微生物轉換D環修飾之異甜菊醇及生物活性評估

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題名:	微生物轉換D環修飾之異甜菊醇及生物活性評估
作者:	周柏宏
貢獻者:	藥學系(博士班)
關鍵詞:	異甜菊醇二萜類化合物微生物轉換
日期:	2010
上傳時間:	2010-10-20 12:58:33 (UTC+8)
摘要:	微生物轉換為利用微生物細胞中的酵素系統進行一連串生物催化反應，將化合物進行構造修飾。值得注意的是，利用此項技術可於各種不同受質中較難以反應之位置進行立體、部位專一性反應。許多研究指出，利用微生物酵素之立體選擇特性於有機合成化合物或天然化合物進行構造修飾，不但可能增加其本身之生理活性，同時也可能產生新的藥理作用。Isosteviol (2)屬於四環二萜類化合物，具有ent-beyerane架構，並於D環上有酮基，其基本骨架與固醇類類似，具有許多生物活性。根據文獻報告指出，固醇類化合物的許多重要藥理活性取決於其構造上D環之變化，而以化學觀點來看，具有bridged ring骨架之isosteviol (2)很適合做為微生物轉換研究之受質，因此，本論文以isosteviol (2)為原料，於構造上進行修飾，以期開發具新的藥理活性之化合物。利用isosterism原理，先將isosteviol (2) D環上的酮基進行化學構造修飾，得到lactone (3)、oxime (4)及lactam (5)之衍生物，再利用微生物轉換技術進行構造修飾3、4及5，得到各種不同的化合物，以進行藥理活性之研究。經由菌種篩選isosteviol衍生物3、4及5發現，Cunninghamella bainieri ATCC 9244、Aspergillus niger BCRC 32720、Absidia pseudocylindrospora ATCC 24169及Mucor recurvatus MR 36可產生許多代謝產物且具再現性，因而進行大量發酵培養，並經由各種萃取及純化等步驟得到代謝產物。研究發現，受質 isosteviol lactone (4-carboxy-13??-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16- lactone) (3)經由與C. bainieri反應得到代謝物4-carboxy-15??-hydroxy-15,16-seco-ent-19-norbeyeran-16-oic acid 15,16-lactone (6)、4-carboxy-7??,13-dihydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (7)、4-carboxy-7,15??-dihydroxy-15,16-seco-ent-19-norbeyeran-16-oic acid 15,16-lactone (8)及15,16-dioxo-16-methoxy-15,16-seco-ent-beyeran-19-oic acid (9)，此生化反應包含異構化反應、羥化反應，以及在環裂解後進一步進行氧化反應與選擇性甲基化反應；另一方面，經由與Asp. niger反應得到羥基取代之代謝物，分別為 6、7、4-carboxy-1,13-dihydroxy-13,16-seco-ent-19-norbeyeran-16- oic acid 13,16-lactone (10)、4-carboxy-1,15-dihydroxy- 15,16-seco-ent-19-norbeyeran-16-oic acid 15,16-lactone (11)、4??-carboxy-1??,7??,13??-trihydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (12)、4??-carboxy-1??,7??,15??-trihydroxy-15,16-seco-ent-19-norbeyeran-16-oic acid 15,16-lactone (13)、4??-carboxy-1??,13??-dihydroxy-7-oxo-13,16-seco-ent-19-norbeyeran- 16-oic acid 13,16-lactone (14)，以及4??-carboxy-1??,7??,11??,13??-tetrahydroxy-13,16-seco-ent-19- norbeyeran- 16-oic acid 13,16-lactone (15)；經由與Abs. pseudocylindrospora反應，得到代謝物 7、4??-carboxy-12??,13??-dihydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (16)、4??-carboxy-12??,13??-dihydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (17)、4??-carboxy-7??,13??-dihydroxy-13,16-seco-ent-19-norbeyeran- 16-oic acid 13,16-lactone (18)、4??-carboxy-13??,17-dihydroxy-13,16-seco-ent-19- norbeyeran-16-oic acid 13,16-lactone (19)、4??-carboxy-7??,13??,17-trihydroxy-13,16-seco-ent-19-norbeyeran- 16-oic acid 13,16-lactone (20)及4??-carboxy-9??,13??,17-trihydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16- lactone (21)；經由與M. recurvatus反應，得到代謝物2、6、7及ent-7??-hydroxy-16-ketobeyeran-19-oic acid (22)，其中6?{21為新化合物。所得之新化合物皆利用各種構造鑑定技術來確認，新化合物 6、7、9、16與18則進一步透過X-ray結晶繞射分析確認結構。由於四環二萜類化合物的結構類似類固醇化合物，因此此系列化合物於藥理活性試驗進行ARE (androgen response element)-mediated luciferase reporter gene assay以及抑制脂多醣刺激類老鼠巨噬細胞株RAW 264.7產生之AP-1的基因表現 [LPS-stimulated AP (activator protein)-1 expression in RAW 264.7 macrophages]作用評估，由結果顯示，6、9及13經ARE-mediated luciferase reporter gene assay篩選均呈有意義結果，其中9的活性比對照化合物testosterone 佳；而3、6、7、11、16、18、19與22對於抑制AP-1基因表現均呈現有意義結果，尤其6的抑制活性比對照化合物dexamethasone佳。受質isosteviol oxime (ent-16-E-hydroxyiminobeyeran-19-oic acid) (4)分別與Asp. niger 及Abs. pseudocylindrospora進行微生物轉換，除了得到D環具有lactone或lactam架構之化合物3與4??-carboxy-13??-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam (5)，及一個已知化合物ent-1,7-dihydroxy- 16-oxobeyeran-19-oic acid (23)，另外也得到五個新化合物ent-7??-hydroxy-16-E-hydroxyl-iminobeyeran-19-oic acid (24)、 ent-1??,7??-dihydroxy-16-E-hydroxyimino-beyeran-19-oic acid (25)、ent-1??-hydroxy-16-E-hydroxyiminobeyeran- 19-oic acid (26)、ent-8??-cyanomethyl-13-methyl-12-podocarpen-19-oic acid (27)及 ent-8??-cyanomethyl- 13-methyl-13-podocarpen-19-oic acid (28)，所得之新化合物均經由高解析ESI質譜以及一維、二維核磁共振光譜等鑑定其結構，化合物24–26則進一步透過X-ray結晶繞射分析確認結構。在藥理活性部分，此系列化合物則針對抑制脂多醣刺激類老鼠巨噬細胞株RAW 264.7產生之NF-?羠及AP-1的基因表現 (LPS-stimulated NF-?羠及AP-1 expressions in RAW 264.7 macrophages)作用進行評估，結果發現5及26於抑制NF-?羠基因表現呈現有意義結果，且26之抑制活性與對照化合物dexamethasone相同；此外，3、23、24、27與28於抑制AP-1基因表現呈現有意義結果，尤其是27對於AP-1的抑制活性比dexamethasone佳。同樣的，受質isosteviol lactam (5)也分別與Asp. niger 及Abs. pseudocylindrospora進行微生物轉換，共得到十一個化合物，27、28、ent-8??-cyanomethyl-7??-hydroxy-13- methyl-13-podocarpen-19-oic acid (29)、ent-8??-cyanomethyl-1??-hydroxy-13-methyl-13- podocarpen-19-oic acid (30)、ent-8??-cyanomethyl-12??-chloro-13(17)-podocarpen-19-oic acid (31)、4??-carboxy-13??-amino-13,16-seco-ent-15??-chloro-19-norbeyeran-16-oic acid 13,16-lactam (32)、ent-8??-cyanomethyl-12??-ethoxy-13-methyl-12-podocarpen-19-oic acid (33)、ent-8??-cyanomethyl-12??-methoxy-13-methyl-13-podocarpen-19-oic acid (34)、ent-8??-cyanomethyl-13-methyl-12-podocarpen-19-hydroxyethyl ester (35)、4??-carboxy-13??- amino-13,16-seco-ent-7??-hydroxy-19-norbeyeran-16-oic acid 13,16-lactam (36)及4??-carboxy-13??-amino-13,16-seco-ent-12??-hydroxy-19-norbeyeran-16-oic acid 13,16- lactam (37)，此生物反應包括: 羥化反應、氯化反應，以及經D環裂解後再進行立體選擇性羥化反應、酯化反應和氯化反應。所得之新化合物均經由高解析ESI質譜以及一維、二維核磁共振光譜等鑑定其結構，其中35則進一步透過X-ray結晶繞射分析確認結構。在生物活性測試方面，此系列化合物則針對抑制脂多醣刺激類老鼠巨噬細胞株RAW 264.7產生之iNOS的基因表現 (LPS-stimulated iNOS expression in RAW 264.7 macrophages)作用進行評估，結果發現化合物29?{31、33?{34，以及37於抑制iNOS基因表現呈現有意義結果，且34與37之活性比對照化合物dexamethasone佳。
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描述:	(一)論文目次 I 目錄目錄…………………………………………………………………………….………I 附表目錄………………………………………………………………...………...…III 附圖目錄…………………………………………………………………………...…V 流程目錄………………………………………………………………………...…XIII 詞彙……………………………………………………………………………………1 中文摘要………………………………………………………………………………2 英文摘要………………………………………………………………………………5 第一章緒論第一節微生物轉換之研究背景…………………………………………………9 第二節藥物代謝研究之回顧…………………………………………………..15 第三節甜菊之成分研究與活性探討…………………………………………..20 第四節微生物轉換異甜菊醇之研究回顧……………………………………..23 第五節研究目的………………………………………………………………..25 第二章微生物轉換異甜菊醇衍生物之化學構造研究 D環修飾異甜菊醇之化學合成步驟…………………………………………….26 第一節 Isosteviol lactone 進行微生物轉換之構造研究……………………….27 第二節 Isosteviol oxime 進行微生物轉換之構造研究………………………...45 第三節 Isosteviol lactam 進行微生物轉換之構造研究………………………..52 第四節微生物轉換D 環修飾之異甜菊醇的代謝路徑推論…………………..61 第三章抗發炎作用機制與生理活性探討第一節微生物轉換D環修飾之異甜菊醇及相關生理活性概況及研究動機...64 第二節微生物轉換D 環修飾之異甜菊醇之藥理活性試驗…………………..66 第三節結果與討論……………………………………………………………128 第四章實驗部份第一節實驗使用之儀器與藥品………………………………………………..73 第二節異甜菊醇之製備………………………………………………………..76 第三節異甜菊醇之化學構造修飾步驟………………………………………..77 II 第四節微生物轉換研究之實驗步驟…………………………………………..79 第五節微生物轉換D 環修飾異甜菊醇之藥理活性試驗步驟………………103 第五章光譜數據第一節微生物轉換isosteviol lactone 成分之光譜數據……………………...105 第二節微生物轉換isosteviol oxime 成分之光譜數據………………………163 第三節微生物轉換isosteviol lactam 成分之光譜數據………………………182 附錄產率附表……………………………………………………………………..215 參考文獻……………………………………………………………………………219 論文發表……………………………………………………………………………229 III 附表目錄 Table 1. 微生物轉換研究歷史……………………………………………………9 Table 2. Mammalian biotransformation of drugs………………………………...17 Table 3. Cytochrome P450 isoforms……………………………………………..19 Table 4. Active components of Stevia rebaudiana…………..…………………...20 Table 5. Biological effects and potential therapeutic applications of stevioside and related compounds……………………………………………………....22 Table 6. Data of tested compounds showing significance on an androgen receptor-mediated luciferase reporter gene assay……………………….66 Table 7. Data of tested compounds showing significance on an AP-1-mediated luciferase reporter gene assay…………………………………………...67 Table 8. Data of tested compounds on an AP-1-mediated luciferase reporter gene assay…………………………………………………………….............68 Table 9. Data of tested compounds on an NF-κB-mediated luciferase reporter gene assay…………………………………………………………….............69 Table 10. Data of tested compounds showing significance on an iNOS-mediated luciferase reporter gene assay…………………………………………...70 Table 11. 13C NMR chemical shifts of compounds 3 and 6−15 (C5D5N, δ values in ppm)a……………………………………………………………………85 Table 12. 1H NMR chemical shifts of compounds 3 and 6−10 (C5D5N, δ values in ppm)a,b…………………………………………………………………...86 Table 13. 1H NMR chemical shifts of compounds 11−15 (C5D5N, δ values in ppm)a,b…………………………………………………………………...87 Table 14. 13C NMR chemical shifts of compounds 3 and 16−21(C5D5N, δ values in ppm)a………………………...………………………………………….90 Table 15. 1H NMR chemical shifts of compounds 16−21 (C5D5N, δ values in ppm)a,b…………………………………………………………………..91 Table 16. 13C NMR chemical shifts of compounds 4 and 24−28(C5D5N, δ values in ppm)a……………………………………………………………………95 Table 17. 1H NMR chemical shifts of compounds 29−31 and 33−35 (C5D5N, δ values in ppm)a,b………………………………………………………...96 Table 18. 13C NMR chemical shifts of compounds 5 and 29−37 (C5D5N, δ values in ppm)a…………………………………………………………………..100 Table 19. 1H NMR chemical shifts of compounds 5, 32 and 36−37 (C5D5N, δ values in ppm)a,b……………………………………………………….101 IV Table 20. 1H NMR chemical shifts of compounds 29−31 and 33−35 (C5D5N, δ values in ppm)a,b……………………………………………………….102 V 附圖目錄 Fig. 1 General strategy for the development of microbial transformations……………………………………………...12 Fig. 2 Methods of conversion of PTBI into rabeprazole…………..13 Fig. 3 The proposed biosynthetic pathway of pregnenolone products of methyl protodioscin………………………………………14 Fig. 4 Metabolic transformation of a drug…………………………16 Fig. 5 Microbial transformation of isosteviol……………………...23 Fig. 6 X-ray crystallography of isosteviol lactone (3)……………..28 Fig. 7 X-ray crystallography of 6…………………………………..29 Fig. 8 X-ray crystallography of 7…………………………………..31 Fig. 9 X-ray crystallography of 9…………………………………..32 Fig. 10 X-ray crystallography of 16…………………………………39 Fig. 11 X-ray crystallography of 18…………………………………41 Fig. 12 X-ray crystallography of 24…………………………………47 Fig. 13 X-ray crystallography of 25…………………………………48 Fig. 14 X-ray crystallography of 26…………………………………50 Fig. 15 X-ray crystallography of 35…………………………………58 Fig. 16 Standard procedure of microbial transformation……………80 Fig. 17 1H NMR spectrum of 3 (pyridine-d5, 400 MHz)…………..105 Fig. 18 13C NMR spectrum of 3 (pyridine-d5, 100 MHz)…………105 Fig. 19 1H NMR spectrum of 6 (pyridine-d5, 400 MHz)…………..106 Fig. 20 13C NMR spectrum of 6 (pyridine-d5, 100 MHz)…………106 Fig. 21 DEPT spectrum of 6 (pyridine-d5, 100 MHz)……………..107 Fig. 22 1H-1H COSY spectrum of 6 (pyridine-d5, 400 MHz)……...107 Fig. 23 HSQC spectrum of 6 (pyridine-d5, 400 MHz)…………….108 Fig. 24 HMBC spectrum of 6 (pyridine-d5, 400 MHz)……………108 Fig. 25 NOESY spectrum of 6 (pyridine-d5, 400 MHz)…………...109 Fig. 26 1H NMR spectrum of 7 (pyridine-d5, 400 MHz)…………..109 VI Fig. 27 13C NMR spectrum of 7 (pyridine-d5, 100 MHz)…………110 Fig. 28 DEPT spectrum of 7 (pyridine-d5, 100 MHz)……………..110 Fig. 29 1H-1H COSY spectrum of 7 (pyridine-d5, 400 MHz)……...111 Fig. 30 HSQC spectrum of 7 (pyridine-d5, 400 MHz)……………..111 Fig. 31 HMBC spectrum of 7 (pyridine-d5, 400 MHz)…………….112 Fig. 32 NOESY spectrum of 7 (pyridine-d5, 400 MHz)…………...112 Fig. 33 1H NMR spectrum of 8 (pyridine-d5, 400 MHz)…………..113 Fig. 34 13C NMR spectrum of 8 (pyridine-d5, 100 MHz)………….113 Fig. 35 DEPT spectrum of 8 (pyridine-d5, 100 MHz)…………......114 Fig. 36 1H-1H COSY spectrum of 8 (pyridine-d5, 400 MHz)……...114 Fig. 37 HSQC spectrum of 8 (pyridine-d5, 400 MHz)……………..115 Fig. 38 HMBC spectrum of 8 (pyridine-d5, 400 MHz)…………….115 Fig. 39 NOESY spectrum of 8 (pyridine-d5, 400 MHz)…………...116 Fig. 40 1H NMR spectrum of 9 (pyridine-d5, 400 MHz)…………..116 Fig. 41 13C NMR spectrum of 9 (pyridine-d5, 100 MHz)………….117 Fig. 42 DEPT spectrum of 9 (pyridine-d5, 100 MHz)……………..117 Fig. 43 1H-1H COSY spectrum of 9 (pyridine-d5, 400 MHz)……...118 Fig. 44 HSQC spectrum of 9 (pyridine-d5, 400 MHz)……………..118 Fig. 45 HMBC spectrum of 9 (pyridine-d5, 400 MHz)…………….119 Fig. 46 NOESY spectrum of 9 (pyridine-d5, 400 MHz)…………...119 Fig. 47 1H NMR spectrum of 10 (pyridine-d5, 400 MHz)…………120 Fig. 48 13C NMR spectrum of 10 (pyridine-d5, 100 MHz)………...120 Fig. 49 DEPT spectrum of 10 (pyridine-d5, 100 MHz)……………121 Fig. 50 1H-1H COSY spectrum of 10 (pyridine-d5, 400 MHz)…….121 Fig. 51 HSQC spectrum of 10 (pyridine-d5, 400 MHz)……………122 Fig. 52 HMBC spectrum of 10 (pyridine-d5, 400 MHz)…………..122 Fig. 53 NOESY spectrum of 10 (pyridine-d5, 400 MHz)………….123 Fig. 54 1H NMR spectrum of 11 (pyridine-d5, 500 MHz)…………123 Fig. 55 13C NMR spectrum of 11 (pyridine-d5, 125 MHz)………...124 VII Fig. 56 DEPT spectrum of 11 (pyridine-d5, 125 MHz)…………….124 Fig. 57 1H-1H COSY spectrum of 11 (pyridine-d5, 500 MHz)…….125 Fig. 58 HMQC spectrum of 11 (pyridine-d5, 500 MHz)…………..125 Fig. 59 HMBC spectrum of 11 (pyridine-d5, 500 MHz)…………..126 Fig. 60 NOESY spectrum of 11 (pyridine-d5, 500 MHz)………….126 Fig. 61 1H NMR spectrum of 12 (pyridine-d5, 400 MHz)…………127 Fig. 62 13C NMR spectrum of 12 (pyridine-d5, 100 MHz)………..127 Fig. 63 DEPT spectrum of 12 (pyridine-d5, 100 MHz)……………128 Fig. 64 1H-1H COSY spectrum of 12 (pyridine-d5, 400 MHz)…….128 Fig. 65 HSQC spectrum of 12 (pyridine-d5, 400 MHz)……………129 Fig. 66 HMBC spectrum of 12 (pyridine-d5, 400 MHz)…………..129 Fig. 67 NOESY spectrum of 12 (pyridine-d5, 400 MHz)………….130 Fig. 68 1H NMR spectrum of 13 (pyridine-d5, 400 MHz)…………130 Fig. 69 13C NMR spectrum of 13 (pyridine-d5, 100 MHz)………..131 Fig. 70 DEPT spectrum of 13 (pyridine-d5, 100 MHz)……………131 Fig. 71 1H-1H COSY spectrum of 13 (pyridine-d5, 400 MHz)…….132 Fig. 72 HSQC spectrum of 13 (pyridine-d5, 400 MHz)……………132 Fig. 73 HMBC spectrum of 13 (pyridine-d5, 400 MHz)…………..133 Fig. 74 NOESY spectrum of 13 (pyridine-d5, 400 MHz)………….133 Fig. 75 1H NMR spectrum of 14 (DMSO-d6, 400 MHz)…….........134 Fig. 76 13C NMR spectrum of 14 (DMSO-d6, 100 MHz)…………134 Fig. 77 DEPT spectrum of 14 (DMSO-d6, 100 MHz)……………..135 Fig. 78 1H-1H COSY spectrum of 14 (DMSO-d6, 500 MHz)……...135 Fig. 79 HSQC spectrum of 14 (DMSO-d6, 400 MHz)…………….136 Fig. 80 HMBC spectrum of 14 (DMSO-d6, 400 MHz)……………136 Fig. 81 NOESY spectrum of 14 (DMSO-d6, 400 MHz)…………...137 Fig. 82 1H NMR spectrum of 15 (pyridine-d5, 400 MHz)…………137 Fig. 83 13C NMR spectrum of 15 (pyridine-d5, 100 MHz)………..138 Fig. 84 DEPT spectrum of 15 (pyridine-d5, 100 MHz)……………138 VIII Fig. 85 1H-1H COSY spectrum of 15 (pyridine-d5, 400 MHz)…….139 Fig. 86 HSQC spectrum of 15 (pyridine-d5, 400 MHz)……………139 Fig. 87 HMBC spectrum of 15 (pyridine-d5, 400 MHz)…………..140 Fig. 88 NOESY spectrum of 15 (pyridine-d5, 400 MHz)………….140 Fig. 89 1H NMR spectrum of 16 (pyridine-d5, 400 MHz)…………141 Fig. 90 13C NMR spectrum of 16 (pyridine-d5, 100 MHz)………..141 Fig. 91 DEPT spectrum of 16 (pyridine-d5, 100 MHz)……………142 Fig. 92 1H-1H COSY spectrum of 16 (pyridine-d5, 400 MHz)…….142 Fig. 93 HSQC spectrum of 16 (pyridine-d5, 400 MHz)……………143 Fig. 94 HMBC spectrum of 16 (pyridine-d5, 400 MHz)…………..143 Fig. 95 NOESY spectrum of 16 (pyridine-d5, 400 MHz)………….144 Fig. 96 1H NMR spectrum of 17 (pyridine-d5, 400 MHz)…………144 Fig. 97 13C NMR spectrum of 17 (pyridine-d5, 100 MHz)………..145 Fig. 98 DEPT spectrum of 17 (pyridine-d5, 100 MHz)…………....145 Fig. 99 1H-1H COSY spectrum of 17 (pyridine-d5, 400 MHz)…….146 Fig. 100 HSQC spectrum of 17 (pyridine-d5, 400 MHz)……………146 Fig. 101 HMBC spectrum of 17 (pyridine-d5, 400 MHz)…………..147 Fig. 102 NOESY spectrum of 17 (pyridine-d5, 400 MHz)………….147 Fig. 103 1H NMR spectrum of 18 (pyridine-d5, 400 MHz)…………148 Fig. 104 13C NMR spectrum of 18 (pyridine-d5, 100 MHz)………..148 Fig. 105 DEPT spectrum of 18 (pyridine-d5, 100 MHz)……………149 Fig. 106 1H-1H COSY spectrum of 18 (pyridine-d5, 400 MHz)…….149 Fig. 107 HSQC spectrum of 18 (pyridine-d5, 400 MHz)……………150 Fig. 108 HMBC spectrum of 18 (pyridine-d5, 400 MHz)…………..150 Fig. 109 NOESY spectrum of 18 (pyridine-d5, 400 MHz)………….151 Fig. 110 1H NMR spectrum of 19 (pyridine-d5, 500 MHz)………....151 Fig. 111 13C NMR spectrum of 19 (pyridine-d5, 125 MHz)………..152 Fig. 112 DEPT spectrum of 19 (pyridine-d5, 125 MHz)……………152 Fig. 113 1H-1H COSY spectrum of 19 (pyridine-d5, 500 MHz)…….153 IX Fig. 114 HSQC spectrum of 19 (pyridine-d5, 500 MHz)……………153 Fig. 115 HMBC spectrum of 19 (pyridine-d5, 500 MHz)…………..154 Fig. 116 HMBC spectrum of 19 (pyridine-d5, 500 MHz)…………..154 Fig. 117 NOESY spectrum of 19 (pyridine-d5, 500 MHz)………….155 Fig. 118 1H NMR spectrum of 20 (pyridine-d5, 500 MHz)…………155 Fig. 119 13C NMR spectrum of 20 (pyridine-d5, 125 MHz)………..156 Fig. 120 DEPT spectrum of 20 (pyridine-d5, 125 MHz)……………156 Fig. 121 1H-1H COSY spectrum of 20 (pyridine-d5, 500 MHz)…….157 Fig. 122 HSQC spectrum of 20 (pyridine-d5, 500 MHz)……………157 Fig. 123 HMBC spectrum of 20 (pyridine-d5, 500 MHz)…………..158 Fig. 124 NOESY spectrum of 20 (pyridine-d5, 500 MHz)………….158 Fig. 125 1H NMR spectrum of 21 (pyridine-d5, 500 MHz)…………159 Fig. 126 13C NMR spectrum of 21 (pyridine-d5, 125 MHz)………..159 Fig. 127 DEPT spectrum of 21 (pyridine-d5, 125 MHz)……………160 Fig. 128 1H-1H COSY spectrum of 21 (pyridine-d5, 500 MHz)…….160 Fig. 129 HSQC spectrum of 21 (pyridine-d5, 500 MHz)……………161 Fig. 130 HMBC spectrum of 21 (pyridine-d5, 500 MHz)…………..161 Fig. 131 NOESY spectrum of 21 (pyridine-d5, 500 MHz)………….162 Fig. 132 1H NMR spectrum of 4 (pyridine-d5, 500 MHz)…………..163 Fig. 133 13C NMR spectrum of 4 (pyridine-d5, 125 MHz)………….163 Fig. 134 1H NMR spectrum of 24 (pyridine-d5, 500 MHz)…………164 Fig. 135 13C NMR spectrum of 24 (pyridine-d5, 125 MHz)………...164 Fig. 136 DEPT spectrum of 24 (pyridine-d5, 125 MHz)……………165 Fig. 137 1H-1H COSY spectrum of 24 (pyridine-d5, 500 MHz)……165 Fig. 138 HSQC spectrum of 24 (pyridine-d5, 500 MHz)……………166 Fig. 139 HMBC spectrum of 24 (pyridine-d5, 500 MHz)…………..166 Fig. 140 NOESY spectrum of 24 (pyridine-d5, 500 MHz)………….167 Fig. 141 1H NMR spectrum of 25 (pyridine-d5, 500 MHz)…………167 Fig. 142 13C NMR spectrum of 25 (pyridine-d5, 125 MHz)………..168 X Fig. 143 DEPT spectrum of 25 (pyridine-d5, 125 MHz)……………168 Fig. 144 1H-1H COSY spectrum of 25 (pyridine-d5, 500 MHz)…….169 Fig. 145 HSQC spectrum of 25 (pyridine-d5, 500 MHz)……………169 Fig. 146 HMBC spectrum of 25 (pyridine-d5, 500 MHz)…………..170 Fig. 147 NOESY spectrum of 25 (pyridine-d5, 500 MHz)………….170 Fig. 148 1H NMR spectrum of 26 (pyridine-d5, 500 MHz)…………171 Fig. 149 13C NMR spectrum of 26 (pyridine-d5, 125 MHz)………..171 Fig. 150 DEPT spectrum of 26 (pyridine-d5, 125 MHz)……………172 Fig. 151 1H-1H COSY spectrum of 26 (pyridine-d5, 500 MHz)…….172 Fig. 152 HSQC spectrum of 26 (pyridine-d5, 500 MHz)……………173 Fig. 153 HMBC spectrum of 26 (pyridine-d5, 500 MHz)…………..173 Fig. 154 NOESY spectrum of 26 (pyridine-d5, 500 MHz)………….174 Fig. 155 1H NMR spectrum of 27 (pyridine-d5, 500 MHz)…………174 Fig. 156 13C NMR spectrum of 27 (pyridine-d5, 125 MHz)………..175 Fig. 157 DEPT spectrum of 27 (pyridine-d5, 125 MHz)……………175 Fig. 158 1H-1H COSY spectrum of 27 (pyridine-d5, 500 MHz)…….176 Fig. 159 HSQC spectrum of 27 (pyridine-d5, 500 MHz)……………176 Fig. 160 HMBC spectrum of 27 (pyridine-d5, 500 MHz)…………..177 Fig. 161 NOESY spectrum of 27 (pyridine-d5, 500 MHz)………….177 Fig. 162 1H NMR spectrum of 28 (pyridine-d5, 500 MHz)…………178 Fig. 163 13C NMR spectrum of 28 (pyridine-d5, 125 MHz)………..178 Fig. 164 DEPT spectrum of 28 (pyridine-d5, 125 MHz)……………179 Fig. 165 1H-1H COSY spectrum of 28 (pyridine-d5, 500 MHz)…….179 Fig. 166 HSQC spectrum of 28 (pyridine-d5, 500 MHz)……………180 Fig. 167 HMBC spectrum of 28 (pyridine-d5, 500 MHz)…………..180 Fig. 168 NOESY spectrum of 28 (pyridine-d5, 500 MHz)………….181 Fig. 169 1H NMR spectrum of 5 (pyridine-d5, 500 MHz)…………..182 Fig. 170 13C NMR spectrum of 5 (pyridine-d5, 125 MHz)………….182 Fig. 171 1H NMR spectrum of 29 (pyridine-d5, 500 MHz)…………183 XI Fig. 172 13C NMR spectrum of 29 (pyridine-d5, 125 MHz)………..183 Fig. 173 DEPT spectrum of 29 (pyridine-d5, 125 MHz)……………184 Fig. 174 1H-1H COSY spectrum of 29 (pyridine-d5, 500 MHz)…….184 Fig. 175 HSQC spectrum of 29 (pyridine-d5, 500 MHz)……………185 Fig. 176 HMBC spectrum of 29 (pyridine-d5, 500 MHz)…………..185 Fig. 177 NOESY spectrum of 29 (pyridine-d5, 500 MHz)………….186 Fig. 178 1H NMR spectrum of 30 (pyridine-d5, 500 MHz)…………186 Fig. 179 13C NMR spectrum of 30 (pyridine-d5, 125 MHz)………..187 Fig. 180 DEPT spectrum of 30 (pyridine-d5, 125 MHz)……………187 Fig. 181 1H-1H COSY spectrum of 30 (pyridine-d5, 500 MHz)…….188 Fig. 182 HSQC spectrum of 30 (pyridine-d5, 500 MHz)……………188 Fig. 183 HMBC spectrum of 30 (pyridine-d5, 500 MHz)…………..189 Fig. 184 NOESY spectrum of 30 (pyridine-d5, 500 MHz)………….189 Fig. 185 1H NMR spectrum of 31 (pyridine-d5, 500 MHz)…………190 Fig. 186 13C NMR spectrum of 31 (pyridine-d5, 125 MHz)………...190 Fig. 187 DEPT spectrum of 31 (pyridine-d5, 125 MHz)……………191 Fig. 188 1H-1H COSY spectrum of 31 (pyridine-d5, 500 MHz)…….191 Fig. 189 HSQC spectrum of 31 (pyridine-d5, 500 MHz)……………192 Fig. 190 HMBC spectrum of 31 (pyridine-d5, 500 MHz)…………..192 Fig. 191 NOESY spectrum of 31 (pyridine-d5, 500 MHz)………….193 Fig. 192 1H NMR spectrum of 32 (pyridine-d5, 500 MHz)…………193 Fig. 193 13C NMR spectrum of 32 (pyridine-d5, 125 MHz)………..194 Fig. 194 DEPT spectrum of 32 (pyridine-d5, 125 MHz)……………194 Fig. 195 1H-1H COSY spectrum of 32 (pyridine-d5, 500 MHz)…….195 Fig. 196 HSQC spectrum of 32 (pyridine-d5, 500 MHz)……………195 Fig. 197 HMBC spectrum of 32 (pyridine-d5, 500 MHz)…………..196 Fig. 198 NOESY spectrum of 32 (pyridine-d5, 500 MHz)………….196 Fig. 199 1H NMR spectrum of 33 (pyridine-d5, 500 MHz)…………197 Fig. 200 13C NMR spectrum of 33 (pyridine-d5, 125 MHz)………...197 XII Fig. 201 DEPT spectrum of 33 (pyridine-d5, 125 MHz)……………198 Fig. 202 1H-1H COSY spectrum of 33 (pyridine-d5, 500 MHz)…….198 Fig. 203 HSQC spectrum of 33 (pyridine-d5, 500 MHz)……………199 Fig. 204 HMBC spectrum of 33 (pyridine-d5, 500 MHz)…………..199 Fig. 205 NOESY spectrum of 33 (pyridine-d5, 500 MHz)………….200 Fig. 206 1H NMR spectrum of 34 (pyridine-d5, 500 MHz)…………200 Fig. 207 13C NMR spectrum of 34 (pyridine-d5, 125 MHz)………..201 Fig. 208 DEPT spectrum of 34 (pyridine-d5, 125 MHz)…………....201 Fig. 209 1H-1H COSY spectrum of 34 (pyridine-d5, 500 MHz)…….202 Fig. 210 HSQC spectrum of 34 (pyridine-d5, 500 MHz)……………202 Fig. 211 HMBC spectrum of 34 (pyridine-d5, 500 MHz)…………..203 Fig. 212 NOESY spectrum of 34 (pyridine-d5, 500 MHz)………….203 Fig. 213 1H NMR spectrum of 35 (pyridine-d5, 500 MHz)………....204 Fig. 214 13C NMR spectrum of 35 (pyridine-d5, 125 MHz)………..204 Fig. 215 DEPT spectrum of 35 (pyridine-d5, 125 MHz)……………205 Fig. 216 1H-1H COSY spectrum of 35 (pyridine-d5, 500 MHz)…….205 Fig. 217 HSQC spectrum of 35 (pyridine-d5, 500 MHz)……………206 Fig. 218 HMBC spectrum of 25 (pyridine-d5, 500 MHz)…………..206 Fig. 219 NOESY spectrum of 35 (pyridine-d5, 500 MHz)………….207 Fig. 220 1H NMR spectrum of 36 (pyridine-d5, 500 MHz)………....207 Fig. 221 13C NMR spectrum of 36 (pyridine-d5, 125 MHz)………..208 Fig. 222 DEPT spectrum of 36 (pyridine-d5, 125 MHz)……………208 Fig. 223 1H-1H COSY spectrum of 36 (pyridine-d5, 500 MHz)…….209 Fig. 224 HSQC spectrum of 36 (pyridine-d5, 500 MHz)……………209 Fig. 225 HMBC spectrum of 36 (pyridine-d5, 500 MHz)…………..210 Fig. 226 NOESY spectrum of 36 (pyridine-d5, 500 MHz)………….210 Fig. 227 1H NMR spectrum of 37 (pyridine-d5, 500 MHz)…………211 Fig. 228 13C NMR spectrum of 37 (pyridine-d5, 125 MHz)………..211 Fig. 229 DEPT spectrum of 37 (pyridine-d5, 125 MHz)……………212 XIII Fig. 230 1H-1H COSY spectrum of 37 (pyridine-d5, 500 MHz)…….212 Fig. 231 HSQC spectrum of 37 (pyridine-d5, 500 MHz)……………213 Fig. 232 HMBC spectrum of 37 (pyridine-d5, 500 MHz)…………..213 Fig. 233 NOESY spectrum of 37 (pyridine-d5, 500 MHz)………….214 XIV 流程目錄 Scheme 1. Structures of stevioside, isosteviol, and metabolites………………….24 Scheme 2. Reagents and conditions………………………………………………26 Scheme 3. Preparation of isosteviol (2) and isosteviol lactone…………………...27 Scheme 4. Microbial transformation of isosteviol lactone (3) by C. bainieri…30 Scheme 5. Microbial transformation of isosteviol lactone (3) by Asp. niger…….33 Scheme 6. Microbial transformation of isosteviol lactone (3) by Abs. pseudocylindrospora…………………………………………………..38 Scheme 7. Microbial transformation of isosteviol lactone (3) by M. recurvatus…44 Scheme 8. Preparation of isosteviol oxime (4)……………………………………45 Scheme 9. Microbial transformation of isosteviol oxime by Abs. Pseudocylindrospora………………………………………………….46 Scheme 10. Microbial transformation of isosteviol oxime by Asp. niger………….49 Scheme 11. Preparation of isosteviol lactam……………………………………….52 Scheme 12. Microbial transformation of isosteviol lactam (5) by Asp. niger……...53 Scheme 13. Microbial transformation of isosteviol lactam by Abs. pseudocylindrospora…………………………………………………..59 Scheme 14. 微生物轉換isosteviol lactone (3)之代謝途徑……………………….62 Scheme 15. 微生物轉換isosteviol oxime (4)之代謝途徑………………………...63 Scheme 16. 微生物轉換isosteviol lactam (5)之代謝途徑………………………..63 (二)參考文獻 1. Pasteur, L. Compt. Rend., 1857, 45, 913. 2. Büchner, E. Ber. Dysch. Chem. Ges. 1897, 30, 117. 3. 蘇遠志及黃世佑, 應用微生物學；華香園出版社, 台北, 1999, 初版. 4. Hanc, O.; Capek, A.; Tadra, M.; Macek, K.; Simek, A. Microbial transformation of steroids. III. Direct conversion of progesterone into testosterone. Arzneimittelforschung, 1957, 7, 175?{176. 5. Marsheck, W. J.; Karim, A. Preparation of metabolites of spironolactone by microbial oxygenation. Appl. Microbial., 1973, 25, 647?{649. 6. Smith, R. V.; Rosazza, J. P. Microbial models of mammalian metabolism. Aromatic hydroxylation. Arch. Biochem. Biophysic., 1974, 161, 551?{558. 7. Marshall, V. P.; McGovren, J. P.; Richard, F. A.; Richard, R. E.; Wiley, P. F. 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