微生物轉換Isostevic acid

Microbial Transformation of Isostevic acid

林文光(Wen-Kuang Lin)

指導教授 ： 林淑娟

臺北醫學大學/藥學院/藥學研究所/碩士(2010年)

摘要

微生物轉換技術具有位置(regio-)及立體(stereo-)選擇性，因此被用來修飾天然及合成化合物的構造。Isosteviol (2)為四環雙萜類化合物，具有ent-beyerane架構，且其活性及微生物轉換已廣泛被研究。文獻指出，將微生物轉換的受質稍微改變其結構，可修飾或引出新的生物活性，因此，isosteviol (2) 經由Wolff-Kishner reduction反應，以製備isostevic acid (ent-beyeran-19-oic acid) (3)，接著再進行微生物轉換研究，產生新的衍生物，以進行新的生物活性之探討。經由菌種篩選實驗發現Aspergillus niger BCRC 32720、Bacillus megaterium ATCC 14581及Mortierella isabellina ATCC 38063具有反應再現性，因此選擇這些菌種進行大量發酵培養，並經由萃取及各種分離純化之方式得到不同代謝產物。研究發現，受質 3經由A. niger代謝得到
ent-1beta,7alpha,11alpha-trihydroxybeyeran-19-oic acid (4)、ent-1beta,7alpha,17-trihydroxybeyeran-19-oic acid (5)、
ent-1beta,17-dihydroxybeyeran-19-oic acid (6)、ent-11alpha-hydroxy-7-oxobeyeran-19-oic acid (7)、ent-1beta-
hydroxy-7-oxobeyeran-19-oic acid (8)、ent-7alpha-hydroxy-1-oxobeyeran-19-oic acid (9)、ent-1beta,7alpha-dihydroxybeyeran-19-oic acid (10) 和ent-7alpha,11alpha-dihydroxybeyeran-19-oic acid (11)；經由B. megaterium代謝得到ent-12beta-hydroxybeyeran-19-oic acid (12)、
ent-12beta,15beta-dihydroxybeyeran-19-oic acid (13)、
ent-17-hydroxybeyeran-19-oic acid (14)和ent-12beta,17-dihydroxybeyeran-19-oic acid (15)；經由M. isabellina代謝得到13、15、ent-9alpha,12beta-dihydroxybeyeran-19-oic acid (16) 和ent-12beta-hydroxy-16-oxobeyeran-19-oic acid (17)。這些生化反應包括選擇性羥化及羥化反應後進一步進行氧化反應，其中4-16為新的化合物，所得之新化合物皆經由低、高解析質譜及一維、二維核磁共振光譜等鑑定其結構，4、10、15和16則進一步透過X-ray單晶繞射分析確認其結構。此外，受質3及分離所得之化合物，其藥理活性實驗，目前正在進行中。

關鍵字

微生物轉換；四環雙萜類；甜菊

並列摘要

Microbial transformation has been used for structurally modification of natural and synthetic compounds due to its significant regio- and stereoselectivities. Isosteviol (2), an ent-beyerane tetracyclic diterpene possessing many biological activities, has been detailed studies of the biotransformation. According to the literature, small modifications in the chemical structure of a compound can modify its biological activities. Therefore, isostevic acid (ent-beyeran-19-oic acid) (3) was prepared by the Wolff-Kishner reduction of 2 and subjected to microbial transformation to produce new derivatives for developing new biological activity. Screening 18 strains of microorganisms revealed that Aspergillus niger BCRC 32720,Bacillus megaterium ATCC 14581, and Mortierella isabellina ATCC 38063 were able to reproducibly produce several compounds. Thus, they were selected for the preparative-scale biotransformation of 3. Incubation of 3 with A. niger afforded ent-1beta,7alpha,11alpha-trihydroxybeyeran-19-oic acid (4), ent-1beta,7alpha,17-trihydroxybeyeran- 19-oic acid (5), ent-1beta,17-dihydroxybeyeran-19-oic acid (6), ent-11alpha- hydroxy-7-oxobeyeran-19-oic acid (7), ent-1beta-hydroxy-7-oxobeyeran- 19-oic acid (8), ent-7alpha-hydroxy-1-oxobeyeran-19-oic acid (9), ent-1beta,7alpha-dihydroxybeyeran-19-oic acid (10), and ent-7alpha,11alpha- dihydroxybeyeran-19-oic acid (11). Incubation of 3 with B. megaterium yielded ent-12beta-hydroxybeyeran-19-oic acid (12), ent-12beta,15beta- dihydroxybeyeran-19-oic acid (13), ent-17-hydroxybeyeran-19-oic acid (14), and ent-12beta,17-dihydroxybeyeran-19-oic acid (15). Incubation of 3 with M. isabellina produced 13, 15, ent-9alpha,12beta-dihydroxybeyeran- 19-oic acid (16), and ent-12beta-hydroxy-16-oxobeyeran-19-oic acid (17). The biochemical reactions involved hydroxylation followed by selective oxidation. Among the isolated compounds, 4-16 were previously unreported and the structures of new compounds were elucidated by LR- and HRMS, and 1D and 2D NMR. Structures of 4, 10, 15, and 16 were further confirmed by a single-crystal diffraction experiment. Besides, the pharmacological testing of these compounds is still in progress.

並列關鍵字

microbial transformation ； tetracyclic diterpene ； stevia

參考文獻

Murakami- Nakai, C.; Kuriyama, I. Structural analysis of

biotransformations. Wiley-VCH: Germany, 2000.

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