CN107653293B - 特异位点羟基化巨大戟烷型二萜衍生物的制备方法 - Google Patents
特异位点羟基化巨大戟烷型二萜衍生物的制备方法 Download PDFInfo
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Abstract
本发明属生物药物领域,涉及巨大戟烷型二萜的转化方法,本发明利用已知微生物对20‑去氧巨大戟醇及13‑O‑正十二烷酸巨大戟酯进行微生物转化,得到多种羟基化的巨大戟烷型二萜衍生物。所述巨大戟烷型二萜骨架中非活性碳位点增加羟基能为该类化合物提供新的化学修饰位点。
Description
技术领域
本发明属生物药物领域,涉及巨大戟烷型二萜化合物的转化方法,具体涉及利用已知微生物对巨大戟烷型二萜衍生物进行微生物转化,制备极性增强的巨大戟烷型二萜衍生物。
背景技术
现有技术公开了巨大戟烷型化合物是一类由5/7/7/3四环稠合而成的大环二萜天然产物。这类二萜来源于各种大戟属植物中[Journal of Natural Products,2002,65(9):1246-1251],多数在其结构母核C-3的羟基或C-13位的羟基连接有长链脂肪酸成酯,如13-O-正十二烷酸巨大戟酯(13-oxyingenol dodecanoat),而巨大戟二萜醇(ingenol)和20-去氧巨大戟二萜醇(20-deoxyingenol)为极性较大的游离型巨大戟二萜醇。巨大戟烷型骨架的化合物大多具有良好的生物活性,例如有抑制肿瘤细胞生长的活性[Phytochemistry,2008,69(3):812-2819]、抑制细胞分裂活性[Journal of Science of Food andAgriculture,2015,44(8):3188-3194]、γ-干扰素调节作用[International Journal ofMolecular Sciences,2012,13(9):11247-11259]等。
现有的巨大戟烷型二萜多数由植物中提取分离得到,或以植物中提取得到的巨大戟烷型二萜为原料再合成新型的巨大戟二萜衍生物。已有专利公开了以大戟植物中萃取得到的20-去氧巨大戟二萜醇衍生物为原料进行化学反应获得巨大戟二萜醇-3-当归酸酯(CN104470888A);另有专利公开了以植物中得到的巨大戟二萜醇为原料,进一步衍生合成多种巨大戟二萜醇-3-酰化物,并从中筛选出可刺激中性粒细胞氧化爆发、可刺激角质形成的细胞IL-8释放、或可在B16小鼠黑色素瘤模型中表现出活性的先导化合物(CN103402977A)。
以上巨大戟二萜衍生物的获取都是以骨架中原生醇羟基的存在为基础,而本发明拟通过微生物转化的方法,在巨大戟烷二萜骨架的非活性碳位点引入羟基,为后续的化学修饰提供活性基团。
微生物转化是利用微生物生长过程中合成的特异酶完成的特定生化反应。据报道,微生物在生长过程中可以合成多种酶,催化不同反应,例如氧化反应、还原反应、水解反应、脱氢反应、缩合反应和开环反应等,这类酶催化反应具有特异性和专属性,相对于化学反应,在底物的非活性碳原子上进行反应更高效环保。
因此,本申请利用微生物对巨大戟烷型二萜类化合物进行转化研究,以期获得一些难以用传统化学合成制备的特异位点(尤其是其C-12和C-16等低活性碳位点)的转化产物,为后续结构修饰提供活性位点。
发明内容
本发明的目的是提供巨大戟烷型二萜非活性碳上进行羟基化的方法,尤其是制备具有生理活性、难以通过传统化学法得到、非活性碳上羟基化的巨大戟烷型二萜的合成方法。
本发明中分别使用拉曼被孢霉Mortierella ramanniana CGMCC 3.03413和藤仓赤霉Gibberella fujikuroi CICC 40272对20-去氧巨大戟醇(20-deoxyingenol)和13-O-正十二烷酸巨大戟醇酯(13-oxyingenol dodecanoat)进行微生物转化,获得以下结构通式的巨大戟烷型二萜衍生物:
经鉴定,所获得的巨大戟烷型二萜衍生物为:
化合物1 16-羟基-20-去氧巨大戟醇 R1=R2=R4=R5=H,R3=OH
化合物2 19-羟基-20-去氧巨大戟醇 R1=R2=R3=R5=H,R4=OH
化合物3 12-羟基-20-去氧巨大戟醇 R2=R3=R4=R5=H,R1=OH
化合物4 12,19-二羟基-20-去氧巨大戟醇 R2=R3=R5=H,R1=R4=OH
化合物5 13-O-10’-羟基正十二烷酸巨大戟酯
R1=R3=R4=R5=H,R2=-OOC(CH2)8CHOHCH2CH3
化合物6 13-O-11’-羟基正十二烷酸巨大戟酯
R1=R3=R4=R5=H,R2=-OOC(CH2)9CHOHCH3
本发明中,所述的微生物转化法包括以下步骤:
(1)将生长在琼脂培养基上的生产菌株划线接种于马铃薯固体培养基上,在28℃的恒温培养箱中培养3-7天,得到试管种;
(2)将试管种中的菌丝接种到250mL的三角瓶中,每瓶含有50mL液体马铃薯培养基,培养温度为28℃,转速为150rpm,培养时间为24小时,获得种子液;
(3)将种子液按2%~5%体积比接入新鲜马铃薯培养基,28℃,130-180rpm条件下培养24-72小时后,加入转化底物20-去氧巨大戟醇或13-O-正十二烷酸巨大戟醇酯,在20-28℃、130-180rpm的条件下转化培养72-240小时;
(4)将发酵液用布氏漏斗减压过滤,获得发酵滤液,按照1:1.5体积比用乙酸乙酯萃取3次,合并乙酸乙酯层,减压蒸干,残渣用多种色谱分离法得到发酵产物。
其中,本发明所述的生产菌株,发酵转化20-去氧巨大戟醇所用菌株为拉曼被孢霉Mortierella ramanniana CGMCC 3.03413,包括其突变体;发酵转化13-O-正十二烷酸巨大戟醇酯的生产菌株为藤仓赤霉Gibberella fujikuroi CICC 40272,包括其突变体;
其中,本发明所述的发酵产物分别指20-去氧巨大戟醇的发酵产物用硅胶柱层析分离法得到16-羟基-20-去氧巨大戟醇(1)、19-羟基-20-去氧巨大戟醇(2)、12-羟基-20-去氧巨大戟醇(3)和12,19-二羟基-20-去氧巨大戟醇(4);13-O-正十二烷酸巨大戟醇酯的发酵产物用硅胶柱层析分离法得到13-O-10’-羟基正十二烷酸巨大戟酯(5)和13-O-11’-羟基正十二烷酸巨大戟酯(6);
其中,所述的液体培养基包括组分:200g马铃薯去皮,切成1立方厘米小丁,1L水微沸煎煮20分钟,随后用8层纱布趁热过滤,放凉后用水将滤液补齐至1L,加入20g葡萄糖,搅拌溶解;分装并经121℃高压灭菌20分钟后,放凉使用;
其中,所述的固体培养基包含组分:上述液体培养基加入1%琼脂,加热溶解并分装,121℃高压灭菌20分钟后,放凉使用。
本发明利用已知微生物对20-去氧巨大戟醇和13-O-正十二烷酸巨大戟醇酯进行微生物转化,分别制备了两化合物非活性碳的羟化衍生物,为两者后继的结构改造提供新的修饰位点。
本发明进一步提供一种药物组合物,其中含有上述获得的巨大戟烷型二萜衍生物化合物或其药学上可接受的溶剂化物及药学上可接受的载体。
具体实施方式
实施例1 检视拉曼被孢霉Mortierella ramanniana转化20-去氧巨大戟醇结果
本发明对20个菌种进行转化筛选,结果表明拉曼被孢霉Mortierella ramannianaCGMCC 3.03413对20-去氧巨大戟醇有较强的转化能力;
本实验中,采用的菌种筛选培养基为马铃薯培养基:将200g去皮马铃薯切成1立方厘米小丁,用1L水煮沸20分钟,马铃薯液过滤后加入20g葡萄糖,分装于250mL三角瓶,每瓶50mL,在121℃、0.15MPa下灭菌20分钟;
菌种接种于斜面固体培养基上,28℃培养7天,保存于4℃冰箱内,采用两步活化法活化菌种,先将菌种接种于马铃薯培养基,28℃,180rpm条件下摇瓶培养48h后,获得种子液;种子液以1%-3%体积比接种于另一马铃薯培养基内,同条件培养48h,每瓶活化后的菌液加入5mg/mL的20-去氧巨大戟醇乙醇溶液,终浓度为0.1mg/mL,同条件培养72h后,抽滤发酵液后得滤液,加乙酸乙酯萃取3次,合并后回收乙酸乙酯,残渣加少量甲醇溶解供薄层色谱鉴别用,其中的空白对照加入纯乙醇;
分别将转化后的样品和空白对照溶液点于硅胶薄层板上,20-去氧巨大戟醇转化后样品以二氯甲烷-甲醇(10:1)上行法展开,取出晾干溶剂后,喷以10%的硫酸-乙醇溶液,加热显色,结果显示,转化产物和底物同样显示砖红色-棕色斑点,转化产物的Rf值小于底物的Rf值。
实施例2 制备转化产物16-羟基-20-去氧巨大戟醇(1)、19-羟基-20-去氧巨大戟醇(2)、12-羟基-20-去氧巨大戟醇和(3)和12,19-二羟基-20-去氧巨大戟醇(4)
采用两步活化法活化拉曼被孢霉Mortierella ramanniana CGMCC 3.03413菌种,获得的种子液以1%体积比接种于装有250mL马铃薯培养基、体积为1L三角瓶中,28℃,150rpm条件下摇瓶培养48h,每瓶活化后的菌液加入5mg/mL的20-去氧巨大戟醇的乙醇溶液,终浓度为0.1mg/mL。同条件培养72h后,发酵液抽滤,滤液加乙酸乙酯萃取3次,合并后回收乙酸乙酯,得到发酵液总提物;
发酵液总提物用少量乙酸乙酯溶解,以0.7g硅胶(100-200目)拌样,二氯甲烷湿法上样于装有30g柱层析硅胶(200-300目)的硅胶柱内,用二氯甲烷-甲醇(30:1-15:1)洗脱,得到三个流分(Fr.1-Fr.3),挥去有机溶剂后,甲醇溶解。Fr.1用高效液相色谱纯化后得到16-羟基-20-去氧巨大戟醇(1)和19-羟基-20-去氧巨大戟醇(2);Fr.2用高效液相色谱纯化后得12-羟基-20-去氧巨大戟醇(3);Fr.3用高效液相色谱纯化后得12,19-二羟基-20-去氧巨大戟醇(4);化合物1-4的NMR结构鉴定数据如表1所示。
表1.转化产物1-4的NMR数据(400MHz,CD3OD)
实施例3 制备转化产物13-O-10’-羟基正十二烷酸巨大戟酯(5)和13-O-11’-羟基正十二烷酸巨大戟酯(6)
采用两步活化法活化藤仓赤霉Gibberella fujikuroi CICC 40272菌种,获得的种子液以1%体积比接种于装有250mL马铃薯培养基、体积为1L三角瓶中,28℃,150rpm条件下摇瓶培养48h,每瓶活化后的菌液加入5mg/mL的13-O-正十二烷酸巨大戟酯的乙醇溶液,终浓度为0.1mg/mL。同条件培养72h后,发酵液抽滤,滤液加乙酸乙酯萃取3次,合并后回收乙酸乙酯,得到发酵液总提物;
发酵液总提物用少量乙酸乙酯溶解,以0.8g硅胶(100-200目)拌样,二氯甲烷湿法上样于装有30g柱层析硅胶(200-300目)的硅胶柱内,用二氯甲烷-甲醇(25:1-18:1)洗脱,得到两个流分(Fr.1-Fr.3),挥去有机溶剂后,甲醇溶解。Fr.1和Fr.2经高效液相色谱纯化(乙腈-水60:40,45min),得到2个转化产物,分别为化合物5和化合物6;化合物5和6的NMR结构鉴定数据如表2所示。
表2:转化产物5和6的NMR数据(400MHz,CDCl3)
Claims (2)
1.特异位点羟基化巨大戟烷型二萜衍生物的制备方法,其特征在于:用拉曼被孢霉Mortierella ramanniana菌种微生物(保藏号为CGMCC 3.03413)对20-去氧巨大戟醇进行微生物转化,获得下列通式中的巨大戟烷型二萜化合物1、化合物2、化合物3和化合物4;用藤仓赤霉Gibberella fujikuroi菌种微生物(保藏号为CICC 40272)对13-O-正十二烷酸巨大戟酯进行微生物转化,获得下列通式中的巨大戟烷型二萜化合物5和化合物6:
其中,
化合物1:16-羟基-20-去氧巨大戟醇,R1=R2=R4=R5=H,R3=OH
化合物2:19-羟基-20-去氧巨大戟醇,R1=R2=R3=R5=H,R4=OH
化合物3:12-羟基-20-去氧巨大戟醇,R2=R3=R4=R5=H,R1=OH
化合物4:12,19-二羟基-20-去氧巨大戟醇,R2=R3=R5=H,R1=R4=OH
化合物5:13-O-10’-羟基正十二烷酸巨大戟酯,
R1=R3=R4=R5=H,R2=-OOC(CH2)8CHOHCH2CH3
化合物6:13-O-11’-羟基正十二烷酸巨大戟酯,
R1=R3=R4=R5=H,R2=-OOC(CH2)9CHOHCH3
所述的微生物转化法中,包括步骤:
(1)将生长在琼脂培养基上的微生物菌丝划线接种于马铃薯固体培养基上,在20-28℃的恒温培养箱中培养3-7天,得到试管种;
(2)将试管种中的菌丝接种到三角瓶中,每瓶含有液体马铃薯培养基,培养温度为25-28℃,转速为130-180rpm,培养时间为24小时,获得种子液;
(3)将种子液按2%~5%体积比接入新鲜马铃薯培养基,28℃,130-180rpm条件下培养24-72小时后,加入转化底物,在20-28℃、130-180rpm的条件下转化培养72-240小时;
(4)将发酵液用布氏漏斗减压过滤,获得发酵滤液,按照1:1.5体积比用乙酸乙酯萃取3次,合并乙酸乙酯层,减压蒸干,发酵产物用硅胶柱层析法分离得到所述巨大戟醇的羟基化产物。
2.根据权利要求1所述的方法,其特征在于,将活化后的微生物活体种子液按一定体积比接入马铃薯培养基,28℃培养24-72小时,再加入转化底物的乙醇溶液。
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