CN113789353A - (2s,3s)-2-氯-3-羟基酯类化合物的制备方法 - Google Patents
(2s,3s)-2-氯-3-羟基酯类化合物的制备方法 Download PDFInfo
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- CN113789353A CN113789353A CN202111125131.9A CN202111125131A CN113789353A CN 113789353 A CN113789353 A CN 113789353A CN 202111125131 A CN202111125131 A CN 202111125131A CN 113789353 A CN113789353 A CN 113789353A
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Abstract
本发明属于生物技术领域，具体为(2S,3S)‑2‑氯‑3‑羟基酯类化合物的制备方法。本发明采用来源于发酵乳酸杆菌的羰基还原酶催化不对称还原2‑氯‑β‑酮酯类化合物生成化合物(2S,3S)‑2‑氯‑3‑羟基酯类化合物；包括：制备含羰基还原酶的工程菌；制备工程菌的静息细胞悬浊液，并经过超声破碎或者加压破碎后得含羰基还原酶细胞上清液，再与底物2‑氯‑β‑酮酯类化合物、葡萄糖脱氢酶、氢供体、辅因子混合，进行不对称还原反应，制得化合物(I)。本发明方法环境友好、操作简便、易于工业放大，并且底物浓度高、底物普适性广、产物收率高，具有很好的工业应用前景。
Description
技术领域
本发明属于生物制药技术领域，具体涉及一种(2S,3S)-2-氯-3-羟基酯类化合物的制备方法。
背景技术
(2S,3S)-2-氯-3-羟基酯类化合物是一种被广泛应用于医药化工中间体，包括地尔硫卓、西拉硫卓等手性化合物，以及活性天然产物结构中的基本骨架，其结构式如下式（I）所示，式中R1为氟、氯、溴、碘等卤素，C1-C8烷基或环烷基，噻吩基，呋喃基，萘基、吡啶基等；R2为C1-C8烷基或环烷基、单取代或多取代芳基；
（I）。
目前关于通过动态还原动力学拆分合成化合物（I）及其结构类似物的化学方法有很多报道，但这些化学方法普遍存在一些不足之处，如需要用到重金属钌以及高压条件等（Synthesis 2003, 15, 2405-2409）。不仅反应条件苛刻，而且严重污染环境，这大大限制了其在工业生产上的应用。其中生物催化动态还原动力学拆分方法由于其高立体选择性以及环境友好被大家所关注，但很少被用于合成化合物(I)及其结构类似物。Azeradzu课题组（Tetrahedron: Asymmetry 1995, 6, 2199-2210）报道了Mucor ambiguus催化还原2-氯-β-酮酯类化合物，选择性地生成(2S,3S)-2-氯-3-羟基酯类化合物，ee值高达99%，但收率仅有38%且处理产物较为复杂，此外催化底物浓度未至克级别，远远不能满足工业的需求。尽管有相关报道，但是通过羰基还原酶催化的动态还原动力学拆分来制备(2S,3S)-2-氯-3-羟基酯类化合物依然很急需，特别是适用于工业生产的方法。
本发明利用羰基还原酶催化还原化合物(II)，高效、高立体选择性（ee>99%，dr>99:1）地生成(2S,3S)-2-氯-3-羟基酯类化合物(I)。与传统化学方法相比，该方法具有反应条件温和、反应收率高，成本低廉等的优点，时空产率可以达到400 g·L-1·d-1，具备良好的工业应用价值。
发明内容
本发明目的在于提供一种底物普适性广、高底物浓度、高立体选择性、高收率的不对称还原反应合成(2S,3S)-2-氯-3-羟基酯类化合物的方法。
本发明提出的手性(2S,3S)-2-氯-3-羟基酯类化合物的制备方法，采用来源于发酵乳酸杆菌（Lactobacillus fermentum）的羰基还原酶催化不对称还原2-氯-β-酮酯类化合物，生成化合物（I）；所述2-氯-β-酮酯类化合物，其结构式为（II）所示：
式中R1为氟、氯、溴、碘等卤素，C1-C8烷基或环烷基，噻吩基，呋喃基，萘基、吡啶基等；R2为C1-C8烷基或环烷基、单取代或多取代芳基；
具体步骤为：
（1）制备含羰基还原酶基因的第一工程菌和含葡萄糖脱氢酶基因的第二工程菌；
（2）分别制备两种工程菌的静息细胞悬浊液；
（3）分别制备含羰基还原酶的细胞上清液和含葡萄糖脱氢酶基因的细胞上清液；
（4）将上述两种细胞上清液混合，然后再与化合物（II）、溶剂、氢供体和辅因子混合，进行不对称还原反应，制得化合物（I）；其反应式，如下：
其中，所述氢供体为葡萄糖，所述辅因子为NADP+/NADPH；所述羰基还原酶（简称LfSDR1）的对应氨基酸序列如SEQ ID NO.1所示。
本发明步骤（1）中，所述工程菌（包括第一工程菌、第二工程菌）含有表达载体pET-28a-LfSDR1，宿主细胞为大肠杆菌BL21(DE3)。
本发明步骤（2）中，所述制备两种工程菌的静息细胞悬浊液，具体步骤为：将第一工程菌或第二工程菌接种到含卡那霉素的培养基上，摇床活化后，扩大培养至OD600值达到0.8-1.2时，加入诱导剂，继续培养，离心收集细胞，用缓冲液重悬，获得第一静息细胞悬浊液或所述第二静息细胞悬浊液。
进一步地，所述诱导剂为IPTG，诱导剂的浓度为0.05 mM-0.8 mM；加入诱导剂后的培养条件为：培养温度为15-25 ℃，培养时间为8-24 h。
进一步地，所述缓冲液为磷酸盐缓冲液，浓度为30-300 mM。
本发明步骤（3）中，所述制备两种细胞上清液，具体步骤为：对所述第一静息细胞悬浊液或所述第二静息细胞悬浊液进行超声破碎，离心，获得所述第一细胞上清液或所述第二细胞上清液。
本发明步骤（4）中，所述两种细胞上清液的混合液中，所述第一细胞上清液和所述第二细胞上清液的体积比为20:1-1:2。更优选两者体积比为10:1-8:1。
本发明步骤（4）的不对称氧化还原反应中，所述化合物（II）的浓度为1.0-300 g/L，所述第一静息细胞悬浊液的细胞浓度为0.1 g湿重/L-25 g湿重/L，所述第二静息细胞悬浊液的细胞浓度为0.1 g湿重/L-25 g湿重/L，所述氢供体的浓度为5-750 g/L，所述辅因子的浓度为0-0.5 mM。
在整个不对称还原反应过程中，一方面羰基还原酶LfSDR1催化还原化合物(II)动态还原动力学拆分生成立体异构纯的化合物(I)；另一方面，葡萄糖脱氢酶（GDH）将葡萄糖氧化为葡萄糖内酯，同时消耗了氧化型辅酶因子NADP+，再生了还原型辅酶因子NADPH，还原型辅酶因子NADPH将提供氢给底物，自身又被氧化成氧化型辅酶因子NADP+，形成了一个辅酶因子消耗与再生的闭合回路，推动主反应的进行。
不对称还原反应中，反应的温度为20-40 ℃，反应缓冲液pH为6.0-9.0。更优选，反应温度为20-25 ℃，反应缓冲液pH为6.5-7.5。缓冲浓度为100-250 mM。
不对称还原反应中，所述溶剂为磷酸盐缓冲液与助溶剂的混合溶剂。其中，助溶剂选自高介电常数溶剂：二甲基亚砜、N，N-二甲基甲酰胺、N，N-二甲基乙酰胺，芳香族溶剂：苯、甲苯、乙苯、氯苯、溴苯，非极性溶剂：正己烷、环己烷，以及极性溶剂：乙腈、乙酸乙酯、二氯甲烷、1， 2-二氯乙烷、甲醇、乙醇、异丙醇中的一种或多种。
优选的溶剂为甲醇、磷酸缓冲液的混合溶剂，体积比为10:90。
在整个反应过程中，待反应至GC检测底物完全耗尽后，用等体积的乙酸乙酯萃取3-4次，合并有机相，用饱和碳酸氢钠洗涤2次，水洗1次，饱和食盐水洗1次，无水硫酸钠干燥，减压蒸馏除去有机溶剂，得到目标产物。
与现有发明技术相比，本技术具有以下有益效果：
本发明构建含羰基还原酶LfSDR1和含葡萄糖脱氢酶GDH的工程菌应用于催化还原化合物（II），为化合物（I）的生产提供了一种新的生物制备途径。相对于其它制备方法，使用本发明方法制备所得的含羰基还原酶LfSDR1和含葡萄糖脱氢酶GDH的工程菌具有对环境友好、操作简便、易于工业放大等优势，且能以底物普适性广、高底物浓度、高立体选择性、高收率得到化合物（I），具有很好的工业应用前景。
附图说明
图1为基因工程菌诱导表达的LfSDR1细胞上清液蛋白质SDS-PAGE电泳图。其中，M:蛋白Marker,1：基因工程菌诱导表达的LfSDR1细胞破碎液上清液。
图2为消旋品2-氯-3-羟基-3-(4-甲氧基苯基)丙酸甲酯（Ia）的HPLC分析图谱。
图3为(2S,3S)-2-氯-3-羟基-3-(4-甲氧基苯基)丙酸甲酯（Ia）的HPLC分析图谱。
图4为(2S,3S)-2-氯-3-羟基-3-(4-甲氧基苯基)丙酸甲酯（Ia）的核磁氢谱。
图5为(2S,3S)-2-氯-3-羟基-3-(4-甲氧基苯基)丙酸甲酯（Ia）的核磁碳谱。
具体实施方式
下面结合具体的实施例进一步阐述本发明。应理解这些实施例仅用于说明本发明而不是限制本发明的范围。
实施例1、基因工程菌的构建及诱导表达
用质粒pET28a-LfSDR1转化表达宿主BL21(DE3)。将该工程菌接种到含卡纳霉素抗性的5mL液体LB试管培养基种，置于37℃的摇床活化8小时，将活化后的培养物按1%转接量转接到含卡纳霉素抗性的液体LB摇瓶培养基中，发酵培养基中恒温振荡培养3小时，培养温度为37℃，转速为200rpm。待菌体浓度长到OD600=0.6-1.0时，加入0.1mM IPTG(终浓度)，18℃诱导16 h，8500 rpm离心15min收集细胞，用pH 7.0，100 mM的磷酸钠缓冲液重悬，55%的超声功率破碎15分钟，离心即得含pET28a-LfSDR1的细胞上清，置于4 ℃冰箱存放。
实施例2、(2S,3S)-2-氯-3-羟基-3-(4-甲氧基苯基)丙酸甲酯（Ia）的制备
取实施例1中的LfSDR1细胞上清液10.5 mL，GDH细胞上清液5mL, 加入含1.65 mol2-氯-3-(-甲氧基苯基)-3-氧代丙酸甲酯（IIa）的甲醇溶液2.5 mL，2 mol葡萄糖，终浓度为0.025mM的NADP+，用pH=7.0、200 mM的磷酸钠缓冲液调节体积至50 mL，然后置于30 ℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为白色固体，产率为95%，通过手性HPLC（Chiracel® OJ-H, 环己烷/异丙醇为流动相）分析，ee值≥99%，dr值≥49:1。1H NMR (400 MHz, CDCl3) δ 7.31 (dd, 2H, J = 2.1, 6.7 Hz), 6.90 (dd, 2 H, J = 2.1,6.7 Hz), 4.99 (dd, 1 H, J =4.7Hz, 8.0 Hz), 4.36 (d, 1H, J=8.0 Hz), 3.81 (s, 6 H), 2.87 (d, 1 H, J=4.7Hz); 13C NMR (100 MHz, CDCl3) δ 169.3, 159.8, 130.7, 128.0, 113.8, 74.8, 59.0,55.2，52.9。
实施例3、(2S,3S)-2-氯-3-(4-氟苯基)-3-羟基丙酸叔丁酯(Ib)的制备
取实施例1中的LfSDR1细胞上清液12.5 mL，GDH细胞上清液5 mL, 加入含2.0mmol 2-氯-3-(-氟)-3-氧代丙酸叔丁酯（IIb）的甲醇溶液5.0 mL，2 mmol葡萄糖，终浓度为0.0125 mM的NADP+，用pH=6.0、150 mM的磷酸钠缓冲液调节体积至50mL，然后置于30℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为淡黄色固体，产率为87%，通过手性HPLC（Chiracel® OD-H, 环己烷/异丙醇为流动相）分析，ee值≥98%，dr值≥49:1。1H NMR (400 MHz, CDCl3) δ 7.50(ddd, , 2H, J = 7.0, 5.7, 1.1 Hz), 7.10 (m, 2H), 5.72 (ddt , 1H, J = 7.1,5.1, 1.0 Hz), 4.79 (d,1H, J = 7.0 Hz), 3.23 (d, 1H, J = 4.9 Hz), 1.43 (s,9H);13C NMR (100 MHz, CDCl3) δ 169.54 , 163.52 , 161.50 , 127.25 , 127.19 ,115.63 , 115.47 , 82.12 , 74.96 , 60.55 , 28.07。
实施例4、(2S,3S)-2-氯-3-羟基-3-(2-萘基)丙酸甲酯(Ic)的制备
取实施例1中的LfSDR1细胞上清液15.5 mL，GDH细胞上清液5 mL, 加入含5 mmol2-氯-3-(2-萘基)-3-氧代丙酸甲酯（IIc）的甲醇溶液2.0 mL，15 mmol葡萄糖，终浓度为0.5mM的NADP+，用pH=7.5、300 mM的磷酸钠缓冲液调节体积至50mL，然后置于30 ℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为白色固体，产率为94%，通过手性HPLC（Chiracel® IA-H, 环己烷/异丙醇为流动相）分析，ee值≥99%，dr值≥56:1。1H NMR (400 MHz, CDCl3) δ 7.92 (d, 1H, J =1.6 Hz), 7.78 (dddd, 3 H, J = 18.4, 5.4,3.7,1.7 Hz), 7.59 (dd, 1 H, J = 7.4,1.5 Hz), 7.50 (m, 2 H), 5.87(dd, 1H, J=7.1,5.0 Hz), 4.82 (d, 1 H, J=7.0 Hz),3.64 (s, 3 H), 3.32 (d, 1 H, J=4.9 Hz); 13C NMR (100 MHz, CDCl3) δ 168.93 ,135.44 , 134.21 , 133.02 , 127.73 , 127.42 , 126.64 , 126.28 , 126.11 ,126.04 , 125.55 , 75.61 , 61.73 , 52.90 。
实施例5、 (2S,3S)-2-氯-3-羟基-3-(4-(三氟甲基)苯基)丙酸苄酯(Id)的制备
取实施例1中的LfSDR1细胞上清液20.5 mL，GDH细胞上清液7.5 mL, 加入含5mmol 2-氯-3-(4-(三氟甲基)苯基)-3-氧代丙酸苄酯（IId）的甲醇溶液10 mL，5 mmol葡萄糖，终浓度为0.05 mM的NADP+，用pH=6.0、100 mM的磷酸钠缓冲液调节体积至50 mL，然后置于30 ℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为淡黄色油状液体，产率为89%，通过手性HPLC（Chiracel® OJ-H, 环己烷/异丙醇为流动相）分析，ee值≥93%，dr值≥56:1。1H NMR (400 MHz,CDCl3) δ 7.64 (d, 2H, J = 7.2 Hz), 7.50 (dd, 2H, J = 7.2, 1.2 Hz), 7.32 (m,5H), 5.60 (m, 1H), 5.44 (d, 1H, J = 12.4 Hz), 5.13 (d, 1H, J = 12.3 Hz), 4.71(d, 1H, J = 7.0 Hz), 3.23 (d, 1H, J = 4.9 Hz); 13C NMR (100 MHz, CDCl3)δ169.56 , 137.97 , 135.75 , 130.02 , 129.76 , 128.37 , 128.32 , 127.97 ,127.71 , 127.69 , 125.71 , 125.68 , 125.64 , 125.61 , 125.42 , 123.27 , 74.96, 66.92 , 60.55。
实施例6、(2S,3S)-3-(5-苯并呋喃基)-2-氯-3-羟基丙酸甲酯(Ie)的制备
取实施例1中的LfSDR1细胞上清液6.5 mL，GDH细胞上清液6.5 mL, 加入含1.0mmol 2-氯-3-(5-苯并呋喃基)-3-氧代丙酸甲酯（IIe）的甲醇溶液5.0 mL，1.5 mmol葡萄糖，终浓度为0.2 mM的NADP+，用pH=6.5、250 mM的磷酸钠缓冲液调节体积至50mL，然后置于30 ℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为白色固体，产率为96%，通过手性HPLC（Chiracel® AD-H,环己烷/异丙醇为流动相）分析，ee值≥98%，dr值≥67:1。1H NMR (400 MHz, CDCl3) δ 7.72(t 1H, J = 1.5 Hz), 7.61 (d, 1 H, J = 7.5), 7.49 (d, 1 H, J = 7.5 Hz), 7.43(dd, 1H, J=7.5, 1.5 Hz), 6.71 (dd, 1H, J=7.4, 1.5 Hz), 5.67 (dd, 1 H, J=5.1Hz), 4.78 (d, 1H, J=7.0), 3.73 (s, 3H), 3.23 (d, 1H, J=4.9 Hz); 13C NMR (100MHz, CDCl3) δ 168.93 , 155.71 , 145.57 , 133.28 , 126.05 , 122.49 , 121.77 ,111.63 , 106.40 , 75.97 , 61.73 , 52.90。
实施例7、 (2S,3S)-3-(5-苯并噻吩基)-2-氯-3-羟基丙酸乙酯(If)的制备
取实施例1中的LfSDR1细胞上清液30 mL，GDH细胞上清液7.5 mL, 加入含5 mmol2-氯-3-(5-苯并噻吩基)-3-氧代丙酸乙酯（IIf）的甲醇溶液5mL，1.5 mmol葡萄糖，终浓度为0.015 mM的NADP+，用pH=7.0、100 mM的磷酸钠缓冲液调节体积至50 mL，然后置于30 ℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为灰色固体，产率为90%，通过手性HPLC（Chiracel® OJ-H, 环己烷/异丙醇为流动相）分析，ee值≥98%，dr值≥48:1。1H NMR (400 MHz, CDCl3) δ 7.93 (s,1H), 7.87 (d, 1H, J = 7.5 Hz), 7.45 – 7.36 (m, 2H), 7.34 (dd, 1H, J = 7.4,1.5 Hz), 5.83 (dd, 1H, J = 7.1, 5.0 Hz), 4.83 (d, 1H, J = 6.9 Hz), 4.42 (dq,1H, J = 12.2, 8.0 Hz), 3.99 (dq, 1H, J = 12.4, 8.0 Hz), 3.23 (d, 1H, J = 4.9Hz), 1.22 (t, 3H, J = 8.0 Hz); 13C NMR (100 MHz, CDCl3) δ 169.13 , 140.50 ,136.42 , 134.39 , 127.09 , 126.61 , 123.01 , 122.34 , 122.29 , 75.97 , 62.25, 60.55 , 14.20 。
实施例8、3-氯-4-甲基苯基（2S,3S）-2-氯-3-羟基-3-(2,3,4-三氟基)丙酸酯(Ig)的制备
取实施例1中的LfSDR1细胞上清液15 mL，GDH细胞上清液5 mL, 加入含3 mmol3-氯-4-甲基苯基2-氯-3-氧代-3-(2,3,4-三氟苯基)丙酸酯（IIg）的甲醇溶液5 mL，3.5 mmol葡萄糖，用pH=6.0、100 mM的磷酸钠缓冲液调节体积至50 mL，然后置于30℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为灰白色固体，产率为85%，通过手性HPLC（Chiracel® OS-H, 环己烷/异丙醇为流动相）分析，ee值≥99%，dr值≥57:1。1H NMR (400 MHz, CDCl3) δ 7.30 (dtd, 1H, J= 6.9, 5.8, 1.0 Hz), 7.07 (dd, 1H, J = 7.4, 1.2 Hz), 6.90 (m, 2H), 6.73 (dd,1H, J = 7.5, 2.0 Hz), 5.94 (ddd, 1H, J = 6.8, 4.9, 1.0 Hz), 5.43 (d, 1H, J =4.9 Hz), 5.04 (d, 1H, J = 6.9 Hz), 2.23 (d, 3H, J = 0.9 Hz);13C NMR (100 MHz,CDCl3) δ 172.67 , 153.22 , 153.16 , 153.00 , 151.24 , 151.20 , 151.14 ,151.04 , 150.98 , 150.00 , 149.94 , 149.78 , 147.98 , 147.92 , 147.76 ,141.51 , 141.35 , 141.19 , 139.49 , 139.33 , 139.17 , 134.46 , 132.36 ,130.12 , 126.54 , 126.52 , 126.48 , 126.45 , 126.42 , 126.39 , 123.88 ,123.82 , 123.64 , 121.47 , 118.74 , 114.01 , 113.99 , 113.95 , 113.93 ,113.85 , 113.83 , 113.79 , 113.77 , 73.21 , 73.17 , 73.13 , 59.80 , 20.54。
实施例9、环丙基（2S,3S）-2-氯-3-羟基-3-苯丙酸酯(Ih)的制备
取实施例1中的LfSDR1细胞上清液20 mL，GDH细胞上清液10 mL, 加入含6.0 mmol2-氯-3-氧代-3-苯基丙酸环丙酯（IIh）的甲醇溶液5.0 mL，8 mmol葡萄糖，终浓度为0.1 mM的NADP+，用pH=6.5、100 mM的磷酸钠缓冲液调节体积至50 mL，然后置于40 ℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为淡黄色固体，产率为87%，通过手性HPLC（Chiracel® OJ-H, 环己烷/异丙醇为流动相）分析，ee值≥96%，dr值≥78:1。1H NMR (400 MHz, CDCl3) δ 7.55 (m, 2 H),7.32 (m, 3 H), 5.51 (m, 1 H), 4.76 (d, 1H, J=7.0 Hz), 3.41 (p, 1H, J=7.0 Hz),3.23 (d, 1 H, J=4.9 Hz),0.50 (tt, 2H, J = 7.2, 4.3 Hz), 0.34 (tt, 2H, J =7.3, 4.3 Hz,); 13C NMR (100 MHz, CDCl3) δ 174.18, 138.87, 128.65, 127.99,126.70, 74.97, 60.35, 57.51, 6.82。
实施例10、(2S,3S)-2-氯-3-羟基-3-(6-异喹啉基)丙酸甲酯(Ii)的制备
取实施例1中的LfSDR1细胞上清液18 mL，GDH细胞上清液5 mL, 加入含5 mmol 2-氯-3-(6-异喹啉基)-3-氧代丙酸甲酯（IIi）的甲醇溶液2 mL，10 mmol葡萄糖，终浓度为0.25 mM的NADP+，用pH=7.0、100 mM的磷酸钠缓冲液调节体积至50mL，然后置于35 ℃的恒温水浴搅拌反应，直至反应完全。反应结束后用乙酸乙酯萃取，有机层结合，用无水硫酸钠干燥，过滤，滤液旋干为灰色固体，产率为72%，通过手性HPLC（Chiracel® IA-H, 环己烷/异丙醇为流动相）分析，ee值≥98%，dr值≥56:1。1H NMR (400 MHz, CDCl3) δ 9.18 (m,1H), 8.49 (d, 1 H, J = 7.5 Hz), 7.86 (t, 1 H, J = 1.5 Hz), 7.75 (dd, 1H, J=7.5, 1.7 Hz), 7.64 (ddd, 2H, J = 7.6, 6.3, 1.5 Hz), 5.71 (dd, 1 H, J=7.0, 5.0Hz),4.79 (d, 1H, J = 7.0 Hz), 3.73 (s, 3H), 3.23 (d, 1H, J = 4.9 Hz) ; 13C NMR(100 MHz, CDCl3) δ168.93 , 152.22 , 143.93 , 138.05 , 134.42 , 128.85 ,128.70 , 125.27 , 125.17 , 120.63 , 75.61 , 61.73 , 52.90 。
以上所述仅为本发明的较佳实施例，只为说明本发明的技术构思和特点，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。凡根据本发明所进行的任何修改、等同替换、改进等，都应涵盖在本发明的保护范围之内。
序列表
<110> 复旦大学
<120> (2S,3S)-2-氯-3-羟基酯类化合物的制备方法
<130> 001
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 247
<212> PRT
<213> 人工序列(Artificial Sequence)
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Glu Glu Asp Trp Gln Asn Ala Thr Lys Ala Val Val Asp Lys Phe Gly
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Leu Thr Gly Thr Met Leu Gly Cys Lys Tyr Gly Val Lys Ala Met Lys
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Leu Leu Thr Lys Ser Val Ala Leu Glu Cys Ala Glu Lys Gly Tyr Ala
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Claims (10)
1.一种(2S,3S)-2-氯-3-羟基酯类化合物的制备方法，采用来源于发酵乳酸杆菌的羰基还原酶催化不对称还原2-氯-β-酮酯类化合物生成化合物(2S,3S)-2-氯-3-羟基酯类化合物（I）；所述2-氯-β-酮酯类化合物的结构式为（II）所示：
式中R1为氟、氯、溴、碘等卤素，C1-C8烷基或环烷基，噻吩基，呋喃基，萘基、吡啶基等；R2为C1-C8烷基或环烷基、单取代或多取代芳基；
制备的具体步骤为：
（1）制备含羰基还原酶的第一工程菌和含葡萄糖脱氢酶的第二工程菌；
（2）分别制备两种工程菌的静息细胞悬浊液；
（3）分别制备含羰基还原酶的细胞上清液和含葡萄糖脱氢酶的细胞上清液；
（4）将上述两种细胞上清液混合，然后再与化合物(II)、溶剂、氢供体和辅因子混合，进行不对称还原反应，制得化合物(I)；其反应式，如下：
其中，所述氢供体为葡萄糖，所述辅因子为NADP+/NADPH；所述羰基还原酶的对应氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的制备方法，其特征在于，所述工程菌含有表达载体pET-28a-LfSDR1，宿主细胞为大肠杆菌BL21(DE3)。
3.如权利要求1所述的制备方法，其特征在于，步骤（2）中所述制备两种工程菌的静息细胞悬浊液，具体步骤为：将第一工程菌或第二工程菌接种到含卡那霉素的培养基上，摇床活化后，扩大培养至OD600值达到0.8-1.2时，加入诱导剂，继续培养，离心收集细胞，用缓冲液重悬，获得第一静息细胞悬浊液或所述第二静息细胞悬浊液。
4. 如权利要求3所述的制备方法，其特征在于，所述诱导剂为IPTG，诱导剂的浓度为0.05 mM-0.8 mM；加入诱导剂后的培养条件为：培养温度为15-25 ℃，培养时间为8-24 h；所述缓冲液为磷酸盐缓冲液，浓度为30-300 mM。
5.如权利要求4所述的制备方法，其特征在于，步骤（3）中所述制备两种细胞上清液，具体步骤为：对所述第一静息细胞悬浊液或所述第二静息细胞悬浊液进行超声破碎，离心，获得所述第一细胞上清液或所述第二细胞上清液。
6.如权利要求5所述的制备方法，其特征在于，步骤（4）中所述两种细胞上清液的混合液中，第一细胞上清液和第二细胞上清液的体积比为20:1-1:2。
7. 如权利要求6所述的制备方法，其特征在于，步骤（4）的不对称氧化还原反应中，所述化合物（II）的浓度为1.0-300 g/L，所述第一静息细胞悬浊液的细胞浓度为0.1 g湿重/L-25 g湿重/L，所述第二静息细胞悬浊液的细胞浓度为0.1 g湿重/L-25 g湿重/L，所述氢供体的浓度为5-750 g/L，所述辅因子的浓度为0-0.5 mM。
8. 如权利要求7所述的制备方法，其特征在于，步骤（4）的不对称氧化还原反应中，反应温度为20-40 ℃，反应缓冲液pH为6.0-9.0；缓冲浓度为100-250 mM。
9. 如权利要求8所述的制备方法，其特征在于，步骤（4）的不对称还原反应中，所述溶剂为磷酸盐缓冲液与助溶剂的混合溶剂；其中，助溶剂选自高介电常数溶剂、芳香族溶剂、非极性溶剂、极性溶剂；所述高介电常数溶剂为二甲基亚砜、N,N-二甲基甲酰胺或N,N-二甲基乙酰胺，所述芳香族溶剂为苯、甲苯、乙苯、氯苯或溴苯，所述非极性溶剂为正己烷或环己烷，所述极性溶剂为乙腈、乙酸乙酯、二氯甲烷、1, 2-二氯乙烷、甲醇、乙醇或异丙醇。
10.如权利要求1-9之一所述的制备方法，其特征在于，在整个反应过程中，待反应至GC检测底物完全耗尽后，用等体积的乙酸乙酯萃取3-4次，合并有机相，用饱和碳酸氢钠洗涤2次，水洗1次，饱和食盐水洗1次，无水硫酸钠干燥，减压蒸馏除去有机溶剂，得到目标产物。
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