异戊烯基化吲哚类生物碱的化学酶合成2009

ISS N 100727626C N 1123870ΠQ

中国生物化学与分子生物学报

Chinese Journal of Biochemistry and M olecular Biology

2009年6月25(6):580～584

收稿日期:2008211205;接受日期:2009203210国家自然科学基金资助(N o 130670019)

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联系人　T el :010*********;E 2mail :liuxq @

Received :N ovember 5,2008;Accepted :M arch 10,2009

Supported by National Natural Science F oundation of China (N o.30670019)

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C orresponding author T el :010*********;E 2mail :liuxq @

异戊烯基化吲哚类生物碱的化学酶合成

王　璐1),　尹文兵2),　李书明2),　刘晓晴

1)3

(1)首都师范大学生命科学学院,北京　100048;

2)

马尔堡大学药物生物研究所,马尔堡　35037,德国)

摘要　异戊烯基化吲哚类生物碱广泛存在于麦角菌、青霉菌和曲霉菌中,具有一定的药理学活性,

与未异戊烯基化的前体在生物活性方面具有明显的差异.曲霉菌中的某些异戊烯基化吲哚类生物碱具有抗癌活性,如烟曲霉毒素C (fumitrem orgin C )、tryprostatin B ,但其天然产量低且不易分离,利用化学酶合成法可很容易地将前体转化为异戊烯基化吲哚类生物碱.异戊烯基转移酶FtmPT 1对二甲丙烯基二磷酸(dimethylallyl diphosphate ,DMAPP )具有专一性,但可以接受不同的芳香族底物.早期研究发现,FtmPT 1能接受含色氨酸的不同环二肽为底物,但以cyclo 2L 2T rp 2L 2T yr 和cyclo 2L 2T rp 2L 2Phe 为底物时,酶的相对活性很低,其产物量少,无法用于合成产物.本实验通过优化酶反应条件来提高其产量.将已构建的含ftmPT 1的质粒在大肠杆菌中诱导表达,经Ni 2NT A 亲和柱纯化后用于酶反应.实验结果表明,通过增加酶量(终浓度218μm ol ΠL )、延长培养时间(37℃,24h ),以cyclo 2L 2T rp 2

L 2T yr 和cyclo 2L 2T rp 2L 2Phe 为底物的酶反应产率分别达到4913%和2113%,产物经1H 2NMR 、1H 21

H 2C OSY 和ESI 2MS 鉴定,其结果与预期吻合.据检索,这2个化合物均为新化合物,分别命名为cyclo 2C221′2DMA 2L 2T rp 2L 2T yr 和cyclo 2C221′2DMA 2L 2T rp 2L 2Phe.关键词　异戊烯基转移酶FtmPT 1;异戊烯基化吲哚类生物碱;化学酶合成中图分类号　Q81

Chemoenzymatic Synthesis of Prenylated I ndole Alkaloids

W ANGLu 1),YI N Wen 2Bing 2),LI Shu 2Ming 2),LI U X iao 2Qing

1)3

(1)College o f Life Science ,Capital Normal Univer sity ,Beijing 100048,China ;

2)

Institut f ür Pharmazeutische Biologie ,Univer sity o f Marburg ,Marburg 35037,G ermany )

Abstract Different from the non 2prenylated precurs ors ,prenylated indole alkaloids produced by Claviceps ,Penicillium and Aspergillus strains exert diverse biological and pharmacological activities ,for exam ples ,fumitrem orgin C and tryprostatin B inhibit the growth of a number of tum or cell lines.H owever ,these com pounds are usually produced at low concentrations in the natural hosts ,thus limits their use in research and development.Chem oenzymatic synthesis has recently been applied to s olve this problem.FtmPT 1,a prenyltrans ferase from Aspergillus f umigatus ,has a high specificity to dimethylallyl diphosphate (DMAPP ),and is als o flexible to tryptophan 2containing cyclic dipeptides as the substrates.H owever ,the yields of FtmPT 1react with cyclo 2L 2T rp 2L 2T yr or cyclo 2L 2T rp 2L 2Phe was not su fficient for success ful production of prenylated

com pounds in previous studies.In this paper ,by increasing the enzyme dose to 218μ

m ol ΠL and extending the incubation time to 24hours ,the conversion rates of cyclo 2L 2T rp 2L 2T yr or cyclo 2L 2T rp 2L 2Phe to their

prenylated derivatives was increased to 4913%and 2113%,respectively.1H 2NMR ,1H 21

H 2C OSY and ESI 2MS analyses con firmed that the products of the enzymatic reactions were cyclo 2C221′2DMA 2L 2T rp 2L 2T yr and cyclo 2C221′2DMA 2L 2T rp 2L 2Phe ,as expected.K ey w ords prenyltrans ferase FtmPT 1;prenylated indole alkaloids ;chem oenzymatic synthesis