微生物轉換

(1)

微生物轉換

steviol-16α,17-epoxide

 Steviol 為 stevioside 的醣 ? 配基，可直接作用於胰臟 β cells 而促進胰島素的分泌。我們曾利用 Muco r recurvatus 轉換 steviol 得到代謝物 ent-13,16β,17-trihydroxy-kauran-19-oic acid (2) ，並且得知代謝物 2 的降血糖活性較 steviol 佳。此外， ent-kaurane 架構的化合物也具有抗 HIV 病毒及抗癌等多樣的生物活性。為了取得更多的代謝物 2 以進行活性試驗及其機轉探討，因此選擇以 steviol-16α,17-epo xide (1) 當作受質進行微生物轉換。經過 15 株菌種篩選，由 Mucor recurvatus 、 Streptomyces griseu s 及 Cunninghamella bainieri 可得到不同的代謝物且具有再現性，因此選擇此三種菌種進行大量發酵培養，經由抽取、分離及純化由 Mucor recurvatus 得到 ent-13,16β,17-trihydroxy-kauran- 19-oic acid (2) 、 ent-13,16β-dihydroxy-17-acetoxy-kauran-19-oic acid (3) 、 ent-11α,13,16α,17-tetrahydroxy-kaura n-19-oic acid (4) 、 ent- 11β,13,16β,17-tetrahydroxy-kauran-19-oic acid (5) 、 ent-11β,13-dihydroxy-16 β,17-epoxy-kauran-19-oic acid (6) 、 ent-1β,17-dihydroxy-16- ketobeyeran-19-oic acid (7) 以及 ent-7α,1 7-dihydroxy-16-ketobeyeran- 19-oic acid (8) ；從 Streptomyces griseus 得到化合物 4 、 ent-13,17-dihyd roxy-kaur-15-en-19-oic acid (9) 、 ent-17-hydroxy-16- ketobeyeran-19-oic acid (10) 、 ent-2α,17-dihydro xy-16-ketobeyeran- 19-oic acid (11) 、 ent-12α,17-dihydroxy-16-ketobeyeran-19-oic acid (12) 、 ent-12β, 17-dihydroxy-16-ketobeyeran-19-oic acid (13) 及 ent- 14α,17-dihydroxy-16-ketobeyeran-19-oic acid (14)

；從 Cunninghamella bainieri 得到化合物 7 、 8 、 ent-7β,17-dihydroxy-16-ketobeyeran-19-oic acid (1 5) 、 ent-9α,13-dihydroxy-16β,17-epoxy-kauran-19-oic acid (16) 、 ent-9α,17-dihydroxy-16-ketobeyeran- 19-oic acid (17) 、 ent-9α,13,16α,17- tetrahydroxy-kauran-19-oic acid (18) 及 ent-9α,13,16β,17-tetrahydro xy- kauran-19-oic acid (19) 。其中化合物 3-8 及 11-19 為新化合物，所得化合物均經由一維、二維核磁共振光譜及低解析、高解析質譜等鑑定其結構。這是首度發現微生物具有將 ent-kaurane 架構進行重排反應而生成 ent-beyerane 架構的能力；此外，也發現 Cunninghamella bainieri 可以專一性的於 st eviol-16α,17-epoxide 的 C-9 進行 hydroxylation ；同時，我們也推測 Mucor recurvatus 中應含有 epoxi dase 及 epoxide hydrolase ，故能將 steviol 先氧化形成 steviol-16α,17-epoxide (1) 後，再進行水解反應產生化合物 2 ；分離所得的代謝物其生物活性試驗目前正在進行中。

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Microbial Transformations of Steviol-16α,17-epoxide

 Steviol, the aglycone of stevioside, could stimulate insulin secretion via directly acting on β cells. Previous ly, we have isolated ent-13,16β,17- trihydroxy-kauran-19-oic acid (2) from transformation of steviol with Mucor recurvatus. The preliminary testing of antihyperglycemic effect showed that it was more potent than steviol. Besides, ent-kaurane possesses many biological activities, such as anti-HIV and anticancer activitie s. In order to generate more amount of 2 for further investigation of action mechanism and for biological te stings, the microbial transformations of steviol-16α,17-epoxide (1) were conducted. By screening fifteen m icroorganisms, Mucor recurvatus, Streptomyces griseus and Cunninghamella bainieri were selected for pre parative-scale transformations of steviol-16α,17-epoxide because they reproducibly formed metabolites. M icrobial transformation of 1 with Mucor recurvatus produced ent-13,16β,17-trihydroxy-kauran-19-oic acid (2), ent-13,16β- dihydroxy-17-acetoxy-kauran-19-oic acid (3), ent-11α,13,16α,17-tetrahydroxy-kauran-1 9-oic acid (4), ent-11β,13,16β,17-tetrahydroxy-kauran- 19-oic acid (5), ent-11β,13-dihydroxy-16β,17-epox y-kauran-19-oic acid (6), ent-1β,17-dihydroxy-16-ketobeyeran-19-oic acid (7), and ent-7α,17- dihydroxy-1 6-ketobeyeran-19-oic acid (8). Microbial transformation of 1 with Streptomyces griseus produced 4, ent-13 ,17-dihydroxy-kaur-15-en- 19-oic acid (9), ent-17-hydroxy-16-ketobeyeran-19-oic acid (10), ent- 2α,17-di hydroxy-16-ketobeyeran-19-oic acid (11), ent-12α,17-dihydroxy- 16-ketobeyeran-19-oic acid (12), ent-12β ,17-dihydroxy-16-ketobeyeran- 19-oic acid (13), and ent-14α,17-dihydroxy-16-ketobeyeran-19-oic acid (1 4). Microbial transformation of 1 with Cunninghamella bainieri produced 7, 8, ent-7β,17-dihydroxy-16-ket obeyeran-19-oic acid (15), ent-9α,13-dihydroxy-16β,17-epoxy-kauran-19-oic acid (16), ent-9α,17- dihydro xy-16-ketobeyeran-19-oic acid (17), ent-9α,13,16α,17-tetrahydroxy-kauran-19-oic acid (18), and ent-9α,1 3,16β,17-tetrahydroxy- kauran-19-oic acid (19). Among them, metabolites 38 and 1119 are the new compo  unds. The structures of metabolites are established on the basis of HRFABMS, IR, and 1D and 2D NMR. T his is the first report that microbe could process the rearrangement of ent-kaurane to ent-beyeran. We also f ound that Cunninghamella bainieri has the ability to regiospecific hydroxylation at C-9 position of steviol- 16α,17-epoxide (1). Besides, the results suggested that steviol was transformed into 2 through 1. The biolo gical testings of isolated metabolites are still in progress.