CN110950788B - 含氟手性胺类化合物的合成方法 - Google Patents
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Abstract
本发明提供了一种含氟手性胺类化合物的合成方法。该合成方法包括:氨基供体与含氟二羟基缩酮类化合物在转氨酶的催化下,反应生成含氟手性胺类化合物;其中,转氨酶来源于多个菌种。本申请的转氨酶对含氟二羟基缩酮类化合物具有底物特异性,能够有效催化此类底物转化为含氟手性胺类化合物,而且,该转氨酶对多种含氟二羟基缩酮类化合物都具有催化活性,反应选择性和活性均较高。该生物酶催化合成方法不仅路线短,产品收率高,而且大大降低了生产成本,减少了有机溶剂和三废产生。
Description
技术领域
本发明涉及含氟化合物的合成领域,具体而言,涉及一种含氟手性胺类化合物的合成方法。
背景技术
含氟化合物是医药及农业化学中非常重要的一类化合物。近十年内,含氟药物以前所未有的速度增长。因此开发一个绿色高效的合成方法有着至关重要的作用。含氟的二羟基缩酮类化合物也是其中一类化合物。通过这类底物可合成重要的含氟手性胺类化合物和。目前通过含氟二羟基缩酮合成含氟手性胺的方法主要有以下几个。
1.根据式1所示的反应路线:在碱性条件下,含氟二羟基缩酮化合物 1的C-C键很容易发生裂解,形成三氟乙酸和相应的含氟烯醇类化合物 2。使用化合物 3作为曼尼希反应受体,经过曼尼希反应后得到化合物 4,进而脱掉亚磺酰基保护得到目标产物,含氟手性胺类化合物。
式1
2.根据式2所示的反应路线:含氟二羟基缩酮化合物 6和苄胺类化合物反应,引入手性氨基,再进而脱掉苄基保护得到目标化合物8。
式2
上述式1所示路线的底物有局限性,且经过三步反应才能得到目标产物,收率偏低。反应路线较长,反应中使用化学试剂产生三废量较大。式2所示路线步骤为两步反应,但反应有时需要使用昂贵的金属配体或催化剂,还会使用到高毒化合物。同样地,反应中使用化学试剂产生三废量较大。
因此,仍需要对此类含氟二羟基缩酮化合物提供一种新的合成方法。
发明内容
本发明的主要目的在于提供一种含氟手性胺类化合物的合成方法,以解决现有技术中含氟手性胺类化合物合成路线长的问题。
为了实现上述目的,本发明提供了一种含氟手性胺类化合物的合成方法,该合成方法包括:氨基供体与含氟二羟基缩酮类化合物在转氨酶的催化下,反应生成含氟手性胺类化合物;其中,转氨酶转氨酶来源于Chromobacterium violaceum DSM30191(CVTA)(NCBIReference Sequence: WP_011135573.1)、Fonsecaea pedrosoi CBS 271.37(NCBIReference Sequence: XP_013286281.1)、Klebsiella pneumoniae subsp. pneumoniaeEcl8(GenBank: CCN29541.1)、Mycobacterium goodii(GenBank: AKS36000.1)、Paracoccus denitrificans(NCBI Reference Sequence: WP_011746975.1)、Penicilliumbrasilianum(GenBank: CEJ55334.1)、Enterobacter sp. TL3(NCBI ReferenceSequence: WP_014885677.1)、Aspergillus terreusNIH2624(NCBI Reference Sequence:XP_001209325.1)、Exophiala spinifera(NCBI Reference Sequence: XP_016233821.1)、Deinococcus geothermalis (strain DSM 11300)(NCBI Reference Sequence: WP_011530545.1)、Geomyces destructans 20631-21 (GdTA) in E. coli(GenBank:ELR05573.1)、Pseudomonas putida KT2440(NCBI Reference Sequence: WP_010954554.1)、Lysinibacillus sphaericus(NCBI Reference Sequence: WP_024363741.1)、Bacillus megaterium DSM 319(NCBI Reference Sequence: WP_013082219.1)、Trichoderma harzianum(GenBank: KKP07030.1)、Aspergillus fumigatusR-ATAs (AspFum)(NCBI Reference Sequence: XP_748821.1)、Geobacillusthermodenitrificans subsp. thermodenitrificans DSM 465(NCBI ReferenceSequence: WP_008879436.1)、Cladophialophora bantiana CBS 173.52(NCBI ReferenceSequence: XP_016617948.1)、Bacillus megaterium(NCBI Reference Sequence: WP_016763026.1)、Burkholderia thailandensis MSMB121 ( BtS -TA )(GenBank:AGK49399.1)、Klebsiella pneumoniae subsp. pneumoniae MGH 78578(NCBI ReferenceSequence: WP_002920226.1)、Geobacillus toebii(NCBI Reference Sequence: WP_062753894.1)和Talaromyces cellulolyticus(GenBank: GAM37533.1)。
进一步地,转氨酶具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4所示的氨基酸序列。
进一步地,含氟二羟基缩酮类化合物具有如下结构式I:
其中,R选自烷基、环烷基、芳基、含杂原子的烷基、含杂原子的环烷基、含杂原子的芳基、酰胺类化合物残基或醚类化合物残基,R1 和 R2 为相同或不同的H原子或卤素原子;
更优选地,含氟二羟基缩酮类化合物具有结构式II所示结构:
进一步地,含氟二羟基缩酮类化合物选自如下任意一种:
进一步地,合成方法包括:将磷酸缓冲液与氨基供体混合,得到第一混合液,向第一混合液中添加含氟二羟基缩酮类化合物,得到第二混合液;向第二混合液中添加转氨酶,得到反应混合液;从反应混合液中分离得到含氟手性胺类化合物。
进一步地,在向第一混合液中添加含氟二羟基缩酮类化合物之前,合成方法还包括:将第一混合液的pH值调节至7.0~9.0;优选地,向第二混合液中添加转氨酶,并调节加入转氨酶后的第二混合液的pH为7.0~9.0,得到反应混合液。
进一步地,从反应混合液中分离得到含氟手性胺类化合物包括:调节反应混合液的酸度使转氨酶变性,优选调节反应混合液的pH值为1~2;将变性后的转氨酶过滤除去,得到滤液;对滤液进行萃取,得到含氟手性胺类化合物;优选地,采用二氯甲烷进行过滤;优选地,采用二氯甲烷进行萃取。
进一步地,萃取为2~5次,萃取之后还包括对萃取所得有机相进行干燥的步骤,优选地,将每次萃取所得有机相合并,得到萃取物;对萃取物进行干燥,得到干燥有机相产物;将干燥有机相产物置于温度<35℃,压力≤-0.06Mpa的条件下浓缩至无馏分,得到含氟手性胺类化合物。
进一步地,转氨酶与含氟二羟基缩酮类化合物质量比为:0.4:1~1.0:1。
进一步地,含氟二羟基缩酮类化合物的浓度为60g/L~100g/L。
进一步地,氨基供体选自异丙胺、异丙胺的盐酸盐、丙氨酸、苯胺或正丁胺。
应用本发明的技术方案,本申请的转氨酶对含氟二羟基缩酮类化合物具有底物特异性,能够有效催化此类底物转化为含氟手性胺类化合物,而且,该转氨酶对多种含氟二羟基缩酮类化合物都具有催化活性,反应选择性和活性均较高。该生物酶催化合成方法不仅路线短,产品收率高,而且大大降低了生产成本,减少了有机溶剂和三废产生。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1示出了本发明的实施例13中不同酶量对相同量的底物的转化率的影响;
图2示出了本发明的实施例14中相同酶量对不同浓度的底物的转化率的影响;
图3示出了本发明的实施例15中不同的氨基供体对同一反应中的底物的转化率的影响;以及
图4示出了本发明的实施例16中不同来源的转氨酶对同一底物均具有转化活性。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如背景技术所提到的,现有技术中,在采用含氟二羟基缩酮类化合物作为起始原料合成含氟手性胺类化合物时,不仅存在反应路线长的缺陷,而且还存在工业三废的问题。为了改善这一状况,本申请的发明人尝试从高效、绿色环保的角度来改进现有合成路线。在研究过程中,发明人发现,申请人自行研制的一种转氨酶除了具有以PLP为辅酶的转氨酶所具有的催化酮类化合物的通用催化活性外,还具有催化含氟二羟基缩酮类化合物的活性。并进一步研究发现采用该转氨酶催化含氟二羟基缩酮类化合物底物时,不仅可以通过一步反应获得目标产物,而且底物类型广泛。由于使用该转氨酶进行生物转化反应选择性很高,大大提升了目标产物的ee值。而且,采用生物酶催化反应大大提高了生产效率,减少了有机溶剂和三废产生。
在上述研究结果的基础上,申请人提出了本申请的技术方案。在本申请一种典型的实施方式中,提供了一种含氟手性胺类化合物的合成方法,该合成方法包括:氨基供体与含氟二羟基缩酮类化合物在转氨酶的催化下,反应生成含氟手性胺类化合物;其中,转氨酶来源于Chromobacterium violaceum DSM30191(CVTA)(NCBI Reference Sequence: WP_011135573.1)、Fonsecaea pedrosoi CBS 271.37(NCBI Reference Sequence: XP_013286281.1)、Klebsiella pneumoniae subsp. pneumoniae Ecl8(GenBank:CCN29541.1)、Mycobacterium goodii(GenBank: AKS36000.1)、Paracoccusdenitrificans(NCBI Reference Sequence: WP_011746975.1)、Penicilliumbrasilianum(GenBank: CEJ55334.1)、Enterobacter sp. TL3(NCBI ReferenceSequence: WP_014885677.1)、Aspergillus terreusNIH2624(NCBI Reference Sequence:XP_001209325.1)、Exophiala spinifera(NCBI Reference Sequence: XP_016233821.1)、Deinococcus geothermalis (strain DSM 11300)(NCBI Reference Sequence: WP_011530545.1)、Geomyces destructans 20631-21 (GdTA) in E. coli(GenBank:ELR05573.1)、Pseudomonas putida KT2440(NCBI Reference Sequence: WP_010954554.1)、Lysinibacillus sphaericus(NCBI Reference Sequence: WP_024363741.1)、Bacillus megaterium DSM 319(NCBI Reference Sequence: WP_013082219.1)、Trichoderma harzianum(GenBank: KKP07030.1)、Aspergillus fumigatusR-ATAs (AspFum)(NCBI Reference Sequence: XP_748821.1)、Geobacillusthermodenitrificans subsp. thermodenitrificans DSM 465(NCBI ReferenceSequence: WP_008879436.1)、Cladophialophora bantiana CBS 173.52(NCBI ReferenceSequence: XP_016617948.1)、Bacillus megaterium(NCBI Reference Sequence: WP_016763026.1)、Burkholderia thailandensis MSMB121 ( BtS -TA )(GenBank:AGK49399.1)、Klebsiella pneumoniae subsp. pneumoniae MGH 78578(NCBI ReferenceSequence: WP_002920226.1)、Geobacillus toebii(NCBI Reference Sequence: WP_062753894.1)和Talaromyces cellulolyticus(GenBank: GAM37533.1)。
更优选地,该转氨酶具有如下SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3或SEQ IDNO:4所示的氨基酸序列:
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWWGYHGSTIGGASLGGFKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMLLAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMDSCGDHIVCAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA(SEQ ID NO:1)。
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWWGYHGSTIGGASLGGFKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMLLAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMDSCGDHIVAAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA(SEQ ID NO:2)。
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWWGYHGSTIGGASLGGFKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMLLAFTLVKNKAKRELFPDFGEIGTLCRDIFQRNNLIMDSCGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA(SEQ ID NO:3)。
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWWGYHGSTIGGASLGGFKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMLLAFTLVKNKAKRELFPDFGEIGTLCRDIFHRNNLIMDSCGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA(SEQ ID NO:4)。
如上述,本申请的上述转氨酶对含氟二羟基缩酮类化合物具有底物特异性,能够有效催化此类底物转化为含氟手性胺类化合物,而且,该转氨酶对多种含氟二羟基缩酮类化合物都具有催化活性,反应选择性和活性均较高。该生物酶催化合成方法不仅路线短,产品收率高,而且大大降低了生产成本,减少了有机溶剂和三废产生。
上述SEQ ID NO:1至4任一所示的转氨酶是一种ω-转氨酶,均来源于Chromobacterium violaceum DSM30191(CVTA)。按照现有方法从Chromobacteriumviolaceum DSM30191(CVTA)中分离得到。
在一种优选的实施例中,含氟二羟基缩酮类化合物具有如下式I所示结构:
其中,R选自烷基、环烷基、芳基、含杂原子的烷基、含杂原子的环烷基、含杂原子的芳基、酰胺类化合物残基或醚类化合物残基,R1 和 R2 为相同或不同的H原子或卤素原子。
在一种更优选的实施例中,R1和R2均为F原子,即含氟二羟基缩酮类化合物具有如下结构式II所示结构:
在一种优选的实施例中,含氟二羟基缩酮类化合物选自如下任意一种:
本申请的转氨酶对上述含氟二羟基缩酮类化合物均具有催化活性。
采用本申请的转氨酶对含氟二羟基缩酮类化合物进行催化转氨基反应的具体步骤与现有转氨反应类似。在一种优选的实施例中,上述合成方法包括:将磷酸缓冲液与氨基供体混合,得到第一混合液;向第一混合液中添加含氟二羟基缩酮类化合物,得到第二混合液;向第二混合液中添加转氨酶,得到反应混合液;从反应混合液中分离得到含氟手性胺类化合物。
该合成方法通过上述分步反应(前面的物料可以改变顺序,转氨酶最后加入是要确保加入后立即进行反应,防止其他操作时间较长导致酶失活),减少了反应体积,使得转氨酶对底物的催化活性和效率较高,从而获得较高的产品收率,提高了生产效率。
在一种优选的实施例中,在向第一混合液中添加含氟二羟基缩酮类化合物之前,合成方法还包括:将第一混合液的pH值调节至7.0~9.0;优选地,向第二混合液中添加转氨酶,并调节加入转氨酶后的第二混合液的pH为7.0~9.0,得到反应混合液。
上述优选实施例中,通过在在向第一混合液中添加含氟二羟基缩酮类化合物之前,对第一混合液进行pH值调节,其目的在于加入酶之前调节到酶反应最适pH,防止酶加入过程中失活。而在向第二混合液中添加转氨酶后也对体系的pH值进行调节,其作用是确保反应在最适pH下进行。上述两次pH值调节的范围最好保持一致,比如,前面调节至7.8,加入转氨酶后也同样调节至7.8。
上述从反应后的混合液中分离得到目的产物的步骤采用已知的分离、提纯方法进行即可。在一种优选的实施例中,从反应混合液中分离得到含氟手性胺类化合物包括:调节反应混合液的酸度使转氨酶变性,优选调节反应混合液的pH值为1~2;将变性后的转氨酶过滤除去,得到滤液;对滤液进行萃取,得到含氟手性胺类化合物;优选地,采用二氯甲烷进行过滤;优选地,采用二氯甲烷进行萃取。
采用调节酸度的方式使反应后的酶蛋白变性,相比其他变性方式(比如高温、调碱或盐析)有变性比较彻底,产物大多在酸性条件下稳定,且之后的操作直接在酸性条件下可将体系其他杂质萃取优势。采用二氯甲烷进行萃取先比采用其他萃取溶剂(比如乙酸乙酯、甲基叔丁基醚、或乙酸异丙酯)进行萃取有萃取效率较高的优势。
为进一步提高纯度和收率,在一种优选的实施例中,萃取为2~5次,萃取后还包括对萃取有机相进行干燥的步骤,优选地,将每次萃取所得有机相合并,得到萃取物;对萃取物进行干燥,得到干燥有机相产物;将干燥有机相产物置于温度<35℃,压力≤-0.06Mpa的条件下浓缩至无馏分(干燥的目的在于减少产品中的含水量,而压力大小影响浓缩速度。
在一种优选的实施例中,转氨酶与含氟二羟基缩酮类化合物的质量比为:0.4:1~1.0:1,在该质量比下对底物的转化率达到95%以上。
在一种优选的实施例中,含氟二羟基缩酮类化合物的浓度为60g/L~100g/L。在该浓度范围下,采用生物酶进行反应,不仅对底物的转化率高达99%,而且有助于提高底物浓度才,从而提高生产效率,减少有机溶剂和三废的产生。
在一种优选的实施例中,氨基供体选自异丙胺、异丙胺的盐酸盐、丙氨酸、苯胺或正丁胺。上述氨基供体度底物的转化率都能达到90%以上。
下面将结合具体的实施例来进一步说明本申请的有益效果。需要说明的是,以下实施例中的室温是指10~25℃的常温范围内的温度,以下实施例1至15中所用到的转氨酶为SEQ ID NO:4所示的转氨酶。
实施例 1
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(5vol),32ml5mol/ L 异丙胺盐酸盐溶液(3.2 vol, 3eq),调pH=7.5~8.0。再加入0.1g 磷酸吡哆醛(1wt%),10g ,搅拌均匀,再加入转氨酶酶液10 ml(0.5wt,0.5 g/ml)调pH=7.5~8.0。升温至29℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用50mL 二氯甲烷萃取。水相调pH=13,再用50ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:98%,ee值:100%,收率:87%。
实施例 2
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(5vol),25ml5mol/ L 异丙胺盐酸盐溶液(2.5 vol, 3eq),调pH=8.0~8.5。再加入0.1g 磷酸吡哆醛(1wt%),10g ,搅拌均匀,再加入转氨酶酶液10 ml(0.5wt,0.5 g/ml),调pH=8.0~8.5。调温至20℃搅拌反应过夜。反应完全后,将体系调酸至pH=2,变性蛋白。过滤后滤液用50mL 二氯甲烷萃取。水相调pH=13,再用50ml 乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<45℃,P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:99%,ee值:100%,收率:86%。
实施例 3
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(5vol),31ml5mol/ L 异丙胺盐酸盐溶液(3.1 vol, 3eq),调pH=7.0~7.5。再加入0.1g 磷酸吡哆醛(1wt%),10g ,搅拌均匀,再加入转氨酶酶液10 ml(0.5wt,0.5 g/ml)升温至45~50℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用50mL 二氯甲烷萃取。水相调pH=12,再用50ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:97%,ee值:100%,收率:83%。
实施例 4
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(5vol),24ml5mol/ L 异丙胺盐酸盐溶液(2.4 vol, 3eq),调pH=8.5~9.0。再加入0.1g 磷酸吡哆醛(1wt%),10g ,搅拌均匀,再加入转氨酶酶液10 ml(0.5wt,0.5 g/ml),调pH=8.5~9.0。调温至10℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用50mL 二氯甲烷萃取。水相调pH=13,再用50ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:98%,ee值:100%,收率:90%。
实施例 5
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(5vol),20ml5mol/ L 异丙胺盐酸盐溶液(2 vol, 3eq),调pH=7.5~8.0。再加入0.1g 磷酸吡哆醛(1wt%),10g ,搅拌均匀,再加入转氨酶酶液10 ml(0.5wt,0.5 g/ml),调pH=7.5~8.0。升温至32℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用50mL 二氯甲烷萃取。水相调pH=13,再用50ml 乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<45℃,P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:98%,ee值:100%,收率:87%。
实施例 6
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(5vol),23ml5mol/ L 异丙胺盐酸盐溶液(2.3 vol, 3eq),调pH=7.0~7.5。再加入0.1g 磷酸吡哆醛(1wt%),10g ,搅拌均匀,再加入转氨酶酶液10 ml(0.5wt,0.5 g/ml)调pH=7.0~7.5,升温至28℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用50mL 二氯甲烷萃取。水相调pH=12,再用50ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:98%,ee值:100%,收率:80%。
实施例 7
室温向500 mL四口瓶内加入200 mL 100 mmol/L 磷酸盐缓冲液(10vol),60ml5mol/ L 异丙胺盐酸盐溶液(3 vol, 3eq),调pH=7.5~8.0。再加入0.2g 磷酸吡哆醛(1wt%),20g ,搅拌均匀,再加入转氨酶酶液20 ml(0.5wt,0.5 g/ml)调pH=7.5~8.0,升温至35℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用100 mL 二氯甲烷萃取。水相调pH=12,再用100 ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:94%,ee值:100%,收率:82%。
实施例 8
室温向250 mL四口瓶内加入80 mL 100 mmol/L 磷酸盐缓冲液(8vol),23ml5mol/ L 异丙胺盐酸盐溶液(2.3 vol, 3eq),调pH=7.0~7.5。再加入0.1g 磷酸吡哆醛(1wt%),10g ,搅拌均匀,再加入转氨酶酶液20 ml(1.0wt,0.5 g/ml)调pH=7.0~7.5,升温至35℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用50 mL 二氯甲烷萃取。水相调pH=12,再用50 ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:97%,ee值:100%,收率:86%。
实施例 9
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(10 vol),16 ml5mol/ L 异丙胺盐酸盐溶液(3.2 vol, 3eq),调pH=7.5~8.0。再加入0.05g 磷酸吡哆醛(1wt%),5g ,搅拌均匀,再加入转氨酶酶液10 ml(1.0 wt,0.5 g/ml)调pH=7.5~8.0,升温至20℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用30mL 二氯甲烷萃取。水相调pH=12,再用30ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经GC检测,纯度:95%,ee值:100%,收率:78%。
实施例 10
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(10 vol),18 ml5mol/ L 异丙胺盐酸盐溶液(3.6 vol, 3eq),调pH=7.5~8.0。再加入0.05g 磷酸吡哆醛(1wt%),5g ,搅拌均匀,再加入转氨酶酶液10 ml(1.0 wt,0.5 g/ml)调pH=7.5~8.0,升温至25℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用40mL 二氯甲烷萃取。水相调pH=12,再用40ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经GC检测,纯度:94%,ee值:100%,收率:76%。
实施例 11
室温向250 mL四口瓶内加入50 mL 100 mmol/L 磷酸盐缓冲液(10 vol),14 ml5mol/ L 异丙胺盐酸盐溶液(2.8 vol, 3eq),调pH=7.5~8.0。再加入0.05g 磷酸吡哆醛(1wt%),5g ,搅拌均匀,再加入转氨酶酶液10 ml(1.0 wt,0.5 g/ml)调pH=7.5~8.0,升温至25℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用40mL 二氯甲烷萃取。水相调pH=12,再用40ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:92%,ee值:100%,收率:79%。
实施例 12
室温向250 mL四口瓶内加入60 mL 100 mmol/L 磷酸盐缓冲液(12 vol),13 ml5mol/ L 异丙胺盐酸盐溶液(2.6 vol, 3eq),调pH=7.5~8.0。再加入0.05g 磷酸吡哆醛(1wt%),5g ,搅拌均匀,再加入转氨酶酶液10 ml(1.0 wt,0.5 g/ml)调pH=7.5~8.0,升温至35℃搅拌反应过夜。反应完全后,将体系调酸至pH=1,变性蛋白。过滤后滤液用40mL 二氯甲烷萃取。水相调pH=12,再用40ml 二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T<35℃,P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物。
经HPLC检测,纯度:95%,ee值:100%,收率:84%。
实施例 13
以实施例1为例,对反应的酶量进行优化具体操作如下:室温向10 mL小瓶内加入1mL 100 mmol/L 磷酸盐缓冲液(5vol),0.64ml 5mol/ L 异丙胺盐酸盐溶液(3.2 vol,3eq),调pH=7.5~8.0。再加入0.002g 磷酸吡哆醛(1wt%),0.2g ,混匀,分别加入转氨酶酶液 0.04ml,0.08ml,0.12ml,0.16ml,0.2ml,0.24ml,0.28ml,0.32ml,0.36ml,0.4ml,0.44ml,0.48ml,0.52ml,0.56ml,0.6ml(酶液浓度为0.5 g/ml,对应质量比分别为0.1wt,0.2wt,0.3wt,0.4wt,0.5wt,0.6wt,0.7wt,0.8wt,0.9wt,1.0wt,1.1wt,1.2wt,1.3wt,1.4wt,1.5wt)调pH=7.5~8.0。摇床转速170rpm,升温至29℃条件下反应过夜。
转化率结果如图1所示,证明0.5~1.0wt底物可转化率大于99%,酶量继续增大也并无显著效果。
实施例 14
以实施例1为例,对底物浓度进行优化具体操作如下:室温向10~50 mL小瓶内(根据反应体积选择合适的摇瓶)分别加入0.3mL,0.38ml,0.50ml,0.62ml,0.78ml,0.96ml,1.16ml,1.46ml,1.82ml,2.28ml,2.96ml,3.96ml,5.56ml,8.96ml 100 mmol/L 磷酸盐缓冲液(分别为1.5vol,1.9vol,2.5vol,3.1vol,3.9vol,4.8vol,5.8vol,7.3vol,9.1vol,11.4vol,14.8vol,19.8vol,27.8vol,44.8vol),0.64ml 5mol/ L 异丙胺盐酸盐溶液(3.2vol, 3eq),调pH=7.5~8.0。再加入0.002g 磷酸吡哆醛(1wt%),0.2g ,混匀,加入转氨酶酶液 0.2ml(0.5wt,酶液浓度为0.5 g/ml)调pH=7.5~8.0。摇床转速170rpm,升温至29℃条件下反应过夜。
转化率结果如图2所示,证明60~100 g/L浓度下,底物可转化率大于99%。继续降低底物浓度会导致反应体积增加,三废量增加。并无显著效果。
实施例 15
以实施例1为例,对氨基供体进行优化具体操作如下:室温向10~50 mL小瓶内(根据反应体积选择合适的摇瓶)加入1ml 100 mmol/L 磷酸盐缓冲液(5vol),分别使用3eq氨基供体的体系(氨基供体分别为异丙胺、异丙胺盐酸盐、丙氨酸、苯胺,正丁胺),调pH=7.5~8.0。再加入0.002g 磷酸吡哆醛(1wt%),0.2g ,混匀,加入转氨酶酶液0.2ml(0.5wt,酶液浓度为0.5 g/ml)调pH=7.5~8.0。摇床转速170rpm,升温至29℃条件下反应过夜。
转化率结果如图3所示,证明异丙胺、异丙胺盐酸盐、丙氨酸、正丁胺作为氨基供体,底物可转化率大于99%。而苯胺作为氨基供体在此反应条件下转化率为93%。
实施例 16
以实施例1底物为例,将来源于Mycobacterium goodii(GenBank: AKS36000.1)的转氨酶编号为TA1,将来源于Fonsecaea pedrosoi CBS 271.37 (NCBI ReferenceSequence: XP_013286281.1)的转氨酶编号TA2,将来源于Klebsiella pneumoniae subsp.pneumoniae Ecl8(GenBank: CCN29541.1)的转氨酶编号TA3,将来源于Chromobacteriumviolaceum DSM30191(CVTA) (NCBI Reference Sequence: WP_011135573.1)的转氨酶编号TA4,将来源于Paracoccus denitrificans (NCBI Reference Sequence: WP_011746975.1)的转氨酶编号TA5,将来源于Penicillium brasilianum (GenBank:CEJ55334.1)的转氨酶编号TA6,将来源于Enterobacter sp. TL3 (NCBI ReferenceSequence: WP_014885677.1)的转氨酶编号TA7,将来源于Aspergillus terreus NIH2624(NCBI Reference Sequence: XP_001209325.1)的转氨酶编号TA8,将来源于Exophialaspinifera (NCBI Reference Sequence: XP_016233821.1)的转氨酶编号TA9,将来源于Deinococcus geothermalis (strain DSM 11300) (NCBI Reference Sequence: WP_011530545.1)的转氨酶编号TA10,将Geomyces destructans 20631-21 (GdTA) in E.coli (GenBank: ELR05573.1)编号TA 11,将来源于Pseudomonas putida KT2440 (NCBIReference Sequence: WP_010954554.1)编号的转氨酶TA12,将来源于Lysinibacillussphaericus (NCBI Reference Sequence: WP_024363741.1)的转氨酶编号TA13,将来源于Bacillus megaterium DSM 319 (NCBI Reference Sequence: WP_013082219.1)的转氨酶编号TA14,将来源于Trichoderma harzianum(GenBank: KKP07030.1)的转氨酶编号TA15,将来源于Aspergillus fumigatus R-ATAs (AspFum) (NCBI Reference Sequence: XP_748821.1)的转氨酶编号TA16,将来源于Geobacillus thermodenitrificans subsp.thermodenitrificans DSM 465(NCBI Reference Sequence: WP_008879436.1)的转氨酶编号TA17,将来源于Cladophialophora bantiana CBS 173.52(NCBI ReferenceSequence: XP_016617948.1)的转氨酶编号TA18,将来源于Bacillus megaterium (NCBIReference Sequence: WP_016763026.1)的转氨酶编号TA19,将来源于Burkholderiathailandensis MSMB121 ( BtS -TA )(GenBank: AGK49399.1)的转氨酶编号TA20,将来源于Klebsiella pneumoniae subsp. pneumoniae MGH 78578(NCBI Reference Sequence:WP_002920226.1)的转氨酶编号TA21,将来源于Geobacillus toebii(NCBI ReferenceSequence: WP_062753894.1)的转氨酶编号TA22,将来源于Talaromyces cellulolyticus(GenBank: GAM37533.1)的转氨酶编号TA23,以Chromobacterium violaceum DSM30191(CVTA) (NCBI Reference Sequence: WP_011135573.1) 来源的转氨酶进化得到的突变体TA4-V1(对应序列为SEQ ID NO:1),TA4-V2(对应序列为SEQ ID NO:2),TA4-V3(对应序列为SEQ ID NO:3),TA4-V4(对应序列为SEQ ID NO:4)进行10mg级别筛选反应,操作如下:
将100 mmol/L 磷酸盐缓冲液,5mol/ L 异丙胺盐酸盐溶液按照体积比5:2配置成50ml溶液,再加入0.005g 磷酸吡哆醛,搅拌混匀。取上述溶液0.3ml分别加入96孔板中,并加入10mg,再分别加入上述酶液 0.2ml(10wt,酶液浓度为0.5g/ml),摇床转速170rpm,升温至30℃条件下反应过夜。
筛选结果如图4所示: 证明所有来源的酶对该底物均有催化活性,以Chromobacterium violaceum DSM30191(CVTA) 来源的转氨酶最好,以Chromobacteriumviolaceum DSM30191(CVTA) 来源的转氨酶进行突变,得到具有转化率更好的四个突变体转化率均大于99%。
从以上的描述中,可以看出,本发明上述的实施例实现了如下技术效果:。
1.目前尚未发现有转化酶转化类似化合物的专利。
2.使用转氨酶进行该生物转化反应可以一步反应直接得到所需目标化合物,底物类型适用广泛。
3.使用转氨酶进行该生物转化反应选择性很高,大大提升了产品ee值。
4.使用自行提取的转氨酶,底物浓度可达到100g/L,大大提高了生产效率,减少了有机溶剂和三废产生。
5.该转氨酶催化效率高,反应体积少,合成路线短,产品收率高,大大降低了生产成本。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 凯莱英医药集团(天津)股份有限公司
<120> 含氟手性胺类化合物的合成方法
<130> PN118430KLY
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Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val
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Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Phe Tyr Lys Thr Thr His Pro Ala Val
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195 200 205
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210 215 220
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225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
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340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
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385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe His Arg Asn Asn Leu Ile Met Asp
405 410 415
Ser Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
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450 455
Claims (13)
2.根据权利要求1所述的合成方法,其特征在于,所述合成方法包括:
将磷酸缓冲液与所述氨基供体混合,得到第一混合液,
向所述第一混合液中添加所述含氟二羟基缩酮类化合物,得到第二混合液;
向所述第二混合液中添加所述转氨酶,得到反应混合液;
从所述反应混合液中分离得到所述含氟手性胺类化合物。
3.根据权利要求2所述的合成方法,其特征在于,在向所述第一混合液中添加所述含氟二羟基缩酮类化合物之前,所述合成方法还包括:将所述第一混合液的pH值调节至7.0~9.0。
4.根据权利要求3所述的合成方法,其特征在于,向所述第二混合液中添加所述转氨酶,并调节加入所述转氨酶后的所述第二混合液的pH为7.0~9.0,得到所述反应混合液。
5.根据权利要求2所述的合成方法,其特征在于,从所述反应混合液中分离得到所述含氟手性胺类化合物包括:
调节所述反应混合液的酸度使所述转氨酶变性;
将变性后的所述转氨酶过滤除去,得到滤液;
对所述滤液进行萃取,得到所述含氟手性胺类化合物。
6.根据权利要求5所述的合成方法,其特征在于,调节所述反应混合液的pH值为1~2。
7.根据权利要求5所述的合成方法,其特征在于,采用二氯甲烷进行所述过滤。
8.根据权利要求5所述的合成方法,其特征在于,采用二氯甲烷进行所述萃取。
9.根据权利要求5所述的合成方法,其特征在于,所述萃取为2~5次,所述萃取之后还包括对萃取所得有机相进行干燥的步骤。
10.根据权利要求5所述的合成方法,其特征在于,
将每次萃取所得有机相合并,得到萃取物;
对所述萃取物进行干燥,得到干燥有机相产物;
将所述干燥有机相产物置于温度<35℃,压力≤-0.06Mpa的条件下浓缩至无馏分,得到所述含氟手性胺类化合物。
11.根据权利要求1至10中任一项所述的合成方法,其特征在于,所述转氨酶与所述含氟二羟基缩酮类化合物质量比为:0.4:1~1.0:1。
12.根据权利要求1至10中任一项所述的合成方法,其特征在于,所述含氟二羟基缩酮类化合物的浓度为60g/L~100g/L。
13.根据权利要求1至10中任一项所述的合成方法,其特征在于,所述氨基供体选自异丙胺、异丙胺的盐酸盐、丙氨酸、苯胺或正丁胺。
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