CN114836490A - 一种亚胺还原酶在催化合成手性2-芳基吡咯烷中的应用 - Google Patents
一种亚胺还原酶在催化合成手性2-芳基吡咯烷中的应用 Download PDFInfo
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- CN114836490A CN114836490A CN202210475631.3A CN202210475631A CN114836490A CN 114836490 A CN114836490 A CN 114836490A CN 202210475631 A CN202210475631 A CN 202210475631A CN 114836490 A CN114836490 A CN 114836490A
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- pmir
- imine reductase
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Abstract
本发明公开了亚胺还原酶PmIR(氨基酸序列如SEQ ID No:1所示)或其活性片段在催化合成手性2‑芳基吡咯烷中的应用。所述亚胺还原酶PmIR能够将5‑芳基‑3,4‑二氢‑2H‑吡咯催化还原为(R)‑2‑芳基吡咯烷,可作为中间体进一步合成手性药物。
Description
技术领域
本发明属于酶催化手性合成领域,具体涉及亚胺还原酶PmIR在催化合成手性2-芳基吡咯烷中的应用。
背景技术
手性药物(chiral drug),是指药物分子结构中引入手性中心后,得到的一对对映异构体。手性药物不同对映体的药效活性存在着普遍差异。通常手性药物的两种异构体具有明显不同的生物活性。选择单一的异构体药物具有更佳的治疗效果,并且可减少剂量和代谢的负担,副作用更小。因此,光学异构体是药物开发和应用的热点。
手性胺是合成农药、医药的重要结构单元,在制药工业中可作为一种或多种活性药物成分(API)的关键中间体。生物酶催化剂具有反应条件温和、专一性、立体选择高、对环境友好等特点,目前,生物酶催化剂不对称催化氢化合成手性胺广泛应用于制药工业领域。用于合成手性胺的酶包括有ω-转氨酶(ω-TA),胺脱氢酶(AmDH),氨基酸脱氢酶(AADH)以及亚胺还原酶(IRED)等。
近些年亚胺还原酶的挖掘以及酶工程改造技术的飞速发展,利用亚胺还原酶合成环状手性胺的效果明显优于拆分法和去消旋化法,具有反应条件温和、催化效率高和优异的立体选择性的优点。Yu-Hui Zhang等(Stereocomplementary Synthesis ofPharmaceutically Relevant Chiral 2-Aryl-Substituted Pyrrolidines Using ImineReductases.Organic Letters,Org.Lett.2020,22,9,3367–3372)通过挖掘一系列天然存在的NADPH依赖型亚胺还原酶(IRED),筛选得到对模式底物(2-(2,5-二氟苯基)-吡咯啉)具有还原活力的IREDs。来自链霉菌(Streptomyces clavuligerus)(ScIR)的亚胺还原酶虽然具有较好的(R)-选择性,但是底物载量很低,反应体系内底物浓度仅为30mM(OrganicLetter,2020,doi:/10.1021/acs.orglett.0c00802)。寻找筛选高比酶活的亚胺还原酶合成高光学纯度的(R)2-芳基吡咯烷,在手性药物合成领域具有产业化应用前景。
发明内容
本发明针对现有技术不足,以5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯为底物,通过筛选构建的亚胺还原酶酶库,筛选得到高(R)立体选择性的亚胺还原酶PmIR。PmIR相对于现有技术公开的亚胺还原酶具有更高的活性。
本发明具体技术方案如下:
亚胺还原酶PmIR或其活性片段在催化合成手性2-芳基吡咯烷中的应用,所述亚胺还原酶PmIR氨基酸序列如SEQ ID No:1所示。
本发明一个具体的示例,所述亚胺还原酶PmIR能够将5-芳基-3,4-二氢-2H-吡咯催化还原为(R)-2-芳基吡咯烷。所述芳基选自取代或非取代的苯基或者取代或非取代的吡啶基。
优选的,所述苯基或吡啶基中的一个或多个取代位被F、Cl、Br、I、C1-C10的烷氧基(例如甲氧基、乙氧基)中的一个或多个取代。
本发明一个具体的应用,利用亚胺还原酶PmIR催化还原5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯,获得(R)-2-(2,5-二氟苯基)吡咯烷,进一步合成拉罗替尼。
本发明优点:
本发明根据已报道的亚胺还原酶(IRED)蛋白序列,通过NCBI的BLAST查询,构建了70个IRED酶库。筛选结果显示其中23个亚胺还原酶具有R构型或S构型的催化活性。其中PmIR具有很高的R构型立体选择性,产物的ee值>96%。同时,PmIR具有优良的热稳定性,30度温育120h仍有35%残余活力。PmIR在8h内可以完全转化31.7g/L的底物,且时空产率为目前相较于已报道催化生成手性2-芳基吡咯烷的亚胺还原酶(Organic Letter,2020,doi:/10.1021/acs.orglett.0c00802))更高。
附图说明
图1为亚胺还原酶IR50和PmIR催化还原5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯产物的HPLC手性分析图谱。
图2为亚胺还原酶PmIR的米氏方程曲线。
图3为亚胺还原酶PmIR最适反应pH曲线(正方形:100mM柠檬酸缓冲液pH4.0、pH5.0;圆形:100mM磷酸盐缓冲液pH5.0、pH6.0、pH7.0;三角:100mM Tris-HCl缓冲液pH7.0、pH7.5、pH8.0;倒三角:100mM三乙醇胺缓冲液pH8.0、pH9、pH10)。
图4为亚胺还原酶PmIR最适反应温度曲线。
图5为亚胺还原酶PmIR的催化稳定性结果。
图6为亚胺还原酶PmIR在最大底物载量下的反应进程曲线。
具体实施方式
以下通过实施例说明本发明的具体步骤,但不受实施例限制。
在本发明中所使用的术语,除非另有说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体实例并参照数据进一步详细描述本发明。应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
在以下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1亚胺还原酶的构建、表达及筛选
(1)亚胺还原酶的构建、表达
培养基配制:液体LB培养基;往LB培养基加入1.5%琼脂粉配制成LB琼脂固体培养基;卡那霉素(工作浓度50μg/ml)抗性液体LB培养基。
根据已报道的亚胺还原酶(IRED)蛋白序列,通过NCBI的BLAST查询(在线网址:https://www.ncbi.nlm.nih.gov/),构建了70个IRED酶库。
IRED基因合成由苏州金唯智生物科技有限公司合成,获得卡那霉素抗性的pET-28a质粒。
亚胺还原酶质粒转化至E.coil BL21(DE3)及进行蛋白表达,得到相应的亚胺还原酶。将0.5g的亚胺还原酶湿菌体使用100mM Tris-HCl Buffer pH8.0 10ml重悬浮破碎,离心后上清液加入5ml甘油混匀,得到亚胺还原酶粗酶液,置于-20℃冻存待用。
(2)亚胺底物的合成
方法1:N-乙烯基吡咯烷酮与带取代基团的苯甲酸甲酯作为原料合成底物亚胺。
将化合物1b(2-6b)加入到三口瓶,加入10倍体积的四氢呋喃,置换氮气后,室温下加入3当量的60%NaH,加完后,移至60℃油浴锅,待反应液温度升至60℃后,滴加使用四氢呋喃溶解的N-乙烯基吡咯烷酮溶液,加完后升温至回流温度,薄层色谱(TLC)点板监测反应是否结束。反应结束后,待反应液冷却至室温,使用6M HCl调pH至2~3,使用乙酸乙酯萃取3次,合并后使用饱和食盐水洗一次,收集到的乙酸乙酯相使用无水硫酸钠干燥,过滤后旋干,获得粗品1c(2-6c)。
将1c(2-6c)加入适量的四氢呋喃溶解,并加入10当量的6M HCl,回流,点板监测反应是否结束。反应结束后,待反应液冷却至室温,使用6M HCl调pH至2~3,使用乙酸乙酯萃取3次,保留水相。水相使用6M氢氧化钠溶液调pH至11左右,使用乙酸乙酯萃取3次,合并,使用无水硫酸钠干燥,过滤后旋干,获得的样品使用石油醚洗涤,收集石油醚相,获得化合物1a(2-6a)。
方法2:1-(叔丁氧基羰基)-2-吡咯烷酮与格氏试剂反应合成亚胺底物
将7b(8-9b)加入到三口瓶,加入10倍体积的四氢呋喃,置换氮气后,置于冰盐浴,待反应液温度降至-10℃,缓慢滴加异丙基氯化镁,反应过程温度不超过0℃,加完后搅拌反应1h。1h后,缓慢滴加使用适量的四氢呋喃溶解的1-(叔丁氧基羰基)-2-吡咯烷酮,滴加完毕后,反应温度缓慢升温至室温,点板检测反应是否结束。反应结束后,使用饱和氯化铵溶液淬灭,使用乙酸乙酯萃取3次,合并后使用饱和食盐水洗一次,收集到的乙酸乙酯相使用无水硫酸钠干燥,过滤后旋干,获得粗品7d(8-9d)。
将1-(叔丁氧基羰基)-2-吡咯烷酮加入到三口瓶,加入10倍体积的四氢呋喃,置换氮气后,置于冰盐浴,待反应液温度降至-10℃,缓慢滴加苯基溴化镁,反应过程温度不超过0℃,点板检测反应是否结束。反应结束后,使用饱和氯化铵溶液淬灭,使用乙酸乙酯萃取3次,合并后使用饱和食盐水洗一次,收集到的乙酸乙酯相使用无水硫酸钠干燥,过滤后旋干,获得粗品10d。
底物7d(8-10d)加入10当量的4M盐酸-二氧六环溶液,反应过夜,脱去-Boc基团。旋掉有机试剂,获得样品,使用丙酮打浆,过滤。将化合物加入适量甲醇,使用6MNaOH溶液调pH至10左右,过夜。反应结束后,旋掉甲醇,使用6M HCl调pH至2~3,使用乙酸乙酯萃取3次,保留水相。水相使用6M氢氧化钠溶液调pH至10左右,使用乙酸乙酯萃取3次,合并,使用无水硫酸钠干燥,过滤后旋干,获得的样品使用石油醚洗涤,收集石油醚相,获得化合物7a(8-10a)。(值得注意的是,化合物9d在脱-Boc基团一步,并没有生成固体,因此,旋干溶剂后,加入适量甲醇,调pH呈碱性,进行关环反应,获得化合物9a。
合成的化合物1-11a核磁数据如下:
3,4-Dihydro-5-(2-methylphenyl)-2H-pyrrole(1a)
棕色油状物.1H-NMR(600MHz,CDCl3):δ(ppm)7.42(d,J=7.7Hz,1H),7.27-7.19(m,3H),4.08(t,J=7.4Hz,2H),2.90(t,J=8.4Hz,2H),2.51(s,3H),2.02-1.95(m,2H)。
13C-NMR(101MHz,CDCl3):δ(ppm)175.15,137.09,135.13,131.22,128.96,128.80,125.55,62.01,38.40,22.79,21.55。
3,4-Dihydro-5-(3-methylphenyl)-2H-pyrrole(2a)
棕色固体.1H-NMR(600MHz,CDCl3):δ(ppm)7.70(s,1H),7.59(d,J=7.6Hz,1H),7.29(t,J=7.6Hz,1H),7.23(d,J=7.5Hz,1H),4.05(t,J=7.3Hz,2H),2.93(t,J=8.2Hz,2H),2.38(s,3H),2.05-1.99(m,2H).13C-NMR(101MHz,CDCl3):δ(ppm)173.51,138.08,134.54,131.10,128.31,128.13,128.86,61.47,34.95,22.67,21.35。
3,4-Dihydro-5-(4-methylphenyl)-2H-pyrrole(3a)
棕色固体.1H-NMR(600MHz,CDCl3):δ(ppm)7.73(d,J=8.0Hz,2H),7.20(d,J=7.9Hz,2H),4.04(t,J=7.3Hz,2H),2.91(t,J=8.2Hz,2H),2.37(s,3H),2.03-1.98(m,2H).
13C-NMR(101MHz,CDCl3):δ(ppm)173.17,140.46,131.95,129.13,127.58,61.44,34.90,22.67,21.43。
5-(3-Fluorophenyl)-3,4-dihydro-2H-pyrrole(4a)
棕色固体.1H-NMR(600MHz,CDCl3):δ(ppm)7.60-7.55(m,2H),7.39-7.35(m,1H),7.13-7.10(m,1H),4.09-4.06(m,2H),2.94-2.90(m,2H),2.08-2.02(m,2H).13C-NMR(101MHz,CDCl3):δ(ppm)172.28,162.83(d,J=164.6Hz),136.88(d,J=4.9Hz),129.95(d,J=5.1Hz),123.36(d,J=1.6Hz),117.18(d,J=14.4Hz),114.36(d,J=15.2Hz),61.62,35.00,22.69。
5-(3-Fluorophenyl)-3,4-dihydro-2H-pyrrole(5a)
棕色固体.1H-NMR(600MHz,CDCl3):δ(ppm)7.77-7.73(m,2H),7.02-6.98(m,2H),3.99-3.95(m,2H),2.85-2.82(m,2H),1.98-1.93(m,2H).13C-NMR(101MHz,CDCl3):δ(ppm)172.13,164.10(d,J=167.2Hz),130.93(d,J=2.3Hz),129.61(d,J=5.7Hz),115.41(d,J=14.4Hz),61.50,34.97,22.75。
5-(3,5-Dimethylphenyl)-3,4-dihydro-2H-pyrrole(6a)
棕色油状物.1H-NMR(600MHz,CDCl3):δ(ppm)7.46(s,1H),7.06(s,1H),4.04(t,J=7.4Hz,2H),2.93-2.98(m,2H),2.34(s,6H),2.03-1.98(m,2H)。13C-NMR(101MHz,CDCl3):δ(ppm)173.63,137.92,134.49,131.97,125.44,61.40,34.96,22.66,21.23。
5-(2-Fluorophenyl)-3,4-dihydro-2H-pyrrole(7a)
棕色油状物.1H-NMR(600MHz,CDCl3):δ(ppm)7.95-7.92(m,1H),7.39-7.36(m,1H),7.18-7.15(m,1H),7.10-7.07(m,1H),4.01(t,J=7.4Hz,2H),3.02-2.98(m,2H),2.04-1.99(m,2H).13C-NMR(101MHz,CDCl3):δ(ppm)170.59,161.42(d,J=169.2Hz),131.75(d,J=5.8Hz),130.06,124.15(d,J=2.3Hz),122.84(d,J=8.1Hz),60.67,37.74,22.92。
Myosmine(8a)
棕色固体.1H-NMR(600MHz,CDCl3):δ(ppm)8.99(s,1H),8.65(d,J=4.7Hz,1H),8.18(d,J=7.9Hz,1H),7.34(dd,J=7.9,4.8Hz,1H),4.08(t,J=7.4Hz,2H),2.96(t,J=8.2Hz,2H),2.12-2.00(m,2H).13C-NMR(101MHz,CDCl3):δ(ppm)171.07,151.23,149.12,134.67,130.24,123.45,61.69,34.80,22.56。
3-(3,4-Dihydro-2H-pyrrol-5-y1)-5-fluoro-2-methoxypyridine(9a)
白色固体.1H-NMR(600MHz,CDCl3):δ(ppm)8.06(d,J=2.5Hz,1H),7.17(d,J=4.9Hz,1H),4.06(t,J=7.5Hz,2H),3.92(s,3H),2.99-2.94(m,2H),2.07-2.01(m,2H).
13C-NMR(101MHz,CDCl3):δ(ppm)169.12(d,J=2.3Hz),160.76,153.42(d,J=167.9Hz),134.75(d,J=18.8Hz),133.07(d,J=8.3Hz),109.66,61.35,54.02,37.44(d,J=3.5Hz),22.71。
2-Phenyl-1-pyrroline(10a)
棕色固体.1H-NMR(600MHz,CDCl3):δ(ppm)7.84–7.83(m,2H),7.43–7.37(m,3H),4.06(t,J=7.3Hz,2H),2.93(t,J=8.2Hz,2H),2.05–2.00(m,2H).13C-NMR(101MHz,CDCl3):δ(ppm)173.30,134.64,130.31,128.43,127.61,61.55,34.93,22.68。
(3)亚胺还原酶的筛选
A.溶液配制
500mM葡萄糖溶液、100mM 5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯(11a)的DMSO或MeOH溶液、2.5mM NADP+溶液、100mM Tris-HCl Buffer pH8.0配制的GDH(100mg/ml)甘油粗酶液。
B.外消旋体2-(2,5-二氟苯基)吡咯烷的制备
使用一定量MeOH溶解化合物11a,冰水浴下加入2当量NaBH4,搅拌至室温,反应过夜。使用6M HCl溶液调pH为弱酸性,旋掉甲醇,使用6M NaOH溶液调pH=10以上,使用乙酸乙酯萃取3遍,乙酸乙酯相合并后,用无水硫酸钠干燥,过滤,旋干,获得外消旋产物。
C.500μl亚胺还原酶酶库筛选反应体系依次往2ml离心管加入亚胺还原酶甘油粗酶液275μl,GDH甘油粗酶液75μl,2.5mM NADP+溶液50μl,500mM葡萄糖溶液50μl,100mM11a的MeOH溶液50μl,于30℃、200rpm下反应30min后加入1ml IPA灭活,使用HPLC检测转化率比较活性。
以5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯(11a)为底物分析70个亚胺还原酶的活性,亚胺还原酶不对称催化还原产物使用HPLC分析,在流动相条件为正己烷(0.1%二乙胺):异丙醇=95:5(V/V),流速为1ml/min,温度为30℃,手性分析柱:AD-H。
筛选结果显示在70个亚胺还原酶里,23个亚胺还原酶具有催化活性,其中SIR、PmIR、BcIR的酶催化产物以R构型为主,其余19个以S构型为主。当中立体选择性最好的为PmIR,它们R产物的ee分别为94%和91%,其中IR53已报道用于不对称催化还原化合物16a(见表2)。结果如表1所示。
表1
PmIR催化还原5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯产物的HPLC手性分析图谱见图1。(图1A:5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯;图1B:化学合成的(±)-2-(2,5-二氟苯基)吡咯烷标准品;图1C:IR50不对称催化获得(S)-2-(2,5-二氟苯基)吡咯烷;图1D:PmIR不对称催化获得(R)-2-(2,5-二氟苯基)吡咯烷)。
实施例2亚胺还原酶PmIR对底物谱活力测定
D.500μl亚胺还原酶酶库筛选反应体系
2ml离心管依次加入实施例1中获得的PmIR粗酶液300μl,GDH甘油粗酶液75μl,500mM葡萄糖溶液50μl,2.5mM NADP+溶液25μl,200mM亚胺底物(1-11a)的甲醇溶液50μl。于30℃、220rpm下反应24h后,加入100μl 6M NaOH溶液,使用乙酸乙酯萃取三次,合并后真空抽干。加入300μl IPA溶解样品,使用HPLC检测ee值,样品的HPLC手性分析条件如下:
手性分析柱=AD-H;流动相=正己烷(0.1%二乙胺):异丙醇(95:5,v/v);流速=1ml/min;温度=30℃,检测波长=254nm。底物谱手性分析条件如表2所示。
表2
[a]流速:1ml/min;温度:30℃;检测波长254nm。
在200μl检测体系下依次往96孔板加入100mM磷酸盐缓冲液pH6.0 135μl、PmIR纯酶液15μl(1.04μg/μL)、5mM NADPH 40μl,紧接着放入30℃酶标仪预测1次,结束后迅速加入50mM亚胺底物(1~11a)的甲醇溶液(终浓度2.5mM),读取340nm处吸光值的变化,以1min以内吸光值变化计算酶活力。结果如表3所示。
表3
[a]nd:no detection。
结果表明,亚胺还原酶PmIR有一定的底物选择性,上述底物中对5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯的活性最高。
实施例3亚胺还原酶PmIR对5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯(11a)的催化活性
配制不同浓度5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯的甲醇溶液配制:75mM、65mM、50mM、35mM、25mM、20mM、12.5mM、6.25mM、3.125mM、1.5625mM。
200μl检测体系下PmIR的动力学参数:依次往96孔板加入100mM磷酸盐缓冲液pH6.0135μl、PmIR纯酶液15μl(1.04μg/μL)、5mM NADPH 40μl,紧接着放入30℃酶标仪预测1次,结束后迅速加入不同浓度5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯的甲醇溶液10μl,读取340nm处吸光值的变化,以1min以内吸光值变化计算酶活力。
根据酶活力、比活力计算公式计算比活力,使用软件Origin9.0作米氏方程曲线,求得KM、Vmax。PmIR的蛋白分子量使用在线工具Expasy测出为Mw(PmIR)=33516.93g/mol,进一步求出Kcat和Kcat/KM比值。结果如图2所示。PmIR的Vmax=0.78±0.03μmol·min-1·mg-1,KM=0.84±0.08mM,Kcat=0.44±0.02s-1,Kcat/KM=0.52±0.25s-1.mM-1。
实施例4亚胺还原酶PmIR对5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯的最适反应pH及温度的测定
(1)最适反应pH的测定
缓冲液配制:100mM柠檬酸缓冲液pH4.0、pH5.0;100mM磷酸盐缓冲液pH5.0、pH6.0、pH7.0;100mM Tris-HCl缓冲液pH7.0、pH7.5、pH8.0;100mM三乙醇胺缓冲液pH8.0、pH9.0、pH10.0
200μl体系下测定PmIR最适反应pH:依次往96孔板加入缓冲液140μl、PmIR纯酶液10μl(3.04μg/μL)、5mM NADPH 40μl,紧接着放入30℃酶标仪预测1次,结束后迅速加入100mM 5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯的甲醇溶液10μl,读取340nm处吸光值的变化,以1min以内吸光值变化计算酶活力。结果如图3所示,通过曲线可知,PmIR当反应缓冲液和pH为100mM磷酸盐缓冲液pH6.0,活性最高。因此,PmIR在不对成催化还原5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯过程中,优选100mM磷酸盐缓冲液pH6.0作为缓冲液。
(2)最适反应温度的测定
200μl体系下测定PmIR最适反应温度:在一个离心管里加入100mM磷酸盐缓冲液pH6.0140μl,5mM NADPH溶液40μl,PmIR纯酶10μl(3.04μg/μL),使用金属加热器在不同温度下预热5min后,迅速取190μl混合液加入到96孔板,并快速加入100mM 5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯的甲醇溶液10μl,读取340nm处吸光值的变化,以1min以内吸光值变化计算酶活力。结果如图4所示。结果显示,在30~45℃之间,PmIR的活性无明显变化的趋势,具有较好的温度适应范围。
实施例5亚胺还原酶PmIR的热稳定性考察
使用100mM磷酸盐缓冲液pH6.0做缓冲液,在30℃下测定亚胺还原酶PmIR的稳定性。在最适温度和最适pH条件下,反应总反应体系为200μl,包含10μL的纯酶(3.04μg/μL),40μl的5mM NADPH,10μl的50mM底物甲醇溶液,140μl PBS缓冲液(100mM,pH6.0),于30℃下温育,间隔24小时测定一次残余酶活,共测定五次。
结果如图5所示。结果显示随着PmIR在30℃下温育时间的延长,虽然对5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯催化活性逐步下降,但在120个小时以后仍对5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯保留35%的酶活力。可见PmIR具备较好的热稳定性,具有较好的工业应用潜力。
实施例6亚胺还原酶PmIR的底物载量考察
使用1.5mL甲醇溶解5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯,葡萄糖1.513g,0.2mMNADP,PmIR湿菌体2.5g,GDH湿菌体0.5g,用PBS(100mM,pH6)定容至40mL,配制不同浓度底物的反应溶液。使用2mol/L Na2CO3溶液调节pH,使反应过程中的pH维持在6.0以上。反应温度30℃,搅拌转速150rpm。每隔2个小时取样,取100μl反应液加入900μl的异丙醇终止反应,HPLC检测。结果如表4所示。结果显示亚胺还原酶PmIR的最大底物载量为174mM(31.7g/L)。该浓度下的反应进程曲线结果如图6所示。结果显示经过反应,当底物浓度为174mM(31.7g/L)时,其在反应八小时内可以将底物完全反应,其分离收率达到75.4%、96%ee。
表4
序列表
<110> 上海健康医学院
<120> 一种亚胺还原酶在催化合成手性 2-芳基吡咯烷中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 310
<212> PRT
<213> unkown
<400> 1
Met Lys Ser Ser Asn Arg Ser Glu Asn Ile Arg Val Gly Thr Glu Asn
1 5 10 15
Thr Val Gly Lys Ser Lys Ser Val Thr Val Ile Gly Leu Gly Pro Met
20 25 30
Gly Lys Ala Met Ala Ala Ala Phe Leu Glu His Gly Tyr Lys Val Thr
35 40 45
Val Trp Asn Arg Thr Ser Asn Lys Ala Asp Glu Leu Ile Thr Lys Gly
50 55 60
Ala Val Arg Ala Ser Thr Val His Glu Ala Leu Ala Ala Asn Glu Leu
65 70 75 80
Val Ile Leu Ser Leu Thr Asp Tyr Asp Ala Met Tyr Thr Ile Leu Glu
85 90 95
Pro Ala Ser Glu Asn Leu Ser Gly Lys Val Leu Val Asn Leu Ser Ser
100 105 110
Asp Thr Pro Asp Lys Ala Arg Glu Ala Ala Lys Trp Leu Ala Asn Arg
115 120 125
Gly Ala Gly His Ile Thr Gly Gly Val Gln Val Pro Pro Ser Gly Ile
130 135 140
Gly Lys Pro Glu Ser Ser Thr Tyr Tyr Ser Gly Pro Lys Glu Val Phe
145 150 155 160
Glu Ala Asn Lys Glu Thr Leu Glu Val Leu Thr Gly Thr Asp Tyr Arg
165 170 175
Gly Glu Asp Pro Gly Leu Ala Ala Leu Tyr Tyr Gln Ile Gln Met Asp
180 185 190
Met Phe Trp Thr Ala Met Leu Ser Tyr Leu His Ala Thr Ala Val Ala
195 200 205
Gln Ala Asn Gly Ile Thr Ala Glu Gln Phe Leu Pro Tyr Ala Ala Glu
210 215 220
Thr Met Ser Ser Leu Pro Lys Phe Ile Glu Phe Tyr Thr Pro Arg Ile
225 230 235 240
Asn Ala Gly Glu Tyr Pro Gly Asp Val Asp Arg Leu Ala Met Gly Met
245 250 255
Ala Ser Val Glu His Val Val His Thr Thr Gln Asp Ala Gly Ile Asp
260 265 270
Ile Thr Leu Pro Thr Ala Val Leu Glu Val Phe Arg Arg Gly Met Glu
275 280 285
Asn Gly His Ala Gly Asn Ser Phe Thr Ser Leu Ile Glu Ile Phe Lys
290 295 300
Lys Ser Asp Ile Arg Pro
305 310
Claims (7)
1.亚胺还原酶PmIR或其活性片段在催化合成手性2-芳基吡咯烷中的应用,其特征在于所述亚胺还原酶PmIR的氨基酸序列如SEQ ID No:1所示。
2.如权利要求1所述的应用,其特征在于所述亚胺还原酶PmIR将5-芳基-3,4-二氢-2H-吡咯催化还原为(R)-2-芳基吡咯烷。
3.如权利要求2所述的应用,其特征在于所述芳基选自取代或非取代的苯基或者取代或非取代的吡啶基。
4.如权利要求3所述的应用,其特征在于所述苯基或吡啶基中的一个或多个取代位被F、Cl、Br、I、C1-C10的烷氧基中的一个或多个取代。
5.如权利要求4所述的应用,其特征在于所述苯基或吡啶基中的一个或多个取代位被F、Cl、Br、I、甲氧基、乙氧基中的一个或多个取代。
7.根据权利要求6所述的应用,其特征在利用亚胺还原酶PmIR催化还原5-(2,5-二氟苯基)-3,4-二氢-2H-吡咯,获得(R)-2-(2,5-二氟苯基)吡咯烷,进一步合成拉罗替尼。
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