微生物轉換ent-13-Hydroxy-kaur-15-en-19-oic acid
Microbial Transformation of ent-13-Hydroxy-kaur-15-en-19-oic acid
中文摘要
微生物轉換是利用微生物細胞中的酵素系統進行構造修飾化合物，因此也具有 酵素反應的立體、部位專一特性，且反應發生於溫和環境。Stevioside (1)為天然 的甜味劑，可自Stevia rebaudiana (Bertoni) Bertoni 的葉子萃取得到，而
stevioside 及其相關化合物具有許多藥理活性，例如：降血糖、降血壓、抗發炎、
抗癌及免疫調節的功能。以化學觀點來看，具bridged ring 架構的 diterpenes 很適
合做為研究微生物轉換的受質，因此許多由stevioside 衍生的化合物已用於微生
物轉換的研究。ent-13-Hydroxy-kaur-15-en-19-oic acid (3)屬於 ent-kaurene diterpene，為 stevioside 以酸水解所得的其中一個產物，然而目前仍無針對此化 合物進行微生物轉換及生物活性試驗之報導。為了產生新的化合物以用於活性試
驗，因此利用微生物轉換技術構造修飾3。由菌種篩選發現， Aspergillus
niger 、Cunninghamella bainieri 及 Mortierella isabellina 可產生各種化合物且具再 現性，因此選擇這些菌種進行大量發酵培養，再經由抽取、分離及純化以得到代 謝物。研究發現經由Asp. niger 代謝得到 ent-13,16beta-dihydroxy-kauran-19-oic acid (4)、ent-13-hydroxy-11alpha,16alpha-epoxy-19-oic acid (5)、steviol (6)及 ent- 7alpha,13-dihydroxy-kaur-15-en-19-oic acid (7)；經由 C. bainieri 代謝得到 7、ent- 7alpha,14beta-dihydroxy-16-oxo-beyeran-19-oic acid (8)、ent-14beta-hydroxy-16- oxo-beyeran-19-oic acid (9)、ent-9alpha,13-dihydroxy-kaur-15-en-19-oic acid (10)及 ent-13,15beta-dihydroxy-kaur-16-en-19-oic acid (11)；經由 M. isabellina 得到 5、10、ent-3alpha,13-dihydroxy-kaur-15-en-19-oic acid (12)及 ent-13,17-dihydroxy- kaur-15-en-19-oic acid (13)，其中 7 及 9 曾以甲基化衍生物被分離出，而 5、8、10
及12 為新的化合物，所得的新化合物皆經由一維、二維核磁共振光譜，低解析、
高解析質譜及X-ray 結晶繞射分析確定結構。此外，受質 3 及分離所得的化合物
其生物活性試驗，目前正在進行中。
英文摘要
Microbial transformation can modify the structure of compounds due to the presence of numerous enzymatic activities in whole cells that act as biocatalysts. The synthesis of optically active compounds by using microbial models has been successfully applied for regio- and stereoselective under mild conditions. Stevioside (1) is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. Stevioside and related compounds have many therapeutic benefits such as anti-hyperglycemic, anti-hypertensive, anti-inflammatory, anti-tumor, and immunomodulatory actions.
From a chemical point of view, the bridged ring skeleton of diterpenes is a suitable substrate for the study of microbial transformation. Thus, a number of compounds
derived from stevioside (1) have been studied on the microbial transformation. ent-13- Hydroxy-kaur-15-en-19-oic acid (3), an ent-kaurene diterpene, is one of compounds from acid hydrolysis of 1. However, no report on the microbial transformation and biological activity of 3 has been described. In an effort to produce new functionalized compounds for biological testings, the microbial transformation of 3 was carried out.
By screening several microorganisms, Aspergillus niger, Cunninghamella bainieri, Mortierella isabellina were selected for preparative-scale transformation of 3 because they reproducibly produced metabolites. Incubation of 3 with Asp. niger produced ent-13,16beta-dihydroxy-kauran-19-oic acid (4), ent-13-hydroxy-11alpha,16alpha- epoxy-kauran-19-oic acid (5), steviol (6), and ent-7alpha,13-dihydroxy-kaur-15-en- 19-oic acid (7). Incubation of 3 with C. bainieri produced 7, ent-7alpha,14beta- dihydroxy-16-oxo-beyeran-19-oic acid (8), ent-14beta-hydroxy-16-oxo-beyeran-19- oic acid (9), ent-9alpha,13-dihydroxy-kaur-15-en-19-oic acid (10), and ent-13,15beta- dihydroxy-kaur-16-en-19-oic acid (11). Incubation of 3 with M. isabellina produced 5, 10, ent-3alpha,13-dihydroxy-kaur-15-en-19-oic acid (12), and ent-13,17-
dihydroxy-kaur-15-en-19-oic acid (13). Among them, 7 and 9 have been isolated as methylated derivatives. Compounds 5, 8, 10, and 12 are new compounds. The
structures of the new compounds were established on the basis of HRESIMS, 1D and 2D NMR, and X-ray crystallographic analyses. The biological testing of these
compounds is still in progress.
