CN102168044B - Streptomyces sp. and process for preparing various antibiotics by using same - Google Patents
Streptomyces sp. and process for preparing various antibiotics by using same Download PDFInfo
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- 239000003242 anti bacterial agent Substances 0.000 title abstract description 9
- 229940088710 antibiotic agent Drugs 0.000 title abstract description 9
- 238000004519 manufacturing process Methods 0.000 title abstract description 6
- MOYOTUKECQMGHE-PDEFJWSRSA-M sodium;(2r)-2-[(2r,3s,6r)-6-[[(2s,4r,5r,6r,7r,9r)-2-[(2r,5s)-5-[(2r,3s,5r)-5-[(2s,3s,5r,6r)-6-hydroxy-6-(hydroxymethyl)-3,5-dimethyloxan-2-yl]-3-methyloxolan-2-yl]-5-methyloxolan-2-yl]-7-methoxy-2,4,6-trimethyl-1,10-dioxaspiro[4.5]decan-9-yl]methyl]-3-met Chemical compound [Na+].C([C@@H]1C[C@H]([C@H]([C@]2([C@@H](C[C@](C)(O2)[C@@H]2O[C@@](C)(CC2)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C)O1)C)OC)[C@H]1CC[C@H](C)[C@H]([C@@H](C)C([O-])=O)O1 MOYOTUKECQMGHE-PDEFJWSRSA-M 0.000 claims abstract description 21
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- DANUORFCFTYTSZ-BIBFWWMMSA-N nigericin Chemical compound C([C@@H]1C[C@H]([C@H]([C@]2([C@@H](C[C@](C)(O2)C2O[C@@](C)(CC2)C2[C@H](CC(O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C)O1)C)OC)[C@H]1CC[C@H](C)C([C@@H](C)C(O)=O)O1 DANUORFCFTYTSZ-BIBFWWMMSA-N 0.000 abstract description 15
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Abstract
The invention discloses Streptomyces sp. and a process for preparing various antibiotics by using the same. Streptomyces sp. SCSIO 1934 is preserved in the China Center for Type Culture Collection (CCTCC) on November 25th, 2010, the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2010317. Nine antibiotics including 17-demethylgeldanamycin, lebstatin, 17-O-demethyllebstatin, nigericin, nigericin sodium salt, abierixin, 29-O-methylabierixin, 11-O-methylelaiophylin and elaiophylin are prepared from a fermentation culture of the Streptomyces sp. SCSIO 1934. Therefore, the invention provides a novel method for producing and preparing the nine antibiotics.
Description
Technical field:
The invention belongs to the industrial microorganism field; Being specifically related to a kind of ability produces multiple antibiotic marine actinomycete bacterial strain streptomycete (Streptomyces sp.) SCSIO 1934 and utilizes this bacterium fermentative prepn 17-demethylation NSC 122750 (17-O-demethylgeldanamycin); Lebstatin, 17-O-demethyllebstatin, polyetherin A (nigericin); Sodium nigericin (nigericin sodium salt); Abierixin, 29-O-methylabierixin, the technology of 11-O-methylelaiophylin and elaiophylin.
Background technology:
Compound 17-O-demethylgeldanamycin (1), lebstatin (2), 17-O-demethyllebstatin (3), nigerincin (4), nigericin sodium salt (5), abierixin (6), 29-O-methylabierixin (7); The structural formula of 11-O-methylelaiophylin (8) and elaiophylin (9) is as shown in Figure 1, digital corresponding behind the compound of the digitized representation among Fig. 1 and the above-claimed cpd bracket.
Compound 17-O-demethylgeldanamycin, lebstatin and 17-O-demethyllebstatin belong to the husky type microbiotic (Ansamycins) of peace, and such microbiotic mainly has antibiotic, antiviral and anti-tumor activity.For example, 17-demethylation NSC 122750 is the most remarkable to the effect of human leukemia cell (K562) model, and lebstatin not only has resistance to herpes simplex virus I-type, also lung carcinoma cell is had and suppresses active.Compound nigerincin, nigericin sodium salt, abierixin and 29-O-methylabierixin all belong to polyether antibiotics (Polyethers).Most of polyether antibioticses can both carry monovalent cation through formation lipophilic mixture and pass microbial film, and are applied in the agriculture prodn as anticoccidiosis medicine and livestock growth stimulant.Nigericin and abierixin can both improve domestic animal to proteinic utilization ratio, and have coccidiostat activity preferably.In addition, nigericin also has resistance to various bacteria, like bacillus acidocaldarius, and bacillus cereus, streptococcus aureus etc.Elaiophylin and 11-O-methylelaiophylin belong to macrolide antibiotics, have better antitumor activity and anti-microbial activity.
Summary of the invention:
First purpose of the present invention provides and a kind ofly can produce 17-demethylation NSC 122750 (17-O-demethylgeldanamycin); Lebstatin; 17-O-demethyllebstatin, polyetherin A (nigericin), Sodium nigericin (nigericin sodium salt); Abierixin; 29-O-methylabierixin, 9 kinds of antibiotic streptomycetes of 11-O-methylelaiophylin and elaiophylin (Streptomyces sp.) SCSIO 1934, this bacterium is preserved in Chinese typical culture collection center (CCTCC) on November 25th, 2010; The address: Chinese Wuhan City Wuhan University, its deposit number is CCTCC NO:M 2010317.
Streptomycete of the present invention (Streptomyces sp.) SCSIO 1934 be northern from China South Sea (111 ° 26.118 of E '; N18 ° 34.363 ') separates in the marine bottom sediment of 200 meters of the depth of waters and obtain; The aerial hyphae of this bacterium is pearl, spore black, and it is abundant to produce spore.Through the 16S rDNA of this bacterium of ordinary method pcr amplification, and order-checking, its sequence is submitted among the GenBank shown in SEQ ID NO.1, obtains sequence number HQ403650.16S rDNA gene sequencing result shows this bacterial strain and streptomycete Streptomyces sp.M4032; The similarity of Streptomyces sp.M4033 and Streptomyces malaysiensis reaches 100%; Clearly disclose the phyletic evolution relation (Fig. 2) of this bacterium and one group of streptomyces species through adjacent method, shown a kind of in streptomyces of this strain Pseudomonas.With this bacterium called after streptomycete (Streptomyces sp.) SCSIO 1934, this bacterium was preserved in Chinese typical culture collection center (CCTCC), address on November 25th, 2010: Chinese Wuhan City Wuhan University, its deposit number is CCTCC NO:M 2010317.
Another object of the present invention provides preparation 17-demethylation NSC 122750 (17-O-demethylgeldanamycin) from the fermenting culture of streptomycete SCSIO 1934 of the present invention; Lebstatin, 17-O-demethyllebstatin, polyetherin A (nigericin); Sodium nigericin (nigericin sodium salt); Abierixin, 29-O-methylabierixin, the technology of 11-O-methylelaiophylin and elaiophylin.
This optimal process prepares 17-demethylation NSC 122750 (17-O-demethylgeldanamycin) through following steps; Lebstatin, 17-O-demethyllebstatin, polyetherin A (nigericin); Sodium nigericin (nigericinsodium salt); Abierixin, 29-O-methylabierixin, 11-O-methylelaiophylin and elaiophylin:
A) fermenting culture of preparation streptomycete SCSIO 1934 separates fermented liquid and the mycelium of this fermenting culture, and fermented liquid is through ethyl acetate extraction, and ethyl acetate layer obtains extractum A through distilling after concentrated; Mycelium use earlier acetone extraction, and the remainder water mixed solution was used ethyl acetate extraction after vat liquor reclaimed acetone, and ethyl acetate layer obtains medicinal extract B through distilling after concentrated;
B) extractum A is through silica gel column chromatography; With volume ratio from 100: 0～0: 100 chloroform-methanol as the eluent gradient wash-out; The chloroform-methanol volume ratio is the fraction A 1 that gradient under elutes at 100: 1; The chloroform-methanol volume ratio is the fraction A 2 that gradient under elutes at 50: 1, and the chloroform-methanol volume ratio is the fraction A 3 that gradient under elutes at 20: 1, and the chloroform-methanol volume ratio is the fraction A 4 that gradient under elutes at 10: 1; The chloroform-methanol volume ratio is that the cut that gradient under elutes at 5: 1 is A5, and the chloroform-methanol volume ratio is the fraction A 6 that gradient under elutes at 2: 1;
Fraction A 2 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, obtain compound 1 through recrystallization again, be 17-O-demethylgeldanamycin;
Fraction A 3 is through gel filtration chromatography, and using volume ratio is 1: 1 chloroform-methanol wash-out, after the preparation thin-layer chromatography, with volume ratio be ETHYLE ACETATE-methyl alcohol of 50: 1 as developping agent, collect the Rf value and be 0.5 cut, obtain compound 2, be lebstatin;
Fraction A 6 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, again through gel filtration chromatography, as the moving phase wash-out,, be 17-O-demethyllebstatin after recrystallization obtains compound 3 with methyl alcohol;
C) medicinal extract B is through silica gel column chromatography; With volume ratio from 100: 0～0: 100 chloroform-methanol as the eluent gradient wash-out; The chloroform-methanol volume ratio is the fraction B 1 that gradient under elutes at 100: 1; The chloroform-methanol volume ratio is gradient under to elute fraction B 2 at 10: 1, and the chloroform-methanol volume ratio is the fraction B 3 that gradient under elutes at 5: 1;
Fraction B 1 obtains fraction A B1 with fraction A 1 merging; Fraction A B1 is through silica gel column chromatography; With volume ratio from 100: 0～20: 1 chloroform-methanol as the eluent gradient wash-out, the chloroform-methanol volume ratio is 100: 1 fraction A B1-1 that elute under the gradient, the chloroform-methanol volume ratio is 50: 1 fraction A B1-2 that elute under the gradient; The chloroform-methanol volume ratio is 30: 1 fraction A B1-3 that elute under the gradient, and the chloroform-methanol volume ratio is 20: 1 fraction A B1-4 that elute under the gradient; Fraction A B1-1 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, again through gel filtration chromatography, with methyl alcohol as the moving phase wash-out; Again through the preparation thin-layer chromatography, with volume ratio be 1: 1 petroleum ether-ethyl acetate as developping agent, collect Rf and be 0.6 cut, obtain compound 5; Be Sodium nigericin, fraction A B1-2 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through gel filtration chromatography; As the moving phase wash-out, again through twice silica gel column chromatography, using for the first time volume ratio is that 2: 1 petroleum ether-ethyl acetate is as moving phase with methyl alcohol; Use for the second time volume ratio be 10: 1 chloroform-methanol as the permanent gradient elution of moving phase, obtain compound 6, be abierixin; Fraction A B1-3 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through silica gel column chromatography; Use volume ratio be 2: 1 petroleum ether-ethyl acetate as the permanent gradient elution of moving phase, obtain compound 4, be polyetherin A; Fraction A B1-4 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through silica gel column chromatography; Use volume ratio be 30: 1 chloroform-methanol as the permanent gradient elution of moving phase, obtain compound 7, be 29-O-methylabierixin;
Fraction B 2 merges with fraction A 4 and obtains fraction A B2, and AB2 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out; Through gel filtration chromatography, use methanol-eluted fractions again, then through the thin layer preparative hplc; With volume ratio is that 5: 1 chloroform-methanol is as developping agent; Collection Rf value is 0.5 cut, obtains compound 8, is 11-O-methylelaiophylin;
Fraction B 3 merges with A5 and obtains fraction A B3, and AB3 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, after obtain compound 9 through recrystallization, be elaiophylin.
The fermenting culture of the preparation streptomycete SCSIO 1934 of described a) step preferably prepares through following method, activatory streptomycete SCSIO 1934 inserted in the seed culture mediums, and 28 ℃, 200rpm; Cultivate 48h and make seed liquor, seed liquor is linked in the fermention medium 28 ℃ with 10% inoculum size; 200rpm, shaking culture 210h, and make fermenting culture; The prescription of described seed culture medium and fermention medium all is to contain in every liter of substratum: starch 10g, glucose 20g, steeping water 3g; Carnis Bovis seu Bubali cream 3g, yeast extract 10g, K
2HPO
40.5g, MgSO
47H
2O 0.5g, CaCO
32g, thick sea salt 30g, surplus is a water, pH 7.0.
Fermented liquid in the described a) step is through ethyl acetate extraction, is fermented liquid with ethyl acetate extraction 5 times, and the amount of institute's solubilizing agent is 2 times of volumes of fermented liquid.
Through structural analysis; 9 compound-compounds 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8 and the compound 9 that from the fermenting culture of streptomycete SCSIO 1934, prepares of the present invention identified that qualification result is following:
Compound 1: yellow needle (methyl alcohol) ESI-MS m/z 545 [M-H]
-.
1H-NMR (CDCl
3, 500MHz) δ: 6.97 (1H, d, J=11.8Hz, H-3), 6.57 (1H, t, J=11.8Hz, H-4), 5.90 (1H, t, J=11.8Hz, H-5); 4.32 (1H, d, J=9.0Hz, H-6), 5.17 (1H, s, H-7), 5.81 (1H, d, J=9.0Hz, H-9), 2.79 (1H, t; J=7.0Hz, H-10), 3.53 (1H, overlap, H-11), 3.51 (1H, overlap, H-12), 1.76 (2H, brs, H-13), 1.76 (1H, overlap; H-14), 2.45 (2H, m, H-15), 7.41 (1H, s, H-19), 0.97 (3H, d, J=6.5Hz, H-22), 2.03 (3H; S, H-23), 1.00 (3H, d, J=6.5Hz, H-24), 1.79 (3H, s, H-25), 3.36 (3H, s, 6-OCH
3), 3.30 (3H, s, 12-OCH
3), 8.96 (1H, s, NH).
13C-NMR (CDCl
3, 125MHz) δ: 168.2 (C-1), 134.6 (C-2), 129.1 (C-3), 126.2 (C-4), 137.0 (C-5), 81.4 (C-6), 56.8 (6-OCH
3), 81.8 (C-7), 156.0 (7-OCONH
2), 133.4 (C-8), 133.1 (C-9), 32.2 (C-10), 72.7 (C-11), 81.0 (C-12), 57.4 (12-OCH
3), 34.4 (C-13), 28.1 (C-14), 32.7 (C-15); 117.4 (C-16), 153.1 (C-17), 183.2 (C-18), 108.2 (C-19); 140.5 (C-20), 184.4 (C-21), 12.3 (C-22), 12.5 (C-23); 23.2 (C-24), 12.9 (C-25). above data and document [Anansiriwattana W, Tanasupawat S; Amnuoypol S, etal.Identification and antimicrobial activities of actinomycetes from soils in Samed Island, andgeldanamycin from strain PC4-3.Thai J Pharm Sci [J]; 2006,30:49-56.] the report unanimity, confirm that compound 1 is 17-demethylation NSC 122750 (17-O-demethylgeldanamycin).
Compound 2: yellow needle (methyl alcohol), ESI-MS m/z 571 [M+Na]
+With m/z 547 [M-H]
-, 583 [M+Cl]
-.1H-NMR (CDCl
3, 500MHz) δ: 5.89 (1H, brs, H-3), 3.30 (1H, m, H-6), 4.95 (1H, d, J=7.5Hz, H-7), 5.31 (1H; D, J=8.5Hz, H-9), 2.49 (1H, d, J=6.5Hz, H-10), 3.55 (1H, d, J=5.5Hz, H-11), 3.13 (1H; D, J=9.5Hz, H-12), 1.15 (1H, m, H-13), 1.72 (1H, m, H-13), 1.95 (1H, m, H-14); 2.54 (1H, dd, J=5.5,13.5Hz, H-15), 2.74 (1H, dd, J=5.5,13.5Hz, H-15), 6.75 (1H; Brs, H-19), 6.35 (1H, s, H-21), 1.79 (3H, s, H-22), 1.51 (3H, s, H-23); 1.03 (3H, d, J=6.5Hz, H-24), 0.83 (3H, d, J=4.5Hz, H-25), 3.46 (3H, s, 6-OCH
3), 3.33 (3H, s, 12-OCH
3), 3.73 (3H, s, 17-OCH
3).
13C-NMR (CDCl
3, 125MHz) δ: 174.6 (C-1), 135.8 (C-2), 136.6 (C-3), 24.8 (C-4), 31.4 (C-5), 81.4 (C-6), 59.8 (6-OCH
3), 83.7 (C-7), 159.2 (7-OCONH
2), 131.6 (C-8), 134.8 (C-9), 35.9 (C-10), 75.5 (C-11), 83.0 (C-12), 57.3 (12-OCH
3), 35.0 (C-13), 32.8 (C-14), 37.2 (C-15), 135.4 (C-16), 145.4 (C-17), 60.9 (17-OCH
3), 133.2 (C-18), 109.6 (C-19), 151.7 (C-20), 118.3 (C-21), 13.7 (C-22), 12.2 (C-23), 17.2 (C-24), 19.8 (C-25).Above data and document [Li MG; Wu SH, Zhao LX, et al.Isolation and structureelucidation of autolytimycin; A new compound produced by Streptomyces autolyticus JX-47.ChinChem Lett [J]; 2001,12 (10): 903-906.] report is consistent, confirms that compound 2 is lebstatin.
Compound 3: colourless sheet brilliant (methyl alcohol), ESI-MS m/z 557 [M+Na]
+With m/z 533 [M-H]
-1H-NMR (CDCl
3, 500MHz) δ: 5.71 (1H, brs, H-3), 2.04 (1H, d, J=6.8Hz, H-4), 2.16 (1H, d, J=6.8Hz, H-4), 1.17 (1H; M, H-5), 1.27 (1H, m, H-5), 3.17 (1H, d, J=6.0Hz, H-6), 4.84 (1H, d, J=9.0Hz, H-7); 5.23 (1H, brs, H-9), 2.36 (1H, m, H-10), 3.36 (1H, brs, H-11), 2.98 (1H, m, H-12), 1.60 (1H; M, H-13), 1.80 (1H, d, J=5.5Hz, H-14), 2.65 (1H, dd, J=5.8,13.0Hz, H-15), 2.29 (1H, dd; J=5.8,13.0Hz, H-15), 6.67 (1H, brs, H-19), 6.16 (1H, s, H-21), 1.66 (3H, s, H-22), 1.39 (3H; S, H-25), 0.90 (3H, d, J=7.0Hz, H-26), 0.75 (3H, s, H-28), 3.31 (3H, s, 12-OCH
3), 3.21 (3H, s, 17-OCH
3), 7.81 (1H, s, Ar-OH), 9.16 (1H, s, Ar-OH), 9.10 (1H, s, NH), 6.41 (2H, s, NH
2).
13C-NMR (CDCl
3, 125MHz) δ: 170.5 (C-1), 130.0 (C-2), 133.8 (C-3), 23.1 (C-4), 29.6 (C-5), 79.2 (C-6), 58.2 (6-OCH
3), 80.8 (C-7), 156.0 (7-OCONH
2), 129.6 (C-8), 133.2 (C-9), 33.7 (C-10), 73.5 (C-11), 80.8 (C-12), 56.2 (12-OCH
3), 35.7 (C-13,15), 30.5 (C-14); 126.6 (C-16), 140.2 (C-17), 144.7 (C-18), 106.5 (C-19); 129.6 (C-20), 116.0 (C-21), 13.2 (C-22), 11.4 (C-23); 16.2 (C-24), 19.2 (C-25). above data and document [Stead P, Latif S, Blackaby AP; Et al.Discovery of novel ansamycins possessing potentinhibitory activity in a cell-based oncostatin M signalling assay.J Antibiot [J], 2000,53 (7): 657-63.] the report unanimity, confirm that compound 3 is 17-O-demethyllebstatin.
Compound 4: white powder, ESI-MS m/z 747 [M+Na]
+With m/z 723 [M-H]
-1H-NMR (CDCl
3, 500MHz) δ: 2.45 (1H, dt, J=4.5,13.5Hz, H-2), 3.45 (1H, d, J=7.5Hz, H-3), 1.75 (1H, m, H-4), 1.41 (1H, m; H-5), 1.97 (1H, m, H-5), 1.09 (1H, m, H-6), 1.95 (1H, m, H-6), 4.03 (1H, m, H-7), 0.92 (1H, m; H-8), 2.11 (1H, dd, J=3.0,11.5Hz, H-8), 4.32 (1H, m, H-9), 1.39 (1H, m, H-10), 2.23 (1H, m, H-10); 3.32 (1H, m, H-11), 1.44 (1H, m, H-12), 2.29 (1H, dd, J=7.0,10.0Hz, H-14), 1.56 (1H, t, J=12.0Hz, H-15); 1.70 (1H, m, H-15), 3.73 (1H, dd, J=2.0,10.3Hz, H-16), 1.79 (1H, m, H-18), 1.25 (1H, m, H-19), 2.17 (1H; M, H-19), 3.48 (1H, m, H-21), 2.20 (1H, m, H-22), 1.09 (1H, m, H-23), 2.32 (1H, m, H-23); 3.92 (1H, dd, J=2.3,10.3 Hz, H-24), 1.32 (1H, m, H-26), 1.36 (1H, m, H-27), 1.76 (1H, m, H-28); 4.11 (1H, dt, J=4.0,11.0Hz, H-30), 0.84 (1H, d, J=6.5Hz, H-31), 0.81 (1H, d, J=6.5Hz, H-32), 0.85 (1H; D, J=6.0Hz, H-33), 1.08 (3H, s, H-34), 1.37 (3H, s, H-35), 0.88 (1H, d, J=7.0Hz, H-36), 1.01 (1H; D, J=7.0Hz, H-37), 0.89 (1H, d, J=6.5Hz, H-38), 0.98 (1H, d, J=7.0Hz, H-39), 3.31 (3H, s, H-40);
13C-NMR (CDCl
3, 125MHz) δ: 177.3 (C-1), 44.1 (C-2), 72.8 (C-3), 27.7 (C-4), 26.0 (C-5); 23.3 (C-6), 68.9 (C-7), 35.3 (C-8), 60.2 (C-9), 32.5 (C-10), 78.0 (C-11); 35.7 (C-12), 108.1 (C-13), 38.9 (C-14), 42.3 (C-15), 81.5 (C-16), 82.3 (C-17); 25.7 (C-18), 30.7 (C-19), 83.4 (C-20), 85.8 (C-21), 35.1 (C-22), 332.3 (C-23); 74.4 (C-24), 77.0 (C-25), 31.8 (C-26), 37.4 (C-27), 37.1 (C-28), 96.9 (C-29); 68.2 (C-30), 16.2 (C-31), 17.3 (C-32), 15.5 (C-33), 22.6 (C-34), 27.4 (C-35); 13.0 (C-36), 12.9 (C-37), 10.7 (C-38), 13.1 (C-39), 57.4 (C-40). above data and document [Stempel A, Westley JW; Benz W.The identity of the antibiotics nigericin, polyetherin A and X-464.J Antibiot [J], 1969,22 (8): 384-5.Berrada R, Dauphin G; David L.Epinigericin, a new polyether carboxylic antibiotic.Structural determination by 2D NMR methods.J Org Chem [J], 1987,52 (12): 2388-91.] the report unanimity, confirm that compound 4 is polyetherin A (nigericin).
Compound 5: colourless needle (methyl alcohol), ESI-MS m/z 769 [M+Na]
+With m/z 723 [M-Na]
-Low Resolution Mass Spectra shows that this compound contains a Na atom.This compound
1H with
13The nuclear magnetic data basically identical of C-NMR data and polyetherin A (nigericin), C-1 shift value (δ 183.8) to low field displacement 6.5, and H-21; The shift value of H-35 and H-40 is respectively 4.33,1.58 and 3.63, with document [Alva R; Lugo-R.JA; Arzt E, et al.Nigericin forms highly stablecomplexes with lithium and cesium.J BioenergBiomembr [J], 1992; 24 (1): 125-9.] the Sodium nigericin characteristic of report is consistent, explains that this compound is Sodium nigericin (nigericin soldium salt).Its nuclear magnetic data is following:
1H-NMR (CDCl
3, 500MHz) δ: 2.36 (1H, m, H-2), 3.68 (1H, m, H-3), 1.74 (1H, m, H-4), 1.44 (1H, m, H-5), 1.99 (1H, m; H-5), 1.06 (1H, m, H-6), 1.99 (1H, m, H-6), 4.04 (1H, d, J=13.5Hz, H-7), 2.58 (1H, dt, J=4.0,13.5Hz; H-8), 0.90 (1H, m, H-8), 4.26 (1H, m, H-9), 1.01 (1H, overlap, H-10), 2.32 (1H, m, H-10), 3.36 (1H, overlap; H-11), 1.78 (1H, m, H-12), 2.19 (1H, m, H-14), 1.63 (1H, t, J=12.0Hz, H-15), 1.79 (1H, m, H-15), 3.66 (1H; D, J=2.0Hz, H-17), 1.82 (2H, m, H-18), 2.17 (1H, m, H-19), 1.25 (1H, m, H-19), 4.33 (1H, d, J=3.5Hz; H-21), 2.28 (1H, m, H-22), 2.38 (1H, m, H-23), 1.42 (1H, t, J=8.0Hz, H-23), 4.36 (1H, dd, J=2.3,8.3Hz; H-24), 3.71 (1H, d, J=2.0Hz, H-25), 1.32 (1H, m, H-26), 1.36 (2H, m, H-27), 1.52 (1H, m, H-28), 3.92 (1H; D, J=11.8Hz, H-30), 3.31 (1H, d, J=11.8Hz, H-30), 0.87 (3H, d, J=6.5Hz, H-31), 0.81 (3H, d, J=4.0Hz, H-32); 0.88 (3H, d, J=6.6Hz, H-33), 1.14 (3H, s, H-34), 1.58 (3H, s, H-35), 0.91 (3H, d, J=7.0Hz, H-36), 1.01 (3H; D, J=7.0Hz, H-37), 0.94 (3H, d, J=7.0Hz, H-38), 0.95 (3H, d, J=7.0Hz, H-39), 3.63 (3H, s, H-40).
13C-NMR (CDCl
3, 125MHz) δ: 183.8 (C-1), 45.8 (C-2), 73.2 (C-3), 27.7 (C-4), 26.3 (C-5), 23.6 (C-6); 68.4 (C-7), 35.8 (C-8), 60.4 (C-9), 32.1 (C-10), 79.5 (C-11), 36.5 (C-12), 107.6 (C-13); 39.6 (C-14), 41.8 (C-15), 82.3 (C-16), 81.5 (C-17), 25.9 (C-18), 29.6 (C-19), 84.8 (C-20); 85.3 (C-21), 35.1 (C-22), 32.4 (C-23), 76.5 (C-24), 76.9 (C-25), 31.9 (C-26), 37.2 (C-27); 36.8 (C-28), 97.2 (C-29), 67.1 (C-30), 16.4 (C-31), 17.0 (C-32), 16.2 (C-33), 22.8 (C-34); 29.0 (C-35), 13.4 (C-36), 13.0 (C-37), 11.5 (C-38), 14.4 (C-39), 59.5 (C-40).
Compound 6: white powder, ESI-MS m/z 747 [M+Na]
+With m/z 723 [M-H]
- 1H-NMR(CDCl
3，500MHz)δ：6.64(1H，d，J＝10.0Hz，H-3)，2.52(1H，m，H-4)，1.76(1H，m，H-5)，1.42(1H，m，H-5)，1.59(2H，m，H-6)，3.86(1H，d，J＝2.5Hz，H-7)，1.40(1H，m，H-8)，1.47(1H，m，H-8)，4.24(1H，t，J＝10.8Hz，H-9)，1.87(2H，brd，J＝14.0Hz，H-10)，3.29(1H，d，J＝2.5Hz，H-11)，1.77(1H，m，H-12)，2.24(1H，m，H-14)，1.95(1H，m，H-15)，1.45(1H，m，H-15)，3.46(1H，d，J＝7.5Hz，H-17)，1.40(1H，m，H-18)，1.77(1H，m，H-18)，2.13(2H，m，H-19)，3.94(1H，d，J＝3.5Hz，H-21)，2.27(1H，m，H-22)，1.47(1H，m，H-23)，2.20(1H，m，H-23)，4.33(1H，dt，J＝2.8，9.3Hz，H-24)，3.84(1H，brs，H-25)，1.41(1H，m，H-26)，1.43(2H，m，H-27)，1.66(1H，m，H-28)，3.53(1H，d，J＝10.8Hz，H-30)，3.61(1H，d，J＝10.8Hz，H-30)，0.87(3H，s，H-31)，0.86(3H，d，J＝6.0Hz，H-32)，0.91(3H，d，J＝7.0，H-33)，1.12(3H，s，H-34)，1.35(3H，s，H-35)，0.89(3H，s，H-36)，1.02(3H，d，J＝2.0Hz，H-37)，1.01(3H，s，H-38)，1.82(3H，s，H-39)，3.35(3H，s，11-OCH
3).
13C-NMR(CDCl
3，125MHz)δ：171.4(C-1)，125.9(C-2)，149.7(C-3)，33.8(C-4)，32.5(C-5)，42.2(C-6)，71.2(C-7)，35.5(C-8)，65.7(C-9)，35.1(C-10)，78.1(C-11)，37.0(C-12)，108.6(C-13)，39.4(C-14)，41.4(C-15)，83.5(C-16)，81.9(C-17)，26.6(C-18)，31.8(C-19)，83.9(C-20)，86.1(C-21)，35.4(C-22)，33.1(C-23)，76.9(C-24)，75.8(C-25)，32.8(C-26)，37.0(C-27)，34.8(C-28)，97.2(C-29)，67.9(C-30)，13.3(C-31)，17.3(C-32)，15.5(C-33)，22.7(C-34)，26.0(C-35)，16.1(C-36)，13.4(C-37)，20.3(C-38)，12.3(C-39)，57.9(11-OCH3)。Above data and document [David L; Leal Ayala H, Tabet JC.Abierixin, anew polyether antibiotic.Production; Structural determination and biological activities.J Antibiot [J]; 1985,38 (12): 1655-63.] report is consistent, confirms that compound 6 is abierixin.
Compound 7: white powder, ESI-MS m/z 761 [M+Na]
+With m/z 737 [M-H]
- 1H-NMR(CDCl
3，500MHz)δ：6.65(dd，J＝1.0，10.0Hz，1H)，4.27(m，2H)，3.83(d，J＝4.5，1H)，3.79(m，1H)，3.68(d，J＝11.0Hz，1H)，3.54(d，J＝11.0Hz，1H)，3.43(dd，J＝3.5，10.0Hz，1H)，3.31(s，3H)，3.27(m，1H)，3.26(s，3H)，2.50(m，1H)，2.33(m，1H)，2.28(dd，J＝5.5，11.5Hz，1H)，2.17(m，1H)，2.12(dd，J＝7.8，12.3Hz，1H)，2.02(m，1H)，1.83-1.90(m，1H)，1.82(s，3H)，1.73(m，1H)，1.70(m，1H)，1.26-1.55(m，15H)，1.25(s，3H)，1.15(s，3H)，1.01(d，J＝7.0Hz，3H)，0.96(d，J＝7.0Hz，3H)，0.95(d，J＝7.0Hz，3H)，0.88(d，J＝7.0Hz，3H)，0.87(d，J＝7.0Hz，3H)，0.86(d，J＝7.0Hz，3H)；
13C-NMR(CDCl
3，125MHz)δ：172.5(C-1)，125.9(C-2)，150.0(C-3)，33.2(C-4)，32.3(C-5)，27.6(C-6)，71.8(C-7)，37.1(C-8)，65.8(C-9)，35.8(C-10)，78.4(C-11)，36.7(C-12)，108.1(C-13)，39.6(C-14)，39.8(C-15)，84.3(C-16)，81.4(C-17)，42.2(C-18)，34.4(C-19)，84.6(C-20)，86.6(C-21)，35.2(C-22)，34.2(C-23)，77.7(C-24)，77.3(C-25)，33.4(C-26)，35.0(C-27)，35.2(C-28)，99.0(C-29)，64.1(C-30)，16.0(C-31)，17.5(C-32)，15.7(C-33)，23.0(C-34)，24.2(C-35)，13.3(C-36)，12.8(C-37)，20.0(C-38)，12.3(C-39)，58.3(C-11-OCH
3)，48.5(C-29-OCH
3)。Above carbon spectrum data and document [Wu ZX; Bai LQ; Wang MZ, et al.Structure-antibacterial relationship of nigericin derivatives.Chem Nat Compd [J], 2009; 45 (3): 333-7.] report is consistent, confirms that compound 7 is 29-O-methylabierixin.
Compound 8:C
55H
90O
18, ESI-MS data (m/z 1061 [M+Na]
+And m/z 1037 [M-H]
-).
1H NMR (CDCl
3, 500MHz) δ: 5.69 (1H, d, J=15.0Hz, H-2,2 '), 6.98 (1H, dd, J=11.3,15.0Hz, H-3,3 '), 6.12 (1H; Dd, J=11.3,15.0Hz, H-4,4 '), 5.62 (1H, m, H-5,5 '), 2.54 (1H, m, H-6,6 '); (4.74 1H, d, J=10.0Hz, H-7,7 '), 1.95 (1H, m, H-8,8 '), 4.10 (1H, d, J=9.0Hz, H-9; 9 '), 1.71 (1H, d, J=7.5Hz, H-10,10 '), 1.03 (1H, m, 2.37 (1H, dd, J=4.3,11.7Hz, H-12; 12 '), 3.93 (1H, m, H-13,13 '), 1.18 (1H, d, J=5.0Hz, H-14,14 '), 3.90 (1H, m, H-15; 15 '), 1.10 (1H, d, J=6.5Hz, H-16,16 '), 1.03 (1H, d, J=7.0Hz, H-17,17 '), 0.81 (1H, d; J=6.5Hz, H-18,18 '), 1.00 (1H, d, J=7.0Hz, H-19,19 '), 1.42 (1H, m, H-20,20 '); (1.62 1H, m, H-20,20 '), 0.84 (1H, t, J=7.5Hz, H-21,21 '), 5.05 (1H, brs; H-22,22 '), 1.78 (1H, d, J=3.5Hz, H-23,23 '), 1.80 (1H, d, J=3.5Hz, H-23; 23 '), 3.97 (1H, m, H-24,24 '), 3.61 (1H, d, J=2.0Hz, H-25,25 '), 3.96 (1H; M, H-26,26 '), 1.23 (1H, d, J=7.0Hz, H-27,27 '), 3.04 (3H, s, 28);
13C NMR (CDCl
3, 125MHz) δ: 170.0 (s, C-1,1 '), 121.0 (d, C-2,2 '), 145.1 (d, C-3,3 '), 132.0 (d, C-4,4 '), 144.4 (d; C-5,5 '), 40.9 (d, C-6,6 '), 77.9 (d, C-7,7 '), 35.9 (d, C-8,8 '), 70.6 (d, C-9,9 '); (41.7 d, C-10,10 '), 99.1 (s, C-11,11 '), 38.9 (t, C-12,12 '), 70.3 (d, C-13,13 '), 48.5 (d, C-14; 14 '), 66.6 (d, C-15,15 '), 19.1 (q, C-16,16 '), 14.9 (q, C-17,17 '), 8.8 (q, C-18,18 '), 7.1 (q; C-19,19 '), 19.4 (t, C-20,20 '), 9.1 (q, C-21,21 '), 93.3 (d, C-22,22 '), 33.5 (t, C-23,23 '); (66.1 d, C-24,24 '), 71.5 (d, C-25,25 '), 66.0 (d, C-26,26 '), 16.8 (q, C-27,27 '), 46.6 (q, C-28).Above carbon spectrum data and document [Ritzau M, Heinze S, Fleck WF; Et al.New macrodiolide antibiotics, 11-O-monomethyl-and11,11 '-O-dimethylelaiophylins; From Streptomyces sp.HKI-0113 and HKI-0114.J Nat Prod [J]; 1998,61 (11): 1337-1339] report is consistent, confirms that compound 8 is 11-O-methylelaiophylin.
Compound 9:C
54H
88O
18, ESI-MS data (m/z 1045 [M+Na]
+And m/z 1023 [M-H]-).
1H NMR (CDCl
3, 500MHz) δ: 5.69 (1H, d, J=15.3Hz, H-2,2 '), 6.98 (1H, dd, J=11.0,15.3 Hz, H-3,3 '), 6.12 (1H; Dd, J=11.0,15.3Hz, H-4,4 '), 5.62 (1H, m, H-5,5 '), 2.55 (1H, m, H-6,6 '); (4.74 1H, d, J=10.0Hz, H-7,7 '), 1.96 (1H, m, H-8,8 '), 4.10 (1H, d, J=8.0Hz, H-9; 9 '), 1.71 (1H, d, J=7.5Hz, H-10,10 '), 1.03 (1H, m, H-9,9 '), 2.37 (1H, dd, J=4.3; 11.7Hz, H-12,12 '), 3.93 (1H, m, H-13,13 '), 1.18 (1H, m, H-14,14 '), 3.90 (1H, m; H-15,15 '), 1.10 (1H, d, J=6.0Hz, H-16,16 '), 1.04 (1H, d, J=6.5Hz, H-17,17 '), 0.82 (1H; D, J=4.5Hz, H-18,18 '), 1.00 (1H, d, J=7.0Hz, H-19,19 '), 1.42 (1H, m, H-20,20 '); (1.62 1H, m, H-20,20 '), 0.85 (1H, t, J=7.5Hz, H-21,21 '), 5.06 (1H, brs; H-22,22 '), 1.78 (1H, d, J=1.5Hz, H-23,23 '), 1.80 (1H, s, H-23,23 '); (3.96 1H, m, H-24,24 '), 3.62 (1H, brs, H-25,25 '), 3.96 (1H, m; H-26,26 '), 1.23 (1H, d, J=6.5Hz, H-27,27 '), 3.04 (3H, s, 28);
13C NMR (CDCl
3, 125MHz) δ: 170.0 (s, C-1,1 '), 121.0 (d, C-2,2 '), 145.1 (d, C-3,3 '), 132.0 (d, C-4,4 '), 144.4 (d; C-5,5 '), 40.9 (d, C-6,6 '), 77.9 (d, C-7,7 '), 35.9 (d, C-8,8 '), 70.7 (d, C-9,9 '); (41.7 d, C-10,10 '), 99.1 (s, C-11,11 '), 38.9 (t, C-12,12 '), 70.3 (d, C-13,13 '), 48.5 (d, C-14; 14 '), 66.6 (d, C-15,15 '), 19.2 (q, C-16,16 '), 14.9 (q, C-17,17 '), 8.8 (q, C-18,18 '), 7.1 (q; C-19,19 '), 19.4 (t, C-20,20 '), 9.2 (q, C-21,21 '), 93.3 (d, C-22,22 '), 33.6 (t, C-23; 23 '), 66.1 (d, C-24,24 '), 71.5 (d, C-25,25 '), 66.0 (d, C-26,26 '), 16.8 (q, C-27,27 ').Above carbon spectrum data and document [Yan Shuling, Huang Weiyi, Wang Shimei; Microbiotic M1 separation in the et al. streptomyces hygroscopicus NND-52 bacterial strain born of the same parents, purifying and structure are confirmed. Chinese microbiotic magazine [J]; 2001,26 (3): 161-203.] report is consistent, confirms that compound 9 is elaiophylin.
The invention provides the multiple antibiotic production bacterium of producing of a kind of marine source-streptomycete SCSIO 1934.Can prepare 17-demethylation NSC 122750 (17-O-demethylgeldanamycin) through streptomycete SCSIO 1934 of the present invention; Lebstatin, 17-O-demethyllebstatin, polyetherin A (nigericin); Sodium nigericin (nigericinsodium salt); Abierixin, 29-O-methylabierixin, 9 kinds of microbiotic of 11-O-methylelaiophylin and elaiophylin.Therefore the present invention provides a kind of new method for these 9 kinds antibiotic production preparations.
Streptomycete of the present invention (Streptomyces sp.) SCSIO 1934, be preserved in Chinese typical culture collection center (CCTCC), address on November 25th, 2010: Chinese Wuhan City Wuhan University, its deposit number is CCTCC NO:M 2010317.。
Description of drawings:
Fig. 1 is a compound 1:17-demethylation NSC 122750 of the present invention (17-O-demethylgeldanamycin); Compound 2:lebstatin; Compound 3:17-O-demethyllebstatin; Compound 4: polyetherin A (nigericin), compound 5: Sodium nigericin (nigericin sodium salt), compound 6:abierixin; Compound 7:29-O-methylabierixin, 9 kinds of antibiotic structural formulas of compound 8:11-O-methylelaiophylin and compound 9:elaiophylin;
Fig. 2 is the systematic evolution tree of streptomycete SCSIO 1934 of the present invention and the nearest kind of sibship with it of rebuilding based on the adjacent method of 16S rDNA sequence.
Embodiment:
Below be to further specify to of the present invention, rather than limitation of the present invention.
Embodiment 1:
One, the evaluation of streptomycete (Streptomyces sp) SCSIO 1934
The separation of the genomic dna that relates to during streptomycete SCSIO 1934 identifies; The pcr amplification of 16S rDNA, the establishment method of sequence alignment and systematic evolution tree etc. are reference [Tian X P, Zhi X Y all; Qiu Y Q; Et al.Sciscionella marinagen.nov., sp.nov., a marine actinomycete isolated from a sediment in the northern South China Sea.Int J Syst Evol Microbiol [J]; 2009,59 (Pt 2): 222-228].
A) bacterium source: separate obtaining in the marine bottom sediment that marine actinomycete streptomycete (Streptomyces sp.) SCSIO 1934 northern from China South Sea (113 ° 44.320 of E ', 21 ° 14.773 of the N ') depth of water is 65 meters.The aerial hyphae of this bacterium is pearl, spore black, and it is abundant to produce spore.
B) strain identification: with reference to the method in the above-mentioned document, the 16S rDNA of pcr amplification streptomycete SCSIO 1934 and order-checking then are submitted among the GenBank, obtain sequence number HQ403650, and its sequence is shown in SEQ ID NO.1.16S rDNA gene sequencing result shows this bacterium and streptomycete Streptomyces sp.M4032, and the similarity of Streptomyces sp.M4033 and Streptomyces malaysiensis reaches 100%.Clearly disclose the phyletic evolution relation (Fig. 2) of this bacterium and one group of streptomyces species through adjacent method, shown a kind of in streptomyces of this strain Pseudomonas.Therefore with this bacterial strain called after: streptomycete (Streptomyces sp.) SCSIO 1934; Be preserved in Chinese typical culture collection center (CCTCC) on November 25th, 2010; The address: Chinese Wuhan City Wuhan University, its deposit number is CCTCC NO:M 2010317.
Two, the scale of streptomycete SCSIO 1934 fermentation
A) preparing culture medium:
The seed culture medium preparation contains in every liter of substratum: starch 10gL
-1, glucose 20gL
-1, steeping water 3gL
-1, Carnis Bovis seu Bubali cream 3gL
-1, yeast extract 10gL
-1, K
2HPO
40.5gL
-1, MgSO
47H
2O 0.5gL
-1, CaCO
32gL
-1, surplus is 3% seawater or Chen Haishui for the sea salt massfraction, 7.0,115 ℃ of pH, and sterilization 30min, subsequent use;
Fermention medium is identical with seed culture medium.
B) fermentation:
Seed culture: will on flat board, insert in the seed culture medium (800mL) by activatory marine streptomyces (Streptomyces sp.) SCSIO 1934,28 ℃, 200rpm cultivates 48h and makes seed liquor;
The scale fermentation culture:
Inoculation: seed liquor is linked into (8L) in the fermention medium with 10% inoculum size, and 28 ℃, 200rpm cultivates 210h, and makes the fermenting culture of streptomycete SCSIO 1934.
Three, marine streptomyces (Streptomyces sp.) SCSIO 1934 separation of antibiotics of producing
1, the extraction of fermented liquid
The fermenting culture of the streptomycete SCSIO 1934 that the scale fermentation culture is obtained carries out spinning (3500rmin earlier
-18min), obtain fermented liquid and mycelium, fermented liquid is with two volumes ethyl acetate extraction 5 times; Underpressure distillation gets fermentation broth extract-extractum A (10.5g); Mycelium is with acetone extraction 3 times, after the underpressure distillation aqueous liquid mixture with two volumes ethyl acetate extraction 4 times, concentrate acquisition mycelium extract-medicinal extract B (5.3g).
2, separation of antibiotics
(a): extractum A (10.5g) is through silica gel column chromatography (100-200 order); With volume ratio from 100: 0～0: 100 chloroform-methanol as the eluent gradient wash-out; The chloroform-methanol volume ratio is the fraction A 1 that gradient under elutes at 100: 1; The chloroform-methanol volume ratio is the fraction A 2 that gradient under elutes at 50: 1, and the chloroform-methanol volume ratio is the fraction A 3 that gradient under elutes at 20: 1, and the chloroform-methanol volume ratio is the fraction A 4 that gradient under elutes at 10: 1; The chloroform-methanol volume ratio is that the cut that gradient under elutes at 5: 1 is A5, and the chloroform-methanol volume ratio is the fraction A 6 that gradient under elutes at 2: 1;
Fraction A 2 is through the LH-20 gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, obtain compound 1 (14.2mg) through recrystallization again, be 17-O-demethylgeldanamycin;
Fraction A 3 is through the LH-20 gel filtration chromatography, and using volume ratio is 1: 1 chloroform-methanol wash-out, after prepare thin-layer chromatography; With volume ratio is that ETHYLE ACETATE-methyl alcohol of 50: 1 is as developping agent; Collection Rf value is 0.5 cut, obtains compound 2 (12.3mg), is lebstatin;
Fraction A 6 is through the LH-20 gel filtration chromatography; Use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, again through the LH-20 gel filtration chromatography, with methyl alcohol as the moving phase wash-out; After recrystallization obtains compound 3 (9.3mg), be 17-O-demethyllebstatin;
(b): medicinal extract B (5.3g) is through silica gel column chromatography (100-200 order); With volume ratio from 100: 0～0: 100 chloroform-methanol as the eluent gradient wash-out; The chloroform-methanol volume ratio is the fraction B 1 that gradient under elutes at 100: 1; The chloroform-methanol volume ratio is gradient under to elute fraction B 2 at 10: 1, and the chloroform-methanol volume ratio is the fraction B 3 that gradient under elutes at 5: 1;
Fraction B 1 obtains fraction A B1 with fraction A 1 merging; Fraction A B1 is through silica gel column chromatography (300-400 order); With volume ratio from 100: 0～20: 1 chloroform-methanol as the eluent gradient wash-out; The chloroform-methanol volume ratio is 100: 1 fraction A B1-1 that elute under the gradient; The chloroform-methanol volume ratio is 50: 1 fraction A B1-2 that elute under the gradient, and the chloroform-methanol volume ratio is 30: 1 fraction A B1-3 that elute under the gradient, and the chloroform-methanol volume ratio is 20: 1 fraction A B1-4 that elute under the gradient; Fraction A B1-1 is through the LH-20 gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through the LH-20 gel filtration chromatography; As the moving phase wash-out, again through the preparation thin-layer chromatography, is that 1: 1 petroleum ether-ethyl acetate is as developping agent with volume ratio with methyl alcohol; Collection Rf is 0.6 cut, obtains compound 5 (9.0mg), is Sodium nigericin; Fraction A B1-2 is through the LH-20 gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through the LH-20 gel filtration chromatography; As the moving phase wash-out, again through twice silica gel column chromatography (300-400 order), using for the first time volume ratio is that 2: 1 petroleum ether-ethyl acetate is as moving phase with methyl alcohol; Use for the second time volume ratio be 10: 1 chloroform-methanol as the permanent gradient elution of moving phase, obtain compound 6 (8.2mg), be abierixin; Fraction A B1-3 is through the LH-20 gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through silica gel column chromatography (300-400 order); Use volume ratio be 2: 1 petroleum ether-ethyl acetate as the permanent gradient elution of moving phase, obtain compound 4 (148.6mg), be polyetherin A; Fraction A B1-4 is through the LH-20 gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through silica gel column chromatography (300-400 order); Use volume ratio be 30: 1 chloroform-methanol as the permanent gradient elution of moving phase, obtain compound 7 (45.3mg), be 29-O-methylabierixin;
Fraction B 2 merges with fraction A 4 and obtains fraction A B2, and AB2 is through the LH-20 gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out; Through the LH-20 gel filtration chromatography, use methanol-eluted fractions again, then through the thin layer preparative hplc; With volume ratio is that 5: 1 chloroform-methanol is as developping agent; Collection Rf value is 0.5 cut, obtains compound 8 (26.5mg), is 11-O-methylelaiophylin
Fraction B 3 merges with A5 and obtains fraction A B3, and AB3 is through the LH-20 gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, after obtain compound 9 (21.8mg) through recrystallization, be elaiophylin.
Claims (4)
1. streptomycete (Streptomyces sp.) SCSIO 1934, its deposit number is CCTCC NO:M 2010317.
2. 17-demethylation NSC 122750, lebstatin, 17-O-demethyllebstatin; Polyetherin A, Sodium nigericin, abierixin; 29-O-methylabierixin; The preparation method of 11-O-methylelaiophylin and elaiophylin is characterized in that, may further comprise the steps:
A) the described deposit number of preparation claim 1 is the fermenting culture of the streptomycete SCSIO 1934 of CCTCC NO:M 2010317; The fermented liquid and the mycelium of this fermenting culture are separated; Fermented liquid is through ethyl acetate extraction, and ethyl acetate layer obtains extractum A through distilling after concentrating; Mycelium use earlier acetone extraction, and the remainder water mixed solution was used ethyl acetate extraction after vat liquor reclaimed acetone, and ethyl acetate layer obtains medicinal extract B through distilling after concentrated;
B) extractum A is through silica gel column chromatography; With volume ratio from 100: 0～0: 100 chloroform-methanol as the eluent gradient wash-out; The chloroform-methanol volume ratio is the fraction A 1 that gradient under elutes at 100: 1; The chloroform-methanol volume ratio is the fraction A 2 that gradient under elutes at 50: 1, and the chloroform-methanol volume ratio is the fraction A 3 that gradient under elutes at 20: 1, and the chloroform-methanol volume ratio is the fraction A 4 that gradient under elutes at 10: 1; The chloroform-methanol volume ratio is that the cut that gradient under elutes at 5: 1 is A5, and the chloroform-methanol volume ratio is the fraction A 6 that gradient under elutes at 2: 1;
Fraction A 2 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, obtain compound 1 through recrystallization again, be 17-demethylation NSC 122750;
Fraction A 3 is through gel filtration chromatography, and using volume ratio is 1: 1 chloroform-methanol wash-out, after the preparation thin-layer chromatography, with volume ratio be ETHYLE ACETATE-methyl alcohol of 50: 1 as developping agent, collect the Rf value and be 0.5 cut, obtain compound 2, be lebstatin;
Fraction A 6 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, again through gel filtration chromatography, as the moving phase wash-out,, be 17-O-demethyllebstatin after recrystallization obtains compound 3 with methyl alcohol;
C) medicinal extract B is through silica gel column chromatography; With volume ratio from 100: 0～0: 100 chloroform-methanol as the eluent gradient wash-out; The chloroform-methanol volume ratio is the fraction B 1 that gradient under elutes at 100: 1; The chloroform-methanol volume ratio is gradient under to elute fraction B 2 at 10: 1, and the chloroform-methanol volume ratio is the fraction B 3 that gradient under elutes at 5: 1;
Fraction B 1 obtains fraction A B1 with fraction A 1 merging; Fraction A B1 is through silica gel column chromatography; With volume ratio from 100: 0～20: 1 chloroform-methanol as the eluent gradient wash-out, the chloroform-methanol volume ratio is 100: 1 fraction A B1-1 that elute under the gradient, the chloroform-methanol volume ratio is 50: 1 fraction A B1-2 that elute under the gradient; The chloroform-methanol volume ratio is 30: 1 fraction A B1-3 that elute under the gradient, and the chloroform-methanol volume ratio is 20: 1 fraction A B1-4 that elute under the gradient; Fraction A B1-1 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, again through gel filtration chromatography, with methyl alcohol as the moving phase wash-out; Again through the preparation thin-layer chromatography, with volume ratio be 1: 1 petroleum ether-ethyl acetate as developping agent, collect Rf and be 0.6 cut, obtain compound 5; Be Sodium nigericin, fraction A B1-2 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through gel filtration chromatography; As the moving phase wash-out, again through twice silica gel column chromatography, using for the first time volume ratio is that 2: 1 petroleum ether-ethyl acetate is as moving phase with methyl alcohol; Use for the second time volume ratio be 10: 1 chloroform-methanol as the permanent gradient elution of moving phase, obtain compound 6, be abierixin; Fraction A B1-3 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through silica gel column chromatography; Use volume ratio be 2: 1 petroleum ether-ethyl acetate as the permanent gradient elution of moving phase, obtain compound 4, be polyetherin A; Fraction A B1-4 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, again through silica gel column chromatography; Use volume ratio be 30: 1 chloroform-methanol as the permanent gradient elution of moving phase, obtain compound 7, be 29-O-methylabierixin;
Fraction B 2 merges with fraction A 4 and obtains fraction A B2, and AB2 is through gel filtration chromatography, and using volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out; Through gel filtration chromatography, use methanol-eluted fractions again, then through the thin layer preparative hplc; With volume ratio is that 5: 1 chloroform-methanol is as developping agent; Collection Rf value is 0.5 cut, obtains compound 8, is 11-O-methylelaiophylin;
Fraction B 3 merges with A5 and obtains fraction A B3, and AB3 is through gel filtration chromatography, use volume ratio be 1: 1 chloroform-methanol as the moving phase wash-out, after obtain compound 9 through recrystallization, be elaiophylin.
3. preparation method according to claim 2 is characterized in that, the fermenting culture of the preparation streptomycete SCSIO 1934 of described a) step prepares through following method, and activatory streptomycete SCSIO 1934 is inserted in the seed culture medium; 28 ℃, 200rpm cultivates 48h and makes seed liquor, and seed liquor is linked in the fermention medium with 10% inoculum size; 28 ℃, 200rpm, shaking culture 210h, and make fermenting culture; The prescription of described seed culture medium and fermention medium all is to contain in every liter of substratum: starch 10g, glucose 20g, steeping water 3g; Carnis Bovis seu Bubali cream 3g, yeast extract 10g, K
2HPO
40.5g, MgSO
47H
2O 0.5g, CaCO
32g, thick sea salt 30g, surplus is a water, pH 7.0.
4. preparation method according to claim 2 is characterized in that, the fermented liquid in the described a) step is through ethyl acetate extraction, is fermented liquid with ethyl acetate extraction 5 times, and the amount of institute's solubilizing agent is 2 times of volumes of fermented liquid.
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| CN103642723B (en) * | 2013-12-02 | 2015-09-09 | 江西师范大学 | Streptomyces and method for preparing two antibiotics by utilizing same |
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