CN107384885A - 亚胺还原酶及其突变体在合成(s)‑1‑芳基‑1,2,3,4‑四氢异喹啉中的应用 - Google Patents
亚胺还原酶及其突变体在合成(s)‑1‑芳基‑1,2,3,4‑四氢异喹啉中的应用 Download PDFInfo
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- CN107384885A CN107384885A CN201710791728.4A CN201710791728A CN107384885A CN 107384885 A CN107384885 A CN 107384885A CN 201710791728 A CN201710791728 A CN 201710791728A CN 107384885 A CN107384885 A CN 107384885A
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Abstract
本发明公开了一种亚胺还原酶及其突变体在合成(S)‑1‑芳基‑1,2,3,4‑四氢异喹啉中的应用,属于酶工程领域。本发明的亚胺还原酶为如下酶中的一种:氨基酸序列如SEQ ID NO.1‑3所示的IR45、IR96、IR99,其对应的编码核苷酸分别优选如SEQ ID NO.4‑6所示;亚胺还原酶突变体,为IR45的突变体IR45‑W191A、IR45‑W191L或IR99的突变体IR99‑L173A中的一种。上述亚胺还原酶或其突变体可催化式I所示化合物合成式II所示化合物(式I、式II中的R为氢、甲氧基或卤素),催化效率高,所得目标产物光学纯度高,后处理简单,环保安全性好。
Description
技术领域
本发明涉及酶工程领域,具体涉及一种亚胺还原酶及其突变体在合成(S)-1-芳基-1,2,3,4-四氢异喹啉中的应用。
背景技术
索非那新(Solifenacin,下式A),商品名为Vesicare,是由日本山之内制药公司研制的一种泌尿系统解痉挛药。它能够选择性作用于M3受体,在临床被广泛用于治疗膀胱过度活动综合症。继2004年首次面市后,索菲那新于2009年获得中国SFDA的许可并在中国上市。由于突出的疗效和安全性,索非那新一经上市便受到了全球患者们的欢迎,占据了很大的市场,仅2014年美国市场的销售额就达到了9.94亿美元。
结构上,索非那新由两部分手性片段组成,即(S)-1-苯基-1,2,3,4-四氢异喹啉(S-1b)和(R)-奎宁醇(R-3-quinuclidnol)。索非那新的化学合成主要是由(S)-1b和(R)-奎宁醇这两个片段通过羰基化合物(如碳酸二甲酯)链接起来实现的(PCT Int.Appl.2014,WO2014005601 A120140109)(上式A),因此手性片段(S)-1b的成本对索菲那新的生产总成本起到了决定作用。在已有的专利或文献中,(S)-1b的合成主要首先以2-苯乙胺和苯甲酰氯反应得到酰胺中间体,然后在多聚磷酸或者三氟甲磺酸酐与2-氯吡啶共同作用下得到脱水环合得到亚胺1a。从亚胺得到目标产物,可通过两个方案(上式B),方案一:在硼氢化钠还原下得到外消旋体胺(rac-1b),再经过手性试剂拆分得到光学纯度的目标化合物(S)-1b(中国专利CN105541712;CN 103159677A);方案二:通过金属配体催化还原亚胺化合物1a的方法一步得到目标化合物(S)-1b(Angew.Chem.Int.Ed.2011,50,10679-10681;Angew.Chem.Int.Ed.2017,56,2725-2729)。金属催化虽然能够达到较高的转化率和e.e.值,但由于贵金属和配体价格高昂,且催化过程需要苛刻的反应条件(无水无氧),因此不适合工业生产,仅在实验室用于小量的合成。目前工业生产主要依赖手性拆分的方法获得光学活性的产物(S)-1b。然而由于拆分的理论产率不超过50%,且拆分过程需要多次重结晶用于提高光学纯度,因此这种方法也存在着步骤繁琐,三废较多且原子不经济等诸多缺点。
酶催化反应以其高度区域、立体选择性、操作简单、环保安全以及可重复利用等优异的性质,已成为工业合成的一个非常瞩目的方面。近年来关于(S)-1a的酶催化合成也有陆续的报道(下式):Turner等人利用了R-选择性的单胺氧化酶(NMO)和硼烷协同反应对消旋的rac-1b进行动力学拆分(下式A),最终以90%的收率,98%的e.e.值获得(S)-1b(J.Am.Chem.Soc.2013,135,10863-10869);随后,Ward等人也发展了一种基于生物素-链霉抗生物素和金属铱催化剂偶联的人造亚胺还原酶(artificial IRED),通过选择性还原亚胺底物1a得到(S)-1b(下式B),但其产物的ee值仅为67%(Chem Cat Chem,2014,6,1010-1014)。尽管这些酶催化反应采用了不同的思路,但核心步骤仍为亚胺的还原。由于单胺氧化酶的动力学拆分需要使用昂贵且剧毒的硼烷,人造亚胺还原酶则需要使用昂贵的铱催化剂且转化率和e.e.都较低,因此这些酶催化方法都存在的较大的缺陷而不适合实际的生产。相应的,直接利用亚胺还原酶(IRED)还原亚胺底物1a合成(S)-1b具有更大的优势;然而由于1a中C1位的苯基取代基的位阻很大,因此长期以来没有找到相应的IRED能够催化化合物1a的还原。直到最近Zheng等人报道了首个来自于微生物Stackebrandtia nassauensis的亚胺还原酶SnIRED(Org.Lett.2017,19,3151-3154),其可以直接还原亚胺化合物1a一步得到(S)-1b(下式C);但是由于其催化转化率和产物的e.e.值仅有81%和51%,仍无法满足实际的生产要求。由此可见,酶催化合成1-苯基-1,2,3,4-四氢异喹啉类化合物虽然已开始吸引人们目光,从目前报道来看,或产物光学纯度不高,或操作复杂条件苛刻,或催化剂不易得,在实际工业生产中的应用大大受限。由此,需要发展出一类具有更高选择性和活性的亚胺还原酶,用于催化合成(S)-1b。除此之外,其他的一些取代的1-苯基-1,2,3,4-四氢异喹啉类化合物,如1-间甲基苯基-1,2,3,4-四氢异喹啉(2b),1-间甲氧基苯基-1,2,3,4-四氢异喹啉(3b),1-间氯苯基-1,2,3,4-四氢异喹啉(4b)类化合物在有机合成和药物研发中也有广泛的用途(Org.Chem.Front.2015,2,288-299;Chem.Rev.2016,116,12369-12465),因此发展出能合成此类结构且具有高选择性和活性的亚胺还原酶具有十分重要的意义。
发明内容
本发明的目的在于克服现有技术存在的缺点与不足,提供一种亚胺还原酶及其在合成(S)-1-芳基-1,2,3,4-四氢异喹啉中的应用。
本发明的目的通过下述技术方案实现:
一种亚胺还原酶,为如下酶中的一种:氨基酸序列如SEQ ID NO.1-3所示的IR45、IR96、IR99,其对应的编码核苷酸分别优选如SEQ ID NO.4-6所示。
上述亚胺还原酶的关键保守氨基酸位点为IR45的191位氨基酸、IR99的173位氨基酸。通过将这些关键保守氨基酸位点突变为体积较小的氨基酸(如丙氨酸)能够提高亚胺还原酶的催化效率。
一种亚胺还原酶突变体,为IR45的突变体IR45-W191A、IR45-W191L或IR99的突变体IR99-L173A中的一种。
一种重组载体,插入有上述亚胺还原酶或其突变体编码核苷酸。优选的,所述重组载体还插入有脱氢酶的编码核苷酸;所述的脱氢酶能够使用NADP+作为辅酶催化氧化还原反应,如葡糖脱氢酶。
上述亚胺还原酶或其突变体可用于催化合成(S)-1-芳基-1,2,3,4-四氢异喹啉,如催化式I所示化合物合成式II所示化合物。
式I、式II中的R为氢、甲氧基或卤素。催化式I所示化合物合成式II所示化合物具体见下式:
上述亚胺还原酶或其突变体催化合成的(S)-1-芳基-1,2,3,4-四氢异喹啉可以用作合成药物等的原料或中间体。
本发明与现有技术相比具有以下优点和有益效果:本发明所述酶催化还原亚胺底物1a-4a的反应,转化率均在78%以上,产品e.e.值达90-99%间,显著优于之前报道的亚胺还原酶催化反应;可以在很大程度上解决现有技术手性拆分造成原料浪费的难题;相对于金属催化,这些酶催化反应还具有成本低廉,产品转化率和光学纯度高,操作简单环保,适用于大规模工业生产等优势。这些酶的催化应用,可以大大的提升产品的质量,简化工业生产后处理步骤,为创制优质原料药和制剂提供了核心竞争力。
附图说明
图1是12%十二烷基磺酸钠聚丙烯酰胺凝胶(SDS-PAGE)电泳检测表达IR45、IR96、IR99和突变体蛋白IR99-L173A、IR45-W191A、IR45-W191L的电泳图。图中M为低分子量蛋白标准品(Marker),其余从左至右依次为IR45、IR96、IR99、IR99-L173A、IR45-W191A、IR45-W191L。
图2是rac-1b的1H谱图。
图3是rac-1b的13C谱图。
图4是rac-2b的1H谱图。
图5是rac-2b的13C谱图。
图6是rac-3b的1H谱图。
图7是rac-3b的13C谱图。
图8是rac-4b的1H谱图。
图9是rac-4b的13C谱图。
图10是rac-1b标品的HPLC谱图。
图11是IR45催化产物(S)-1b的HPLC谱图。
图12是rac-2b标品的HPLC谱图。
图13是IR45催化产物(S)-2b的HPLC谱图。
图14是rac-3b标品的HPLC谱图。
图15是IR45催化产物(S)-3b的HPLC谱图。
图16是rac-4b标品的HPLC谱图。
图17是IR45催化产物(S)-4b的HPLC谱图。
图18是IR96催化产物(S)-1b的HPLC谱图。
图19是IR99催化产物(S)-1b的HPLC谱图。
图20是IR45模拟结构和亚胺底物1a相互作用图。
图21是IR45催化亚胺底物1a的动力学数据分析结果图。
图22是IR45-W191L催化亚胺底物1a的动力学数据分析结果图。
图23是IR45-W191A催化亚胺底物1a的动力学数据分析结果图。
图24是IR99催化亚胺底物1a的动力学数据分析结果图。
图25是IR99-L173A催化亚胺底物1a的动力学数据分析结果图。
具体实施方式:
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 本发明亚胺还原酶的获得
(1)编码亚胺还原酶的基因的获得
本发明获得的三个具有催化能力的亚胺还原酶(IR45、IR96和IR99)是通过筛选亚胺还原酶酶库催化化合物1a-4a的反应(下式)所获得的。亚胺还原酶酶库是申请人前期所构建的100个可能编码亚胺还原酶的基因,根据大肠杆菌密码子偏好性对这100条基因的DNA序列进行优化,并送交公司合成。密码子优化后的基因,分别在5’端添加NdeⅠ(CATATG)酶切位点、在3’端添加HindⅢ(TTCACT)酶切位点,克隆至pET28a质粒上得到重组质粒pET28a-IR45、pET28a-IR96、pET28a-IR99。
亚胺还原酶IR45、IR97和IR99的氨基酸序列分别如SEQ ID NO.1-3所示,对应的优化后编码核苷酸的序列如SEQ ID NO.4-6所示。
(2)表达亚胺还原酶工程菌的获得
将携带有编码亚胺还原酶基因的pET28a质粒转化入大肠杆菌BL21(DE3)感受态细胞,涂布于含有50μg/mL卡那霉素的LB选择性平板上,37℃倒置培养约12小时,能在含卡那霉素的LB选择性平板上长出来的就是转化有重组质粒的转化子,即为表达亚胺还原酶的工程菌。
(3)工程菌发酵培养
从LB选择性培养平板上挑单菌落,接种至3mL含50μg/mL卡那霉素的LB液体培养基中,于37℃、220rmp摇床培养过夜;取8mL培养过夜的培养液,接种至800mL含50μg/mL卡那霉素的LB液体培养基中,于37℃、220rpm培养至OD600=0.6,菌液冷却至室温,加入异丙基硫代半乳糖苷(IPTG)使其终浓度为0.1mM,18℃、220rmp培养18小时。
(4)亚胺还原酶的纯化
步骤(3)培养的菌液6000rpm、4℃离心10分钟,菌体重悬于15mL破菌缓冲液(25mMHEPES,pH 7.5,300mM NaCl,5mM咪唑,10%甘油),超声破菌,细胞裂解液12000rpm、4℃离心60分钟。按照每1L菌液用2mL Ni-IDA琼脂糖树脂的比例加入1.6mL Ni-IDA琼脂糖树脂于细胞裂解上清液中,4℃结合1小时。蛋白树脂结合物在重力作用下依次用25mM、50mM、100mM、300mM咪唑缓冲液A(25mM HEPES,pH 7.5,300mM NaCl,10%甘油)洗脱,收集洗脱液,用12%SDS-PAGE胶检测蛋白大小及纯度(图1),将蛋白纯度大于90%的洗脱液合并并回收。纯化之后的亚胺还原酶,按照GE Healthcare公司的操作说明,用PD-10的凝胶柱换到蛋白保存缓冲液B(25mM HEPES,pH 7.5,50mMNaCl,10%甘油),然后用10KDa(GE Healthcare)超滤管浓缩,之后用液氮速冻保存在-80℃。得到目标的亚胺还原酶。经NanoDrop 2000分光光度计(Thermo Scientific)测定蛋白浓度。
实施例2 亚胺底物的合成
(1)1-间甲基苯基-3,4-二氢异喹啉的合成
合成路线如下:
氮气保护下,在事先烘干的250mL圆底烧瓶中加入苯乙胺2.5mL(20mmol,1.0equiv)、60mL无水二氯甲烷,降温至0℃,同时缓慢滴加三乙胺4.2mL(30mmol,1.5equiv)和3-甲基苯甲酰氯2.9mL(22mmol,1.1equiv),滴加完后在0℃反应30分钟,然后升至室温反应24小时,反应结束后旋蒸除去溶剂,加入50mL 1M HCl,用乙酸乙酯萃取三次,每次50mL,合并乙酸乙酯相,用水洗一次,饱和食盐水洗一次并用无水硫酸镁干燥1小时,旋蒸除去溶剂既得粗产物3-甲基-N-苯乙基苯甲酰胺,该产物不需纯化直接用于下一步反应。
在25mL圆底烧瓶中,加入3-甲基-N-苯乙基苯甲酰胺1.2g(5mmol,1equiv),加入多聚磷酸(大约84%五氧化磷)6.9g,130℃保持30分钟,升温至200℃并保持3小时,反应结束后降至室温,倒入细冰中,用25%氨水调节pH=9,用二氯甲烷萃取3次,每次30mL,合并二氯甲烷相,用饱和食盐水洗一次并用无水硫酸镁干燥1小时,旋蒸除去溶剂即得粗产物1-间甲基苯基-3,4-二氢异喹啉(2a),粗产物经硅胶柱色谱分离得到纯产品为亮黄色油状物,收率为89%。
化合物2a核磁质谱数据:1H NMR(400MHz,Chloroform-d)δ7.48(d,J=1.8Hz,1H),7.44–7.37(m,2H),7.34(d,J=7.4Hz,1H),7.31–7.23(m,4H),3.91–3.83(m,2H),2.85–2.80(m,2H),2.43(s,3H).13C NMR(101MHz,Chloroform-d)δ167.4,138.9,138.8,137.9,130.6,130.1,129.3,128.9,128.0,127.9,127.4,126.6,126.0,47.6,26.4,21.5.HRMS(ESI)m/zcalcd.for C16H16N:222.1277[M+H],found:222.1279。
(2)按照上述(1)的方法,将3-甲基苯甲酰氯替换为3-氯苯甲酰氯,其余条件不变,得到粗产物1-间氯苯基-3,4-二氢异喹啉(3a),粗产物经硅胶柱色谱分离得到纯产品为亮黄色油状物,收率为83%。
化合物3a核磁质谱数据:1H NMR(400MHz,Chloroform-d)δ7.64(t,J=1.9Hz,1H),7.50(dt,J=7.4,1.5Hz,1H),7.47–7.34(m,3H),7.32–7.24(m,3H),3.93–3.82(m,2H),2.89–2.79(m,2H).13C NMR(101MHz,Chloroform-d)δ166.1,140.8,138.8,134.3,131.0,129.4,129.4,128.9,128.4,127.6,127.6,127.0,126.8,47.7,26.2.HRMS(ESI)m/zcalcd.for C15H13ClN:242.0731[M+H],found:242.0731。
(3)1-(间甲氧基苯基)-3,4-二氢异喹啉的合成
合成路线如下:
氮气保护下,在事先烘干的250mL圆底烧瓶中加入苯乙胺2.5mL(20mmol,1.0equiv)、60mL无水二氯甲烷,降温至0℃,同时缓慢滴加三乙胺4.2mL(30mmol,1.5equiv)和3-甲氧基苯甲酰氯2.9mL(22mmol,1.1equiv),滴加完后在0℃反应30分钟,然后升至室温反应24小时,反应结束后旋蒸除去溶剂,加入50mL 1M HCl,用乙酸乙酯萃取三次,每次50mL,合并乙酸乙酯相,用水洗一次,饱和食盐水洗一次并用无水硫酸镁干燥1小时,旋蒸除去溶剂既得粗产物3-甲氧基-N-苯乙基苯甲酰胺,该产物不需纯化直接用于下一步反应。
在事先烘干的100mL圆底烧瓶中,加入3-甲氧基-N-苯乙基苯甲酰胺(4mmol,1.0equiv)、超干二氯甲烷30mL、2-氯吡啶0.45mL(4.8mmol,1.2equiv),降温至-78℃,缓慢滴加三氟甲磺酸酐0.74mL(4.4mmol,1.1equiv),-78℃反应15分钟,缓慢升至室温并搅拌过夜,用饱和碳酸氢钠淬灭反应,二氯甲烷萃取三次,每次50mL,合并二氯甲烷相,用饱和食盐水洗,无水硫酸镁干燥1小时,旋蒸除去溶剂即得粗产物1-间甲氧基苯基-3,4-二氢异喹啉(4a),粗产物经硅胶柱色谱分离得到纯产品为白色固体,收率77%。
化合物4a核磁质谱数据:1H NMR(400MHz,Chloroform-d)δ7.41(m,1H),7.35m,1H),7.32–7.24(m,3H),7.22–7.15(m,2H),7.02(ddd,J=8.2,2.6,1.0Hz,1H),3.90–3.85(m,5H),2.86–2.79(m,2H).13C NMR(101MHz,Chloroform-d)δ167.2,159.5,140.3,138.8,130.7,129.1,128.8,128.0,127.4,126.6,121.4,115.6,113.7,55.4,47.6,26.3.HRMS(ESI)m/z calcd.for C16H16NO:238.1226[M+H],found:242.1227。
实施例3亚胺还原产品外消旋体的合成
(1)1-苯基-1,2,3,4-四氢异喹啉的合成
由以下路线合成:
具体操作如下所述:氮气保护条件下,在10mL事先烘干的圆底烧瓶中,加入0.5mmol 1-苯基-3,4-二氢异喹啉(1a),加入无水甲醇5mL,降温至0℃,加入0.6mmolNaBH4,0℃搅拌30分钟后,升至室温搅拌2小时,加入20mL水淬灭反应,用乙酸乙酯萃取三次,每次20mL,合并乙酸乙酯相并用无水硫酸镁干燥30分钟,旋干除去溶剂外消旋体的1-苯基-1,2,3,4-四氢异喹啉(rac-1b)为白色固体,收率96%。
化合物rac-1b核磁质谱数据(1H和13C谱图见图2、3):1H NMR(400MHz,Chloroform-d)δ7.38–7.28(m,5H),7.18(m,2H),7.06(m,1H),6.79(d,J=8.0Hz,1H),5.13(s,1H),3.33–3.26(m,1H),3.15–3.03(m,2H),2.90–2.82(m,1H),2.08(s,1H).13C NMR(101MHz,Chloroform-d)δ144.9,138.3,135.5,129.1,129.0,128.5,128.2,127.4,126.3,125.7,62.1,42.3,29.8.HRMS(ESI)m/z calcd.for C15H16N:210.1277[M+H],found:210.1277.
(2)按照上述(1)的方法,将1-苯基-3,4-二氢异喹啉(1a)替换为1-间甲基苯基-3,4-二氢异喹啉(2a),其余条件不变,得到产物外消旋体的1-间甲基苯基-1,2,3,4-四氢异喹啉(rac-2b)为白色固体,收率为98%。
化合物rac-2b核磁质谱数据(1H和13C谱图见图4、5):1H NMR(400MHz,Chloroform-d)δ7.25(m,1H),7.22–7.17(m,2H),7.16–7.12(m,2H),7.12–7.05(m,2H),6.83–6.77(m,1H),5.13(s,1H),3.36–3.28(m,1H),3.17–3.07(m,2H),3.00(s,1H),2.89(m,1H),2.36(s,3H).13C NMR(101MHz,Chloroform-d)δ144.2,138.2,137.9,135.2,129.8,129.1,128.4,128.4,128.2,126.4,126.3,125.8,62.0,42.2,29.6,21.5.HRMS(ESI)m/z calcd.forC16H18N:224.1434[M+H],found:224.1434.
(3)按照上述(1)的方法,将1-苯基-3,4-二氢异喹啉(1a)替换为1-间氯苯基-3,4-二氢异喹啉(3a),其余条件不变,得到产物外消旋体的1-间氯苯基-1,2,3,4-四氢异喹啉(rac-3b)为白色固体,收率为96%。
化合物rac-3b核磁质谱数据(1H和13C谱图见图6、7):1H NMR(400MHz,Chloroform-d)δ7.29(m,3H),7.22–7.16(m,3H),7.09(m,1H),6.77(d,J=7.7Hz,1H),5.12(s,1H),3.31–3.24(m,1H),3.15–3.03(m,2H),2.91–2.83(m,1H),2.44(s,1H).13C NMR(101MHz,Chloroform-d)δ146.8,137.3,135.4,134.4,129.8,129.3,129.2,128.1,127.7,127.4,126.7,125.9,61.6,42.1,29.6.HRMS(ESI)m/z calcd.for C15H15ClN:244.0887[M+H],found:244.0887.
(3)按照上述(1)的方法,将1-苯基-3,4-二氢异喹啉(1a)替换为1-间甲氧基苯基-3,4-二氢异喹啉(4a),其余条件不变,得到产物外消旋体的1-间甲氧基苯基-1,2,3,4-四氢异喹啉(rac-4b)为白色固体,收率为94%。
化合物rac-4b核磁质谱数据(1H和13C谱图见图8、9):1H NMR(400MHz,Chloroform-d)δ7.30–7.26(m,1H),7.18(m,2H),7.08(m,1H),6.9–6.79(m,4H),5.12(s,1H),3.80(s,3H),3.34–3.26(m,1H),3.15–3.03(m,2H),2.91–2.83(m,1H),2.71(s,1H).13C NMR(101MHz,Chloroform-d)δ159.8,146.1,137.8,135.3,129.5,129.1,128.2,126.4,125.8,121.5,114.7,112.9,61.9,55.3,42.1,29.6.HRMS(ESI)m/z calcd.for C16H18NO:240.1382[M+H],found:240.1382.
实施例4 亚胺还原酶催化亚胺的反应
(1)亚胺还原酶催化反应
反应体系为500μL,催化用缓冲液为100mM pH 7.0的磷酸钾缓冲液,其中含有20mMD-葡萄糖、0.2mg/mL葡萄糖脱氢酶(来源于枯草芽孢杆菌)、5mM烟酰胺腺嘌呤二核苷酸磷酸(NADP+)、亚胺底物2mM以及5%(v/v)助溶剂DMSO和0.5mg/mL纯化的亚胺还原酶,置于30℃、200rpm反应24小时,反应结束后加入30μL 10M NaOH,用乙酸乙酯萃取三次,每次300μL,合并乙酸乙酯相,经饱和食盐水洗,用无水硫酸镁干燥1小时。
(2)亚胺还原酶催化转化率测定
在(1)所述的反应体系经乙酸乙酯萃取干燥后,将乙酸乙酯过滤出,HPLC直接进样分析。HPLC条件为:
仪器:Shimadzu LC-20A system HPLC;
色谱柱:C18,4.6×150mm,5μm;
流动相:流动相A乙腈(添加0.01%三乙胺)、流动相B水(添加0.01%三乙胺);
流速:1mL/min;
检测波长:220nm处的UV吸光度。
转化率用产物的峰面积计算,通过与产物标品配制不同浓度测定峰面积绘制的标准曲线对比求算出转化率。
转化率结果见表2,其中IR45对1a-4a的转化率均为99%;IR96和IR99对1a的转化率分别为78%和97%,对2a转化率分别为29%和24%,。
(3)亚胺还原酶催化产物光学纯度鉴定
亚胺还原产物的光学纯度鉴定是用反应产物经乙酸酐衍生化后测定的,如(1)中所述的反应体系经乙酸乙酯萃取干燥后,将乙酸乙酯过滤出,置于2mL EP管中,挥发去除乙酸乙酯,再经冷冻干燥去除助溶剂DMSO,加入100μL HPLC级二氯甲烷、10μL乙酸酐、2μL三乙胺,封口置于30℃、200rpm反应2小时,在水浴42℃下旋蒸,除去未反应的乙酸酐、三乙胺以及反应产生的乙酸。之后,将衍生化产物用正相HPLC分析光学纯度。
正相HPLC条件为:
仪器:Agilent 1260Infinity Series HPLC System;
色谱柱:DaicelOJ-H 250mm×4.6mm,5μm;
流动相:流动相A正己烷:流动相B异丙醇;
流速:1mL/min
检测波长:220nm或254nm处的UV吸光度。
其中绝对构型是通过与文献对照(Angew.Chem.Int.Ed.2011,50,10679-10681;Angew.Chem.Int.Ed.2017,56,2725-2729)相同HPLC条件出峰顺序得到。
用于各个1-芳基-1,2,3,4-四氢异喹啉合成亚胺还原产物光学纯度分析所用液相条件如表1所示:
表1.分析亚胺还原产物的手性柱和HPLC条件
rac-1b、rac-2b、rac-3b、rac-4b标品以及IRED还原合成的1-芳基-1,2,3,4-四氢异喹啉的光学纯度分析HPLC谱图见图10-19,转化率及光学纯度统计见下表2。
表2.IRED催化还原亚胺底物1a-4a反应的转化率以及产物的e.e.结果
imine | IR45 | IR96 | IR99 |
1a | 99;99S | 78;90S | 97;96S |
2a | 99;99S | 29;47R | 24;16R |
3a | 99;91S | 74;81R | 50;21S |
4a | 99;99S | 26;8S | 16;2R |
备注:左边数值为转化率,右边粗体为产物的e.e.值,右上角的S表示产物构型为S型。
实施例5 突变蛋白改造
IR45的酶工程改造,保守位点W191的鉴定,以及突变蛋白IR45-W191A的获得及活性的测试。
(1)IR45的晶体结构模拟和与底物1a的对接
通过序列比对IR45与PDB库中的亚胺还原酶Q1EQE0(PDB:3zhb)有64%的同源性。利用Discovery Studio 4.1软件包中Modeler模块,以Q1EQE0为模板模拟出IR45的蛋白结构。而后再利用LibDock模块将亚胺化合物1a和模拟的三维结构对接获得底物和蛋白相互作用的关系。IR45与1a对接关系见图20。从图20中可以看到IR45中的W191正对着1a中的苯基取代基,两者之间的距离很近,形成了排斥作用。
(2)IR45 W191突变成Alanine和Leucine,以及IR45-W191A和IR45-W191L突变体的表达纯化和测活。
由于W191的位阻太大,将其变成更小的残基,有可能增加底物的结合能力,进而提高酶催化的效率。因此利用滚环PCR将其突变成较小的氨基酸Ala和Leu。其中,
引物1:5'-GGATTTCTTCCTGACCAGCATGAGCGGTCT-3',
引物2:5'-TCATGCTGGTCAGGAAGAAATCCAGCATGC-3',用于突变W191至L;
引物3:5'-GGATTTCTTCGCGACCAGCATGAGCGGTCT-3',
引物4:5'-TCATGCTGGTCGCGAAGAAATCCAGCATGC-3',用于突变W191至A。
滚环PCR的操作是非常经典的方法,具体步骤可以参考分子克隆第三版,这里不在赘述。突变之后的质粒,经过DpnI消化去除原始质粒之后转化大肠杆菌DH5α。提取的质粒经过测序后验证无误,转化至大肠杆菌BL21(DE3)进行表达。
IR45-W191A和IR45-W191L的表达和纯化参照IR45的相应实验完成。纯化之后的IR45-W191A和IR45-W191L用于标准底物1a的转化率和动力学测试。采用和IR45相同的条件,IR45-W191A的转化与IR45相当,也达到了99%,IR45-W191L则在90%。动力学测试的结果见表3和图21-23:IR45-W191A的Km为野生型IR45的1/170,Kcat为原先的1/21,综合起来Kcat/Km是野生型IR45的7.7倍。IR45-W191L的Kcat/Km则比IR45低了4倍。动力学数据表明IR45-W191A相对于野生型IR45具有更好的催化效率。
表3.IR45及突变蛋白的动力学参数
Enz. | Km(mM) | kcat(s-1) | kcat/Km(s-1mM-1) |
IR45 | 0.17 | 0.32 | 1.88 |
IR45-W191L | 0.027 | 0.013 | 0.48 |
IR45-W191A | 0.001 | 0.015 | 15.00 |
IR99 | 0.592 | 5.932 | 10.02 |
IR99-L173A | 0.047 | 1.396 | 29.7 |
(3)191大位阻氨基酸在绝大多数IRED呈现高度的保守性
IR45中的W191将其改成体积较小的Alaine后,能够显著的提高IRED的催化效率。
为了验证策略在其他IRED中同样有效,IR99中对应的173L被突变成Alaine。利用引物5’-GGACATCTTCGCGAACAGCCTGA-3’、5’-TCAGGCTGTTCGCGAAGATGTCC-3’对质粒pET28a-IR99进行PCR滚环突变,具体的操作方式同上描述。突变质粒经过测序确认后,转化至大肠杆菌BL21(DE3)进行表达。蛋白表达和纯化步骤同上,蛋白SDS-PAGE分析见图1。
纯化之后的IR99-L173A和对照IR99用于标准底物1a的转化率和动力学测试。采用和IR99相同的条件,IR99-L173A的转化率从97%提高到了99%。动力学测试的结果见表3和图24、25:IR99-L173A的Km为野生型IR96的1/12,kcat/Km为野生型的2.97倍,催化性能具有一定程度的提高,证实了缩小该位点氨基酸残基体积对提高底物结合能力的重要作用。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 武汉大学
<120> 亚胺还原酶及其突变体在合成(S)-1-芳基-1,2,3,4-四氢异喹啉中的应用
<130> 2017-08-25
<141> 2017-09-05
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 303
<212> PRT
<213> Artificial Sequence
<400> 1
Met Ala Thr Thr Thr Asn Ser Thr Asn Pro Thr Thr Ala Ser Thr Leu
1 5 10 15
Thr Pro Val Thr Val Ile Gly Leu Gly Ala Met Gly Gln Ala Leu Ala
20 25 30
Gly Ala Phe Leu Lys Ala Gly His Pro Thr Thr Ile Trp Asn Arg Ser
35 40 45
Pro Gly Lys Gly Glu Asp Leu Val Ala Arg Gly Ala Thr Arg Ala Ala
50 55 60
Thr Pro Ala Glu Ala Val Arg Ala Gly Glu Val Val Val Val Cys Val
65 70 75 80
Val Asp Tyr Glu Ala Ser Gln Ser Ile Leu Glu Pro Ile Ala Ala Asp
85 90 95
Leu Ala Gly Arg Val Leu Val Asn Val Thr Ser Asp Ala Pro Glu Arg
100 105 110
Ala Arg Glu Ala Gly Glu Trp Ala Ala Glu His Asp Ile Ala Tyr Leu
115 120 125
Asp Gly Ala Val Met Ile Pro Thr Val Met Ile Gly Thr Pro Asp Ala
130 135 140
Leu Leu Phe Tyr Ser Gly Asp Lys Ala Ala Tyr Asp Lys His Glu Gly
145 150 155 160
Leu Leu Lys Ser Leu Gly Gly Gln Ser Ala Tyr Val Gly Ala Asp His
165 170 175
Gly Leu Ala Ala Val Tyr Asp Leu Ser Met Leu Asp Phe Phe Trp Thr
180 185 190
Ser Met Ser Gly Leu Val His Gly Tyr Ala Leu Ala Ala Lys Asp Gly
195 200 205
Val Pro Ala Ala Ser Ile Ala Pro Phe Leu Lys Ser His Ile Ser Leu
210 215 220
Leu Ser Leu Leu Val Glu Glu Thr Ala Lys Asn Leu Asp Glu Gly Ala
225 230 235 240
Tyr Pro Gly Ala Glu Ala Asn Leu Ala Met Glu Val Glu Gly Ile Glu
245 250 255
His Ile Leu His Ala Ala Glu Arg Arg Gly Leu Asp Val Ser Val Leu
260 265 270
Arg Gly Val Arg Asp Val Ala Gln Arg Ala Val Asp Leu Gly His Gly
275 280 285
Ala Asp Ser Trp Ser Ala Thr Val Glu Gly Ala Arg Asn Pro Ala
290 295 300
<210> 2
<211> 290
<212> PRT
<213> Artificial Sequence
<400> 2
Met Ser Glu Asn Ala Ser Gly Ile Ser Phe Ile Gly Leu Gly Pro Met
1 5 10 15
Gly Gln Ala Met Val Arg Thr Phe Leu Asp Asn Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Thr Ala Ala Arg Ala Asp Gly Val Val Ala Ser Gly
35 40 45
Ala Val Leu Ala Gly Thr Val Ala Glu Val Leu Lys Ala Asn Glu Leu
50 55 60
Val Ile Leu Ser Leu Thr Asp Tyr Gln Ala Met Tyr Asp Ile Leu Gly
65 70 75 80
Gln Ala Glu Asp Ser Leu Ser Gly Arg Val Val Val Asn Leu Ser Ser
85 90 95
Asp Thr Pro Glu Glu Thr Arg Ala Ala Ala Ala Trp Leu Ala Glu Arg
100 105 110
Gly Ala Glu Leu Val Val Gly Gly Val Met Val Pro Pro Glu Leu Val
115 120 125
Gly Lys Glu Asn Ala Tyr Val Phe Tyr Ser Gly Pro Arg Ala Val Phe
130 135 140
Asp Ala His Glu Pro Val Leu Lys Leu Ile Gly Arg Pro Asp Tyr Arg
145 150 155 160
Gly Glu Asp His Ile Leu Ala Gln Leu Phe Tyr Gln Ala Gln Leu Asp
165 170 175
Ile Phe Leu Thr Thr Leu Ser Ala Tyr Met His Ala Val Ala Leu Val
180 185 190
Gly Ser Ala Gly Val Ala Ala Glu Thr Phe Gln Pro Tyr Ala Val Ala
195 200 205
Leu Phe Asp Glu Met Ser Phe Phe Leu Asp Gly Thr Gly Arg Gln Leu
210 215 220
Asp Asn Gly Asp Tyr Pro Gly Glu Leu Gly Asn Ala Ala Met Met Gly
225 230 235 240
Ala Thr Ala Asp His Ile Val Gly Ala Ser Glu Asp Ala Gly Ile Asp
245 250 255
Leu Val Leu Pro Lys Ala Val Lys Ala His Tyr Asp Arg Ala Ile Ala
260 265 270
Ala Gly His Gly Lys Asp His Trp Thr Ser Leu Phe Glu Val Ile Lys
275 280 285
Lys Pro
290
<210> 3
<211> 286
<212> PRT
<213> Artificial Sequence
<400> 3
Met Ile Thr Leu Ile Gly Leu Gly Pro Met Gly Gln Ala Met Val Arg
1 5 10 15
Val Leu Leu Glu Asn Gly His Gly Val Thr Val Trp Asn Arg Thr Ala
20 25 30
Ser Arg Ala Asp Gly Val Val Ala Ala Gly Ala Val Arg Ala Glu Thr
35 40 45
Pro Ala Asp Ala Val Ala Ala Ser Glu Leu Val Leu Leu Ser Leu Thr
50 55 60
Asp Tyr Ala Ala Met Tyr Asp Ile Leu Gly Lys Ala Gly Glu Thr Leu
65 70 75 80
Ala Gly Lys Val Val Val Asn Leu Ser Ser Asp Thr Pro Glu Lys Thr
85 90 95
Arg Glu Ala Ala Glu Trp Val Lys Ala Arg Gly Gly Gln Phe Ile Ala
100 105 110
Gly Gly Val Met Val Pro Ala Pro Leu Val Gly Lys Glu Glu Ala Tyr
115 120 125
Val Phe Tyr Ser Gly Pro Thr Glu Val Phe Glu Lys His Arg Glu Val
130 135 140
Leu Ala Leu Ile Gly Arg Ala Asp Phe Leu Gly Glu Asp Val Arg Leu
145 150 155 160
Ala Gln Leu Phe Tyr Gln Ala Gln Leu Asp Ile Phe Leu Asn Ser Leu
165 170 175
Ser Ala Phe Met His Ala Ser Ala Leu Val Arg Ser Ala Gly Val Pro
180 185 190
Leu Glu Lys Phe Leu Pro Tyr Ala Lys Asp Asn Phe Ala Met Met Gly
195 200 205
Phe Tyr Leu Glu Ala Ala Val Glu Gln Ile Glu Lys Gly Asp His Pro
210 215 220
Gly Asp Glu Ala Asp Val Ile Met Met Gly Ala Ser Ala Asp His Ile
225 230 235 240
Val Gln Ala Ser Arg Asp Ala Gly Ile Asp Val Ala Leu Pro Glu Ala
245 250 255
Val Lys Ser His Tyr Asp Arg Ala Ile Ala Ala Gly His Gly Arg Ser
260 265 270
Ser Trp Thr Ser Leu Phe Glu Ile Ile Lys Ala Asp Gly Arg
275 280 285
<210> 4
<211> 912
<212> DNA
<213> Artificial Sequence
<400> 4
atggccacca caaccaacag caccaacccg accaccgcca gtaccctgac accggtgacc 60
gtgatcggtc tgggtgccat gggtcaggcc ctggcaggtg catttctgaa agccggccac 120
cctacaacca tttggaatcg tagcccgggc aaaggtgaag atctggttgc ccgcggtgcc 180
acacgtgcag caacaccggc agaagccgtg cgtgcaggtg aagtggttgt ggtgtgcgtg 240
gttgattacg aggccagcca gagtattctg gaaccgattg cagcagacct ggccggccgt 300
gtgctggtta atgtgaccag cgatgcaccg gaacgtgccc gcgaagcagg tgaatgggca 360
gcagagcatg atatcgccta cctggacggc gcagttatga ttccgaccgt gatgatcggc 420
accccggatg cactgctgtt ttacagcggc gataaagccg cctacgataa acacgaaggc 480
ctgctgaaaa gcctgggtgg tcagagcgcc tatgtgggtg ccgatcatgg cctggcagcc 540
gtttatgatc tgagcatgct ggatttcttc tggaccagca tgagcggtct ggtgcatggc 600
tacgccctgg cagcaaagga tggtgtgccg gccgcaagca ttgccccttt cctgaagagc 660
cacattagcc tgctgagcct gctggtggag gaaaccgcca aaaacctgga tgagggtgca 720
tatccgggcg ccgaagccaa tctggccatg gaagtggagg gcatcgaaca tattctgcat 780
gccgccgaac gtcgcggctt agatgttagc gtgctgcgcg gcgttcgtga tgtggcacag 840
cgtgccgttg atctgggcca cggtgccgat agctggagtg caaccgtgga aggtgcccgc 900
aatccggcct aa 912
<210> 5
<211> 873
<212> DNA
<213> Artificial Sequence
<400> 5
atgagcgaga acgccagcgg tatcagcttt attggtctgg gcccgatggg tcaggcaatg 60
gtgcgcacct ttctggacaa cggccaccct accacagttt ggaaccgtac cgccgcacgt 120
gcagatggcg tggtggcaag tggcgcagtg ctggcaggta ccgttgccga agtgctgaaa 180
gccaacgaac tggtgatcct gagcctgacc gactatcagg ccatgtacga tatcctgggc 240
caggcagaag atagcctgag cggtcgcgtt gtggtgaatc tgagcagtga taccccggaa 300
gaaacccgtg cagcagcagc atggttagcc gaacgcggtg cagaactggt ggttggcggt 360
gttatggttc cgccggagct ggttggcaaa gaaaacgcct acgtgtttta tagcggcccg 420
cgtgccgtgt ttgatgccca tgaacctgtg ctgaagctga tcggtcgccc ggattatcgt 480
ggcgaagacc atatcctggc ccagctgttc taccaggccc agctggacat ctttctgacc 540
accctgagcg cctacatgca tgccgtggca ctggtgggta gcgcaggtgt ggcagcagaa 600
acctttcagc cgtacgccgt ggccctgttc gacgagatga gctttttcct ggatggcacc 660
ggccgtcagc tggataatgg cgattatccg ggtgaactgg gcaatgccgc catgatgggt 720
gcaaccgccg atcatattgt gggtgccagc gaagatgccg gcattgatct ggtgctgccg 780
aaagccgtga aagcccacta cgatcgtgca attgccgccg gtcatggcaa agatcactgg 840
accagcctgt ttgaggtgat caaaaaaccg taa 873
<210> 6
<211> 861
<212> DNA
<213> Artificial Sequence
<400> 6
atgattaccc tgattggtct gggcccgatg ggtcaggcca tggttcgcgt gctgctggaa 60
aacggtcatg gcgttaccgt ttggaatcgc accgccagcc gtgccgatgg tgtggttgca 120
gcaggtgccg tgcgcgcaga aacccctgca gatgccgtgg cagccagtga actggtgctg 180
ctgagcctga ccgactacgc cgcaatgtac gatatcctgg gcaaagcagg tgaaaccctg 240
gccggtaaag tggtggtgaa tctgagcagc gacaccccgg aaaaaacccg tgaagcagcc 300
gaatgggtga aagcccgcgg tggccagttt attgcaggcg gtgtgatggt gccggcacct 360
ctggttggta aagaagaggc ttatgtgttt tacagcggcc cgaccgaagt tttcgaaaaa 420
caccgcgagg ttttagccct gattggtcgt gccgatttcc tgggcgaaga tgttcgcctg 480
gcccagctgt tttatcaggc ccagctggac atcttcctga acagcctgag cgcctttatg 540
catgcaagcg ccctggttcg tagcgccggt gttccgctgg aaaaattcct gccgtatgca 600
aaagataatt ttgccatgat gggcttctat ctggaggccg ccgtggagca aatcgagaaa 660
ggcgatcatc cgggtgatga ggcagacgtg attatgatgg gcgccagcgc cgaccatatt 720
gtgcaggcaa gtcgtgatgc aggcattgat gttgcactgc cggaagccgt gaagagccat 780
tacgatcgtg ccattgcagc aggccatggt cgcagtagct ggaccagcct gttcgagatc 840
attaaagccg atggccgcta a 861
Claims (10)
1.一种亚胺还原酶,其特征在于:为氨基酸序列如SEQ ID NO.1-3所示的IR45、IR96、IR99中的一种。
2.根据权利要求1所述的亚胺还原酶,其特征在于:IR45、IR96、IR99的编码核苷酸分别如SEQ ID NO.4-6所示。
3.权利要求1所述的亚胺还原酶的关键保守氨基酸位点在提高其催化效率中的应用,其特征在于:IR45的关键保守氨基酸位点为191位,IR99的关键保守氨基酸位点为173位。
4.根据权利要求3所述的应用,其特征在于:将IR45的191位氨基酸、IR99的173位氨基酸突变为体积较小的氨基酸。
5.一种亚胺还原酶突变体,其特征在于:为权利要求1中所述IR45的突变体IR45-W191A或IR45-W191L,或为权利要求1中所述IR99的突变体IR99-L173A。
6.一种重组载体,其特征在于:插入有权利要求1所述的亚胺还原酶或权利要求5所述的突变体的编码核苷酸。
7.根据权利要求6所述的重组载体,其特征在于:插入有脱氢酶的编码核苷酸。
8.根据权利要求7所述的重组载体,其特征在于:所述的脱氢酶为葡糖脱氢酶。
9.权利要求1或2所述的亚胺还原酶或权利要求5所述的突变体在催化合成(S)-1-芳基-1,2,3,4-四氢异喹啉中的应用。
10.根据权利要求9所述的应用,其特征在于:催化式I所示化合物合成式II所示化合物,
式I、式II中的R为氢、甲氧基或卤素。
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CN110317849A (zh) * | 2018-03-30 | 2019-10-11 | 浙江大学 | 一种制备(s)-1,2,3,4-四氢异喹啉-1-甲酸及其衍生物的方法 |
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CN114836490A (zh) * | 2022-04-29 | 2022-08-02 | 上海健康医学院 | 一种亚胺还原酶在催化合成手性2-芳基吡咯烷中的应用 |
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EP4431612A1 (de) * | 2023-03-17 | 2024-09-18 | Universität Graz | Verfahren zur herstellung von corytuberin und salutaridin |
CN116200357A (zh) * | 2023-03-21 | 2023-06-02 | 中国科学院天津工业生物技术研究所 | 亚胺还原酶突变体及其在合成手性四氢异喹啉中的应用 |
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CN117230091B (zh) * | 2023-11-16 | 2024-01-19 | 四川大学华西第二医院 | 一种亚胺还原酶ir11或其突变体及应用 |
CN117965479A (zh) * | 2023-12-13 | 2024-05-03 | 华南理工大学 | 一种亚胺还原酶突变体及其应用 |
CN117965479B (zh) * | 2023-12-13 | 2024-08-06 | 华南理工大学 | 一种亚胺还原酶突变体及其应用 |
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