海洋鏈霉菌L211次生代謝產物分離和結構鑒定

郭書舉 , 蘇 華, 李憲璀 史大永 朱校斌

(1. 中國科學院 海洋研究所, 山東 青島 266071; 2. 中國科學院 海洋研究所 海洋科學與技術研究與發展中心, 江蘇 南通 226006; 3. 中國科學院 青島生物能源與過程研究所, 山東 青島 266101)

海洋放線菌是一類重要的海洋微生物, 可以產生多種多樣的代謝產物, 如含氮雜環類、多肽類、萜類、糖苷類及大環內酯類化合物[1-2], 鏈霉菌作為放線菌的重要組成, 也是新結構化合物的重要來源。本研究以海洋鏈霉菌Streptomyces sp. L211為研究對象,對其發酵液的乙酸乙酯相提取物進行了分離, 先期分離了15個化合物, 并通過1H-NMR、13C-NMR等方法對其中的 10個進行了結構鑒定, 分別為:Kalamycin (1), Medermycin (2), Menoxymycin-B (3),spatozoate (4), 鄰氨基苯甲酸(5), 3-吲哚乙醇(6), 1-乙?；沁?7), 對羥基苯基乙醇(8), 吲哚-3-乙酸(9),吲哚-3-甲酸(10)。

1 材料與方法

1.1 儀器與材料

BRUKER AVANCE-500型核磁共振儀; 三氯甲烷、二氯甲烷、四氯化碳、石油醚、乙酸乙酯、冰乙酸、三氟乙酸酐、溴素、香草醛、N-溴代丁二酰亞胺、乙醇鈉、乙醇均為分析純; 柱色譜硅膠(160～200 目)和薄層色譜硅膠 GF254(60型)為青島海洋化工廠產品。

1.2 實驗方法

1.2.1 菌株培養

取適量菌種接種到固體培養基(麥芽提取物10 g/L,酵母提取物4 g/L, 葡萄糖4 g/L, 瓊脂粉15 g/L, 海水, pH 7.8)的平板上, 28℃培養48 h后, 作為種子接種到液體培養基(麥芽提取物10 g/L, 酵母提取物4 g/L,葡萄糖4 g/L, 海水, pH 7.8)中, 共接種20 L, 28℃,110 r/min于搖床培養4 d。

1.2.2 提取分離

發酵物離心將菌體和發酵液分離, 發酵液用同體積的乙酸乙酯萃取3次后, 減壓蒸干; 菌體在丙酮中超聲破碎提取3次后, 減壓蒸干; 將兩部分粗提物TLC對比, 發現成分相似, 合并后得到7.50g浸膏。浸膏經硅膠柱色譜分離(石油醚: 乙酸乙酯, 氯仿:甲醇梯度洗脫), 得到8個組分, 各組再經過凝膠柱、硅膠柱及制備薄層色譜分離后, 得到 15個化合物,利用核磁共振波譜和質譜, 鑒定了其中的10個化合物, 見圖1。

2 波譜數據與結果鑒定

化合物(1), 黃色固體, C16H12O6。1H-NMR(CDCl3,300MHz) δ: 11.8 (s, 9-OH), 7.30 (dd, 7.5, 2.1 Hz, H-8),7.73-7.65 (overlapping, H-6, 7), 5.27 (d, 3.0 Hz, H-4),5.09 (q, 6.9 Hz, H-1), 4.70 (dd, 5.1, 3.0 Hz, H-5), 2.97(dd, 17.7, 5.2 Hz, H-13β), 2.71 (d, 17.7, H-13α), 1.57(d, 6.9 Hz, 1-CH3)。 以上數據與Kalamycin波譜數據一致[3]。

圖1 化合物1-10的結構Fig.1 Structure of the Compounds 1-10

化合物(2), 黃色固體, C24H27NO8。1H-NMR(CDCl3, 300MHz) δ: 7.91(1H, d, J=7.8), 7.71(1H, d,J=7.8), 5.25(1H, d, J=2.9), 5.08(1H, q, J=7.0),4.87(1H, dd, J=10.9, 2.0), 4.69(1H, dd, J=5.1, 2.9),3.53(1H, dp, J=8.9, 6.2), 3.2(1H, dd, J=9.5, 8.9),2.97(1H, dd, J=17.6, 5.1), 2.78(1H, ddd, J=12.5, 9.5,3.8), 2.69(1H, d, J=17.6), 2.34(6H, s), 2.26(1H, ddd,J=12.4, 3.8, 2.0), 1.57(3H, d, J=7.0), 1.43(3H, d,J=6.2), 1.30(1H, ddd, J=12.5, 12.4, 10.9);13C-NMR(CDCl3, 75 MHz) δ: 187.8, 180.8, 173.5, 157.6, 149.1,138.5, 134.8, 133.5, 129.6, 119.5, 117.1, 77.5, 72.1,71.3, 68.6, 67.1, 66.4, 66.2, 40.2, 36.9, 28.1, 18.9,18.8。FAB-MS m/z: 458 [M+H]+。以上數據與Medermycin波譜數據一致[4]。

化合物(3), 黃色固體, C25H31NO9。1H-NMR(CDCl3, 300MHz) δ: 7.87(1H, d, J=7.7, ), 7.67(1H, d,J=7.7), 5.02(1H, q, J=6.9), 4.86(1H, dd, J=10.9, 2.07),4.66(1H, d, J=2.5), 4.35(1H, dt, J=6.5, 2.5), 3.75(3H,s), 3.52(1H, dp, J=9.0, 6.0), 3.18(2H, dd, J=9.0, 9.0),2.82(2H, d, J=6.5), 2.73(1H, ddd, J=12.4, 9.0, 3.5),2.32(6H, s), 2.25(1H, ddd, J=12.4, 3.5, 2.0), 1.56(3H, d,J=6.9), 1.42(3H, d, J=6.0), 1.28(1H, ddd, J=12.4, 12.4,10.7).13C-NMR (CDCl3, 75 MHz) δ: 189.3, 182.7,171.8, 157.4, 146.5, 141.2, 138.1, 133.1, 130.1, 119.2,114.1, 77.3, 72.0, 71.3, 67.7, 67.1, 66.7, 59.2, 51.8,40.0, 35.5, 28.3, 17.6, 17.5。FAB-MS m/z: 490[M+H]+。以上數據與Menoxymycin-B波譜數據一致[4]。

化合物(4), 無色油狀, C19H20O4。1H-NMR(CD3OD,300MHz) δ: 7.21 (1H, m, H-2), 7.63～7.70 (3H, m, H-3,H-4, H-5), 4.23 (2H, m, H2-8), 1.43～1.63 (4H, m, H2-9,H2-10), 0.97 (3H, m, H3-11), 5.55 (2H, s, H2-12), 8.03(1H, m, H-15), 7.44 (2H, m, H-16, H-18), 7.51 (1H, m,H-17), 7.95 (1H, m, H-19);13C-NMR (CD3OD, 75 MHz)δ: 138.0 (s, C-1), 121.7(d, C-2), 129.1(d, C-3), 126.5(d,C-4), 130.0(d, C-5), 132.4(s, C-6), 166.2 (s, C-7),66.1(t, C-8), 31.3 (t, C-9), 19.0(t, C-10), 13.0(q, C-11),63.0(t, C-12), 167.3(s, C-13), 127.0(s, C-14), 129.9(d,C-15), 128.0(d, C-16), 131.1(d, C-17), 128.0(d, C-18),129.9(d, C-19); EIMS m/z: 312.0 [M]+。以上數據與Spatozoate波譜數據一致[5]。

化合物(5), 黃色粉末, C7H7NO2。1H-NMR(CD3OD,300 MHz) δ: 7.33(1H, dd, J= 5.9, 6.9Hz, H-3), 6.65(2H,m, H-4, H-5), 7.91(1H, d, J= 5.9 Hz, H-6);13C-NMR(CD3OD, 75 MHz) δ: 170.3(s, CO), 110.8(s, C-1),152.6(s, C-2), 134.7(d, C-), 132.3(d, C-), 117.2(d, C-),115.8(d, C-); EIMS: m/z(%) 137.0(65), 119.1(100),93.0(4), 92.1(50), 71.1(3), 66.0(2), 65.0(11), 58.0(5),43.0(7), 41.0(3)。以上數據與鄰氨基苯甲酸波譜數據一致[6]。

化合物(6), 無色油狀物, C10H11NO。1H-NMR(CD3OD, 300 MHz) δ: 8.05 (1H, br s, NH), 7.11(1H, s,H-2), 7.66(1H, d, J= 8.2 Hz, H-4), 7.15(1H, dd, J= 8.2,1.5 Hz, H-5), 7.23 (1H, dd, J= 8.2, 1.5 Hz, H-6),7.40(1H, d, J=8.2 Hz, H-7), 3.00(2H, t, J= 6.6Hz,H2-8), 3.90(2H, t, J= 6.6 Hz, H2-9);13C-NMR (CD3OD,75 MHz)δ: 123.1(d, C-2), 112.0(s, C-3), 127.2 (s,C-3a), 121.0(d, C-4), 123.2 (d, C-5), 124.5(d, C-6),1120.1 (d, C-7), 133.0(s, C-7a), 28.6 (t, C-8), 66.6(t,C-9)。EIMS m/z: 161.1 [M]+。以上數據與3-吲哚乙醇波譜數據一致[7]。

化合物(7), 無色油狀物, C13H10N2O。1H-NMR(CD3OD, 300 MHz)δ: 8.68(1H, d, J= 6.0Hz, H-4),8.23(2H, m, H-8, H-9), 7.29(1H, m, H-6), 7.60(2H, m,H-7, H-5), 2.95(3H, s, H3-12);13C-NMR (CD3OD,75MHz)δ: 135.4(s, C-2), 123.3(s, C-3), 123.4(s, C-3a),122.2(d, C-4), 122.1(d, C-5), 121.8 (d, C-6), 111.9(d,C-7), 140.0(sC-7a), 120.5(d, C-8), 137.1(d, C-9),141.5(s, C-10), 201.1(s, C-11), 26.0(q, C-12)。EIMS m/z: 210.0 [M]+。以上數據與1-乙?；沁ㄗV數據一致[8-10]。

化合物(8), 淡黃色油狀物, C8H10O2;1H-NMR(CD3OD, 300 MHz)δ: 7.14(2H, J= 8.0 Hz, H-2, H-6),6.80(2H, d, J= 8.0 Hz, H-3, H-5), 2.81(2H, t, J= 6.2 Hz,H2-7), 3.80(2H, t, J= 6.2 Hz, H2-8);13C-NMR (CD3OD,75 MHz)δ: 156.9(s, C-1), 118.0(d, C-2), 129.8(d, C-3),138.7 (s, C-4), 129.8(d, C-5), 118.0(d, C-6), 39.3(t,C-7), 63.5(t, C-8); EIMS m/z: 138.1 [M]+。以上數據與對羥基苯基乙醇波譜數據一致[11-12]。

化合物(9), 淡黃色油狀物, C10H9NO2。1H-NMR(CD3OD, 300 MHz)δ: 7.30(1H, s, H-2), 7.64(d1H, d,J= 1.8, 7.5 Hz, H-4), 7.00(1H, m, H-5), 7.17(1H, m,H-6), 7.30(1H, dd, J= 1.8, 7.5 Hz, H-7), 3.77(1H, s,H-8);13C-NMR (CD3OD, 75 MHz)δ: 124.9(d, C-2),109.0 (s, C-3), 128.0(s, C-3a), 120.1 (d, C-4), 120.3(d,C-5), 122.0(d, C-6), 112.3(d, C-7), 137.5(s, C-7a),32.4(t, C-8), 173.9(s, C-9); EIMS m/z: 175.1 [M]+。以上數據與吲哚-3-乙酸波譜數據一致[13]。

化合物(10), 淡黃色油狀物, C9H7NO2。1H-NMR(CD3OD, 300 MHz)δ: 8.10(1H, m, H-4), 7.92(1H, s,H-2), 7.41(1H, m, H-7), 7.20(2H, m, H-5,H-6);13C-NMR (CD3OD, 75.5 MHz)δ: 168.3(s, CO),129.0 (d, C-2), 112.0(s, C-3), 133.2(s, C-3a), 122.0(d,C-4), 123.2(d, C-5), 123.1(d, C-6), 113.7(d, C-7),137.9 (s, C-7a)。以上數據與吲哚-3-甲酸波譜數據一致[14-16]。

3 結論

本研究以海洋鏈霉菌Streptomyces sp. L211為研究對象, 先期從中分離了 15個化合物, 并鑒定了其中的 10個, 研究表明化合物 Kalamycin (1),Medermycin (2), Menoxymycin-B (3)均表現出不同的抗腫瘤活性[3-4]。目前, 本課題組正在對其他海洋放線菌次生代謝產物進行深入研究, 相信會有更多有活性的代謝產物被發現。

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