海洋鏈霉菌L211次生代謝產(chǎn)物分離和結(jié)構(gòu)鑒定

郭書舉 , 蘇 華, 李憲璀 史大永 朱校斌

(1. 中國科學(xué)院 海洋研究所, 山東 青島 266071; 2. 中國科學(xué)院 海洋研究所 海洋科學(xué)與技術(shù)研究與發(fā)展中心, 江蘇 南通 226006; 3. 中國科學(xué)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(chǎn)生多種多樣的代謝產(chǎn)物, 如含氮雜環(huán)類、多肽類、萜類、糖苷類及大環(huán)內(nèi)酯類化合物[1-2], 鏈霉菌作為放線菌的重要組成, 也是新結(jié)構(gòu)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(fā)酵液的乙酸乙酯相提取物進(jìn)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(jìn)行了結(jié)構(gòu)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-乙酰基咔啉(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(chǎn)品。

1.2 實驗方法

1.2.1 菌株培養(yǎng)

取適量菌種接種到固體培養(yǎng)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(yǎng)48 h后, 作為種子接種到液體培養(yǎng)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(yǎng)4 d。

1.2.2 提取分離

發(fā)酵物離心將菌體和發(fā)酵液分離, 發(fā)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(fā)現(xiàn)成分相似, 合并后得到7.50g浸膏。浸膏經(jīng)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(jīng)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(zhì)譜, 鑒定了其中的10個化合物, 見圖1。

2 波譜數(shù)據(jù)與結(jié)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(shù)據(jù)與Kalamycin波譜數(shù)據(jù)一致[3]。

圖1 化合物1-10的結(jié)構(gòu)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(shù)據(jù)與Medermycin波譜數(shù)據(jù)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(shù)據(jù)與Menoxymycin-B波譜數(shù)據(jù)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(shù)據(jù)與Spatozoate波譜數(shù)據(jù)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(shù)據(jù)與鄰氨基苯甲酸波譜數(shù)據(jù)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(shù)據(jù)與3-吲哚乙醇波譜數(shù)據(jù)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(shù)據(jù)與1-乙?；沁ㄗV數(shù)據(jù)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(shù)據(jù)與對羥基苯基乙醇波譜數(shù)據(jù)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(shù)據(jù)與吲哚-3-乙酸波譜數(shù)據(jù)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(shù)據(jù)與吲哚-3-甲酸波譜數(shù)據(jù)一致[14-16]。

3 結(jié)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(xiàn)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(chǎn)物進(jìn)行深入研究, 相信會有更多有活性的代謝產(chǎn)物被發(fā)現(xiàn)。

[1] Amagata T, Xiao J, Chen Y P, et al. Creation of an HDAC-Based Yeast Screening Method for Evaluation of Marine-Derived Actinomycetes: Discovery of Streptosetin A[J]. Journal of Natural Products, 2012,75(12): 2193-2199.

[2] Sato S, Iwata F, Yamada S, et al. Neomaclafungins A–I:Oligomycin-Class Macrolides from a Marine-Derived Actinomycete[J]. Journal of Natural Products, 2012,75(11): 1974-1982.

[3] Zhang Y H, Qin M, Li F C, et al. Isolation and identification of metabolites produced by marine Streptomyces sp.S007[J]. Nat Prod Res Dev, 2011, 23:410-414.

[4] Yoichi H, Ishigami K, Shin-Ya K, et al. Menoxymycins A and B, antitumor antibiotics generating active oxygen in tumor cells[J]. J Antibiot, 1994, 47: 1344-1347.

[5] Atta-ur-Rahman M, Iqbal C, Safdar H, et al. Spatozoate and varninasterol from the brown alga Spatoglossum variabile[J]. Phytochemistry, 1999, 52: 495-499.

[6] Sindler-Kulyk M, Neckers D C. Photochemistry of 2-phenylbenzothiazole with ethoxyacetylene and ethoxypropyne. Synthesis of 1, 5-benzothiazepines[J]. Journal of Organic Chemistry, 1982, 47(25): 4914-4919.

[7] Gordon B B, Arthur C G. Indole Compounds Synthesized by Diplodia natalensis[J]. Plant physiology, 1962,37: 439-445.

[8] Zhang Y, Morikawa T, Nakamura S, et al. Bioactive constituents from chinese natural medicines. XXV.New flavonol bisdesmosides, sarmenosides I, II, III,and IV, with hepatoprotective activity from Sedum sarmentosum (Crassulaceae) [J]. Heterocycles, 2007,71(7): 1565-1576.

[9] Morikawa T, Zhang Y, Nakamura S, et al. Bioactive constituents from Chinese natural medicines. XXII.Absolute structures of new megastigmane glycosides,sedumosides E1, E2, E3, F1, F2, and G, from Sedum sarmentosum (Crassulaceae)[J]. Chemical & Pharmaceutical Bulletin, 2007, 55(3): 435-441.

[10] Ohmoto T, Koike K. Studies on the constituents of Picrasma quassioides Bennet. I. On the alkaloidal constituents[J]. Chemical & Pharmaceutical Bulletin,1982, 30(4): 1204-1209.

[11] Huang Q, Tezuka Y, Hatanaka Y, et al. Studies on metabolites of mycoparasitic fungi. III. new sesquiterpene alcohol from Trichoderma koningii[J]. Chemical & Pharmaceutical Bulletin, 1995, 43: 1035-1038.

[12] William A A, Latchezar S T, Phenolic and polyketide metabolites of the aspen blue stainfungus Ophiostoma crassivaginata[J]. Phytochemistry, 1995, 38: 371-372.

[13] Alejandro F B, Juan E O, Juan A P. Acidic metabolites from Phycomyces blakesleeanus[J]. Phytochemistry,1996, 42: 1427-1433.

[14] 劉海河, 林文翰, 趙喜, 等.南海紅樹內(nèi)生真菌#HA-094化學(xué)成分的研究[J].黑龍江醫(yī)藥, 2007, 20(6): 564-565.

[15] Shaaban K A, Shaaban M, Facey P, et al. Electrospray ionization mass spectra of piperazimycins A and B and butyrolactones from a marine-derived Streptomyces sp.[J]. Journal of Antibiotics, 2008, 61(12): 736-746.

[16] Eggers M E, Jog P V, Bates D K. Intramolecular sulfoxide electrophilic sulfenylation in 2- and 3-indoleanilides[J]. Tetrahedron, 2007, 63(49): 12185-12194.