CN113789310B - 一种转氨酶及其在制备莫西沙星或其中间体中的应用 - Google Patents
一种转氨酶及其在制备莫西沙星或其中间体中的应用 Download PDFInfo
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- CN113789310B CN113789310B CN202110098981.8A CN202110098981A CN113789310B CN 113789310 B CN113789310 B CN 113789310B CN 202110098981 A CN202110098981 A CN 202110098981A CN 113789310 B CN113789310 B CN 113789310B
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- transaminase
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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Abstract
本发明提供了一种转氨酶,其氨基酸序列如SEQ ID NO:1所示。本发明还公开了该转氨酶在制备莫西沙星或其中间体中的应用。本发明转氨酶的酶活和比酶活均较高;将本发明的转氨酶用于制备莫西沙星或其中间体时,产率显著提高,并且还可进一步用于无DMSO的反应体系,例如使用乙醇代替DMSO作为助溶剂,后处理更简单,更有利于工业化生产。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种转氨酶及其在制备莫西沙星或莫西沙星中间体中的应用。
背景技术
莫西沙星(moxifloxaxin)是第三代喹诺酮类广谱抗菌药物,广泛应用于治疗呼吸道感染如获得性肺炎、慢性支气管炎急性发作、急性细菌性鼻窦炎等,具有抗菌性强、抗菌谱广、不易产生耐药并对常见耐药菌有效、半衰期长、不良反应少等优点。
(S,S)-2,8-二氮杂双环[4.3.0]壬烷(如下列化合物1所示)及其N-保护衍生物是合成应用广泛的喹诺酮类抗菌药莫西沙星的关键手性中间体,又称莫西沙星侧链或莫西小环:
由于(S,S)-2,8-二氮杂双环[4.3.0]壬烷含有两个手性中心,其合成方法通常有拆分法、不对称合成法和手性源法,这些方法都涉及到高压氢化技术和成本高昂的问题,工艺成本高且对环境不友好。
近来以转氨酶为催化剂制备莫西沙星中间体的方法被研究和公布。因其成本低,工艺流程短,对环境友好,能够工业化应用的合成方法成为当下的研究趋势。
CN104262225B中公开了一种莫西沙星中间体的制备方法,采用化合物Ⅲb(R为氨基保护基,Y为氯、溴、碘或羟基磺酸酯)为原料,在ω-转氨酶作用下得到化合Ⅱb,Ⅱb进行关环反应得到化合物Ιb,Ιb再脱保护,当R限定为-COOC4H9,Y为氯时,三步收率73.3%,ee值96.8%。但是并没有公布转氨酶的序列,且转化率和ee值都还有待提高。
CN106399418A也公开了底物3-(3-氯丙基)-4-羰基吡咯烷-1-甲酸苯甲酯(4C)在转氨酶(CN106399418A中的SEQ ID NO:20)作用下进行转氨反应,接着关环反应制得化合物2C,转化率99.8%,ee值99.9%,de值99.9%,2C经脱保护制得(S,S)-2,8-二氮杂双环[4,3,0]壬烷,但是该合成方法需要用到二甲基亚砜(DMSO)作为助溶剂,而DMSO在后期难以从产物中去除,后处理困难,不适合工业化生产。
本领域仍然需要一种更适合工业化生产、并且能以较高的产率和生产效率制备莫西沙星中间体的方法;尤其是不使用DMSO做助溶剂,也能够实现转氨酶催化制备莫西沙星中间体的方法。
发明内容
本发明所要解决的技术问题是为了克服现有技术中以转氨酶为催化剂制备莫西沙星或其中间体的方法中使用DMSO作为助溶剂使得后处理困难、且产率和ee、de值不高等缺陷,本发明提供了一种转氨酶、将其用于制备莫西沙星或其中间体的方法及其应用。本发明转氨酶的酶活和比酶活均较高;将本发明的转氨酶用于制备莫西沙星或其中间体例如(S,S)-2,8-二氮杂双环[4.3.0]壬烷、如本发明的式III或II所示的化合物时,产率显著提高,并且还可进一步将其用于无DMSO的反应体系,例如使用乙醇代替DMSO作为助溶剂时产率、ee值和de值均较高,且后处理更简单,更有利于工业化生产。
为了解决上述技术问题,本发明第一方面提供了一种转氨酶,所述转氨酶的氨基酸序列如SEQ ID NO:1所示。
较佳地,编码所述转氨酶的核苷酸序列如SEQ ID NO:2所示。
为了解决上述技术问题,本发明第二方面提供了一种分离的核酸,其编码如本发明第一方面所述的转氨酶。
在某一较佳实施例中,所述核酸的序列如SEQ ID NO:2所示。
为了解决上述技术问题,本发明第三方面提供了一种包含如本发明第二方面所述的核酸的重组表达载体。
较佳地,所述重组表达载体的骨架为质粒pET21a。
为了解决上述技术问题,本发明第四方面提供了一种包含如本发明第二方面所述的核酸或如本发明第三方面所述的重组表达载体的转化体。
较佳地,所述转化体的宿主为大肠杆菌,优选大肠杆菌BL21。
为了解决上述技术问题,本发明第五方面提供了一种如式III所示的化合物的制备方法,所述如式III所示的化合物为:
其中,所述制备方法包括以下步骤:在氨基供体存在时,在反应溶剂中用如本发明第一方面所述的转氨酶催化如式IV所示的化合物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮得如式III所示的化合物;
其中,所述如式IV所示的化合物为:
较佳地,所述制备方法的反应体系还包含助溶剂。所述助溶剂可为本领域常规,例如可以为DMSO和/或醇类例如乙醇。
较佳地,所述反应溶剂为水。
较佳地,所述氨基供体为异丙胺或其盐,所述的盐优选为异丙胺盐酸盐。
较佳地,所述制备的反应体系中还包括转氨酶的辅助因子例如吡哆醛磷酸(PLP),其与底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的质量比为1:10000~1:100例如1:160或1:200。
较佳地,所述异丙胺或其盐与所述底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的摩尔比为1:2~5:1,例如2:1,3:1等。
较佳地,所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的浓度为20g/L~200g/L,例如54g/L,96g/L,113g/L等。
较佳地,所述转氨酶以转氨酶菌泥(在某一较佳实施例中,比酶活为0.08U/mg)的形式存在,所述转氨酶菌泥与所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的质量比为1:3~5:1,例如为1:1,1:2等。
较佳地,所述反应的pH为7.0-8.5。
较佳地,所述反应的温度为20-45℃,例如40℃。
在本发明某一较佳实施例中,所述如式Ⅲ所示的化合物的制备方法的反应体系和反应条件可以如本发明实施例的表4或表6所示。
为了解决上述技术问题,本发明第六方面提供了一种如式II所示的化合物的制备方法,所述式II所示的化合物为:
其中,所述制备方法包括:根据如本发明第五方面所述的制备方法制备如式III所示的化合物的步骤和使如式III所示的化合物自发闭环形成化合物II的步骤。
为了解决上述技术问题,本发明第七方面提供了一种制备莫西沙星中间体(S,S)-2,8-二氮杂双环[4.3.0]壬烷的方法,所述方法包括根据如本发明第六方面所述的制备方法制备所述如式II所示的化合物的步骤和使如式II所示的化合物脱保护基的步骤。
为了解决上述技术问题,本发明第八方面提供了一种制备莫西沙星的方法,所述方法包括根据如本发明第七方面所述的方法制备莫西沙星中间体(S,S)-2,8-二氮杂双环[4.3.0]壬烷的步骤。
为了解决上述技术问题,本发明第九方面提供了一种如本发明第一方面所述的转氨酶在制备如式III所示的化合物、如式II所示的化合物、莫西沙星中间体(S,S)-2,8-二氮杂双环[4.3.0]壬烷和/或莫西沙星中的用途,
所述如式III所示的化合物为:
所述如式II所示的化合物为:
本发明另一方面还提供了一种酶制剂,其包括如本发明第一方面所述的转氨酶。
较佳地,所述酶制剂还包括转氨酶的辅助因子例如吡哆醛磷酸。
本发明中,所述的酶制剂通常可以是从培养物中获得的含转氨酶的转化体宿主细胞或其培养液得到的酶制剂,或者用其加工后得到的制品;其中,所述制品是指由转化体宿主细胞得到的提取物,通过对提取物中的转氨酶进行分离或纯化得到的分离产品,或通过固定化转化体细胞及其提取物或提取物的分离产品而得到的固定化制品。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:本发明转氨酶的酶活和比酶活均较高;将本发明的转氨酶用于制备莫西沙星或其中间体例如(S,S)-2,8-二氮杂双环[4.3.0]壬烷、如本发明的式III或II所示的化合物时,产率显著提高,并且还可进一步用于无DMSO的反应体系,例如使用乙醇代替DMSO作为助溶剂时产率、ee值和de值均较高,且后处理更简单,更有利于工业化生产。在本发明某一较佳实施例中,将本发明的转氨酶用于莫西沙星中间体的制备时,产物的产率可达98.8%,产物ee值可达99.7%,de值可达91.8%。
附图说明
图1为化合物2消旋体对照品的核磁图谱。
图2为化合物3的质谱图。
图3为化合物2经Enz.1催化制得的产率检测图谱。
图4为化合物1经化合物2脱保护制得的产率检测图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
异丙胺购买自国药集团化学试剂有限公司,醋酸异丙酯购买自上海处泰化工科技有限公司,底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮为本公司实验室参考CN108863886A方法自制,产物化合物2消旋体对照品为本公司实验室自制,合成方法参考CN103044418A将手性辅基改为苄胺所得,其核磁图谱见图1。化合物2经酯基脱保护得化合物1消旋体对照品。
本发明中GC检测方法如下:
色谱柱:Agilent DB-1,30m×0.53mm×1.5μm,流速:1.5mL/min;分流比:30:1,进样口温度:250℃;检测器温度:270℃;柱温箱温度:75℃(保持2min)到240℃(保持10min);升温速率:15℃/min;进样体积:0.5μL。
底物对照品保留时间:16.676min;产物化合物2消旋体对照品保留时间:15.762min;产物化合物1消旋体对照品保留时间:10.886min。
ee值分析方法:
NBD-Cl衍生后检测;色谱柱:Daicel CHIRALPAK IC 4.6mm×250mm5μm;流动相:甲醇:乙醇:二乙胺=500:500:1;流速:0.6mL/min;柱温:35℃;波长:340nm;进样量:10μl。
保留时间:(S,S)-构型23.2min,(R,R)-构型25.9min,非对映异构体27.3min和40.4min。
实施例1转氨酶的制备
Enz.1来自于本公司转氨酶酶库,Enz.2、Enz.3、Enz.4来自于专利号CN106399418A中的转氨酶氨基酸序列SEQ ID NO:19、SEQ ID NO:18、SEQ ID NO:20。Enz.2、Enz.3、Enz.4在苏州金唯智生物科技有限公司(江苏省南京市江北新区研创园浦滨路211号)全基因合成,酶切位点NdeI、HindIII,载体pET21a。
将含有转氨酶基因的载体转化宿主大肠杆菌BL21感受态细胞,得到分别含有各转氨酶的工程菌株。将分别含有转氨酶基因的工程菌在经平皿划线活化后,挑单菌落接种至含100μg/mL氨苄抗生素的5mL LB液体培养基中,37℃震荡培养12h。按2%接种量转接至150mL同样含100μg/mL氨苄抗生素的新鲜LB液体培养基中,37℃震荡至OD600达到0.8左右时,加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)至其终浓度为0.5mM,25℃诱导培养16h。培养结束后,将培养液4000rpm离心20min,弃上清液,收集菌体,置于-20℃超低温冰箱中保存,待用。
将培养结束后收集到的菌体10g,用50mM pH 8.0三乙醇胺缓冲液洗涤菌体两次,之后将菌体重悬于40mL pH8.0的三乙醇胺缓冲液中,定容50mL,加入0.1g PLP粉末,搅拌至完全溶解。0~4℃条件下,500~700bar均质破碎,得粗酶液。将粗酶液置于37℃、220rpm摇床中震荡处理1h。12000rpm离心3min,得到的上清液为含转氨酶的粗酶液,用Bradford试剂盒(采购自上海捷瑞生物工程有限公司)测定蛋白浓度。
酶活测定方法:粗酶液稀释5倍后用于酶活测定。
底物溶液配制:反应容器中加入20mL 0.2M三乙醇胺缓冲溶液,1.18g异丙胺,24.7mg PLP,盐酸或异丙胺调pH至8.5,0~4℃下,加入15mL1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的DMSO溶液(含底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮0.8g),0.2M三乙醇胺缓冲溶液定容至50mL。
反应容器中,加入4.5mL底物溶液,于摇床45℃,200rpm下预热5min,加入0.5mL稀释过的酶液,于摇床45℃,200rpm下反应。反应1h后终止反应,向反应液中加入5mL醋酸异丙酯萃取,取上层液体气相色谱进行检测,根据产物的标准曲线计算出产物浓度,计算出酶活。
酶活定义:45℃反应条件下,1min内生成1μmol产物所需的酶量为一个活力单位(U)。
表1
由表1可以看出,Enz.1的酶活和比酶活较Enz.2-4均显著提高。
实施例2DMSO助溶剂体系下转氨酶催化1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的反应
反应容器中加入4g底物化合物4(1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮,结构如上所示),10mL DMSO,35mL三乙醇胺缓冲液,2.5mL异丙胺,调pH到8.5,再加入0.025gPLP,控制温度40-45℃,加入酶液2.5mL(0.25g菌泥加缓冲液均质得到),200rpm条件下反应,反应过程中控制pH。反应24小时后,取样0.5ml加1ml乙酸异丙酯萃取后,取上层GC测产物化合物2(结构如上所示)产量。检测结果见下表2。可见Enz.1催化底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮所得产率显著优于Enz.2-Enz.4。
表2
酶编号 | 产率 |
Enz.1 | 26% |
Enz.2 | 12% |
Enz.3 | 5% |
Enz.4 | 5% |
实施例3乙醇助溶剂体系下转氨酶催化1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的反应
反应容器中加入4g底物化合物4(1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮),3mL乙醇,10mL 4M三乙醇胺缓冲液,8.5mL 4M异丙胺盐酸盐,调pH到8.5,再加入0.025g PLP,控制温度40-45℃,加入酶液20mL(4g菌泥加缓冲液均质得到),200rpm条件下反应,反应过程中控制pH。反应24小时后,取样0.5ml加1ml乙酸异丙酯萃取后,取上层GC测产物化合物2产量。检测结果见下表3。可见Enz.1催化底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮所得产率显著优于Enz.2-Enz.4。
表3
酶编号 | 产率 |
Enz.1 | 90% |
Enz.2 | 36% |
Enz.3 | 5% |
Enz.4 | 6% |
实施例4Enz.1催化1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的反应
反应体系如表4所示,在1L的反应瓶中依次加入0.2M三乙醇胺缓冲液280ml,pH8.5的4M异丙胺盐酸溶液70ml,0.1g磷酸吡哆醛(PLP),调节pH到8.5,温度到40-45℃后,加入10g实施例1中所得Enz.1的菌体(酶菌泥),流加底物化合物4(1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮)乙醇溶液(20g底物用20ml乙醇溶解),5h左右流加完底物,用异丙胺溶液控制pH为8.5进行反应,催化获得化合物2(结构式见实施例6)。GC检测产率结果如下表5所示。由表5可以看出,Enz.1催化底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮制备化合物2的产率较高。反应过程中经GCMS检测,可以检测到化合物3,图谱见图2。
表4
表5
反应时间 | 产率Enz.1 |
16h | 97.0% |
24h | 98.8% |
实施例5Enz.1催化1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的反应
反应体系如表6所示,在1L的反应瓶中加入250ml水,85ml pH8.5的4M异丙胺盐酸溶液,0.2g磷酸吡哆醛(PLP)调节pH到8.5,温度到40-45℃后,加入20g实施例1中所得Enz.1的菌体(酶菌泥),流加底物,其中取40g底物(1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮)先用20ml乙醇溶解,5h左右流加完底物,用异丙胺溶液控制pH为8.5进行反应。经过GC检测,Enz.1催化获得化合物2(结构式见实施例6)的24h产率为98.8%。检测图谱见图3。
表6
实施例6莫西沙星中间体的制备
脱保护反应如下所示:
取实施例4或5中转氨反应的最终包含化合物2的反应液,加入硅藻土,50℃下搅拌灭活1h,抽滤,硅藻土加少量水洗涤。用1N稀盐酸调节至反应液pH=1-2,加入醋酸异丙酯萃取杂质,取水相。水相加入30%氢氧化钠水溶液调节至pH=9-10,加入醋酸异丙酯萃取3次,合并有机相并旋干得到深色油状物化合物2。取1g深色油状物化合物2中加入10ml浓盐酸,置于95℃反应48h。取样GC测产率(检测图谱见图4)以及HPLC检测de值、ee值。所得结果如表7所示。
表7
产率 | de值 | ee值 | |
Enz.1 | 97% | 91.8% | 99.7% |
由表7可以看出,Enz.1催化底物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮制备化合物2、并由化合物2制得化合物1的产率、de值和ee值高。
SEQUENCE LISTING
<110> 上海予君生物科技发展有限公司
<120> 一种转氨酶及其在制备莫西沙星或其中间体中的应用
<130> P20012871C
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.1氨基酸序列
<400> 1
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 2
<211> 993
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.1核苷酸序列
<400> 2
atggcattct cagcagacac gccggaaatt gtttacaccc acgatacggg cctggactac 60
attacctaca gcgactacga actggacccg gcaaacccgc tggctggcgg tgcagcatgg 120
attgagggtg cgtttgtgcc gccgagtgaa gcccgtattt ccatctttga tcagggtttc 180
tatacgtctg acgcaaccta caccacgttt catgtttgga acggtaatgc tttccgtctg 240
ggcgaccaca ttgaacgcct gttcagcaat gcagaatcta ttcgcctgat cccgccgctg 300
acgcaagatg aagtcaaaga aatcgcgctg gaactggtgg ccaagaccga actgcgtgaa 360
gcccaggtca ccgtgacgat tacccgcggc tatagctcta cgccgtttga acgtgatatc 420
accaaacatc gcccgcaggt gtatatgagt gcgtgcccgt accaatggat tgttccgttc 480
gatcgtatcc gcgacggtgt gcacctgatg gttgcacaga gcgtccgtcg caccccgcgt 540
agttccattg atccgcaggt gaagaacttt caatggggcg acctgattcg tgcaatccaa 600
gaaacccatg atcgcggttt cgaactgccg ctgctgctgg attgtgacaa cctgctggct 660
gaaggtccgg gctttaatgt ggttgtcatc aaagatggtg tggttcgtag cccgggtcgt 720
gcagctctgc cgggtattac gcgcaagacc gttctggaaa tcgcggaatc tctgggccac 780
gaagcgattc tggccgatat cacgccggca gaactgtacg atgctgacga agttctgggt 840
tgctcaaccg gcggtggcgt ctggccgttc gtttcggtcg atggtaattc aatttcggac 900
ggtgtgccgg gtccggttac ccagagcatt atccgtcgtt actgggaact gaatgtggaa 960
ccgtcgtcgc tgctgacccc ggtgcaatac tga 993
Claims (7)
1.一种如式II所示的化合物的制备方法,所述如式II所示的化合物为:
,
其特征在于,所述制备方法包括以下步骤:在氨基供体存在时,在反应溶剂中用氨基酸序列如SEQ ID NO: 1所示的转氨酶催化如式IV所示的化合物1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮得如式III所示的化合物;如式III所示的化合物自发闭环形成如式II所示的化合物;
其中,所述如式IV所示的化合物为:
;
所述如式III所示的化合物为:
所述制备方法的反应体系包含助溶剂,所述助溶剂为乙醇;
所述氨基供体为异丙胺或其盐;
所述制备方法的反应体系中还包括转氨酶的辅助因子吡哆醛磷酸;
所述转氨酶以转氨酶菌泥的形式存在,所述转氨酶菌泥与所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的质量比为1:2~1:1。
2.如权利要求1所述的制备方法,其特征在于,编码所述转氨酶的核苷酸序列如SEQ IDNO: 2所示;
和/或,所述反应溶剂为水;
和/或,所述的盐为异丙胺盐酸盐;
和/或,所述吡哆醛磷酸与所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的质量比为1:10000~1:100;
和/或,所述异丙胺或其盐与所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的摩尔比为1:2~5:1;
和/或,所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的浓度为20 g/L~200 g/L;
和/或,所述反应的pH为7.0-8.5;
和/或,所述反应的温度为20-45℃。
3.如权利要求2所述的制备方法,其特征在于,所述吡哆醛磷酸与所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的质量比为1:160或1:200;
和/或,所述氨基供体与所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的摩尔比为2:1或3:1;
和/或,所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的浓度为54、96或113 g/L;
和/或,所述反应的温度为40℃。
4.一种制备莫西沙星中间体(S,S)-2,8-二氮杂双环[4.3.0]壬烷的方法,其特征在于,所述方法包括根据如权利要求1~3任一项所述的制备方法制备如式II所示的化合物的步骤和使如式II所示的化合物脱保护基的步骤。
5.一种制备莫西沙星的方法,其特征在于,所述方法包括根据如权利要求4所述的方法制备莫西沙星中间体(S,S)-2,8-二氮杂双环[4.3.0]壬烷的步骤。
6. 一种氨基酸序列如SEQ ID NO: 1所示的转氨酶在制备如式II所示的化合物、莫西沙星中间体(S,S)-2,8-二氮杂双环[4.3.0]壬烷和/或莫西沙星中的用途,
所述如式II所示的化合物为:;
所述制备的底物为1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮;
所述制备的反应体系包括助溶剂,所述助溶剂为乙醇;
所述制备的反应体系中,氨基供体为异丙胺或其盐;
所述制备的反应体系中,转氨酶的辅助因子为吡哆醛磷酸;
所述转氨酶以转氨酶菌泥的形式存在,所述转氨酶菌泥与所述1-甲酸乙酯基-4-(3-氯丙基)-3-吡咯烷酮的质量比为1:2~1:1。
7.如权利要求6所述的用途,其特征在于,编码所述转氨酶的核苷酸序列如SEQ ID NO:2所示。
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