CN113930404B - 一种酶法合成手性枸橼酸托法替布中间体的方法 - Google Patents
一种酶法合成手性枸橼酸托法替布中间体的方法 Download PDFInfo
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Abstract
本发明公开了一种酶法合成手性枸橼酸托法替布中间体的方法，本发明以4‑甲基‑1‑(苯基甲基)‑3‑哌啶酮(TOF15)为原料，通过生物催化得到手性的中间体TOF20‑A，再进行甲基化反应得到中间体TOF25‑A，也即游离的(3R,4R)‑1‑苄基‑N,4‑二甲基哌啶‑3‑胺，中间体TOF25‑A可进一步成盐酸盐而得到(3R,4R)‑1‑苄基‑N,4‑二甲基哌啶‑3‑胺二盐酸盐(TOF30)。本发明是一种成本低、收率高、对环境友好的合成技术，适合大规模工业化生产，具有巨大的市场应用价值。
Description
技术领域
本发明涉及酶催化技术领域，具体涉及一种转氨酶催化剂，还涉及一种酶法合成一种手性枸橼酸托法替布中间体((3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐)的方法，以及一种手性枸橼酸托法替布中间体((3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐)的生产方法。
背景技术
(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐是重要的医药及有机合成的中间体，尤其用于合成枸橼酸托法替布。
枸橼酸托法替布由美国辉瑞制药公司开发，是首个作用机制的JAK通路抑制剂，是一种新型的口服蛋白酪氨酸激酶抑制剂。该产品于2012年在美国首先批准上市，已在美国、日本、中国、俄罗斯、澳大利亚、加拿大等全球50多个国家和地区获得上市批准。
现有的(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐的合成方法均是化学合成法，从4-甲基-1-(苯基甲基)-3-哌啶酮出发(TOF15)，经过氨甲基化得到 TOF20，TOF20是一个混旋体，需要经过L-DTTA成盐拆分，才能得到手性的 TOF25，再将TOF25游离、成盐酸盐，从而得到(3R,4R)-1-苄基-N,4-二甲基哌啶 -3-胺二盐酸盐(TOF30)，合成路线如下：
然而，现有的化学合成方法涉及较多试剂，且操作复杂，工序繁琐，尤其是最后拆分有50％的产物要丢弃，导致收率很低，原子经济性特别差，因此成本较高，并且无法得到高光学纯度的目标产物。
发明内容
针对现有技术中存在的种种技术缺陷，本发明旨在提供一种全新的酶法合成路线，该路线能高效的制得目标产物----(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐(TOF30)，而且产物ee值高达99.7％以上。
为实现上述目的，本发明采取的技术方案包括：
本发明第一方面提供了一种转氨酶催化剂，其核苷酸序列如SEQ ID NO.1 所示，并且其氨基酸序列如SEQ ID NO.2所示，具体参见下表1：
表1转氨酶催化剂的核苷酸序列与氨基酸序列
本发明第二方面提供了一种酶法合成(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺或其二盐酸盐(TOF30)的方法：
1)在转氨酶催化剂的存在下，反应底物TOF15与氨基供体反应生成手性的 TOF20-A，
2)TOF20-A再与甲基化试剂进行甲基化反应，加入缚酸剂得到TOF25-A，也即游离的(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺，或者TOF25-A进一步成盐酸盐得到(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐(TOF30)；
其中，所述转氨酶催化剂的核苷酸序列如SEQ ID NO.1所示，所述转氨酶催化剂的氨基酸序列如SEQ ID NO.2所示。
其反应方程式为：
优选的，在上述酶法合成TOF20-A的方法中，所述氨基供体选自以下任意一种，异丙胺，叔丁胺，丙氨酸，丁氨酸；进一步优选地，所述氨基供体为异丙胺。所述的溶剂为水与有机溶剂的混合溶剂或水，所述有机溶剂是二甲亚砜、四氢呋喃或DMF，所述转氨酶反应的pH值为7-10，所述转氨酶反应的反应温度为10-50摄氏度；进一步优选地，所述的溶剂为水与二甲亚砜的混合溶剂，所述转氨酶反应的pH值为7-8，所述转氨酶反应的反应温度为15-25摄氏度。
TOF20-A到TOF25-A的合成方法中，所述甲基化反应所用的甲基化试剂选自以下任意一种，碘甲烷，硫酸二甲酯；所述缚酸剂选自以下任意一种，碳酸钾，碳酸钠，碳酸氢钠；所用反应溶剂选自以下任意一种，二氯甲烷，乙酸乙酯，乙醇，甲醇；进一步优选地，所述甲基化试剂为碘甲烷，所述缚酸剂为碳酸钾，所用反应溶剂为二氯甲烷，反应温度为15-25℃。
本发明以4-甲基-1-(苯基甲基)-3-哌啶酮(TOF15)为原料，通过生物催化得到手性的中间体TOF20-A，再进行甲基化反应得到中间体TOF25-A，也即游离的(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺，或者进一步成盐酸盐而得到(3R,4R)-1- 苄基-N,4-二甲基哌啶-3-胺二盐酸盐(TOF30)。本发明是一种成本低、收率高、对环境友好的合成技术，适合大规模工业化生产，具有巨大的市场应用价值。
具体实施方式
下面结合具体实施方式对本发明做进一步阐述和说明。本发明中各个实施方式的技术特征在没有相互冲突的前提下，均可进行相应组合。
实施例一转氨酶菌体冻干粉的制备
将合成的转氨酶催化剂基因DNA片段在35-37℃用限制性内切酶NdeI和 AvrⅡ双酶切6h，经琼脂糖凝胶电泳纯化，利用琼脂糖凝胶DNA回收试剂盒回收目标片段(SEQ IDNO.1)；然后，将目标片段在T4DNA连接酶的作用下，与同样经NdeI和EcoRI酶切后的质粒pACYCDuet-B，在25-27℃下连接过夜得到重组表达质粒；将重组表达质粒转化到大肠埃希氏菌感受态细胞中，转化条件为： 40-45℃，热击80-90秒，在含有氯霉素的抗性平板上对阳性重组体进行筛选，挑取单克隆，培养重组菌，待质粒扩增后提取质粒，重新转化至感受态细胞中，转化液涂布到含有氯霉素抗性的LB平板上，35-37℃培养过夜，即获得阳性重组转化体(即重组大肠杆菌)。将上述重组大肠杆菌接种于含有氯霉素抗性的LB 固体培养基，35-37℃培养18-20h；挑取单菌落接种于含有氯霉素抗性的100mL LB液体培养基，振荡培养18-20h，培养结束后移取菌液于500mL TB液体培养基，培养2.5h后加入0.1mM IPTG诱导蛋白表达，28-30℃振荡培养18-20h， 10000rpm离心收集菌体，菌体经过冷冻干燥即得含转氨酶的菌体冻干粉，-70℃下保存待用。
实施例二TOF20-A的制备
将异丙胺100g溶于100ml水中，在冰水浴冷却下，用盐酸水溶液调节pH 值到7.0-8.0，并加入10ml二甲亚砜，然后用0.1M的Tris-HCl缓冲液稀释至700ml，并预热到30℃，再加入含有4-甲基-1-(苯基甲基)-3-哌啶酮(TOF15)50g的二甲亚砜溶液100ml，最后加入1g转氨酶菌体冻干粉和PLP(磷酸吡哆醛)0.8g，反应用20％的异丙胺水溶液控制pH值为7.0-8.0，温度在15-25℃转化12小时以上， TLC监控反应完毕。过滤去除固体，母液用二氯甲烷萃取3次，合并后的有机相用无水硫酸钠干燥，浓缩得油状物TOF20-A 45g，收率约为90％。
实施例三(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺(TOF25-A)的制备
将实施例一中的油状物TOF20-A 10g(约50mmol)，溶解于100ml二氯甲烷中，加入粉末状的碳酸钾7g(约50mmol)，在冰水浴冷却下降温至0-5℃，开始滴加碘甲烷(7.1g，约50mmol)，滴加完成后，缓慢升温至15-25℃保温反应2 小时，TLC监控反应完毕。过滤去除固体，二氯甲烷层水洗3遍，二氯甲烷相用无水硫酸钠干燥，浓缩得油状物TOF25-A 10g，收率约为95％。
实施例四(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐(TOF30)的制备
将实施例一中的油状物TOF25-A 10g，溶解于20ml无水乙醇中，室温下开始滴加10ml 30％盐酸乙醇溶液，滴加完成后即开始析出白色固体，再在0-5℃下搅拌1小时，过滤得到白色固体，烘干后即得(3R,4R)-1-苄基-N,4-二甲基哌啶-3- 胺二盐酸盐TOF30约12g，收率约为90％，ee值99.8％。
1H-NMR(400M,D2O)δ：7.53(m,5H),4.45(s,2H),3.67(m,1H),3.36-3.67(m,2H),3.23-3.36(m,2H),2.76(s,3H),2.58(m,1H),1.99(m,2H),1.12(d,3H)。
HRMS(ESI+)m/z calcd for 219.1856；found：[M+H]+219.1859。
综合分析以上实验结果可知，本发明所使用的转氨酶催化剂的催化活性较高，能够生成光学纯度极高的(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐，且对环境友好。因此，该生产方法有利于实现手性枸橼酸托法替布中间体的大规模工业化生产，具有巨大的市场应用价值。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明的保护范围应以所附权利要求为准。
序列表
<110> 浙江乐普药业股份有限公司
<120> 一种酶法合成手性枸橼酸托法替布中间体的方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 622
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcttcga tggacaaagt cttctcaggt tactacgccc gtcaaaaact gctggaacgc 60
tcagataatc cgttctcaaa aggtattgcc tatgtcgaag gctgccgagt aaatatcctg 120
tcatgcgata aaatgcgtct gaaatttccg ctggcactga gctctgtcaa gtaaactggt 180
ctacgacgtt gcagaaatgg tggctaaaag cggcattcgt tcgaagtgat cgttacccgc 240
gatgcgcgca ttccgctgct ggacgaaggc tttatgcata gtgatctgac gacgctttcg 300
ctggtgctgc cgtatatctg ccggaaaatc agctgcatgg cattctggaa cggtgaagct 360
ggcctgacgg ctctaaaccg gaagatctgt ataacaataa catttacctg atctccgtct 420
gggacggccg tttctttcgc ctggatgacc acctgcagcg gtgttcgtgg gctgatggca 480
attatcaccc gtacggtgcg tcgcaccccg ccgggtgcct ttgatccgac gatcaaaaac 540
taccaagaaa atttgggatg gctattggga aatgcactac aacccggctt attcgtttcc 600
ggtggattat ggcagcggtt ag 622
<210> 2
<211> 62
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr Tyr Ala Arg Gln Lys
1 5 10 15
Leu Leu Gln Arg Ser Asp Asn Pro Phe Ser Lys Gly Ala Tyr Val Glu
20 25 30
Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Gly Arg Phe Phe Asn Asp
35 40 45
Gly Gln Val Gly Tyr Ser Phe Pro Val Asp Tyr Gly Ser Gly
50 55 60
Claims (8)
1.一种酶法合成手性枸橼酸托法替布中间体的方法，其特征在于：
1)在转氨酶催化剂的存在下，反应底物TOF15与氨基供体反应生成手性的TOF20-A，
2)TOF20-A再与甲基化试剂进行甲基化反应，加入缚酸剂得到TOF25-A，也即游离的(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺，或者TOF25-A进一步成盐酸盐得到(3R,4R)-1-苄基-N,4-二甲基哌啶-3-胺二盐酸盐(TOF30)；
其中，所述转氨酶催化剂的核苷酸序列如SEQ ID NO.1所示，所述转氨酶催化剂的氨基酸序列如SEQ ID NO.2所示；
其反应方程式为：
。
2.根据权利要求1所述的方法，其特征在于：所述氨基供体为异丙胺、叔丁胺、丙氨酸或丁氨酸中的一种或多种。
3.根据权利要求1所述的方法，其特征在于：所述步骤1)中，反应在溶剂中进行，溶剂为水、有机溶剂或水与合溶剂的混合溶剂，所述有机溶剂是二甲亚砜、四氢呋喃或DMF中的一种或多种。
4.根据权利要求1所述的方法，其特征在于：所述步骤1)中反应体系的pH值为7-10，反应温度为10-50摄氏度。
5.根据权利要求3所述的方法，其特征在于：所述步骤1)中，溶剂为水与二甲亚砜的混合溶剂，反应体系的pH值为7-8，反应温度为15-25摄氏度。
6.根据权利要求1所述的方法，其特征在于：所述步骤2)中，甲基化反应所用的甲基化试剂选自碘甲烷或硫酸二甲酯，所述缚酸剂为碳酸钾、碳酸钠或碳酸氢钠的一种或多种；步骤2)反应在溶剂中进行，溶剂为二氯甲烷、乙酸乙酯、乙醇、甲醇中的一种或多种。
7.根据权利要求6所述的方法，其特征在于：所述步骤2)中，所述甲基化试剂为碘甲烷，所述缚酸剂为碳酸钾，所用反应溶剂为二氯甲烷。
8.根据权利要求1所述的方法，其特征在于：步骤2)中，甲基化反应温度为15-25℃。
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