CN113881727B - 一种阿托伐他汀酸的合成方法 - Google Patents
一种阿托伐他汀酸的合成方法 Download PDFInfo
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- CN113881727B CN113881727B CN202111030461.XA CN202111030461A CN113881727B CN 113881727 B CN113881727 B CN 113881727B CN 202111030461 A CN202111030461 A CN 202111030461A CN 113881727 B CN113881727 B CN 113881727B
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- enzyme
- atorvastatin acid
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- BJGBAZAEWKCPHZ-MQSFZEHASA-N (3r,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1.C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 BJGBAZAEWKCPHZ-MQSFZEHASA-N 0.000 title claims abstract description 22
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- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 6
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Abstract
本发明涉及药物合成领域,特别是涉及阿托伐他汀酸的合成方法。针对阿托伐他汀酸的化学合成中存在对环境不友好,以及容易产生杂质的问题,本发明公开了一种阿托伐他汀酸的合成方法,通过在含有式II化合物的溶剂中添加酶LP001实现由式II化合物生物合成式I化合物,该方法与现有技术中的单纯化学合成法相比,具有对环境友好、专一性强、不易生成脱酰胺杂质的优势,通过本发明公开的技术方案合成式I化合物,可以显著提高产物纯度,无需复杂的纯化工艺,具有广阔的工业化应用前景。
Description
技术领域
本发明涉及药物合成领域,特别是涉及阿托伐他汀酸的合成方法。
背景技术
阿托伐他汀钙(结构式如下所示)为羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂。主要通过抑制HMGCoA还原酶的合成,从而抑制体内胆固醇的合成,降低血清低密度脂蛋白胆固醇、甘油三酯的含量。由于阿托伐他汀钙抑制了细胞合成胆固醇,干扰了脂蛋白的生成,使血清总胆固醇水平下降,能有效降低血清甘油三酯水平,还能升高血清高密度脂蛋白胆固醇水平。能降低某些纯合子家族性高胆固醇血症患者的血浆低密度脂蛋白胆固醇水平,而这一类型的人群,对其他降脂药治疗很少有应答。临床用于原发性高胆固醇血症、混合型高脂血症、高甘油三酯血症、纯合子家族性高胆固醇血症及防治动脉粥样硬化。他汀类药物成为上世纪70年代的研究热点,1987年世界上首个他汀类药物洛伐他汀获FDA批准上市,之后辛伐他汀、普伐他汀、阿托伐他汀等新药如同雨后春笋一般相继问世。
阿托伐他汀酸为阿托伐他汀钙成盐的前体,目前现有技术中所采用的的方法为化学合成方法,主要通过强碱进行制备。化学合成中需要使用大量化学试剂,这些试剂在工业生产中具有对环境不友好的问题,不符合现代绿色生产需要。同时,在目前现有的化学制备过程中且容易生成如下结构式的杂质,这也同时增加了后续分离纯化的成本。
生物合成法相较于化学合成法,反应条件更加温和,反应产物专一性更高。因此,是一种符合现代绿色合成理念的工业化合成方法。作为生物合成法中的重要参与者酶,具有特异性的高效生物效率。但是,具体到一个合成路线中,找到能够适用于该合成路线的特异性酶则是生物合成领域的研究核心内容。这也是本领域技术人员在针对不同合成路线开发其生物合成法中的研究重点和需要攻克的技术难点。
发明内容
本发明所要解决的技术问题是在阿托伐他汀酸的化学合成中存在对环境不友好,以及容易产生杂质的问题。
为了解决上述技术问题,本发明公开了一种阿托伐他汀酸的合成方法,包括以下步骤,式II化合物在酶LP001的作用下酶催化反应生成式I化合物,其反应式如下:
进一步优选地,所述酶LP001的氨基酸序列如SEQ ID NO:1所示。
更为优选的是,所述酶LP001的核苷酸序列如SEQ ID NO:2所示。
进一步优选地,所述化合物II与酶LP001的质量比为1:(1-30)。
进一步优选地,所述阿托伐他汀酸的酶催化反应在醇与缓冲液组成的混合溶液中进行,所述醇为异丙醇、甲醇或乙醇中的一种或几种,所述醇与缓冲溶液的体积比为1:10-20;所述化合物II与混合溶剂的质量体积比为1:10-100g/mL。
进一步优选地,所述缓冲液为Tris-HCl缓冲液。
在一个优选的技术方案中,所述酶LP001是通过构建LP001基因工程菌株,并通过发酵培养,收集菌体得到。
具体而言,所述酶LP001的制备方法如下:将全合成的LP001酶序列进行PCR 扩增后,导入质粒pET30a(+)的NdeI和XhoI酶切位点获得重组表达载体,将重组表达载体电转入LP001表达细胞中,得到LP001表达工程菌,经过抗生素抗性平板涂布筛选后,获得克隆菌株LP001-11,送检测序确认无误,进行活化后发酵培养,离心收集菌体,洗涤并重悬,超声破碎,冻干得到LP001冻干粉。
本发明通过在含有式II化合物的溶剂中添加酶LP001实现由式II化合物生物合成式I化合物,该方法与现有技术中的单纯化学合成法相比,具有对环境友好、专一性强、不易生成脱酰胺杂质的优势,通过本发明公开的技术方案合成式I化合物,可以显著提高产物纯度,无需复杂的纯化工艺,具有广阔的工业化应用前景。
附图说明
图1为重组表达载体示意图。
具体实施方式
为了更好的理解本发明,下面我们结合具体的实施例对本发明进行进一步的阐述。
实施例1制备酶LP001
步骤1:LP001基因工程菌的制备
按照人工序列SEQ ID NO:2所示的核苷酸序列全序列合成酶LP001。将该和合成后的序列进行PCR扩增后克隆载入表达质粒pET-30a(+)的NdeI和XhoI双酶切位点。引物分别如SEQ ID NO:3-4所示:
F:cgccatatgactggtggacagcaaatg
R:ccgctcgagttacaggcaggtgccaatcaga
将重组表达质粒转入E.coli BL21(DE3)感受态菌株中,挑取阳性转化子并测序鉴定后,获得LP001表达工程菌。
步骤2:LP001的制备
将获得的LP001工程菌株接种到含有抗生素卡那抗性的LB液体培养基中,于 37℃过夜培养,得到种子培养液。取1ml种子液接种至100ml TB发酵液培养基中。然后置于37℃下培养至OD600值为0.6,加入终浓度为0.05mol的IPTG,置于 16℃继续培养16h后,12000rmp,4℃下离心收集菌体,即为实施例2和实施例3 中所用的酶LP001,所述酶LP001的氨基酸序列如SEQ ID NO:1所示。
将所获得的酶LP001加入液氮罐中速冻5min,冻干,获得冻干粉,备用。
实施例2化合物I的制备
于250ml锥形瓶中,加入30mL pH8.0 Tris-HCl缓冲液,然后依次加入8g酶 LP001,1g化合物II,2mL异丙醇,10mMCaCl2于220rpm下进行搅拌,于35℃下反应24h,得到化合物I。反应结果经HPLC检测,转化率为61%,重结晶纯度94%。
实施例3化合物I的制备
于250ml锥形瓶中,加入30mL pH8.0 Tris-HCl缓冲液,依次加入8g酶LP001, 1g化合物II,2mL异丙醇,于220rpm下进行搅拌,于35℃下反应24h,得到化合物I。反应结果经HPLC检测,转化率为53%,重结晶纯度90%。
通过实施例2和实施例3的实验结果可以看出,本发明添加Ca2+后,LP001催化效果更佳。
本发明制得的酶具有优异的立体选择性,可以有效提高产率和产品的光学纯度,降低杂质的产生量,具有很好的应用前景。
以上所述是本发明的具体实施方式。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也视为本发明的保护范围。
序列表
<110> 江苏福瑞康泰药业有限公司
<120> 一种阿伐他汀酸的合成方法
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Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys
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ggtaaaaata atgatgcccc ggcaggtacc aatattacct gtaccggtaa tgcctgcccg 180
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gttaccggct ttctggcact ggataatacc aataagctga ttgttctgag ttttcgtggc 300
agccgcagca ttgaaaattg gattggtaat ctgaatttcg atctgaaaga aattaacgat 360
atctgtagtg gctgtcgtgg ccatgatggt tttaccagta gttggcgtag cgttgcagat 420
accctgcgtc agaaagttga agatgccgtt cgcgaacatc cggattatcg tgtggtgttt 480
accggccata gtctgggcgg cgcactggcc accgtggctg gtgcagatct gcgtggtaat 540
ggttatgata ttgatgtttt tagctacggc gccccgcgtg tgggtaatcg cgcctttgcc 600
gaatttctga ccgttcagac cggtggtacc ctgtatcgta ttacccatac caatgatatt 660
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Claims (10)
1.一种阿托伐他汀酸的合成方法,其特征在于,包括以下步骤,式II化合物在酶LP001的作用下酶催化反应生成式I化合物,其反应式如下:
;
所述酶LP001的氨基酸序列如SEQ ID NO:1所示。
2. 根据权利要求1所述的阿托伐他汀酸的合成方法,其特征在于,所述酶LP001的核苷酸序列如SEQ ID NO:2所示。
3.根据权利要求1所述的阿托伐他汀酸的合成方法,其特征在于,所述化合物II与酶LP001的质量比为1:(1-30)。
4.根据权利要求1所述的阿托伐他汀酸的合成方法,其特征在于,所述阿托伐他汀酸的酶催化反应在醇与缓冲液组成的混合溶液中进行,所述醇为异丙醇、甲醇或乙醇中的一种或几种,所述醇与缓冲溶液的体积比为1:10-20。
5.根据权利要求4所述的阿托伐他汀酸的合成方法,其特征在于,所述化合物II与混合溶剂的质量体积比为1:10-100g/mL。
6.根据权利要求4所述的阿托伐他汀酸的合成方法,其特征在于,所述缓冲液为Tris-HCl缓冲液。
7.根据权利要求1所述的阿托伐他汀酸的合成方法,其特征在于,所述酶LP001是通过构建LP001基因工程菌株,并通过发酵培养,收集菌体得到。
8.根据权利要求1所述的阿托伐他汀酸的合成方法,其特征在于,所述酶催化反应中还添加有Ca2+。
9.根据权利要求8所述的阿托伐他汀酸的合成方法,其特征在于,所述Ca2+通过氯化钙的形式加入反应体系。
10. 根据权利要求8所述的阿托伐他汀酸的合成方法,其特征在于,所述Ca2+与酶LP001的添加比例为8g酶LP001添加Ca2+ 5-15mM。
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CN109536468A (zh) * | 2019-01-04 | 2019-03-29 | 浙江宏元药业股份有限公司 | 二羰基还原酶及其在他汀类药物中间体合成中的应用 |
CN109776686A (zh) * | 2019-03-27 | 2019-05-21 | 云南师范大学 | 一种热稳性提高的融合型脂肪酶及其制备方法和应用 |
CN111647591A (zh) * | 2020-06-24 | 2020-09-11 | 湖州颐盛生物科技有限公司 | 利用固定化酶制备他汀中间体的方法 |
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CN109536468A (zh) * | 2019-01-04 | 2019-03-29 | 浙江宏元药业股份有限公司 | 二羰基还原酶及其在他汀类药物中间体合成中的应用 |
CN109776686A (zh) * | 2019-03-27 | 2019-05-21 | 云南师范大学 | 一种热稳性提高的融合型脂肪酶及其制备方法和应用 |
CN111647591A (zh) * | 2020-06-24 | 2020-09-11 | 湖州颐盛生物科技有限公司 | 利用固定化酶制备他汀中间体的方法 |
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