学法的不足,但是消旋体水解后得到的目标产品只有 50% 的理论得率,意味着一半的底物被浪费,不符合绿色化学和原子经济性的原则,而且该方法的底物投料量较低,只有 20 mmol/L ,不具有工业应用的价值。
发明内容
本发明所要解决的技术问题是克服现有技术的不足,提供一种罗素伐他汀中间体 (R)-3- 羟基戊二酸单甲酯和羟基被保护的 (R)-3- 羟基戊二酸单甲酯的制备方法,实现了接近 100% 的转化率和高反应底物浓度。
为解决以上技术问题,本发明采取如下技术方案: 一种 (R)-3- 羟基戊二酸单甲酯的制备方法,该方法以 (R)-4- 氰基 -3- 羟基丁酸甲酯为底物,使其在酶的作用下反应生成所述 (R)-3- 羟基戊二酸单甲酯,反应在 pH 值为 7.0~9.0 的水相缓冲液中以及温度 25 ℃ ~35℃下进行。特别地,反应中使用的酶为重组腈水解酶,该重组腈水解酶的制备方法为:将含有腈水解酶基因的重组大肠杆菌单菌落接种到含氨苄青霉素抗性的液体LB 培养基中,于 35~40℃下活化8~12 小时,将活化后得到的培养物接种到含氨苄青霉素抗性的液体 LB 培养基中,于 35~40℃下振荡培养,培养至OD600 值达到 0.6~0.8 时,加入诱导剂,于 25~33℃下继续培养8~12 小时,离心,收集沉淀物,加入磷酸盐缓冲液得悬浮液,将悬浮液置于冰水浴中超声破碎 8~12 分钟,再离心,将上清液预冻至温度降至 -10 ℃ ~-25℃,然后冷冻干燥24~48 小时,即得冻干的粉状重组腈水解酶。
根据本发明,所述的诱导剂可以且优选为异丙基 - b -D- 硫代半乳糖苷。所述的水相缓冲液可以为磷酸盐缓冲液或 Tri-HCl 缓冲液。重组腈水解酶与底物的投料质量比可以为 0.01~0.1:1 ,优选为 0.02~0.05:1 例如 0.03:1 。反应起始时,底物浓度可以为 0.8~1.5mol/L ,优选为 1.0~1.5mol/L 。
根据本发明的一个具体方面,所述方法具体实施如下:将重组腈水解酶加入到水相缓冲液中,然后采用恒流泵流加底物,待底物加毕,使反应体系在温度 25 ℃ ~35℃下以及搅拌下反应,至底物转化率大于等于99.0% ,终止反应。
进一步地,终止反应后,用盐酸调节体系至酸性(例如 pH 值为 2~6 ,优选 pH 值为 2~3 ),然后用硅藻土过滤,用乙酸乙酯冲洗滤渣;滤液用乙酸乙酯萃取,合并有机相;有机相用无水硫酸钠干燥,浓缩后得到黄褐色油状物即为 (R)- 3- 羟基戊二酸单甲酯。
本发明采取的又一技术方案是:一种羟基被保护的 (R)-3- 羟基戊二酸单甲酯的制备方法,该羟基被保护的 (R)-3- 羟基戊二酸单甲酯为 (R)-3- 叔丁基二甲基硅氧基戊二酸单甲酯,其包括如下步骤:
( 1 )按照上述的方法制备得到 (R)-3- 羟基戊二酸单甲酯;
( 2 )使 (R)-3- 羟基戊二酸单甲酯与叔丁基二甲基氯硅烷( TBSCl )在有机溶剂中,催化剂和有机碱缚酸剂存在下,于常温下搅拌反应,得到 3- 叔丁基二甲基硅氧基 - 戊二酸甲酯叔丁基二甲基硅酯,其中, (R)-3- 羟基戊二酸单甲酯、叔丁基二甲基氯硅烷以及有机碱缚酸剂的投料摩尔比为 0.4~0.5:1:1.05~1.2 。
( 3 )使 3- 叔丁基二甲基硅氧基 - 戊二酸甲酯叔丁基二甲基硅酯在碳酸钾存在下、溶剂中发生水解反应生成所述 (R)-3- 叔丁基二甲基硅氧基戊二酸单甲酯。
优选地,步骤( 2 )中,所述催化剂为 4- 二甲氨基吡啶( DMAP ),其与 (R)-3- 羟基戊二酸单甲酯的投料摩尔比为 0.002~0.01:1 。
具体地,有机碱缚酸剂可以为咪唑、 N,N- 二异丙基乙胺、吡啶、三乙胺等,可以是其中的一种,也可以是多种的组合。
优选地,步骤( 3 )中,采用甲醇、四氢呋喃以及水的组合作为溶剂,所述碳酸钾与 3- 叔丁基二甲基硅氧基 - 戊二酸甲酯叔丁基二甲基硅酯的投料摩尔比为 4.5~5:1 。
由于以上技术方案的实施,本发明与现有技术相比具有如下优点:
本发明方法通过采用特定的重组腈水解酶,解决了现有生物法中底物浓度过低,转化率低的问题。该方法反应条件温和,反应效率高,操作简便,特别是,反应底物的浓度可以有大幅提高,提高了制备 (R)- 3- 羟基戊二酸单甲酯的效率和降低了反应的成本,具有重要的工业应用价值。
本发明提供的羟基被保护的 (R)- 3- 羟基戊二酸单甲酯的制备方法,操作简单,条件温和,且所得羟基被保护的 (R)- 3- 羟基戊二酸单甲酯的收率高。
附图说明
图 1 为实施例 4 所制备的产物的氢核磁谱图。
具体实施方式
本发明提供一种 利用重组腈水解酶制备 (R)-3- 羟基戊二酸单甲酯的 方法,反应方程式如下:
Figure PCTCN2013083294-appb-I000001
反应所用的物质除重组腈水解酶外,均可通过商购获得。下面结合具体实施例对本发明做进一步详细的说明,但本发明并不限于以下实施例。
实施例 1 重组腈水解酶的制备
从甘油管或转化平板将含有腈水解酶基因的重组大肠杆菌单菌落接种到 4 mL 含氨苄青霉素抗性的液体 LB 培养基活化过夜( 37 ℃ , 200 rpm )。从过夜培养物以 1/100 接种量转接 100 mL 含氨苄青霉素抗性的液体 LB 培养基, 37 ℃ 、 200 rpm 振荡培养至 OD600 值达到 0.6-0.8 ,加入 IPTG 于 30 ℃ 继续培养过夜。离心收集细胞,用 10 mL 磷酸缓冲液( 2 mM , pH 值为 7.0 )悬浮细胞。细胞悬浮液置于冰浴中超声波破碎 10 分钟,离心,上清液预冻过夜,冻干 24h-48h ,即得冻干粉状的重组腈水解酶。
实施例 2 克 级 (R)-3- 羟基戊二酸单甲酯的制备
将 85 ml 磷酸盐缓冲液 (0.1M , pH 值为 8.0) 加至 250 mL 圆底烧瓶中,加入 450 mg 重组腈水解酶;称取 15 g (R)-4- 氰基 -3- 羟基丁酸甲酯并采用恒流泵流加至体系中,在 30 ℃ 下磁力搅拌反应 24 h 后取样进行 LC-MS 分析,转化率 >99% ,终止反应,用盐酸调节反应液 pH 值,至酸性( pH 值 =2~3 ),硅藻土过滤,用乙酸乙酯冲洗滤渣;滤液用乙酸乙酯萃取,合并有机相;无水硫酸钠干燥,浓缩后得 14 g 黄褐色油状物即为 (R)- 3- 羟基戊二酸单甲酯,收率 81.4% , GC 纯度 88.2% 。
实施例 3 公斤 级 (R)-3- 羟基戊二酸单甲酯的制备
在反应器中加入 8.5 L 磷酸盐缓冲液 (0.1M , pH 值为 8.0) ,再加入 45 g 重组腈水解酶;称取 1.5 Kg (R)-4- 氰基 -3- 羟基丁酸甲酯并采用恒流泵流加至体系中,在 30 ℃ 下机械搅拌反应 24 h 后取样进行 LC-MS 分析,转化率 >99% ,终止反应。盐酸调节反应液 pH 值,至酸性( pH 值 =2~3 ),硅藻土过滤,用乙酸乙酯冲洗滤渣;滤液用乙酸乙酯萃取,合并有机相;无水硫酸钠干燥,浓缩后得 1.4 Kg 左右黄褐色油状物即为 (R)- 3- 羟基戊二酸单甲酯,收率 75% , GC 纯度 88% 。
实施例 4 羟基被保护的 (R)-3- 羟基戊二酸单甲酯( (R)-3- 叔丁基二甲基硅氧基戊二酸单甲酯,化合物 1 )的制备
合成路线如下:
Figure PCTCN2013083294-appb-I000002
具体实施如下:
( 1 )、 (R)-3- 羟基戊二酸单甲酯 (203 g , 1.25 mol , 1 eq) 加至 5 000mL 圆底烧瓶中,加入 1700 mL 二氯甲烷,加入咪唑 (201 g , 2.95 mol , 2.35 eq) ;反应液用冰浴冷却,缓慢加入 TBSCl (430 g , 2.85 mol , 2.3 eq) ,加入 DMAP (0.8 g , 6.6 mmol , 0.005 eq) ;反应液于常温下搅拌, LC-MS 监控,搅拌 30 h 后反应完全,终止反应。向反应液中加入水 (1500 mL) 与氯化钠 (300 g) ,搅拌至完全溶解后分出有机相;水相用乙酸乙酯 (250 mL×2) 萃取,合并有机相浓缩得到 543 g 化合物 2 的粗品直接用于下一步反应。
( 2 )、将步骤( 1 )所得粗品加至 5 000ml 圆底烧瓶中,加入甲醇 (330 mL) 、四氢呋喃 (330 mL) 、水 (660 mL) ,加入碳酸钾 (850 g , 6.16 mol , 4.93 eq) ,于常温下搅拌, TLC 监控,反应 12 h 后反应完全,终止反应。向反应体系中加入水 (1500 mL) 与石油醚 (1200 mL) ,溶液分三相,取出中间相,浓缩除去甲醇与四氢呋喃;加水稀释后用石油醚洗涤 (500 mL×3) ,水相用盐酸调节 pH 值至 1 ,用乙酸乙酯萃取 (1000 mL×3) ,合并有机相浓缩除去溶剂得到深色油状物,向该油状物中加入石油醚 (1 000mL) ,加入无水硫酸钠,活性炭( 50 克 )搅拌 3 h ,过滤后浓缩得到淡黄色油状物 265 g ,即为 (R)-3- 叔丁基二甲基硅氧基戊二酸单甲酯(化合物 1 ),收率 76.6 % 。产物光学纯度大于 99.6% 。 (R)-3- 叔丁基二甲基硅氧基戊二酸单甲酯的氢核磁谱图参见图 1 。
上述实施例只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内 。

Claims (10)

1 、一种 (R)-3- 羟基戊二酸单甲酯的制备方法,该方法以 (R)-4- 氰基 -3- 羟基丁酸甲酯为底物,使其在酶的作用下反应生成所述 (R)-3- 羟基戊二酸单甲酯,其特征在于:所述的反应在 pH 值为 7.0~9.0 的水相缓冲液中以及温度 25 ℃ ~35℃下进行,且反应中所利用的酶为重组腈水解酶,该重组腈水解酶的制备方法为:将含有腈水解酶基因的重组大肠杆菌单菌落接种到含氨苄青霉素抗性的液体LB 培养基中,于 35~40℃下活化8~12 小时,将活化后得到的培养物接种到含氨苄青霉素抗性的液体 LB 培养基中,于 35~40℃下振荡培养,培养至OD600 值达到 0.6~0.8 时,加入诱导剂,于 25~33℃下继续培养8~12 小时,离心,收集沉淀物,加入磷酸盐缓冲液得悬浮液,将悬浮液置于冰水浴中超声破碎 8~12 分钟,再离心,将上清液预冻至温度降至 -10 ℃ ~-25℃,然后冷冻干燥24~48 小时,即得冻干的粉状重组腈水解酶;。
2 、根据权利要求 1 所述的 (R)-3- 羟基戊二酸单甲酯的制备方法,其特征在于:诱导剂为异丙基 - b -D- 硫代半乳糖苷。
3 、根据权利要求 1 所述的 (R)-3- 羟基戊二酸单甲酯的制备方法,其特征在于:水相缓冲液为磷酸盐缓冲液或 Tri-HCl 缓冲液。
4 、根据权利要求 1 所述的 (R)-3- 羟基戊二酸单甲酯的制备方法,其特征在于:重组腈水解酶与底物的投料质量比为 0.01~0.1:1 ,反应起始时,底物浓度为 0.8~1.5mol/L 。
5 、根据权利要求 4 所述的 (R)-3- 羟基戊二酸单甲酯的制备方法,其特征在于:重组腈水解酶与底物的投料质量比为 0.02~0.05:1 ,反应起始时,底物浓度为 1.0~1.5mol/L 。
6 、根据权利要求 1 至 5 中任一项所述的 (R)-3- 羟基戊二酸单甲酯的制备方法,其中,该制备方法具体实施如下:将所述重组腈水解酶加入到水相缓冲液中,然后采用恒流泵流加底物,待底物加毕,使反应体系在温度 25 ℃ ~35℃下以及搅拌下反应,至底物转化率大于等于99.0% ,终止反应。
7 、根据权利要求 6 所述的 (R)-3- 羟基戊二酸单甲酯的制备方法,其特征在于:终止反应后,用盐酸调节体系至酸性,然后用硅藻土过滤,用乙酸乙酯冲洗滤渣;滤液用乙酸乙酯萃取,合并有机相;有机相用无水硫酸钠干燥,浓缩后得到黄褐色油状物即为 (R)- 3- 羟基戊二酸单甲酯。
8 、一种羟基被保护的 (R)-3- 羟基戊二酸单甲酯的制备方法,所述羟基被保护的 (R)-3- 羟基戊二酸单甲酯为 (R)-3- 叔丁基二甲基硅氧基戊二酸单甲酯,其特征在于所述制备方法包括如下步骤:
( 1 )按照权利要求 1 至 7 中任一项权利要求所述的方法制备得到 (R)-3- 羟基戊二酸单甲酯;
( 2 )使 (R)-3- 羟基戊二酸单甲酯与叔丁基二甲基氯硅烷在有机溶剂中,催化剂和有机碱缚酸剂存在下,于常温下搅拌反应,得到 3- 叔丁基二甲基硅氧基 - 戊二酸甲酯叔丁基二甲基硅酯,其中, (R)-3- 羟基戊二酸单甲酯、叔丁基二甲基氯硅烷以及有机碱缚酸剂的投料摩尔比为 0.4~0.5:1:1.05~1.2 。
( 3 )使 3- 叔丁基二甲基硅氧基 - 戊二酸甲酯叔丁基二甲基硅酯在碳酸钾存在下、溶剂中发生水解反应生成所述 (R)-3- 叔丁基二甲基硅氧基戊二酸单甲酯。
9 、根据权利要求 8 所述的羟基被保护的 (R)-3- 羟基戊二酸单甲酯的制备方法,其特征在于:步骤( 2 )中的催化剂为 4- 二甲氨基吡啶,其与 (R)-3- 羟基戊二酸单甲酯的投料摩尔比为 0.002~0.01:1 ,有机碱缚酸剂为选自咪唑、 N,N- 二异丙基乙胺、吡啶及三乙胺中的一种或多种的组合。
10 、根据权利要求 8 或 9 所述的羟基被保护的 (R)-3- 羟基戊二酸单甲酯的制备方法,其特征在于:步骤( 3 )中,采用甲醇、四氢呋喃以及水的组合作为溶剂,碳酸钾与 3- 叔丁基二甲基硅氧基- 戊二酸甲酯叔丁基二甲基硅酯的投料摩尔比为 4.5~5:1 。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024049229A1 (ko) * 2022-08-31 2024-03-07 동국대학교 산학협력단 뽕나무 유래 화합물을 유효성분으로 포함하는 피부 질환 예방 또는 개선용 화장료 조성물

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104561142A (zh) * 2013-10-25 2015-04-29 中国科学院天津工业生物技术研究所 (r)-3-羟基戊二酸单酯的生物合成方法
CN104370953B (zh) * 2014-08-24 2017-01-18 浙江新东港药业股份有限公司 一种(r)‑叔丁基二甲基硅氧基‑戊二酸单酯的制备方法
CN104193776A (zh) * 2014-08-24 2014-12-10 浙江新东港药业股份有限公司 一种(3r)-羟基保护-戊二酸单酯(i)的制备方法
CN104152500A (zh) * 2014-08-27 2014-11-19 中国科学院天津工业生物技术研究所 一种生物合成(r)-3-羟基戊二酸单酯的新方法
CN104292252B (zh) * 2014-09-19 2017-01-18 浙江科技学院 一种1‑乙氧羰基‑5‑甲基‑(3r)‑叔丁基二甲硅氧基戊二酸酯的合成工艺
CN104630297B (zh) * 2014-12-10 2018-07-06 江南大学 一种酶法非水相催化合成(r)-3-tbdmso戊二酸甲单酯及其衍生物
CN105385714A (zh) * 2015-12-26 2016-03-09 尚科生物医药(上海)有限公司 生物酶-化学法制备瑞舒伐他汀中间体
CN112941122B (zh) * 2021-02-23 2022-12-09 浙江工业大学 一种(s)-3-氰基-5-甲基己酸的制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101072871A (zh) * 2004-08-16 2007-11-14 纳幕尔杜邦公司 使用腈水解酶突变体生产3-羟基羧酸
CN101805756A (zh) * 2010-01-30 2010-08-18 浙江工业大学 生物催化法生产他汀类药物中间体
CN102181493A (zh) * 2011-01-12 2011-09-14 江苏阿尔法药业有限公司 一种瑞苏伐他汀钙侧链中间体的制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK1404642T3 (da) * 2001-07-06 2010-05-17 Basf Se Fremgangsmåde til fremstillimg af 7-amino-syn-3,5-dihydroxyheptansyrederivater, mellemprodukter derfor og fremgangsmåder til deres fremstilling

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101072871A (zh) * 2004-08-16 2007-11-14 纳幕尔杜邦公司 使用腈水解酶突变体生产3-羟基羧酸
CN101805756A (zh) * 2010-01-30 2010-08-18 浙江工业大学 生物催化法生产他汀类药物中间体
CN102181493A (zh) * 2011-01-12 2011-09-14 江苏阿尔法药业有限公司 一种瑞苏伐他汀钙侧链中间体的制备方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024049229A1 (ko) * 2022-08-31 2024-03-07 동국대학교 산학협력단 뽕나무 유래 화합물을 유효성분으로 포함하는 피부 질환 예방 또는 개선용 화장료 조성물

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