CN1553959A - 制备取代的2-氨基-3-(2-氨基-苯硫基)-丙酸的酶催化方法 - Google Patents
制备取代的2-氨基-3-(2-氨基-苯硫基)-丙酸的酶催化方法 Download PDFInfo
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- CN1553959A CN1553959A CNA02817772XA CN02817772A CN1553959A CN 1553959 A CN1553959 A CN 1553959A CN A02817772X A CNA02817772X A CN A02817772XA CN 02817772 A CN02817772 A CN 02817772A CN 1553959 A CN1553959 A CN 1553959A
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- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 230000002255 enzymatic effect Effects 0.000 title description 5
- MYHSTGDTIRTLEG-UHFFFAOYSA-N 2-amino-3-(2-aminophenyl)sulfanylpropanoic acid Chemical class OC(=O)C(N)CSC1=CC=CC=C1N MYHSTGDTIRTLEG-UHFFFAOYSA-N 0.000 title 1
- 150000001875 compounds Chemical class 0.000 claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
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- 108091005804 Peptidases Proteins 0.000 claims abstract description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Natural products CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 62
- 238000006243 chemical reaction Methods 0.000 claims description 43
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 20
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 20
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical group C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 20
- 125000000217 alkyl group Chemical group 0.000 claims description 18
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 13
- 125000006239 protecting group Chemical group 0.000 claims description 11
- 125000003368 amide group Chemical group 0.000 claims description 10
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 10
- 229910052736 halogen Inorganic materials 0.000 claims description 9
- 102000035195 Peptidases Human genes 0.000 claims description 8
- 125000005092 alkenyloxycarbonyl group Chemical group 0.000 claims description 8
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- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- 239000001257 hydrogen Substances 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims description 5
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 5
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 claims description 5
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 4
- FHGWEHGZBUBQKL-UHFFFAOYSA-N 1,2-benzothiazepine Chemical compound S1N=CC=CC2=CC=CC=C12 FHGWEHGZBUBQKL-UHFFFAOYSA-N 0.000 claims description 3
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- APSBXTVYXVQYAB-UHFFFAOYSA-M sodium docusate Chemical compound [Na+].CCCCC(CC)COC(=O)CC(S([O-])(=O)=O)C(=O)OCC(CC)CCCC APSBXTVYXVQYAB-UHFFFAOYSA-M 0.000 claims description 2
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 abstract description 2
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- 239000011734 sodium Substances 0.000 description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 14
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- -1 isobutyl- Chemical group 0.000 description 12
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 10
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- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 10
- 239000001488 sodium phosphate Substances 0.000 description 10
- 229910000162 sodium phosphate Inorganic materials 0.000 description 10
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 10
- 238000005160 1H NMR spectroscopy Methods 0.000 description 9
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 125000004432 carbon atom Chemical group C* 0.000 description 7
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- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 5
- BLORRZQTHNGFTI-ZZMNMWMASA-L calcium-L-ascorbate Chemical compound [Ca+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] BLORRZQTHNGFTI-ZZMNMWMASA-L 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 5
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Abstract
式(I)化合物可用于制备1,5-苯并硫氮杂类,1,5-苯并硫氮杂类可用作酶(如蛋白酶、白介素-1β-转化酶、弹性蛋白酶或血管紧张肽-转化酶)抑制剂,或者GPCR拮抗剂(缩胆囊素、血管紧张肽II受体)。本发明涉及一种制备式(I)化合物(其中R1、R2、R3和n如说明书中所描述的)的新方法,该方法包括在含有一种有机共溶剂的水性系统中使式(II)化合物(其中R1、R2、R3、n和R4如说明书中所描述的)与一种蛋白酶反应。
Description
本发明涉及一种制备式I化合物的新的酶催化方法,
其中
R1是氢或烷基;
R2是氨基保护基团;
每个R3独立地是卤素、羧基、烷氧羰基、链烯氧基羰基或苄氧基羰基;
n是1或2。
在EP0407033A中描述了将不是2-卤素丙酸的2-取代的酸的酯外消旋混合物经酶催化的立体选择性地水解成相应的对映异构酸。在皱落假丝酵母(Candida rugosa)脂肪酶同工酶、有机溶剂(例如甲苯)和还原剂的存在下进行该反应。该方法对立体选择性地生产S-酮洛芬、S-布洛芬、S-非诺洛芬、S-2-苯基丙酸和S-吲哚洛芬尤其有用。
在EP0178553中描述了用胰凝乳蛋白酶通过选择性水解相应的烷基酯制备芳族取代的L-氨基酸的方法。
在DE2804892中描述了用丝氨酸蛋白酶、特别是枯草蛋白酶Carlsberg水解N-酰基-DL-苏氨酸酯制备光学纯的N-酰基-L-苏氨酸的方法。
但是,迄今为止,文献中还没有描述过制备式I化合物的酶催化反应方法。
通过化学水解合成式I化合物的尝试导致低产量和起始物质的分解。
所以,本发明的目的是提供一种新的、创造性的制备取代的2-氨基-3-(2-氨基-苯硫基)-丙酸的方法。
可通过本发明的方法生产式I化合物,本发明提供了一种新的制备式I的取代的2-氨基-3-(2-氨基-苯硫基)-丙酸的方法,
其中
R1是氢或烷基;
R2是氨基保护基团;
每个R3独立地是卤素、羧基、烷氧羰基、链烯氧基羰基或苄氧基羰基;
n是1或2,
该方法包括在含有一种有机共溶剂的含水体系中使式II化合物与蛋白酶反应,
其中R1、R2、R3和n如上所定义,R4是烷基或苄基。
使用本文描述的酶催化方法使得在温和的反应条件下选择性地水解酯成为可能。
本文所用术语“烷基”表示含有1到12个碳原子、任选取代的直链或支链的烃残基,如甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、戊基、己基、庚基、辛基、壬基、癸基、十一烷基和十二烷基,包括它们的不同异构体。
烷基链的适宜的取代基可以是选自1-3个卤素原子(如氟或氯)和C1-4-烷氧基(如甲氧基或乙氧基)。取代的烷基的实例是2-氯乙基、2-氟乙基、2,2,2-三氟乙基或2-甲氧基乙基。
R1中烷基如上定义并且优选是含有1到7个碳原子的直链或支链烃残基。R1中烷基更优选甲基、乙基、丙基、异丙基、正丁基、仲丁基、异丁基或叔丁基。
R4中烷基如上定义并且优选是含有1到7个碳原子的任选取代的直链或支链烃残基。烷基链的适宜取代基可以选自1-3个卤素原子(如氟或氯)和C1-4-烷氧基(如甲氧基或乙氧基)。取代的烷基的实例是2-氯乙基、2-氟乙基、2,2,2-三氟乙基或2-甲氧基乙基。R4中烷基更优选是含有1到7个或1到4个碳原子的直链烃残基。其实例是甲基、乙基、丙基、正丁基、正戊基、正己基、正庚基。优选的实例是甲基、乙基或丙基。R4中最优选的烷基是甲基。
本文所用术语“氨基保护基团”指如在“有机合成中的保护基团”(GreenT.Protective Groups in Organic Synthesis,第5章,John Wiley和Sons,Inc.(1981),218-287页)中所描述的肽化学中所用的那些基团,如烯丙氧基羰基基团(ALLOC)、低级烷氧基羰基基团(例如叔丁氧基羰基(t-BOC))、取代的低级烷氧基羰基基团(例如三氯乙氧基羰基)、任选取代的芳氧基羰基基团(例如对-硝基苄氧基羰基、苄氧基羰基(Z)或苯氧基羰基)、烷酰基基团(例如甲酰基、乙酰基)、芳酰基基团(例如苯甲酰基)、卤代烷酰基基团(例如三氟乙酰基)或甲硅烷基保护基团(例如叔丁基二甲基甲硅烷基)。优选的氨基保护基团是苄氧基羰基、叔丁氧基羰基、烯丙氧基羰基或苯甲酰基,特别优选的氨基保护基团是叔丁氧基羰基。
术语“烷氧基羰基”表示连接到羰基基团(>C=O)的C1-7-烷氧基。烷氧基的实例是甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、1-仲丁氧基、异丁氧基、叔丁氧基、戊氧基、己氧基和庚氧基,包括它们的不同异构体。烷氧基羰基基团的实例是甲氧基羰基、乙氧基羰基、丙氧基羰基、叔丁氧基羰基等。优选的低级烷氧基羰基是叔丁氧基羰基。
术语“链烯氧基羰基”表示连接到羰基基团(>C=O)的链烯氧基。本文所用术语“链烯基”表示具有2到8个碳原子、优选2到4个碳原子并且具有至少一个烯烃双键的未取代或取代的烃链基,包括它们的不同异构体。实例是乙烯基、烯丙基或异丙烯基。
R3的优选的“链烯氧基羰基”是烯丙氧基羰基或异丙烯氧基羰基,更优选的是烯丙氧基羰基。
术语卤素表示氟、氯、溴或碘。优选的卤素是溴。
数字n可以是1或2,优选数字n是1。
取代基R3可以在任何可能的位置连接到苯环。如果n是1,R3可以在苯环的3、4、5或6位。优选地,R3取代基处于4位。如果n是2,两个R3取代基可以相互独立地位于苯环的3、4、5或6位。
本发明的一个优选的实施方案是一种制备式I化合物的方法,其特征是式II化合物是
其中R1、R2、R3、R4和n如上定义。
在本发明进一步优选的实施方案中,R1是氢或烷基。
更优选R1是氢;
R2是氨基保护基团。
更优选R2是氨基保护基团;
每个R3独立地是卤素、羧基或链烯氧基羰基;
R4是烷基或苄基。
更优选R4是烷基;
n是1或2。
更优选n是1。
在本发明另一个优选的实施方案中,在含有有机共溶剂的含水体系中,在pH 4.0-10、优选6.0-8.5下用蛋白酶实施该方法。
在将式II的底物酶催化水解后,通过酸化和随后萃取分离对映体纯的式I产物。
式II化合物可用于L或D对映异构体的任何可能的混合物中。优选用外消旋混合物或者只含有L-异构体的混合物完成转化。
当使用异构体混合物时,在酸化反应介质之前通过萃取步骤除去未反应的D-酯并随后萃取L-酸。
催化反应的适宜的酶是蛋白酶,优选微生物来源的廉价散装蛋白酶,更优选为芽孢杆菌蛋白酶(象Novo Nordisk生产的Savinase)或枯草蛋白酶例如Novo Nordisk的枯草蛋白酶Carlsbeg(Alkalase)或者Solvay生产的枯草蛋白酶(蛋白酶-L)或者曲霉蛋白酶(象Amano的Prozyme 6)或者Tritachium蛋白酶(Fluka的Proteinase K)。该反应的作为催化剂的最优选的酶是枯草蛋白酶Carlsberg(例如Norvo Nordisk的Alcalase)。
作为备选方案,这些酶可以以固定的形式使用。
反应在含有有机共溶剂如不水混溶的溶剂或者水可混合的有机共溶剂的含水体系中进行。水不混溶的溶剂可以以任何比例用于水相中,优选的比例是25-75%(v/v)。水混溶的有机共溶剂的用量可以高至酶的耐受量,通常是5-25%(v/v),但是不能超过50%(v/v),例如对于枯草蛋白酶Carlsberg就是如此。
反应的反应温度为0℃到50℃,优选反应温度是15℃到40℃之间,最优选的反应温度是15℃到25℃之间。
对于水相,可以使用本领域进行生化转化的常用缓冲溶液,例如最高1M,优选约5mM到50mM的磷酸钠或磷酸钾缓冲液。这种缓冲溶液还可以含有一种常用盐如氯化钠或氯化钾,以及LiSCN、Na2SO4或一种多羟基醇例如糖;该盐浓度最高1M。
适宜的有机共溶剂是技术上常用的溶剂。实例是醚(例如四氢呋喃(THF)、二噁烷或叔丁基甲基醚(TBME))、低级醇、酯(例如乙酸乙酯)、极性非质子溶剂(例如二甲亚砜(DMSO)、二甲基乙酰胺、N,N-二甲基甲酰胺(DMF)或丙酮)。优选的有机共溶剂是四氢呋喃(THF)、叔丁基甲基醚(TBME)和乙酸乙酯。
本文所用术语“低级醇”指具有一个羟基基团、含有1到8个碳原子的直链或支链烷基残基,例如甲醇、乙醇、丙醇、异丙醇、丁醇、异丁醇、叔丁醇、戊醇、己醇、庚醇或辛醇,优选甲醇、乙醇、丙醇、异丙醇、丁醇、异丁醇、叔丁醇,更优选的醇是甲醇或乙醇。
底物适于应用的总浓度为0.1-25%(w/w)的溶液。更优选的总浓度是1-10%。
加入酶后,在剧烈搅拌下,通过可控地加入碱使反应混合物的pH保持在选定的pH值。优选的碱是水性NaOH或KOH溶液。
终止反应后,首先进行相分离,然后通过用适宜的酸酸化、随后用适宜的有机溶剂萃取来对式I的对映异构纯的产物进行处理。
根据本发明合成高对映异构纯的式I化合物,高对映异构纯指对映体过量90%或更高,优选对映体过量95%或更高。
根据下面的反应方案或者本领域技术人员已知的常用方法制备式II化合物。
反应方案1:
其中R1、R2、R3和R4如式I和式II化合物所描述的。
在反应方案1中,式a)的取代的2-硝基氟代芳香族化合物和式b)的受保护的半胱氨酸(例如BOC-半胱氨酸-甲基酯)反应,得到相应的式c)的硝基-苯基取代的受保护的半胱氨酸。通常在碱性条件下(含有一种二异丙胺,例如乙基二异丙胺),合适的有机溶剂(如己烷、二异丙基醚、乙酸乙酯、甲醇、乙醇、丙醇、二氯甲烷、DMF或DMSO)中进行该反应。反应温度优选在-30℃到+150℃之间。最优选地,在110℃的温度下、在乙基二异丙胺存在下、于乙醇中与BOC-半胱氨酸-甲基酯进行该反应。
式a)化合物和式b)的受保护的半胱氨酸(例如BOC-半胱氨酸-甲基酯)可通过商业途径得到或者按照有机化学教科书(例如J.March(1992),“Advanced Organic Chemistry:Reactions,Mechanisms,andStructure”,第四版,John Wiley & Sons)中已知的方法合成。
进行该反应的第二步,在该步骤中式c)的硝基-苯基取代的受保护的半胱氨酸的硝基被还原成相应的式II的氨基-苯基取代的受保护的半胱氨酸。根据本领域中已知的方法例如有机化学教科书(例如J.March(1992),“Advanced Organic Chemistry:Reactions,Mechanisms,and Structure”,第四版,John Wiley & Sons)中已知方法进行还原反应。优选在含有有机溶剂(如己烷、二异丙基醚、乙酸乙酯、甲醇、乙醇、丙醇、二氯甲烷、DMF或DMSO,优选甲醇)的酸性介质中与合适的还原剂(例如锌和任选的NH4Cl)进行该反应。反应温度优选在-30℃到+150℃之间。
可以用R1Hal烷基化式II的氨基-苯基取代的受保护的半胱氨酸化合物的NH2-基团,其中R1如上定义,Hal为氯或溴。
式II-a和II-b化合物(见下面)是新的中间产物,因此也是本发明的主题。
或
式I化合物是合成1,5-苯并硫氮杂类的通用结构单元。这种苯并硫氮杂骨架被用作各种酶(蛋白酶、白介素-1β-转化酶、弹性蛋白酶或血管紧张肽-转化酶抑制剂,但是也作为GPCR拮抗剂(缩胆囊素、血管紧张肽II受体)中的受限二肽模拟物。可能的不同用途在G.C.Morton等,Tet.Lett.,41(2000)3029-3033中描述。
根据反应方案2制备苯并硫氮杂类:
其中,R1、R2、R3、R4和n如式I和式II化合物所描述的。
加热或者在适当试剂的存在下(例如在G.C.Morton等,Tet.Lett.,41(2000)3029-3033中所描述的)进行式I化合物的环化反应以得到式III的苯并硫氮杂类。优选在合适的溶剂如己烷、二甲苯、二异丙基醚、乙酸乙酯、甲醇、乙醇、丙醇、二氯甲烷、DMF或DMSO中,用碳二亚胺进行该反应。反应温度优选-30℃到+150℃。优选在EDAC(1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐)的存在下、在DMF中、于室温下进行该反应。
在下面的实施例中所用的缩写具有下面的含义。
ISP-MS 离子喷雾阳性质谱
ISN-MS 离子喷雾阴性质谱
EI-MS 电子冲击质谱
SFC 超临界液相色谱
NMR 核磁共振谱
IR 红外光谱
HV 高真空
min 分钟
实施例1(起始物质的制备)
(S)-3-(4-溴-2-硝基-苯硫基)-2-叔丁氧基羰基氨基-丙酸甲基酯
按照M.K.Schwarz等(J.Org.Chem.1999,64,2219-2231)描述的方法进行反应。向市售BOC-半胱氨酸-甲基酯(27.38g,116.35mmol,1eq)和N-乙基二异丙基胺(DIPEA)(4.4mL,25.75mmol,2.5eq)的EtOH(310ml)溶液加入5-溴-2-氟硝基苯(25.62g;116.45mmol;1eq)并在110℃下加热3h。通过HPLC监视反应的进程。蒸发溶剂得到红褐色油状物,其在水(600ml)和乙酸乙酯(3×300ml)之间分配。干燥(Na2SO4)并蒸发得到粗产物,用环己烷将其重结晶得到黄色晶体(38.75g,77%)。分析数据:1H-NMR(CDCl3,400MHz):1.430(s,9H,O(CH3));3.350(dd,1H,H(4′),J4’-4″=5.2Hz,J4′-5=14.4Hz);3.500(dd,1H,H(4″),J4″-4′=5.2Hz,J4″-5=14.4Hz);3.755(s,3H,OCH3);4.621(m,1H,H(5));5.301(d,1H,H(6),J6-5=6.4Hz);7.449(d,1H,H(1),J1-2=8.8Hz);7.670(dd,1H,H(2),J1-2=8.8Hz,J2-3=2.0Hz);8.278(d,1H,H(3),J2-3=2Hz).IR:3353cm-1(NH);1765cm-1(酯-C=O);1686cm-1(氨基甲酸酯-C=O);1546cm-1(酰胺-C=O);1460和1329cm-1(NO2);1220cm-1(酯).MS:m/z=452.3[M+NH4 +],用79Br;m/z=454.3[M+NH4 +],用81Br;m/z=457.3[M+Na+],用79Br;m/z=459.3[M+Na+],用81Br。Rf=0.38,在乙酰乙酯/己烷(1∶2)中,在SiO2上。
实施例2
(S)-3-(2-氨基-4-溴-苯硫基)-2-叔丁氧基羰基氨基-丙酸甲基酯
按照J.Slade等(J.Slade et.al.,J.Med.Chem.1985,28,1571-1521)所描述的方法进行反应。加入上面硝基化合物(40.34g,92.6mmol,1eq)和NH4Cl(19.81g,370.6mmol,4eq)的混合物并且将置于MeOH(950mL)中的Zn(79.37g,1.204mmol,13eq)回流加热16h,所得混合物通过硅藻土过滤并用沸腾的MeOH洗涤。浓缩后,粗产物在乙酸乙酯和NaHCO3(aq.)之间分配。层析(用乙酸乙酯/己烷/3%三乙胺)所得油得到产物(21.11g,56%)。分析数据:1H-NMR(CDCl3,400MHz):1.395(s,9H,O(CH3);3.185(d,2H,H(4),J4-5=4Hz);3.617(s,3H,OCH3);4.446(s,2H,H(7));4.528(m,1H,H(5));5.502(d,1H,H(6),J6-5=8Hz);6.793(dd,1H,H(3),J3-2=2Hz,J3-1=8.4Hz);6.868(d,1H,H(3),J3-2=2Hz),7.225(d,1H,H(1),J1-2=8.4Hz).IR:3356cm-1(-NH2,-NH);1744cm-1酯-C=O);1707cm-1(氨基甲酸酯-C=O);1540cm-1(酰胺);1250cm-1(酯).MS:m/z=405.3[M+H+],用79Br;m/z=407.3[M+H+],用81Br。Rf=0.47,在乙酰乙酯/己烷(1∶2)中,在SiO2上。
实施例3.1(大规模酶促水解)
(S)-3-(2-氨基-4-溴-苯硫基)-2-叔丁氧基羰基氨基-丙酸
将19.9g(48.02mmol)N-叔丁氧基羰基-3-(2-氨基-4-溴苯硫基)-L-丙氨酸甲基酯(97.8%)溶于750ml TBME中并在剧烈搅拌下在3L缓冲液(0.1M氯化钠,20mM磷酸钠,pH 7.5)中乳化。加入12.0ml Alcalase 2.4L和30mg枯草蛋白酶A[两种酶制剂都是Novo Nordisk生产的枯草蛋白Carlsberg]并通过可控地加入1.0N氢氧化钠溶液在剧烈搅拌下保持pH在7.5(pH-恒定)。7天后,消耗掉44.75ml 1.0N氢氧化钠溶液,转化率>99%(HPLC分析)。分离两相反应混合物。迅速用1LTBME洗涤水相以便分离少量亲脂性杂质和痕量剩余底物。用pH 7.6的1×250ml 0.1M磷酸钾缓冲液萃取合并的有机相。用32%盐酸将合并的水相酸化到pH 2并用1.5L乙酸乙酯萃取。通过搅拌下加入10%Dicalite并过滤分离所得乳液。用2×1L乙酸乙酯萃取水相。经无水硫酸钠干燥合并的乙酸乙酯相并蒸发。将所得残渣溶于二氯甲烷、蒸发并在HV中干燥得到灰黄色固态18.79g N-叔丁氧基羰基-3-(2-氨基-4-溴苯硫基)-L-丙氨酸(产率81.8%)。分析数据:1H-NMR(CDCl3,400MHz)1.399(s,9H,OC(CH3));3.184(dd,1H,SCH2,J=14.2Hz,J=6.6Hz);3.235(dd,1H,SCH2,J=14.2Hz,J=6.6Hz);4.515(m,1H,COCHNH);5.517(d,1H,CONHCH,J=6.8Hz);6.796(dd,1H,arom.,J=2Hz,J=8.4Hz);6.876(d,1H,arom.,J=2Hz);7.250(d,1H,arom.,J=8.4Hz)。IR(ATR-IR)3445和3355cm-1(-NH,-NH2);2978和2929cm-1宽峰(-COOH);1693cm-1(COOH-C=O,氨基甲酸酯-C=O);1508cm-1(酰胺-CO-NH);1247和1157cm-1(COOH)。MS(ESI-阳性离子化)m/z=391.1[M+H+],用79Br;m/z=393.1[M+H+],用81Br;m/z=413.2[M+Na+],用79Br;m/z=415.2[M+Na+],用81Br。OR[α]D=+53.3°(CHCl3;c=1.0).HPLC分析:柱子:ABZ+plus;流动相:A:0.1%TFA水溶液;B:MeCN;梯度B:30-80%0-15min,80-30%15-16min,30%16-19.5min;流速:1ml/min;压力:50-80巴;检测:UV,300nm;保留时间:12.1min(产物-酸)13.5min(底物-酯)。
实施例3.2(小规模酶促水解)
(S)-3-(2-氨基-4-溴-苯硫基)-2-叔丁氧基羰基氨基-丙酸
将1.65g(3.99mmol)N-叔丁氧基羰基-3-(2-氨基-4-溴苯硫基)-L-丙氨酸甲基酯(98%)溶于65ml TBME并在剧烈搅拌下将其在240缓冲液中(0.1M氯化钠;20mM磷酸钠,pH 7.5)乳化。加入0.75ml Alcalase2.4L[NovoNordisk生产的一种枯草蛋白酶Carlsberg]并通过可控地加入1.0N氢氧化钠溶液在剧烈搅拌下保持pH在7.5(pH-恒定)。33h后消耗4.23ml 1.0N氢氧化钠溶液,转化率>99%(HPLC分析)。用32%的盐酸酸化反应混合物到pH 2,在Decalite上过滤。相分离后用3×275ml乙酸乙酯萃取水相。合并的有机相经无水硫酸钠干燥并蒸发。将残渣溶解于二氯甲烷、蒸发并在HV中干燥得到1.56g灰黄色固态N-叔丁氧基羰基-3-(2-氨基-4-溴苯硫基)-L-丙氨酸(产率:99.8%)。
实施例3.3(用不同溶剂酶促水解)
(S)-3-(2-氨基-4-溴-苯硫基)-2-叔丁氧基羰基氨基-丙酸
剧烈搅拌下将100mg(0.242mmol)N-叔丁氧基羰基-3-(2-氨基-4-溴苯硫基)-L-丙氨酸甲基酯(98%)加入到由20%有机共溶剂(见下表)和80%缓冲溶液(0.1M氯化钠;3mM磷酸钠,pH 7.5)组成的系统中。加入100μlAlcalase 2.4L[一种Novo Nordisk生产的枯草蛋白酶Carlsberg]并通过可控地加入0.1N氢氧化钠溶液在剧烈搅拌下保持pH在7.5(pH-恒定)。反应结束时用HPLC测定转化率。
有机共溶剂,总反应体积 | 转化百分数a | 注释 |
乙酸乙酯,20ml | ~59% | 碱消耗殆尽(>200%),反应中断 |
THF,50ml | >97% | 24mg(0.058mmol)底物和50μl Alcalase |
TBME,25ml | >99% |
a转化从300nm处HPLC分析的峰面积计算。
实施例3.4(用其他酶酶促水解)
(S)-3-(2-氨基-4-溴-苯硫基)-2-叔丁氧基羰基氨基-丙酸
剧烈搅拌下将200mg(0.493mmol)N-叔丁氧基-3-(4-溴-2-氨基苯硫基)-L-丙氨酸甲基酯(93.8%)加到由5ml TBME和20ml缓冲液(0.1M氯化钠;3mM磷酸钠,pH 7.0)组成的系统中。加入酶[见下表]并通过可控地加入0.1N氢氧化钠溶液在剧烈搅拌下保持pH在7.0(pH-恒定)。终止反应后将反应混合物用25ml TBME洗涤两次,用25%HCl酸化到pH 2.5并用3×25ml乙酸乙酯萃取。在硫酸钠上干燥合并的有机相,蒸发溶剂并在高真空中干燥残渣。
酶(公司) | 酶量 | 时间h | 消耗的滴定试剂ml | 产物(量;HPLC纯) |
Prozyme 6(AmanoPharma-ceuticals) | 42mg | 44 | 4.76 | 191mg;92.2% |
Savinase 16L(NovoNordisk) | 100μl | 43 | 4.969 | 195mg;90.7% |
实施例4
(S)-(7-溴-4-氧代-2,3,4,5-四氢-苯并[b][1,4]硫氮杂-3-基)-氨基甲酸叔丁基
酯
按照G.C.Morton等(G.C.Morton et.al,Tet.Lett.,41(2000)3029-3033)所描述的方法进行反应。将酶水解产物(15.2g,38.8mmol,1eq)和EDAC(1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐)(7.44g,38.8mmol,1eq)的DMF(500mL)溶液在室温下搅拌16h,浓缩并将残渣溶解于乙酸乙酯(500ml)中并用NaHCO3(1M)水溶液(500mL)和水(500mL)萃取。干燥(Na2SO4)并浓缩得到产物(14.5g,定量的)。分析数据:1H-NMR(CDCl3,400MHz):1.409(s,9H,O(CH3));2.939(t,1H,H(4′),J=11.6Hz);3.794(dd,1H,H(4″),J4″-5=11Hz,J4″-4′=6.4Hz);4.439(m,1H,H(5));5.567(d,1H,H(6),J6-5=8Hz);7.306(d,1H,H(2),J2-1=8Hz,J2-3=2Hz);7.337(d,1H,H(3),J3-2=2Hz);7.748(d,1H,H(1),J1-2=8Hz);8.042(s,1H,H(7))。IR:3284和3184cm-1(-NH);1729cm-1(氨基甲酸酯-C=O);1678cm-1(酰胺-C=O);1574cm-1(酰胺);1251cm-1(酯)。MS:m/z=373.3[M+H+],用79Br;m/z=375.3[M+H+],用81Br;m/z=395[M+Na+],用79Br;m/z=397[M+Na+],用81Br。Rf=0.23,在乙酸乙酯/己烷(1∶2)中,在SiO2上。
实施例5
(S)-4-(2-叔丁氧基羰基氨基-2-甲氧基羰基-乙硫基)-3-硝基-苯甲酸烯丙
基酯
将4-氟-3-硝基苯甲酸(49.95g,269.8mmol,1eq)溶于甲醇(350mL),加入Cs2CO3(44.06g,135.2mmol,0.5eq)并蒸发溶剂。然后将Cs盐溶于DMF,加热到50℃并用烯丙基溴(22.8mL,269.8mmol,1eq)处理。5分钟后蒸发溶剂并将所得固体在Et2O中研磨。过滤并蒸发得到定量的粗烯丙基酯。根据M.K.Schwarz等(M.K.Schwarz et al.,J.Org.Chem.1999,64,2219-2231)描述的方法进行下一步。将烯丙基酯(30g,133.2mmol,1eq)、BOC-半胱氨酸-甲基酯(31.35g,133.2mmol,1eq)和DIPEA(68.3mL,399.6mmol,3eq)缓慢加入EtOH中(观察到放热反应)并在室温搅拌3小时,浓缩得到固体,将其用二异丙基醚(46.16g,79%)重结晶。分析数据:1H-NMR(CDCl3,400MHz):1.443(s,9H,O(CH3));3.401(dd,1H,H(7′),J7′-7″=2.4Hz,J7’-8=6Hz);3.506(dd,1H,H(7″),J7″-7′=2.4Hz,J7″-8=6Hz);3.777(s,3H,OCH3);4.608(m,1H,H(8));4.860(dt,2H,H(3′,3″),J3-2=5.6Hz;J3-1=1.6Hz);5.322(q,1H,H(1′),J=0.8Hz);5.347(t,1H,H(1″),J=0.8Hz);5.406(d,1H,H(9),J9-8=1.6Hz);6.032(m,1H,H(2));7.625(d,1H,H(5),J5-4=8.4Hz);8.201(dd,1H,H(4),J4-5=8.4Hz,J4-6=1.6Hz);8.830(d,1H,H(6),J6-4=1.6Hz).IR:3350cm-1(-NH);1742cm-1(酯-C=O);1719cm-1(Conj.酯-C=O);1686cm-1(氨基甲酸酯-C=O);1523和1334cm-1(-NO2);1245cm-1(酯).MS:m/z=441.3[M+H+];m/z=458.4[M+NH4+];m/z=463.2[M+Na+].Rf=0.40,在乙酸乙酯/己烷(1∶2)中,在SiO2上.
实施例6
(S)-3-氨基-4-(2-叔丁氧基羰基氨基-2-甲氧基羰基-乙硫基)-苯甲酸烯丙基酯
按照J.Slade等(J.Slade et.al.,J.Med.Chem.1985,28,1517-1521)描述的方法进行反应。将上面产物(506mg,1.15mmol,1eq)含水NH4Cl(20mL)和Zn(976mg,14.9mmol,13eq)在DME(15mL)中搅拌,将其在80℃下加热16h。蒸发溶剂并将残渣溶解于乙酸乙酯(250mL)并用(含水)NaHCO3(1M)(3×50mL)萃取。用Na2SO4干燥有机相并蒸发得到黄色油,其在几天后结晶(276mg,60%)。分析数据:1H-NMR(CDCl3,400MHz):1.380(s,1H,O(CH3));3.283(d,2H,H(7′,7″),J7-8=4.4Hz);3.579(s,3H,OCH3);4.445(s,2H,H(10′,10″));4.554(m,1H,H(8));4.787(ddd,2H,H(2′,2″),J=1.2Hz);5.283(dd,1H,H(1′),J1’-2=10.6Hz,J1’-1”=1.2Hz);5.390(dd,1H,H(1″),J1”-2=17.8Hz,J1”-1’=1.2Hz);5.506(d,1H,H(9),J9-8=7.6Hz);6.015(m,1H,H(8));7.360(dd,1H,H(2),J4-5=8Hz,J4-6=1.6Hz);7.398(d,1H,H(6),J6-4=1.6Hz);7.425(d,1H,H(5),J5-4=8Hz).IR:3378cm-1(-NH,-NH2);1751cm-1(酯-C=O);1713cm-1(Conj.酯-C=O);1685cm-1(氨基甲酸酯-C=O);1511cm-1(-酰胺);1220cm-1(酯).MS:m/z=411.3[M+H+];m/z=433.3[M+Na+].Rf=0.27,在乙酸乙酯/己烷(1∶2)中,在SiO2上。
实施例7.1(大规模酶促水解)
(S)-3-氨基-4-(2-叔丁氧基羰基氨基-2-羧基-乙硫基)-苯甲酸烯丙基酯
将6.4g(15.658mmol)N-叔丁氧基羰基-3-(4-烯丙氧基羰基-2-氨基苯硫基)-L-丙氨酸甲基酯(99%)溶解于480ml TBME并在剧烈搅拌下在1.5升缓冲液(0.1M氯化钠;160mM磷酸钠,pH 7.5)中乳化。加入12.0ml Alcalase 2.4L[Novo Nordisk的一种枯草蛋白酶Carlsberg]并通过可控地加入1.0N氢氧化钠溶液在剧烈搅拌下保持pH在7.5(pH-恒定)。48.5h后消耗16.6ml1.0N氢氧化钠溶液,转化率>97%(HPLC分析)。相分离后用1l TBME萃取水相。用2×0.4L 0.1M磷酸钾缓冲液(pH 7.6)萃取合并的TBME相。用32%盐酸酸化合并的水相到pH 2并用3×0.5l乙酸乙酯萃取。如果形成稳定的乳液,则通过在Dicalite上过滤完成相分离。经无水硫酸钠干燥合并的乙酸乙酯相并蒸发。将残渣溶于二氯甲烷、蒸发、HV中干燥得到5.85g黄色高粘性油状N-叔丁氧基羰基-3-(4-烯丙氧基羰基-2-氨基苯硫基)-L-丙氨酸(产率:94.6%)。分析数据:1H-NMR(DMSO,400MHz)1.338(s,9H,OC(CH3));3.077(dd,1H,SCH2,J=12.8Hz,J=6.8Hz);3.238(dd,1H,SCH2,J=13.8Hz,J=4.6Hz);3.867(m,1H,COCHNH);4.753(d,2H,COOCH2,J=5.2Hz);5.526(d,1H,CH=CH2,J=10Hz);5.538(d,1H,CH=CH2,J=17.6Hz);5.595(m,2H,NH2);6.018(ddtr,1H,CH2CH=CH2,J=16.6Hz,J=10.6Hz,J=5.6Hz);6.519(m,1H,CONHCH);6.876(d,1H,arom.CSCH,J=8.4Hz);7.330(d,1H,arom.CSCHCH,J=8.4Hz);7.338(s,1H,arom.CHCNH2).IR(ATR-IR)3420和3355cm-1(-NH,-NH2);2980和2935cm-1宽峰(-COOH);1705cm-1(COOH-C=O,氨基甲酸酯-C=O);1510cm-1(酰胺-CO-NH);1486cm-1 aromate;1229和1160cm-1(COOH);982和932cm-1(乙烯基,-C=CH2).MS(ESI-阳性离子化)m/z=397.1[M+H+];m/z=419.4[M+Na+].OR[α]D=+16.16°(CHCl3;c=1.0).HPLC分析:柱子:ABZ+plus;流动相:A:0.1%TFA水溶液;B:MeCN;梯度B:30-80%0-15min,80-30%15-16min,30%16-19.5min;流速:1ml/min;压力:50-80巴;检测:UV,300nm;保留时间:11.2min(产物-酸);12.7min(底物-酯)。
实施例7.2(小规模酶促水解)
(S)-3-氨基-4-(2-叔丁氧基羰基氨基-2-羧基-乙硫基)-苯甲酸烯丙基酯
将0.769g(1.658mmol)N-叔丁氧基羰基-3-(4-烯丙氧基羰基-2-氨基苯基-硫代)-L-丙氨酸甲基酯溶于40ml TBME并在剧烈搅拌下在220ml 0.1M氯化钠溶液、3mM磷酸钠缓冲液(pH7.5)中乳化。加入0.1ml Alcalase2.4L[Novo Nordisk的一种枯草蛋白酶Carlsberg]并通过可控地加入1.0N氢氧化钠溶液在剧烈搅拌下保持pH在7.5(pH-恒定)。45.4h后消耗23.6ml1.0N氢氧化钠溶液,转化率>97%(HPLC分析)。用100ml TBME萃取反应混合物。相分离后用32%盐酸酸化合并的水相到pH2.3并用3×250ml乙酸乙酯萃取。如果形成稳定的乳液,则通过在Dicalite上过滤完成相分离。经无水硫酸钠干燥合并的乙酸乙酯相并蒸发。将残渣溶于二氯甲烷、蒸发、HV中干燥得到604mg灰黄色固态N-叔丁氧基羰基-3-(4-烯丙氧基羰基-2-氨基苯硫基)-L-丙氨酸(产率:93.9%,纯度:95%)。
实施例7.3(用不同溶剂的酶促水解)
(S)-3-氨基-4-(2-叔丁氧基羰基氨基-2-羧基-乙硫基)-苯甲酸烯丙基酯
将200mg(0.487mmol)N-叔丁氧基羰基-3-(4-烯丙氧基羰基-2-氨基苯硫基)-L-丙氨酸甲基酯(96.7%)在剧烈搅拌下加入到由2到5ml有机共溶剂(见下表)和20ml缓冲液(0.1M氯化钠;3mM磷酸钠,pH 7.0)组成的系统中。加入Alcalase 2.4L[Novo Nordisk的一种枯草蛋白酶Carlsberg,用量见下表]并通过可控地加入0.1N氢氧化钠溶液在剧烈搅拌下保持pH在7.5(pH-恒定)。通过滴定剂的消耗监控反应,用HPLC确定产物的形成。
有机共溶剂,用量 | Alcalase,用量 | 时间h | 注释 |
乙醇,2.0ml | 100μl | 72 | 反应结束时滴定剂的消耗:115% |
丙酮,5.0ml | 200μl | 171 | 反应结束时滴定剂的消耗:103% |
THF,5.0ml | 100μl | 146 | 反应结束时滴定剂的消耗:98% |
TBME,5.0ml | 100μl | 72 | 反应结束时滴定剂的消耗:119% |
实施例7.4(用其他酶的酶促水解)
(S)-3-氨基-4-(2-叔丁氧基羰基氨基-2-羧基-乙硫基)-苯甲酸烯丙基酯
将200mg(0.487mmol)N-叔丁氧基羰基-3-(4-烯丙氧基羰基-2-氨基苯硫基)-L-丙氨酸甲基酯(96.7%)在剧烈搅拌下加入到由5ml TBME和20ml缓冲液(0.1M氯化钠;3mM磷酸钠,pH 7.0)组成的系统中。加入酶[见下表]并通过可控地加入0.1N氢氧化钠溶液在剧烈搅拌下保持pH在7.0(pH-恒定)。通过滴定剂的消耗监控反应,用HPLC确定产物的形成。
酶(公司) | 酶量 | 时间h | 注释 |
Prozyme 6(AmanoPharmaceuticals) | 50mg | 42 | 反应结束时滴定剂的消耗:112% |
蛋白酶K(Fluka) | 25mg | 137 | 慢:137h后只有40%(理论上)的滴定剂被消耗 |
实施例7.5(较高温度的酶促水解)
(S)-3-氨基-4-(2-叔丁氧基羰基氨基-2-羧基-乙硫基)-苯甲酸烯丙基酯
40℃下将200mg(0.487mmol)N-叔丁氧基羰基-3-(4-烯丙氧基羰基-2-氨基苯硫基)-L-丙氨酸甲基酯(96.7%)在剧烈搅拌下加入到由5ml TBME和20ml缓冲液(0.1M氯化钠;3mM磷酸钠,pH 7.0)组成的系统中。加入100μl Alcalase 2.4L[Novo Nordisk的一种枯草蛋白酶Carlsberg]并通过可控地加入(pH-恒定)0.1N氢氧化钠溶液在剧烈搅拌下于40℃保持pH在7.0。消耗5.44ml滴定剂(理论值的112%;17.4h后)后,将反应混合物用25mlTBME洗涤,用1N HCl酸化到pH 2.5并用3×25ml乙酸乙酯萃取。经无水硫酸钠干燥合并的有机相,蒸发溶剂并将残渣在高真空中干燥得到190mg(0.479mmol;98%)94.5%HPLC-纯(面积-%)的产物酸。
实施例8
(S)-3-叔丁氧基羰基氨基-4-氧代-2,3,4,5-四氢-苯并[b][1,4]硫氮杂-7-
羧酸烯丙基酯
按照G.C.Morton等(G.C.Morton et al.,Tet.Lett.,41(2000)3029-3033)描述的方法进行反应。将酶水解产物(5.6g,14.1mmol,1eq)溶于DMF(75ml)并用EDAC(2.70g,14.1mmol,1eq)处理并在室温下搅拌16h。浓缩后,将残渣溶于乙酸乙酯(500mL)中并用(含水的)NaHCO3(1M)(3×150mL)和水(3×150mL)萃取。干燥有机相(Na2SO4)并蒸发得到黄色油。用乙酸乙酯/己烷(1∶2)层析后得到黄色晶体(4.96g,93%)。分析数据:1H-NMR(CDCl3,400MHz):1.400(s,1H,O(CH3));3.014(t,1H,H(7′或7″),J=12Hz);3.815(dd,1H,H(7′或7″),J7-8=12Hz,J7’-7”=6.4Hz);4.466(m,1H,H(8));4.835(d,2H,H(3′,3″),J3-2=5.7Hz);5.316(dd,1H,H(1′),J1′-2=10.5Hz,J1′-1″=1.2Hz);5.413(dd,1H,H(1″),J1″-2=17.2Hz,J1″-1′=1.2Hz);5.581(d,1H,H(9),J9-8=8Hz);6.013(m,1H,H(8));7.338(d,1H,H(5),J5-4=11.1Hz);7.704(s,1H,H(10));7.751(d,1H,H(6),J6-4=1.8Hz);7.338(dd,1H,H(2),J4-5=11.1Hz,J4-6=1.8Hz).IR:3206cm-1(-NH);1720cm-1(氨基甲酸酯-C=O);1671cm-1(酰胺-C=O);1500cm-1(酰胺-CONH);1209cm-1(酯)。MS:m/z=379.3[M+H+];m/z=401.4[M+Na+].Rf=0.36,在乙酸乙酯/己烷(1∶2)中,在SiO2上.
实施例9
(S)-3-氨基-4-(2-叔丁氧基羰基氨基-2-羰基-乙硫基)-苯甲酸
将200mg(0.540mmol)N-叔丁氧基羰基-3-(4-羧基-2-氨基苯硫基)-L-丙氨酸甲基酯加入由5ml TBME和20ml缓冲液(0.1M氯化钠;3mM磷酸钠,pH 7.0)组成的系统中。加入100μl Alcalase 2.5L[Novo Nordisk的一种枯草蛋白酶Carlsberg]并通过可控地加入0.1N氢氧化钠溶液在剧烈搅拌下保持pH在7.0(pH-恒定)。消耗5.847ml滴定剂(理论值的108%;1.5h后)后,将反应混合物用25ml TBME洗涤两次,用25%HCl酸化到pH 2.5并用3×25ml乙酸乙酯萃取。经无水硫酸钠干燥合并的有机相,蒸发溶剂并将残渣在HV中干燥得到187mg(0.479mmol)黄色晶体的N-叔丁氧基羰基-3-(2-氨基-4-羧基苯硫基)-L-丙氨酸(产率:93.4%)。分析数据:HPLC纯度:96.4%(面积%).1H-NMR(DMSO,400MHz)1.378(s,9H,OC(CH3));2.982(dd,1H,SCH2,J=13.2Hz,J=9.6Hz);3.150(dd,1H,SCH2,J=13.2Hz,J=4.4Hz);3.950(m,1H,COCHNH);5.542(m,2H,-NH2);7.067(dd,1H,CONHCH,J=8.0Hz,J=1.6Hz);7.201(d,1H,arom.SCCH,J=8.0Hz);7.298(d,1H,arom.SCCHCH,J=8.0Hz);7.323(d,1H,arom.CHCNH2,J=1.6Hz);12.73(bs,2H,2-COOH).IR:3445和3347cm-1(-NH,-NH2);2977和2930cm-1宽峰(-COOH);1720和1692cm-1(COOH-C=O,氨基甲酸酯-C=O);1608cm-1(-COO-);1509cm-1(酰胺-CO-NH);1486cm-1(aromate);1247和1163cm-1(COOH).ISN-MS:m/z=355.1[M-H+].OR[α]D=+13.00°(EtOH;c=1.0)。
Claims (9)
1.一种制备式I化合物的方法
其中
R1是氢或烷基;
R2是氨基保护基团;
每个R3独立地是卤素、羧基、烷氧羰基、链烯氧基羰基或苄氧基羰基;
n是1或2,
该方法包括:
在含有一种有机共溶剂的水性系统中,使式II化合物与一种蛋白酶反应:
其中R1、R2、R3和n如上定义,R4是烷基或苄基。
2.一种根据权利要求1的方法,其特征是式II化合物是:
其中R1、R2、R3、R4和n如权利要求1所定义。
3.一种根据权利要求1或2的方法,其特征是:
R1是氢或烷基;
R2是氨基保护基团;
每个R3独立地是卤素、羧基或链烯氧基羰基;
R4是烷基或苄基;
n是1或2。
4.一种根据权利要求1-3中任一项的方法,其特征是
R1是氢;
R2是氨基保护基团;
R3是卤素、羧基或链烯氧基羰基;
R4是烷基;
n是1。
5.一种根据权利要求1-4中任一项的方法,其特征是在6.0-8.5的pH下进行反应。
6.一种根据权利要求1-5中任一项的方法,其特征是用芽孢杆菌蛋白酶、枯草杆菌蛋白酶、曲霉蛋白酶或Tritachium蛋白酶进行反应。
7.一种根据权利要求1-6中任一项的方法,其特征是所述有机共溶剂是四氢呋喃、二噁烷、叔丁基甲基醚、C1-8-醇、乙酸乙酯、二甲亚砜、二甲基乙酰胺或丙酮。
9.一种根据权利要求1-7中任一项的方法,其特征是式I化合物被进一步处理成式III的苯并硫氮杂:
其中R1、R2、R3、R4和n如式I和式II所描述的。
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CN105392891A (zh) * | 2013-11-01 | 2016-03-09 | 株式会社糖锁工学研究所 | 具有硫醇基的d-型或l-型氨基酸衍生物的制造方法 |
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DE3438189A1 (de) * | 1984-10-18 | 1986-04-24 | Hoechst Ag, 6230 Frankfurt | Verfahren zur herstellung aromatisch substituierter l-aminosaeuren |
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US5106750A (en) * | 1988-08-30 | 1992-04-21 | G. D. Searle & Co. | Enantio- and regioselective synthesis of organic compounds using enol esters as irreversible transacylation reagents |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105392891A (zh) * | 2013-11-01 | 2016-03-09 | 株式会社糖锁工学研究所 | 具有硫醇基的d-型或l-型氨基酸衍生物的制造方法 |
TWI685480B (zh) * | 2013-11-01 | 2020-02-21 | 日商糖鎖工學研究所股份有限公司 | 具有硫醇基之d-體或l-體胺基酸衍生物的製造方法 |
CN104404098A (zh) * | 2014-12-15 | 2015-03-11 | 苏州汉酶生物技术有限公司 | 一种硫辛酸的生物制备方法 |
Also Published As
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CN100413972C (zh) | 2008-08-27 |
IL160527A (en) | 2010-05-31 |
US20030119152A1 (en) | 2003-06-26 |
DK1434870T3 (da) | 2007-04-30 |
CA2461296A1 (en) | 2003-04-10 |
ES2278061T3 (es) | 2007-08-01 |
IL160527A0 (en) | 2004-07-25 |
HUP0401500A3 (en) | 2005-11-28 |
DE60217145T2 (de) | 2007-10-25 |
HU228360B1 (en) | 2013-03-28 |
US7011960B2 (en) | 2006-03-14 |
DE60217145D1 (de) | 2007-02-08 |
JP4359142B2 (ja) | 2009-11-04 |
EP1434870A1 (en) | 2004-07-07 |
KR20040044979A (ko) | 2004-05-31 |
MXPA04002694A (es) | 2004-06-18 |
WO2003029477A1 (en) | 2003-04-10 |
KR100882972B1 (ko) | 2009-02-12 |
EP1434870B1 (en) | 2006-12-27 |
JO2426B1 (en) | 2008-04-17 |
JP2005503830A (ja) | 2005-02-10 |
CA2461296C (en) | 2011-07-05 |
HUP0401500A2 (hu) | 2005-02-28 |
ATE349545T1 (de) | 2007-01-15 |
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