新型强效免疫抑制剂西罗莫司的微生物转化
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摘要
西罗莫司(Sirolimus, SRL)是由吸水链霉菌产生的36元环含氮三烯大环内酯具有很强的抗真菌、抗增殖和抗肿瘤作用的新型强效免疫抑制剂。由于它的多功能作用及其应用价值,西罗莫司结构改造已成为人们的研究热点。通过化学半合成法已获得一些新的mTOR酶抑制剂如RAD001、CCI-779和AP23573,已被开发为防治血管再狭窄的涂层支架药物、被开发为毒副作用极低的靶向抗肿瘤药物。除化学半合成外,人们还尝试利用微生物或微生物产生的酶对西罗莫司进行结构改造的另一条途径,企图通过微生物转化方法来获取新化合物。本研究应用不同类型的66株微生物对西罗莫司进行微生物转化试验,获得15株能转化西罗莫司的小单孢菌和1株游动放线菌。我们选取了生物转化率较高的小单孢菌FIM03-712作为代表菌株进行了深入的研究。菌种分类研究表明它与旱地小单孢菌Micromonospora chersina类似。研究了小单孢菌FIM03-712对西罗莫司的生物转化条件并从转化发酵液中分离、纯化出微生物转化产物FJ900。理化性质和LC-MS,NMR等波谱分析研究表明FJ900与14-去氧雷帕霉素同质。它的抗真菌和免疫抑制活性比西罗莫司低,它的潜在抗肿瘤作用将有待于进一步研究。
Sirolimus produced by Streptomyces hygroscopicus FC904 is a 36-membered macrolide immunosuppressant with antifungal, antiproliferative and antitumor activity. The structural modification of sirolimus has attracted great interest for its multifunctional effect and applied value. Several novel mTOR inhibitors such as RAD001, CCI-779 and AP23573 made by chemical synthesis have been used as coating for vascular stents to prevent restenosis of coronary arteries and are being developed into anticancer targeting drugs. Besides the chemical synthesis, microbial conversion is performed as an another approach for searching novel analogs of sirolimus. In this paper, it showed that different types of 66 culture were screened for the biotransformation of sirolimus and 15 strains of Micromonospora and 1 strain of Actinoplanes were found to be capable of transforming sirolimus. Micromonospora sp. FIM03-712 as a candidate was selected and classified as Micromonospora chersina. The conversion conditions of sirolimus by Micromonospora sp. FIM03-712 were explored. One conversion product, FJ900, from the broth of the strain was isolated and purified. By means of physico-chemical properties and spectral analyses such as HPLC, UV, IR, LC-MS and NMR, FJ900 was turned out to be identical with 14-deoxorapamycin. In comparation with sirolimus, FJ900 had less activity in antifungal and immunosuppressive actions. Whereas, the anticancer activity will be investigated further.
Sirolimus produced by Streptomyces hygroscopicus FC904 is a 36-membered macrolide immunosuppressant with antifungal, antiproliferative and antitumor activity. The structural modification of sirolimus has attracted great interest for its multifunctional effect and applied value. Several novel mTOR inhibitors such as RAD001, CCI-779 and AP23573 made by chemical synthesis have been used as coating for vascular stents to prevent restenosis of coronary arteries and are being developed into anticancer targeting drugs. Besides the chemical synthesis, microbial conversion is performed as an another approach for searching novel analogs of sirolimus. In this paper, it showed that different types of 66 culture were screened for the biotransformation of sirolimus and 15 strains of Micromonospora and 1 strain of Actinoplanes were found to be capable of transforming sirolimus. Micromonospora sp. FIM03-712 as a candidate was selected and classified as Micromonospora chersina. The conversion conditions of sirolimus by Micromonospora sp. FIM03-712 were explored. One conversion product, FJ900, from the broth of the strain was isolated and purified. By means of physico-chemical properties and spectral analyses such as HPLC, UV, IR, LC-MS and NMR, FJ900 was turned out to be identical with 14-deoxorapamycin. In comparation with sirolimus, FJ900 had less activity in antifungal and immunosuppressive actions. Whereas, the anticancer activity will be investigated further.
引文
[1] 程元荣. 微生物产生的免疫抑制剂[J]. 中国抗生素杂志, 1996;3 (21) 增刊:24~33.
[2] C. Vezina, A. Kudelski, S. N. Sehgal. Rapamycin (AY22989), a new antifungal antibiotic I. Taxonomy of the producing streptomycete and isolation of the active principle[J]. J Antibiot, 1975; 28(10): 721~726.
[3] S. N. Sehgal, H. Baker, C. Vezina. Rapamycin(AY-22989), a new antifungal an- tibiotic II. Fermentation, isolation and characterization[J]. J Antibiot, 1975; 28 (10): 727~731.
[4] 程元荣.新型强效大环内酯免疫抑制剂—雷帕霉素, 第四届全国化疗及药理学术会议, 1994.
[5] 程元荣, 林文良, 黄捷等. 新型强效免疫抑制剂西罗莫司F904的研究[J]. 中国抗生素杂志, 2002; 27(12): 709～712.
[6] 程元荣, 郑卫.新分子靶位mTOR和新抗肿瘤靶向药物雷帕霉素及其衍生物[J].中国处方药, 2006; 12(57): 16～19.
[7] 宋欣. 微生物酶转化技术[M]. 北京: 化学工业出版社, 2004.
[8] 陈代杰, 朱宝泉. 微生物转化技术在现代医药工业中的应用[J]. 中国抗生素杂志, 2006; 31(2): 112～118.
[9] 徐诗伟. 微生物转化在药物合成中的应用前景[J]. 中国医药工业杂志,1996, 27(9): 422～430.
[10] 张玉彬. 生物催化的手性合成[M]. 北京: 化学工业出版社, 2002.
[11] H. Nishida, T. Sakakibara, et al. Generation of novel rapamycin structures by microbial manipulations[J]. J Antibiot, 1995; 48(7): 657～666.
[12] M. A . Guadliana, S. J. Truesdell. U. S. Patent 5849730, 1998.
[13] M. Kuhnt, F. Bitsch, et al. Microbial conversion of rapamycin[J]. Enzyme Microb.Technol, 1997; 21: 405～412.
[14] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immu- nosuppressive compounds I. Desmythylation of FK506 and immunomycin (FR-900520) by Actinoplanes sp. ATCC 53771[J]. J Antibiot, 1992; 45(1):118～123.
[15] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immu- nosuppressive compounds II. Specific desmethylation of 13-methoxy group of FK506 and FR900520 by Actinomycete sp. ATCC 53828 [J] . J Antibiot,1992; 45(4): 577～580.
[16] 李夸良,黄捷,程元荣. 游动放线菌ATCC 12065生物转化强效免疫抑制剂西罗莫司[J]. 海峡药学, 2008; 20(4): 98～101.
[17] E . B . Shirling, D.Gottlieb. Methods for characterization of Streptomyces species[J]. International Journal of Systematic Bacteriology, 1966; 16: 313～340.
[18] S. A. Waksman. Classification, identification and description of genera and spe- cies. The actinomycetes [M], The Williams and Wilkins Co. Baltimore, 1961(2).
[19] 王平. 测定放线菌菌体中氨基酸和单糖的快速方法—薄层层析法[J]. 微生物学通报, 1986, 13(5): 228～231.
[20] 邓子新, 唐纪良译. 链霉菌遗传操作实验手册( D. A. Hopwood )[M]. 长沙:湖南科学技术出版社: 1988, 57～58.
[21] 杨国新, 江丹云, 黄捷等. 西罗莫司异构体Rap-D1的分离与鉴定[J]. 中国抗生素杂志, 2008, 2; 33(2): 80～83.
[22] 黄文强主编. 吸附分离材料[M]. 北京: 化学工业出版社: 2005, 34～58.
[23] 杨国新, 江丹云, 黄捷等. 高纯度西罗莫司的制备[J]. 海峡药学, 2007; 19(7): 25～26.
[24] 林文良, 吴晓华, 程元荣等. 新型强效免疫抑制剂F904的结构测定[J]. 中国抗生素杂志, 2002, 12; 27(12): 713～716.
[25] N. L. Paiva, A. L. Demain. In corporation of acetate, propionate, and methio- nine into rapamycin by Streptomyces hygroscopicus[J]. J Natural Products, 1991; 54(1): 167～177.
[26] 张祝兰, 林善, 唐文力等. 新的免疫调节剂筛选模型的建立[J]. 海峡药学, 1999; 11(1): 13～14.
[1] C. Vezina, A. Kudelski, S. N. Sehgal. Rapamycin(AY22989), a new antifungal antibiotic I. Taxonomy of the producing streptomycete and isolation of the active principle[J]. J Antibiot, 1975; 28(10): 721.
[2] S. N. Sehgal, H. Baker, C. Vezina. Rapamycin(AY-22989), a new antifungal antibiotic II. Fermentation, isolation and characterization [J]. J Antibiot, 1975; 28(10): 731.
[3] F. L. Luan , R. Ding, V. K. Sharma, et al. Rapamycin is an effevtive inhibitor of human renal cancer metastasis. Kidney Int, 2003; 63(3): 917.
[4] 程元荣,林文良,黄捷等.新型强效免疫抑制剂-西罗莫司F904的研究[J].中国抗生素杂志,2002; 27(12): 709~712.
[5] N. L. Paiva, A. L. Demain. In corporation of acetate, propionate, and methionine into rapamycin by streptomyces hygroscopicus[J]. J Natural Produccts, 1991; 54(1): 167～177.
[6] D. A. Holt, A. L. Konialian, et al. Structure-activity studies of nonmacrocyclic rapamycin derivatives[J]. Medicinal Chemistry Letters, 1993; 3(10): 1977～1980.
[7] 陆群. 雷帕霉素生物合成、化学修饰及作用机制的研究进展[J]. 抗生素信息, 1997; 20(2): 1～6.
[8] H. Nishida, T. Sakakibara, et al. Generation of novel rapamycin structures by microbial manipulations[J]. J Antibiot , 1995; 48(7): 657～666.
[9] 张亚,王明伟.新型免疫抑制剂构效关系的研究进展[J]. 中国新药杂志, 2005; 14(9): 1109~1113.
[10] S. Cottens, R. Sedrani. U . S . Patent 5665772, 1997.
[11] W. Kao, R. L.Vogel. U. S. Patent 5349060, 1994.
[12] S. Cottens, R. Sedran. U. S. Patent 6200985B1.
[13] C. C. Shaw, J. Sellstedt, et al. U. S. Patent 6277983B1, 2001.
[14] D. C. Hu, A. A. Greenfield, et al. U. S. Patent 5922730, 1999.
[15] 程元荣, 郑卫. 新分子靶位mTOR和新抗肿瘤靶向药物雷帕霉素及其衍生物[J]. 中国处方药, 2006; 12(57): 16～19.
[16] V. S. Bernan, E. I. Graziani. U. S. Patent 0115879 A1.
[17] G. E. Caufield , J. H. Musser, et al. U.S.Patent 5023262, 1991.
[18] D. A. Holt, J. I. Luengo, et al. U. S. Patent RE37421E, 2001.
[19] F. V. Ritacco, E. I. Graziani, et al. production of novel rapamycin analogs by precursor-directed biosynthesis[J]. Applied and Environmental Microbiology, 2005; 74(4): 1971～1976.
[20] 陈代杰, 朱宝泉. 微生物转化技术在现代医药工业中的应用[J]. 中国抗生素 杂志,2006; 31(2): 112～118.
[21] 徐诗伟.微生物转化在药物合成中的应用前景[J]. 中国医药工业杂志, 1996; 27(9): 422～430.
[22] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immunosuppressive compounds I. Desmythylation of FK506 and immunomycin (FR-900520) by Actinoplanes sp. ATCC 53771 [J]. J Antibiot, 1992; 45(1): 118～123.
[23] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immu- nosuppressive compounds II. Specific desmethylation of 13-methoxy group of FK506 and FR-900520 by Actinomycete sp. ATCC 53828[J]. J Antibiot, 1992; 45(4): 577～580.
[24] A. Shafiee, B. H. Arison, et al. U. S. Patent 5352783, 1994.
[25] B. R. Petuch, B. Arison, A. Hsu, et al. Microbial transformation of immunosup- pressive compounds III. Glucosylation of immunomycin (FR900520) and FK506 by Bacillus subtilis ATCC 55060[J]. J Industrial Microbiology, 1994;(13): 131～135.
[26] T. S. Chen, B. petuch, R. White, et al. Microbialtransformation of immunosu- ppressive compounds IV. Hydroxylation and hemiketal formation of ascomycin (immunomycin) by streptomyces sp. MA 6970 (ATCC NO.55281) [J]. J Anti- biot, 1994; 47(12): 1557～1559.
[27] 宋欣. 微生物酶转化技术[M]. 北京: 化学工业出版社, 2004.
[28] 张玉彬. 生物催化的手性合成[M]. 北京: 化学工业出版社,2002.
[29] M. Kuhnt, F. Bitsch, et al. Microbial conversion of rapamycin [J]. Enzyme Microb. Technol, 1997; 21: 405～412.
[30] M. A . Guadliana, S. J. Truesdell. U. S. Patent 5849730, 1998.
[2] C. Vezina, A. Kudelski, S. N. Sehgal. Rapamycin (AY22989), a new antifungal antibiotic I. Taxonomy of the producing streptomycete and isolation of the active principle[J]. J Antibiot, 1975; 28(10): 721~726.
[3] S. N. Sehgal, H. Baker, C. Vezina. Rapamycin(AY-22989), a new antifungal an- tibiotic II. Fermentation, isolation and characterization[J]. J Antibiot, 1975; 28 (10): 727~731.
[4] 程元荣.新型强效大环内酯免疫抑制剂—雷帕霉素, 第四届全国化疗及药理学术会议, 1994.
[5] 程元荣, 林文良, 黄捷等. 新型强效免疫抑制剂西罗莫司F904的研究[J]. 中国抗生素杂志, 2002; 27(12): 709～712.
[6] 程元荣, 郑卫.新分子靶位mTOR和新抗肿瘤靶向药物雷帕霉素及其衍生物[J].中国处方药, 2006; 12(57): 16～19.
[7] 宋欣. 微生物酶转化技术[M]. 北京: 化学工业出版社, 2004.
[8] 陈代杰, 朱宝泉. 微生物转化技术在现代医药工业中的应用[J]. 中国抗生素杂志, 2006; 31(2): 112～118.
[9] 徐诗伟. 微生物转化在药物合成中的应用前景[J]. 中国医药工业杂志,1996, 27(9): 422～430.
[10] 张玉彬. 生物催化的手性合成[M]. 北京: 化学工业出版社, 2002.
[11] H. Nishida, T. Sakakibara, et al. Generation of novel rapamycin structures by microbial manipulations[J]. J Antibiot, 1995; 48(7): 657～666.
[12] M. A . Guadliana, S. J. Truesdell. U. S. Patent 5849730, 1998.
[13] M. Kuhnt, F. Bitsch, et al. Microbial conversion of rapamycin[J]. Enzyme Microb.Technol, 1997; 21: 405～412.
[14] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immu- nosuppressive compounds I. Desmythylation of FK506 and immunomycin (FR-900520) by Actinoplanes sp. ATCC 53771[J]. J Antibiot, 1992; 45(1):118～123.
[15] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immu- nosuppressive compounds II. Specific desmethylation of 13-methoxy group of FK506 and FR900520 by Actinomycete sp. ATCC 53828 [J] . J Antibiot,1992; 45(4): 577～580.
[16] 李夸良,黄捷,程元荣. 游动放线菌ATCC 12065生物转化强效免疫抑制剂西罗莫司[J]. 海峡药学, 2008; 20(4): 98～101.
[17] E . B . Shirling, D.Gottlieb. Methods for characterization of Streptomyces species[J]. International Journal of Systematic Bacteriology, 1966; 16: 313～340.
[18] S. A. Waksman. Classification, identification and description of genera and spe- cies. The actinomycetes [M], The Williams and Wilkins Co. Baltimore, 1961(2).
[19] 王平. 测定放线菌菌体中氨基酸和单糖的快速方法—薄层层析法[J]. 微生物学通报, 1986, 13(5): 228～231.
[20] 邓子新, 唐纪良译. 链霉菌遗传操作实验手册( D. A. Hopwood )[M]. 长沙:湖南科学技术出版社: 1988, 57～58.
[21] 杨国新, 江丹云, 黄捷等. 西罗莫司异构体Rap-D1的分离与鉴定[J]. 中国抗生素杂志, 2008, 2; 33(2): 80～83.
[22] 黄文强主编. 吸附分离材料[M]. 北京: 化学工业出版社: 2005, 34～58.
[23] 杨国新, 江丹云, 黄捷等. 高纯度西罗莫司的制备[J]. 海峡药学, 2007; 19(7): 25～26.
[24] 林文良, 吴晓华, 程元荣等. 新型强效免疫抑制剂F904的结构测定[J]. 中国抗生素杂志, 2002, 12; 27(12): 713～716.
[25] N. L. Paiva, A. L. Demain. In corporation of acetate, propionate, and methio- nine into rapamycin by Streptomyces hygroscopicus[J]. J Natural Products, 1991; 54(1): 167～177.
[26] 张祝兰, 林善, 唐文力等. 新的免疫调节剂筛选模型的建立[J]. 海峡药学, 1999; 11(1): 13～14.
[1] C. Vezina, A. Kudelski, S. N. Sehgal. Rapamycin(AY22989), a new antifungal antibiotic I. Taxonomy of the producing streptomycete and isolation of the active principle[J]. J Antibiot, 1975; 28(10): 721.
[2] S. N. Sehgal, H. Baker, C. Vezina. Rapamycin(AY-22989), a new antifungal antibiotic II. Fermentation, isolation and characterization [J]. J Antibiot, 1975; 28(10): 731.
[3] F. L. Luan , R. Ding, V. K. Sharma, et al. Rapamycin is an effevtive inhibitor of human renal cancer metastasis. Kidney Int, 2003; 63(3): 917.
[4] 程元荣,林文良,黄捷等.新型强效免疫抑制剂-西罗莫司F904的研究[J].中国抗生素杂志,2002; 27(12): 709~712.
[5] N. L. Paiva, A. L. Demain. In corporation of acetate, propionate, and methionine into rapamycin by streptomyces hygroscopicus[J]. J Natural Produccts, 1991; 54(1): 167～177.
[6] D. A. Holt, A. L. Konialian, et al. Structure-activity studies of nonmacrocyclic rapamycin derivatives[J]. Medicinal Chemistry Letters, 1993; 3(10): 1977～1980.
[7] 陆群. 雷帕霉素生物合成、化学修饰及作用机制的研究进展[J]. 抗生素信息, 1997; 20(2): 1～6.
[8] H. Nishida, T. Sakakibara, et al. Generation of novel rapamycin structures by microbial manipulations[J]. J Antibiot , 1995; 48(7): 657～666.
[9] 张亚,王明伟.新型免疫抑制剂构效关系的研究进展[J]. 中国新药杂志, 2005; 14(9): 1109~1113.
[10] S. Cottens, R. Sedrani. U . S . Patent 5665772, 1997.
[11] W. Kao, R. L.Vogel. U. S. Patent 5349060, 1994.
[12] S. Cottens, R. Sedran. U. S. Patent 6200985B1.
[13] C. C. Shaw, J. Sellstedt, et al. U. S. Patent 6277983B1, 2001.
[14] D. C. Hu, A. A. Greenfield, et al. U. S. Patent 5922730, 1999.
[15] 程元荣, 郑卫. 新分子靶位mTOR和新抗肿瘤靶向药物雷帕霉素及其衍生物[J]. 中国处方药, 2006; 12(57): 16～19.
[16] V. S. Bernan, E. I. Graziani. U. S. Patent 0115879 A1.
[17] G. E. Caufield , J. H. Musser, et al. U.S.Patent 5023262, 1991.
[18] D. A. Holt, J. I. Luengo, et al. U. S. Patent RE37421E, 2001.
[19] F. V. Ritacco, E. I. Graziani, et al. production of novel rapamycin analogs by precursor-directed biosynthesis[J]. Applied and Environmental Microbiology, 2005; 74(4): 1971～1976.
[20] 陈代杰, 朱宝泉. 微生物转化技术在现代医药工业中的应用[J]. 中国抗生素 杂志,2006; 31(2): 112～118.
[21] 徐诗伟.微生物转化在药物合成中的应用前景[J]. 中国医药工业杂志, 1996; 27(9): 422～430.
[22] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immunosuppressive compounds I. Desmythylation of FK506 and immunomycin (FR-900520) by Actinoplanes sp. ATCC 53771 [J]. J Antibiot, 1992; 45(1): 118～123.
[23] T. S. Chen, B. H. Arison, L. S. Wicker, et al. Microbial transformation of immu- nosuppressive compounds II. Specific desmethylation of 13-methoxy group of FK506 and FR-900520 by Actinomycete sp. ATCC 53828[J]. J Antibiot, 1992; 45(4): 577～580.
[24] A. Shafiee, B. H. Arison, et al. U. S. Patent 5352783, 1994.
[25] B. R. Petuch, B. Arison, A. Hsu, et al. Microbial transformation of immunosup- pressive compounds III. Glucosylation of immunomycin (FR900520) and FK506 by Bacillus subtilis ATCC 55060[J]. J Industrial Microbiology, 1994;(13): 131～135.
[26] T. S. Chen, B. petuch, R. White, et al. Microbialtransformation of immunosu- ppressive compounds IV. Hydroxylation and hemiketal formation of ascomycin (immunomycin) by streptomyces sp. MA 6970 (ATCC NO.55281) [J]. J Anti- biot, 1994; 47(12): 1557～1559.
[27] 宋欣. 微生物酶转化技术[M]. 北京: 化学工业出版社, 2004.
[28] 张玉彬. 生物催化的手性合成[M]. 北京: 化学工业出版社,2002.
[29] M. Kuhnt, F. Bitsch, et al. Microbial conversion of rapamycin [J]. Enzyme Microb. Technol, 1997; 21: 405～412.
[30] M. A . Guadliana, S. J. Truesdell. U. S. Patent 5849730, 1998.