CN102219815A - Six tiacumicins compounds, preparation method and use of six tiacumicins compounds in preparing antibacterial drugs - Google Patents
Six tiacumicins compounds, preparation method and use of six tiacumicins compounds in preparing antibacterial drugs Download PDFInfo
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Abstract
本发明公开了六种台勾霉素类化合物及其制备方法和在制备抗菌药物中的应用。本发明以指孢囊菌NRRL 18085作为原始菌，对该菌的tiaS5-氧甲基转移酶基因实行敲除，获得tiaS5-氧甲基转移酶基因敲除突变株(TCM70)。对突变株发酵产物粗提物采用正相硅胶、反相硅胶、凝胶等柱层析色谱技术和制备薄层层析色谱技术等，从突变株TCM70得到化合物1-6。这六种化合物对金黄色葡萄球菌，苏云金芽孢杆菌，粪肠球菌等都具有较好的抑制活性。与母体化合物台勾霉素B相比较，化合物1和6均对粪肠球菌和金黄色葡萄球菌具有较低的MIC值，表明它们具有更好的抑菌活性，特别是化合物6，其针对金黄色葡萄球菌的活性比母体化合物台勾霉素B提高了8倍，因此有望研发成为抗菌新药。
The invention discloses six tiacumicins compounds, their preparation methods and their application in the preparation of antibacterial drugs. In the present invention, the fungus NRRL 18085 is used as the original bacterium, and the tiaS5-oxymethyltransferase gene of the bacterium is knocked out to obtain the knockout mutant strain (TCM70) of the tiaS5-oxymethyltransferase gene. For the crude extract of the fermentation product of the mutant strain, the compound 1-6 was obtained from the mutant strain TCM70 by using column chromatography techniques such as normal phase silica gel, reverse phase silica gel, gel chromatography and preparative thin layer chromatography techniques. These six compounds have good inhibitory activity against Staphylococcus aureus, Bacillus thuringiensis, Enterococcus faecalis and the like. Compared with the parent compound tiacumicin B, both compounds 1 and 6 had lower MIC values against Enterococcus faecalis and Staphylococcus aureus, indicating that they had better antibacterial activity, especially compound 6, which had The activity of Staphylococcus aureus is 8 times higher than that of the parent compound tiacumicin B, so it is expected to be developed as a new antibacterial drug.
Description
Technical field:
The present invention relates to six kinds of platforms and collude mould chlorins compound and preparation method thereof and the application in the preparation antibacterials.
Background technology:
Macrocylc compound is the important treatment microbiotic of a class.Platform colludes the general name that mycin (Tiacumicins) is a series of 18 yuan of ring macrolide antibiotics.It is a primary product in producing the bacterial strain that platform colludes mycin (Tiacumicins) that platform colludes mycin B (Tiacumicin B), it has one 18 yuan ring macrolides, two deoxidation glycosyls and an aromatic nucleus side chain, it is produced bacterial strain and mainly comprises, actinomycetes refer to sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085, actinoplanes Actinoplanes deccanensisATCC 21983 and little spore chain bacterium Catellatospora sp.Bp3323-81 etc.
Refer to that sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 is Micromonosporaceae (Micromonosporaceae) actinomycetes, can produce multiple and collude mould chlorins compound.
Summary of the invention:
The purpose of this invention is to provide six kinds of platforms and collude mould chlorins compound and preparation method thereof and the application in the preparation antibacterials.
The present invention is by the gene knockout sudden change to referring to that sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 carries out at Tiacumicin B oxygen methyltransgerase, obtain this oxygen methyl transferase gene deletion mutantion strain, from the culture of this mutant strain, be separated to six kinds of new platforms and colluded mould chlorins compound, and find that they are to streptococcus aureus Staphylococcus aureus ATCC 29213, bacillus thuringiensis Bacillus thuringensis, enterococcus faecalis Enterococcus faecalis ATCC 29212 has bacteriostatic activity preferably, can be used to prepare antibacterials, thereby realize purpose of the present invention.
Six kinds of platforms of the present invention collude mould chlorins compound, its structure as the formula (1):
Formula (1)
Compound 1:R wherein
1=Z, R
2=H, R
3=ET; Compound 2:R
1=Z, R
2=OH, R
3=ET; Compound 3:R
1=X, R
2=H, R
3=ET; Compound 4:R
1=Y, R
2=H, R
3=ET; Compound 5:R
1=Z, R
2=OH, R
3=Me; Compound 6:R
1=Z, R
2=Me, R
3=Et.
The present invention is to refer to that sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 is as original bacterium, tiaS5-oxygen methyl transferase gene implementation to this bacterium knocks out, and obtains tiaS5-oxygen methyl transferase gene knockout mutant strain (TCM70).Mutant strain tunning crude extract is adopted column chromatography chromatogram technology such as purification on normal-phase silica gel, reverse phase silica gel, gel and preparation thin-layer chromatography chromatographic technique etc., obtain compound 1-6 from mutant strain TCM70.
Compound 1-6 of the present invention separates to obtain from the fermented liquid of tiaS5-oxygen methyl transferase gene knockout mutant strain (TCM70).Further preferred in the fermentation culture process of mutant strain, add macroporous resin as sorbent material, enrichment adsorption compound 1-6 separates obtaining compound 1-6 then from this sorbent material.Compound 1-6 preferred manufacturing procedure is: macroporous resin is added in the substratum of tiaS5-oxygen methyl transferase gene knockout mutant strain TCM70, after fermentation culture, isolate the macroporous resin in the fermented liquid, through ethanol elution, ethanol extraction is after concentrating recovery ethanol, use ethyl acetate extraction, ethyl acetate layer gets the meta-bolites crude extract after concentrating, with this crude extract through silica gel column chromatography, with volume ratio from 100: 0～5: 1 chloroform-methanol as the eluent gradient elution, collecting the chloroform-methanol volume ratio is 100: 1-20: the cut that elutes under 1 gradient, again through gel filtration chromatography, with 1: 1 chloroform-methanol of volume ratio as the eluent wash-out, and then with the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution 180min of volume fraction from 10%-85%, obtain 6 cuts, what wherein get off from the acetonitrile/water gradient elution of 55%-60% with volume fraction is cut 1, what get off from the acetonitrile/water gradient elution of 60%-65% with volume fraction is cut 2, what get off from the acetonitrile/water gradient elution of 65%-70% with volume fraction is cut 3, what get off from the acetonitrile/water gradient elution of 70%-75% with volume fraction is cut 4, what get off from the acetonitrile/water gradient elution of 75%-80% with volume fraction is cut 5, and what get off from the acetonitrile/water gradient elution of 80%-85% with volume fraction is cut 6; Cut 1 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, with the acetonitrile/water gradient elution 60min of volume fraction from 10%-65%, the cut that the acetonitrile/water gradient elution of collected volume mark 60-65% gets off, the recycle silicon plastic column chromatography, with volume ratio is that 8: 1 chloroform-methanol is the eluent wash-out, collects cut, obtains compound 5 (26.5mg), cut 2 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution 160min of volume fraction from 10%-68%, the cut that the acetonitrile/water gradient elution of collected volume mark 65-68% gets off, again with the preparation thin-layer chromatography, with volume ratio is that 8: 1 chloroform-methanol launches as developping agent, collects the RF value and be 0.45 cut, obtains compound 2 (171.5mg).Cut 3 is that 8: 1 chloroform-methanol launches as developping agent through the preparation thin-layer chromatography with volume ratio, collects the RF value and be 0.5 cut, and after gel filtration chromatography as the eluent wash-out, obtains compound 3 (36.4mg) with 1: 1 chloroform-methanol of volume ratio.Cut 4 is through the preparation thin-layer chromatography, with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collecting the RF value and be 0.5 cut, after half preparative high-performance liquid chromatographic, is 65% acetonitrile/water wash-out with volume ratio, flow velocity 3ml/min, collecting retention time is the cut of 40.5min, after the preparation thin-layer chromatography, is that 10: 1 chloroform-methanol launches as developping agent with volume ratio, collection RF value is 0.55 cut, obtains compound 4 (7.8mg).Cut 5 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution 180min of volume fraction from 10%-75%, the cut that the acetonitrile/water gradient elution of collected volume mark 70-75% gets off, again with the preparation thin-layer chromatography, with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collects the RF value and be 0.6 cut, obtains compound 1 (285.0mg).Cut 6 is through the preparation thin-layer chromatography, with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collection RF value is 0.65 cut, after the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity are 15ml/min, with the acetonitrile/water gradient elution 60min of volume fraction from 10%-78%, the cut that the acetonitrile/water gradient elution of collected volume mark 75-78% gets off, use silica gel column chromatography then, the ethyl acetate-methyl alcohol that with volume ratio is 50: 1 obtains compound 6 (39.7mg) as the moving phase wash-out.
Table 1 is the LC-MS analytical results of compound 1-6:
Table 1: the LC-MS analytical results of compound 1-6
aThe HPLC condition: chromatographic column is phenomex 150 * 4.6mm (SphereClone SAX), moving phase comprise flow A mutually with mobile B mutually, mobile phase A phase: the trifluoroacetic acid of the acetonitrile of 10% (volume fraction)+0.08% (volume fraction), solvent is a water, the B phase flows: the acetonitrile of 90% (volume fraction), solvent are water; Sample introduction program: 0-20min, the moving phase ratio is A phase/B phase (volume ratio): 95: 5-0: 100,20-21min, the moving phase ratio is A phase/B phase (volume ratio): 0: 100, and 21-22min, the moving phase ratio is A phase/B phase (volume ratio): 0: 100-95: 5,22-30min, the moving phase ratio is A phase/B phase (volume ratio): 95: 5, detect wavelength 254nm, flow velocity 1ml/min.Retention time obtains with this understanding.
Nd
b: do not detect valid data.
Compound 1: white powder, C
51H
72Cl
2O
17,
1H NMR (500MHz, CD
3OD) δ 7.16 (d, J=12.0Hz, 1H), 6.53 (dd, J=12.0,14.0Hz, 1H), 5.81 (m, 1H), 5.75 (s, 1H), 5.44 (t, J=7.3Hz, 1H), 5.24 (t, J=9.8Hz, 1H), 5.00 (d, J=9.0Hz, 1H), 4.99 (d, J=9.8Hz, 1H), 4.81 (m, 1H), 4.64 (s, 1H), 4.61 (d, J=11.5Hz, 1H), 4.59 (s, 1H), 4.47 (d, J=11.5Hz, 1H), 4.26 (s, 1H), 4.03 (d, J=2.8Hz, 1H), 4.02 (d, J=3.0Hz, 1H), 3.70 (dd, J=2.8,9.8Hz, 1H), 3.66 (dd, J=3.0,9.8Hz, 1H), 3.64 (d, J=9.5Hz, 1H), 3.53 (m, 1H), 3.04 (m, 1H), 2.96 (m, 1H), 2.70 (m, 1H), 2.58 (pentet, J=7.0Hz, 1H); 2.70 (m, 1H), 2.46 (m, 2H), 2.28 (m, 1H), 1.83 (m, 2H), 1.78 (s, 3H), 1.69 (s, 3H), 1.66 (m, 1H), 1.62 (s, 3H), 1.31 (d, J=6.0Hz, 3H), 1.17 (m, 1H), 1.16 (s, 3H), 1.16 (t, J=7.3Hz, 3H), 1.16 (d, J=7.0Hz, 3H), 1.15 (d, J=7.0Hz, 3H), 1.09 (s, 3H), 0.94 (t, J=7.3Hz, 3H), 0.80 (t, J=7.3Hz, 3H);
13C NMR (125MHz, CD
3OD) δ 177.2s, 169.8s, 167.6s, 157.2s, 152.6s, 144.4d, 142.8s, 140.8d, 136.2s, 135.3s, 134.4s, 133.9d, 128.3d, 125.6d, 124.7s, 122.7d, 113.7s, 107.3s, 107.0s, 99.0d, 94.7d, 92.5d, 76.0d, 75.2d, 74.6d, 73.3s, 72.5d, 72.0d, 71.5d, 70.8d, 70.0d, 69.8d, 62.4t, 41.1d, 36.3t, 34.1d, 31.2t, 27.9q, 26.4t, 26.0t, 25.9t, 19.0q, 18.7q, 18.2q, 17.6d, 17.1q, 14.9q, 13.8q, 13.3q, 10.8q, 10.0q.In sum, the structure of knowing compound 1 by inference as the formula (1), R wherein
1=Z, R
2=H, R
3=ET, reference J Antibiot (Tokyo), 1987,40 (5): 575-88; J.Chem.Soc., Perkin Trans.1,1987,1353-1359.
Compound 2: white solid, C
51H
72Cl
2O
18,
1H NMR (500MHz, CDCl
3) δ 7.12 (d, J=11.8Hz, 1H), 6.55 (dd, J=11.8,14.0Hz, 1H), 5.84 (s, 1H), 5.79 (m, 1H), 5.44 (t, J=7.8Hz, 1H), 5.23 (t, J=9.3Hz, 1H), 4.98 (d, J=10.0Hz, 1H), 4.96 (d, J=11.5Hz, 1H), 4.68 (m, 1H), 4.65 (m, 1H), 4.63 (d, J=12.0Hz, 1H), 4.60 (s, 1H), 4.45 (d, J=12.0Hz, 1H), 4.24brs, 4.08 (dq, J=2.5,6.3Hz, 1H), 4.05 (d, J=2.3Hz, 1H), 4.00 (d, J=2.5Hz, 1H), 3.71 (dd, J=2.3,9.3Hz, 1H), 3.65 (dd, J=2.5,10.0Hz, 1H), 3.62 (d, J=9.5Hz, 1H), 3.56 (m, 1H), 3.05 (m, 1H), 2.98 (m, 1H), 2.77 (m, 1H), 2.68 (m, 2H), 2.59 (pentet, J=7.0Hz, 1H), 2.46 (m, 1H), 2.24 (m, 1H), 1.91 (s, 3H), 1.87 (m, 1H), 1.79 (s, 3H), 1.65 (s, 3H), 1.31 (d, J=6.0Hz, 3H), 1.19 (d, J=6.3Hz, 1H), 1.18 (m, 1H), 1.17 (t, J=6.0Hz, 1H), 1.17 (d, J=7.0Hz, 1H), 1.16 (d, J=7.0Hz, 1H), 1.13 (s, 3H), 1.08 (s, 3H), 0.80 (t, J=7.5Hz, 3H);
13C NMR (125MHz, CDCl
3) δ 177.3s, 169.8s, 168.7s, 157.1s, 152.6s, 144.6d, 142.8s, 141.0d, 136.6s, 136.1s, 134.3s, 134.2d, 128.9d, 127.8d, 124.7s, 123.1d, 113.8s, 107.4s, 107.1s, 99.5d, 94.6d, 92.4d, 79.3d, 76.0d, 74.7d, 73.3s, 72.6d, 71.9d, 71.5d, 70.8d, 70.0d, 69.8d, 68.6d, 62.8t, 41.6d, 36.7t, 34.2d, 28.1t, 28.0q, 26.0t, 25.6t, 19.0q, 18.7q, 18.3q, 18.3q, 17.6d, 16.9q, 15.3q, 13.9q, 13.6q, 10.9q.In sum, the structure of knowing compound 2 by inference as the formula (1), R wherein
1=Z, R
2=OH, R
3=ET, reference J Antibiot (Tokyo), 1987,40 (5): 575-88; J.Chem.Soc., Perkin Trans.1,1987,1353-1359.
Compound 3: white solid, C
49H
68Cl
2O
17,
1H NMR (500MHz, CDCl
3) δ 7.16 (d, J=11.8Hz, 1H), 6.54 (dd, J=11.8,14.0Hz, 1H), 5.81 (m, 1H), 5.75 (s, 1H), 5.44 (t, J=7.8Hz, 1H), 5.24 (t, J=9.5Hz, 1H), 5.01 (d, J=10.0Hz, 1H), 5.00 (d, J=9.8Hz, 1H), 4.82 (m, 1H), 4.64 (s, 1H), 4.60 (s, 1H), 4.60 (d, J=11.5Hz, 1H), 4.47 (d, J=11.5Hz, 1H), 4.26 (s, 1H), 4.05 (d, J=3.0Hz, 1H), 4.01 (d, J=3.0Hz, 1H), 3.71 (dd, J=3.0,9.5Hz, 1H), 3.66 (dd, J=3.0,9.8Hz, 1H), 3.64 (d, J=9.5Hz, 1H), 3.53 (m, 1H), 3.05 (m, 1H), 2.97 (m, 1H), 2.69 (m, 2H), 2.48 (m, 1H), 2.33 (m, 1H); 2.48 (m, 1H), 2.09 (s, 3H), 1.85 (m, 1H), 1.83 (m, 1H), 1.78 (s, 3H), 1.69 (s, 3H), 1.66 (m, 1H), 1.62 (s, 3H), 1.31 (d, J=6.0Hz, 3H), 1.23 (m, 1H), 1.18 (t, J=7.5Hz, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.94 (t, J=7.5Hz, 3H), 0.81 (t, J=7.3Hz, 3H);
13C NMR (125MHz, CDCl
3) δ 171.2s, 169.8s, 167.5s, 157.2s, 152.6s, 144.4d, 142.8s, 140.8d, 136.2s, 135.3s, 134.4s, 133.9d, 128.3d, 125.6d, 124.3s, 122.7d, 113.8s, 107.2s, 107.0s, 99.0d, 94.7d, 92.4d, 76.0d, 75.2d, 74.9d, 73.3s, 72.4d, 72.0d, 71.5d, 70.7d, 69.9d, 69.8d, 62.4t, 41.1d, 36.3t, 31.2t, 27.9q, 26.4t, 25.9t, 21.1q, 18.0q, 17.4q, 17.1q, 14.8q, 13.8q, 13.2q, 10.8q, 10.0q.In sum, the structure of knowing compound 3 by inference as the formula (1), R wherein
1=X, R
2=H, R
3=ET, reference J Antibiot (Tokyo), 1987,40 (5): 575-88; J.Chem.Soc., Perkin Trans.1,1987,1353-1359.
Compound 4: white solid, C
50H
70Cl
2O
17,
1H NMR (500MHz, CDCl
3) δ 7.18 (d, J=11.8Hz, 1H), 6.55 (dd, J=11.8,14.3Hz, 1H), 5.82 (m, 1H), 5.76 (s, 1H), 5.46 (t, J=7.8Hz, 1H), 5.25 (t, J=9.8Hz, 1H), 5.02 (d, J=9.0Hz, 1H), 5.02 (d, J=10.0Hz, 1H), 4.84 (m, 1H), 4.68 (s, 1H), 4.64 (d, J=11.5Hz, 1H), 4.63 (s, 1H), 4.48 (d, J=11.5Hz, 1H), 4.28 (s, 1H), 4.06 (d, J=3.2Hz, 1H), 4.02 (d, J=4.3Hz, 1H), 3.71 (dd, J=3.2,9.8Hz, 1H), 3.66 (d, J=9.5Hz, 1H), 3.63 (dd, J=4.3,9.0Hz, 1H), 3.55 (m, 1H), 3.09 (m, 1H), 3.01 (m, 1H), 2.67 (m, 2H), 2.51 (m, 1H), 2.36 (m, 1H); 2.51 (m, 1H), 2.36 (pentet, J=7.4Hz, 1H), 1.85 (m, 1H), 1.83 (m, 1H), 1.80 (s, 1H), 1.71 (s, 1H), 1.68 (m, 1H), 1.64 (s, 1H), 1.33 (d, J=6.0Hz, 3H), 1.25 (m, 1H), 1.25 (t, J=7.3Hz, 3H), 1.16 (s, 3H), 1.16 (t, J=7.4Hz, 3H), 1.10 (s, 3H), 0.96 (t, J=7.5Hz, 3H), 0.84 (t, J=7.5Hz, 3H);
13C NMR (125MHz, CDCl
3) δ 174.7s, 169.9s, 167.5s, 157.3s, 152.4s, 144.2d, 142.9s, 140.7d, 136.5s, 135.3s, 134.4s, 133.9d, 128.4d, 125.7d, 124.8s, 122.7d, 113.1s, 107.5s, 107.0s, 99.1d, 94.7d, 92.5d, 76.1d, 75.3d, 74.8d, 73.4d, 72.5d, 72.0d, 71.5d, 70.7d, 70.0d, 69.8d, 62.5t, 41.1d, 36.3t, 31.3t, 28.0q, 27.8t, 26.4t, 26.0t, 25.9t, 18.3q, 17.7q, 17.1q, 15.0q, 13.9q, 13.3q, 10.9q, 10.1q, 9.2q.In sum, the structure of knowing compound 4 by inference as the formula (1), R wherein
1=Y, R
2=H, R
3=ET, reference J Antibiot (Tokyo), 1987,40 (5): 575-88; J.Chem.Soc., Perkin Trans.1,1987,1353-1359.
Compound 5: white solid, C
50H
70Cl
2O
18,
1H NMR (500MHz, CDCl
3) δ 7.12 (d, J=12.0Hz, 1H), 6.56 (dd, J=12.0,14.0Hz, 1H), 5.85 (s, 1H), 5.80 (m, 1H), 5.45 (t, J=7.8Hz, 1H), 5.23 (t, J=9.5Hz, 1H), 4.99 (d, J=9.5Hz, 1H), 4.96 (d, J=8.5Hz, 1H), 4.66 (m, 1H), 4.64 (s, 1H), 4.63 (d, J=12.0Hz, 1H), 4.61 (s, 1H), 4.46 (d, J=12.0Hz, 1H), 4.23 (s, 1H), 4.10 (m, 1H), 4.06 (d, J=2.3Hz, 1H), 4.01 (d, J=3.0Hz, 1H), 3.76 (dd, J=2.3,9.5Hz, 1H), 3.66 (dd, J=3.0,9.5Hz, 1H), 3.62 (d, J=10.0Hz, 1H), 3.57 (m, 1H), 2.78 (m, 1H), 2.68 (m, 2H), 2.58 (pentet, J=7.0Hz, 1H), 2.54 (s, 1H), 2.49 (m, 1H), 2.24 (m, 1H), 1.91 (s, 3H), 1.88 (m, 1H), 1.79 (s, 3H), 1.65 (s, 3H), 1.30 (d, J=6.0Hz, 3H), 1.24 (m, 1H), 1.19 (d, J=6.5Hz, 3H), 1.17 (d, J=7.0Hz, 3H), 1.16 (d, J=7.0Hz, 3H), 1.13 (s, 3H), 1.08 (s, 3H), 0.80 (t, J=7.3Hz, 3H);
13C NMR (125MHz, CDCl
3) δ 177.3s, 170.3s, 168.7s, 157.5s, 152.7s, 144.6d, 141.0d, 137.5s, 136.6s, 136.2s, 134.3s, 134.2d, 128.9d, 127.8d, 124.7s, 123.1d, 114.2s, 107.3s, 106.8s, 99.4d, 94.6d, 92.4d, 79.3d, 76.0d, 74.7d, 73.3s, 72.6d, 72.0d, 71.5d, 70.8d, 70.1d, 69.9d, 68.6d, 62.8t, 41.7d, 36.7t, 34.2d, 29.7q, 28.0t, 25.7t, 19.7q, 19.1q, 18.8q, 18.3q, 18.3q, 17.6q, 16.9q, 15.3q, 13.6q, 10.9q.In sum, the structure of knowing compound 5 by inference as the formula (1), R wherein
1=Z, R
2=OH, R
3=Me, and reference J Antibiot (Tokyo Hz, 1H), 1987,40 (5): 575-88; J.Chem.Soc., Perkin Trans.1,1987,1353-1359.
Compound 6: white solid, C
52H
74Cl
2O
17,
1H NMR (500MHz, CDCl
3) δ 7.17 (d, J=11.5Hz, 1H), 6.54 (dd, J=11.5,14.0Hz, 1H), 5.81 (m, 1H), 5.76 (s, 1H), 5.42 (t, J=8.0Hz, 1H), 5.25 (t, J=9.5Hz, 1H), 5.00 (d, J=10.0Hz, 1H), 4.99 (d, J=9.5Hz, 1H), 4.64 (s, 1H), 4.63 (m, 1H), 4.62 (d, J=11.5Hz, 1H), 4.60 (s, 1H), 4.49 (d, J=11.5Hz, 1H), 4.26 (s, 1H), 4.04 (d, J=2.5Hz, 1H), 4.01 (d, J=2.5Hz, 1H), 3.70 (dd, J=2.5,9.5Hz, 1H), 3.66 (dd, J=2.5,9.5Hz, 1H), 3.65 (d, J=9.5Hz, 1H), 3.54 (m, 1H), 3.06 (m, 1H), 2.96 (m, 1H), 2.69 (m, 2H), 2.60 (pentet, J=7.0Hz, 1H), 2.47 (m, 1H), 2.40 (m, 1H), 2.08 (m, 1H), 1.85 (m, 1H), 1.78 (s, 3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.32 (d, J=5.5Hz, 3H), 1.22 (m, 1H), 1.17 (s, 3H), 1.17 (t, J=7.5Hz, 3H), 1.17 (d, J=7.0Hz, 3H), 1.17 (d, J=7.0Hz, 3H), 1.09 (s, 3H), 0.97 (d, J=6.5Hz, 3H), 0.95 (d, J=6.5Hz, 3H), 0.82 (t, J=7.5Hz, 3H);
13CNMR (125MHz, CDCl
3) δ 177.2s, 169.9s, 167.7s, 157.2s, 152.6s, 144.3d, 142.8s, 140.8d, 136.2s, 135.2s, 134.4s, 133.8d, 128.4d, 125.5d, 124.7s, 122.7d, 113.7s, 107.3s, 107.0s, 99.0d, 94.8d, 92.5d, 78.8d, 76.0d, 74.6d, 73.3s, 72.5d, 72.0d, 71.6d, 70.8d, 69.9d, 69.8d, 62.5t, 41.0d, 36.3t, 34.1d, 30.5d, 28.7t, 28.0q, 26.0t, 25.9t, 19.5q, 19.0q, 18.7q, 18.2q, 18.0q, 17.6q, 17.1q, 14.9q, 13.8q, 13.3q, 10.8q.In sum, the structure of knowing compound 6 by inference as the formula (1), R wherein
1=Z, R
2=Me, R
3=Et, reference JAntibiot (Tokyo), 1987,40 (5): 575-88; J.Chem.Soc., PerkinTrans.1,1987,1353-1359.
Compound 1-6 belongs to platform and colludes mould chlorins compound, is the new compound that platform colludes the mycin class.
With streptococcus aureus Staphylococcus aureus ATCC 29213, bacillus thuringiensis Bacillusthuringensis, 29,212 three kinds of bacteriums of enterococcus faecalis Enterococcus faecalis ATCC are as indicator, adopt doubling dilution to carry out the MIC pH-value determination pH, its result is as shown in table 2.
Table 2: each compound is to the minimal inhibitory concentration (μ g/ml) of three kinds of bacteriums
The clear and definite demonstration of result in the table 2, compound 1-6 all has bacteriostatic activity preferably, the MIC value is between 1-64 μ g/ml, colluding mycin B with the parent compound platform compares, compound 1 and 6 all has lower MIC value to enterococcus faecalis and streptococcus aureus, shows that they have better bacteriostatic activity, and particularly compound 6, its specific activity parent compound platform at streptococcus aureus colludes mycin B and has improved 8 times, and therefore being expected to research and development becomes antibiotic new drug.
The present invention has been separated to six kinds of new platforms and has colluded mould chlorins compound from the mutant strain that refers to sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085, enriched platform and colluded the mycin structure diversity.And find that these six kinds of compounds are to streptococcus aureus Staphylococcus aureus ATCC 29213, bacillus thuringiensis Bacillusthuringensis, enterococcus faecalis Enterococcus faecalis ATCC 29212 has and suppresses active, and being expected to be used to research and develop becomes new antibiotic new drug.
Be used for finger sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 of the present invention, openly be recorded in United States Patent (USP) in the past in the application, its patent No. is in the patent of US4918174.Record according to this patent documentation, refer to that sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensisNRRL 18085 is preserved in american agriculture research (the Agricultural Research Service Culture Collection of DSMZ, write a Chinese character in simplified form: NRRL), its accession number is NRRL 18085.
Description of drawings:
Fig. 1 is the peak position that goes out of the HPLC contrast figure of the standard substance platform secondary metabolite that colludes mycin B and mutant strain TCM70 and compound 1-6, the HPLC condition: chromatographic column is phenomex 150 * 4.6mm (SphereClone SAX), moving phase comprise flow A mutually with mobile B mutually, mobile phase A phase: the trifluoroacetic acid of the acetonitrile of 10% (volume fraction)+0.08% (volume fraction), solvent is a water, the B phase flows: the acetonitrile of 90% (volume fraction), solvent are water; Sample introduction program: 0-20min, the moving phase ratio is A phase/B phase (volume ratio): 95: 5-0: 100,20-21min, the moving phase ratio is A phase/B phase (volume ratio): 0: 100, and 21-22min, the moving phase ratio is A phase/B phase (volume ratio): 0: 100-95: 5,22-30min, the moving phase ratio is A phase/B phase (volume ratio): 95: 5, detect wavelength 254nm, flow velocity 1ml/min.Wherein 1 expression compound, 1,2 expression compound, 2,3 expression compounds, 3,4 expression compounds 4,5 are represented compounds 5,6 expression compounds 6.
Fig. 2 is the structure synoptic diagram of the mutant strain TCM70 that knocks out of tiaS5-oxygen methyl transferase gene, Fig. 2-A is that tiaS5-oxygen methyl transferase gene knocks out synoptic diagram: by the PCR-targetting technology, 810bpDNA fragment in the tiaS5 gene with including the 1369bp dna fragmentation displacement of shifting homing sequence oriT and A Baila mycin resistant gene acc3 (IV), is filtered out double exchange mutant strain TCM70; Shown position and the metathetical clip size and the PCR stripe size of primer among the figure, wherein the pcr amplification band of 1176bp comes from wild-type D.aurantiacum NRRL 18085, and the pcr amplification band of 1735bp comes from mutant strain TCM70.Fig. 2-B is the electrophoretic analysis of PCR product: dna profiling comes from respectively: mutant strain TCM70 (band 1); Distilled water (band 3), negative contrast; Wild-type D.aurantiacum NRRL 18085 (band 2); Dna molecular amount marker (band M).
Embodiment:
Below be to further specify to of the present invention, rather than limitation of the present invention.
Embodiment 1:tiaS5-oxygen methyl transferase gene knocks out the acquisition of mutant strain (TCM70)
Utilize the method for PCR-targeting to obtain external knockout mutant strain.Collude mycin synthetic gene bunch sequence according to the platform that obtains, the PCR-targeting system of reference literature report, design paired t iaS5 gene knock out primer
70PTs：5′-GCCGACGTACGCATCCACCAGGACCTCTCGGCATTCGCCattccggggatccgtcgacc-3′
70PTa：5′-CACCGGATAGTCGGGCATGAGATAGCGAGGCCCCTGGCG?tgtaggctggagctgcttc-3′
Wherein 5 end 39bp capitalization partial sequences of primer and platform collude mycin tiaS5 gene and are complementary, and the lowercase partial sequence of 3 ends respectively with plasmid pIJ773 in one comprise and shift consensus dna sequence or the complementation that homing sequence and A Bo draw the fragment both sides of resistant gene.Knocking out plasmid outward with reference to the method construct of PCR-targeting then is transferred to then in conjunction with in the donor bacterium that shifts.Concrete steps are as follows: (1) changes cosmid plasmid pCSG10 over to and obtains E.coli BW25113/pIJ790/pCSG10 among the intestinal bacteria E.coliBW25113/pIJ790, induce λ/red recombination system to express with the L-arabinose of 10mmol/L, and it is stand-by that its preparation is become electricity commentaries on classics competent cell.(2) with restriction endonuclease EcoR I and Hind III digested plasmid pIJ773, then reclaim about 1.4kb and contain the dna fragmentation that shifts initial point and apramycin resistant gene, as pcr template, go out the PCR product of 1.4kb, the PCR reaction system of 50 μ l: high-fidelity DNA polymerase 3U by pcr amplification with this with 70PTs and 70PTa primer, 10 * Buffer, 5 μ l, dNTPs 0.5mmol/L, DMSO 2.5 μ l, each 0.5 μ mol/L of primer, the about 1ng of dna profiling adds water to 50 μ l.The PCR reaction conditions is: pre-94 ℃ of 5min of sex change; Amplification cycles is 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, and 72 ℃ are extended 90s, 30 circulations; Last 72 ℃ are extended 10min.The PCR product recovery purifying of 1.4kb is stand-by.(3) changing PCR product electricity over to prepare in (1) step competent cell recombinates it, (contain 100 μ g/ml penbritins with the LB screening is dull and stereotyped, 50 μ g/ml kantlex, 50 μ g/ml apramycins) go up in 37 ℃ of incubated overnight, choose positive monoclonal from flat board, the extracting plasmid, called after pCSG70, the dna fragmentation of the 810bp of the tiaS5 gene in this plasmid is contained the transfer initial point by 1369bp and apramycin resistant gene fragment replaces.(4) the recombination mutation plasmid pCSG70 electricity that builds is forwarded among the E.coli ET12567/pUZ8002, be built into E.coliET12567/pUZ8002/pCSG70, as the donor bacterium of conjugal transfer.
Wild-type refers to that sporangiocyst bacterium Dactylosporangium aurantiacum subsp.Hamdenensis NRRL 18085 bacterial strains cultivated 3 days in 50ml YMS liquid nutrient medium, the centrifugal 10min of 4000r/min collects thalline, abandon supernatant, clean mycelium 3 times with same substratum, be suspended in the 4ml YMS substratum, as the recipient bacterium of conjugal transfer.Donor bacterium E.coliET12567/pUZ8002/pCSG70 contains 25 μ g/ml kantlex at 50ml, grows to OD in 37 ℃ in the LB liquid nutrient medium of 25 μ g/ml paraxin and 50 μ g/ml apramycins
600Value is about at 0.8 o'clock, and (4000r/min 10min), cleans thalline 3 times with LB to centrifugal collection thalline, is suspended in the 300 μ l LB substratum, as the donor bacterium of conjugal transfer.Above-mentioned recipient bacterium and donor bacterium are respectively got 100 μ l mix to coat and do not contain on any antibiotic 2CMY solid medium, after drying up, cultivate 16h in 30 ℃.Then will flat board take out the back and cover with the 3ml sterilized water, be coated with gently strike-off stick surface of rod with sterilization, sucking-off is wiped off contains bacteria liquid.After cleaning finishes, cover flat board with containing antibiotic water, its final concentration is 35 μ g/ml apramycins and 50 μ g/ml trimethoprims, after drying up, places 30 ℃ of incubators, cultivates after 7 days and observes.
After growing small colonies on the conjugal transfer flat board, with syringe needle it is transferred on the YMS rich medium flat board that contains 35 μ g/ml apramycins and 50 μ g/ml trimethoprims, cultivate after 3 days for 30 ℃, single mutant strain is inoculated into respectively contains in the same antibiotic 2.5ml YMS liquid nutrient medium, cultivated 5 days in 30 ℃.Extract the genomic dna of each mutant strain, utilize and detect primer
70s：5′-GGGAGAAGCTGGTCGTGCAG-3′
70a：5′-GCCGTCACTTGCTGCTTGTC-3′
Be diagnosis PCR and judge the screening-gene knockout mutant strain, can and only can amplify the segmental positive clone of 1735bp size by above-mentioned PCR primer amplification and (see Fig. 2-B), promptly obtain tiaS5-oxygen methyl transferase gene and knock out mutant strain (TCM70).
Embodiment 2: the fermentation of mutant strain TCM70 and the extraction of tunning
1, substratum:
(1) seed culture medium: contain yeast extract 4g in every liter of seed culture medium, Zulkovsky starch 4g, maltose 10g, CoCl
26H
2O 5mg, apramycin 35mg, surplus is a water, PH7.2.
(2) fermention medium: contain glucose 20g in every liter of fermention medium, fish meal 10g, yeast extract 2.5g, acid hydrolyzed casein 2.5g, K
2HPO
40.5g, MgSO
47H
2O 0.5g, KCl 1g, CaCO
33g, macroporous resin (XAD-16) 40g, surplus is a water, pH 7.0.
2, fermentation:
To refer to that sporangiocyst bacterium mutant strain TCM70 is inoculated in the 600ml seed culture medium, 50ml * 12 bottle, 28 ℃, 200rpm cultivates 3d, be inoculated in the 12L fermented liquid by 5% inoculum size then, 200ml * 60 bottle, 28 ℃, 200rpm cultivates 7d, obtains tunning.
3, the extraction of tunning:
The tunning that refers to sporangiocyst bacterium mutant strain TCM70 is centrifugal, isolate macroporous resin, macroporous resin 12L ethanol elution 4 times, with 4L ethyl acetate room temperature extraction 4 times, the ethyl acetate layer concentrating under reduced pressure gets the meta-bolites crude extract of mutant strain TCM70 behind the ethanol extraction decompression recycling ethanol.The meta-bolites crude extract detects through HPLC, and its result as shown in Figure 1.
Embodiment 3: the separation of compound 1-6
The meta-bolites crude extract of getting embodiment 2 middle finger sporangiocyst bacterium mutant strain TCM70 is through silicagel column (300-400mesh, 150g), with volume ratio from 100: 0-5: 1 chloroform-methanol gradient elution, the chloroform-methanol volume ratio is 100: 1-20: the cut of wash-out contains target compound 1-6 under 1 gradient, collect this cut, this cut is through gel sephadex LH-20 column chromatography chromatogram (30g), with volume ratio is that 1: 1 chloroform-methanol is as the moving phase wash-out, eluate is after concentrating, and then with the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution 180min of volume fraction from 10%-85%, obtains 6 cuts.
What get off from the acetonitrile/water gradient elution of 55%-60% with volume fraction is cut 1, through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, with the acetonitrile/water gradient elution 60min of volume fraction from 10%-65%, the cut that the acetonitrile/water gradient elution of collected volume mark 60～65% gets off, recycle silicon plastic column chromatography (300-400 order) is that 8: 1 chloroform-methanol is the eluent wash-out with volume ratio, collect cut, obtain compound 5 (26.5mg).
What get off from the acetonitrile/water gradient elution of 60%-65% with volume fraction is cut 2, through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution 160min of volume fraction from 10%-68%, the cut that the acetonitrile/water gradient elution of collected volume mark 65～68% gets off is again with preparation thin-layer chromatography (silica gel G, 20 * 20cm), with volume ratio is that 8: 1 chloroform-methanol launches as developping agent, collects the RF value and be 0.45 cut, obtains compound 2 (171.5mg).
What get off from the acetonitrile/water gradient elution of 65%-70% with volume fraction is cut 3, through preparation thin-layer chromatography (silica gel G, 20 * 20cm), with volume ratio is that 8: 1 chloroform-methanol launches as developping agent, collect the RF value and be 0.5 cut, and after gel filtration chromatography (Sephadex LH-20), with 1: 1 chloroform-methanol of volume ratio as the eluent wash-out, collect cut, obtain compound 3 (36.4mg).
What get off from the acetonitrile/water gradient elution of 70%-75% with volume fraction is cut 4, through preparation thin-layer chromatography (silica gel G, 20 * 20cm), with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collection RF value is 0.5 cut, after half preparative high-performance liquid chromatographic (YMC*GEL ODS-A, 120A S-5 μ m, 250 * 10mm), it with volume ratio 65% acetonitrile/water wash-out, flow velocity 3ml/min, collecting retention time is the cut of 40.5min, after preparation thin-layer chromatography (silica gel G, 20 * 20cm), with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collects the RF value and be 0.55 cut, obtains compound 4 (7.8mg).
What get off from the acetonitrile/water gradient elution of 75%-80% with volume fraction is cut 5, through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution 180min of volume fraction from 10%-75%, the cut that the acetonitrile/water gradient elution of collected volume mark 70～75% gets off is again with preparation thin-layer chromatography (silica gel G, 20 * 20cm), with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collects the RF value and be 0.6 cut, obtains compound 1 (285.0mg).
What get off from the acetonitrile/water gradient elution of 80%-85% with volume fraction is cut 6, through preparation thin-layer chromatography (silica gel G, 20 * 20cm), with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collection RF value is 0.65 cut, after the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity are 15ml/min, with the acetonitrile/water gradient elution 60min of volume fraction from 10%-78%, the cut that the acetonitrile/water gradient elution of collected volume mark 75～78% gets off is used silica gel column chromatography (300-400 order) then, and the ethyl acetate-methyl alcohol that with volume ratio is 50: 1 is as the moving phase wash-out, collect cut, obtain compound 6 (39.7mg).
Above-claimed cpd 1,2,3,4,5 and 6 is identified through structure, its structure as the formula (1), compound 1:R wherein
1=Z, R
2=H, R
3=ET; Compound 2:R
1=Z, R
2=OH, R
3=ET; Compound 3:R
1=X, R
2=H, R
3=ET; Compound 4:R
1=Y, R
2=H, R
3=ET; Compound 5:R
1=Z, R
2=OH, R
3=Me; Compound 6:R
1=Z, R
2=Me, R
3=Et.
Embodiment 4: the bacteriostatic activity of compound 1-6 is measured
Adopt streptococcus aureus Staphylococcus aureus ATCC 29213, bacillus thuringiensis Bacillusthuringensis, 29,212 three kinds of bacteriums of enterococcus faecalis Enterococcus faecalis ATCC carry out the MIC pH-value determination pH as indicator.
Strain golden look staphylococcus Staphylococcus aureus ATCC 29213 and bacillus thuringiensis Bacillus thuringensis bacterial strain are cultivated as indicator and carry out MIC mensuration in Mueller-Hinton (MH) broth cultures, and enterococcus faecalis Enterococcus faecalis ATCC 29212 bacterial strains are cultivated the heart leach liquor substratum of requiring mental skill (Brain heart infusion broth).The mensuration of MIC uses micro-broth dilution method (broth microdilution method) to measure the MIC (antimicrobial spectrum mensuration) of compound to each indicator with reference to the National Committee for Clinical Laboratory Standards method.
The preparation of antibacterials stock solution: each sample precision takes by weighing more than the 5mg, uses the DMSO through 0.22 μ m filtering with microporous membrane to make solvent, is configured to 5.12mg/ml.The antibacterials stock solution for preparing should be preserved in 4 ℃ of refrigerators.
MIC plate preparation: aseptic technique, the compound solution of different concns behind 2 doubling dilutions is added to respectively in the 96 aseptic hole transparent polystyrene microwell plates, the 1st to the 11st hole adds compound, and the 12nd not dosing of hole is as growth control.The final concentration that makes the 1st hole compound is 128 μ g/ml.
Inoculum preparation: prepare concentration with growth method and be equivalent to 5 * 10
8The bacteria suspension of/ml, after meat soup dilution in 1: 1000, it is 100 μ l that every hole joins final volume, makes the final concentration of the 1st hole to the 11 hole compounds be respectively 128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/ml.In the rearmounted 35 ℃ of incubators of sealing, hatch the 16-20h observations.
The result judges: to detect by an unaided eye, the lowest concentration of drug of bacteria growing inhibiting is MIC fully in aperture, the results are shown in Table 2.The demonstration compound 1-6 that its result is clear and definite has still kept antibiotic vigor, and the MIC value is between 1-64ug/ml.This shows that compound 1-6 has better bacteriostatic activity, being expected to research and development becomes antibiotic new drug.
Claims (4)
1. six kinds of platforms as the formula (1) collude mould chlorins compound:
Formula (1)
Compound 1:R wherein
1=Z, R
2=H, R
3=ET; Compound 2:R
1=Z, R
2=OH, R
3=ET; Compound 3:R
1=X, R
2=H, R
3=ET; Compound 4:R
1=Y, R
2=H, R
3=ET; Compound 5:R
1=Z, R
2=OH, R
3=Me; Compound 6:R
1=Z, R
2=Me, R
3=Et.
2. the preparation method of the described compound 1-6 of claim 1, it is characterized in that described compound 1-6 separates the fermenting culture of the tiaS5-oxygen methyl transferase gene knockout mutant strain TCM70 after the tiaS5-oxygen methyl transferase gene that refers to sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 is knocked out to obtain.
3. preparation method according to claim 2 is characterized in that, the preparation method of described compound 1-6 may further comprise the steps:
Macroporous resin is added in the substratum of tiaS5-oxygen methyl transferase gene knockout mutant strain TCM70, after fermentation culture, isolate the macroporous resin in the fermented liquid, through ethanol elution, ethanol extraction is after concentrating recovery ethanol, use ethyl acetate extraction, ethyl acetate layer gets the meta-bolites crude extract after concentrating, with this crude extract through silica gel column chromatography, with volume ratio from 100: 0～5: 1 chloroform-methanol as the eluent gradient elution, collecting the chloroform-methanol volume ratio is 100: 1-20: the cut that elutes under 1 gradient, again through gel filtration chromatography, with 1: 1 chloroform-methanol of volume ratio as the eluent wash-out, and then with the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution of volume fraction from 10%-85%, what wherein get off from the acetonitrile/water gradient elution of 55%-60% with volume fraction is cut 1, what get off from the acetonitrile/water gradient elution of 60%-65% with volume fraction is cut 2, what get off from the acetonitrile/water gradient elution of 65%-70% with volume fraction is cut 3, what get off from the acetonitrile/water gradient elution of 70%-75% with volume fraction is cut 4, what get off from the acetonitrile/water gradient elution of 75%-80% with volume fraction is cut 5, and what get off from the acetonitrile/water gradient elution of 80%-85% with volume fraction is cut 6;
Cut 1 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, 120A aperture, 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, with the acetonitrile/water gradient elution of volume fraction from 10%-65%, the cut that the acetonitrile/water gradient elution of collected volume mark 60-65% gets off, the recycle silicon plastic column chromatography, with volume ratio is that 8: 1 chloroform-methanol is the eluent wash-out, collects cut, obtains compound 5; Cut 2 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution of volume fraction from 10%-68%, the cut that the acetonitrile/water gradient elution of collected volume mark 65-68% gets off, again with the preparation thin-layer chromatography, with volume ratio is that 8: 1 chloroform-methanol launches as developping agent, collects the RF value and be 0.45 cut, obtains compound 2; Cut 3 is that 8: 1 chloroform-methanol launches as developping agent through the preparation thin-layer chromatography with volume ratio, collects the RF value and be 0.5 cut, and after gel filtration chromatography as the eluent wash-out, obtains compound 3 with 1: 1 chloroform-methanol of volume ratio; Cut 4 is through the preparation thin-layer chromatography, with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collecting the RF value and be 0.5 cut, after half preparative high-performance liquid chromatographic, is 65% acetonitrile/water wash-out with volume ratio, flow velocity 3ml/min, collecting retention time is the cut of 40.5min, after the preparation thin-layer chromatography, is that 10: 1 chloroform-methanol launches as developping agent with volume ratio, collection RF value is 0.55 cut, obtains compound 4; Cut 5 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, with the acetonitrile/water gradient elution of volume fraction from 10%-75%, the cut that the acetonitrile/water gradient elution of collected volume mark 70-75% gets off, again with the preparation thin-layer chromatography, with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collects the RF value and be 0.6 cut, obtains compound 1; Cut 6 is through the preparation thin-layer chromatography, with volume ratio is that 10: 1 chloroform-methanol launches as developping agent, collection RF value is 0.65 cut, after the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-type filler, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity are 15ml/min, with the acetonitrile/water gradient elution of volume fraction from 10%-78%, the cut that the acetonitrile/water gradient elution of collected volume mark 75-78% gets off, use silica gel column chromatography then, the ethyl acetate-methyl alcohol that with volume ratio is 50: 1 obtains compound 6 as the moving phase wash-out.
4. the described six kinds of platforms of claim 1 collude the application of mould chlorins compound in the preparation antibacterials.
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| CN103275152A (en) * | 2013-05-29 | 2013-09-04 | 华北制药集团新药研究开发有限责任公司 | Preparation method of high-purity fidaxomicin |
| WO2014111254A1 (en) | 2013-01-15 | 2014-07-24 | Astellas Pharma Europe Ltd | Composition of tiacumicin compounds |
| CN104418925A (en) * | 2013-09-05 | 2015-03-18 | 重庆乾泰生物医药有限公司 | Method for preparing high-purity fidaxomicin |
| CN104513286A (en) * | 2013-09-27 | 2015-04-15 | 博瑞生物医药技术(苏州)有限公司 | Method for separating and purifying fidaxomicin |
| WO2015169451A1 (en) | 2014-05-09 | 2015-11-12 | Astellas Pharma Europe Ltd | Treatment regimen tiacumicin compound |
| CN107236686B (en) * | 2017-06-21 | 2019-10-15 | 杭州华东医药集团新药研究院有限公司 | A kind of cyst fungus citrus finger and its application in regulating the microbial metabolite fidaxomicin |
| CN117024611A (en) * | 2023-03-01 | 2023-11-10 | 中国科学院南海海洋研究所 | Construction and activity application of oligosaccharide antibiotic everninomicin high-yield strain |
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| CN102115757B (en) * | 2010-12-14 | 2015-10-28 | 中国科学院南海海洋研究所 | Platform hooks biological synthesis gene cluster and the application thereof of mycin |
| CN103160521A (en) * | 2011-12-15 | 2013-06-19 | 上海来益生物药物研究开发中心有限责任公司 | Biosynthesis gene cluster of arylomycins A |
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| CN106916834B (en) * | 2015-12-24 | 2022-08-05 | 武汉合生科技有限公司 | Biosynthetic gene cluster of compounds and application thereof |
| CN106916835B (en) * | 2015-12-24 | 2022-08-12 | 武汉合生科技有限公司 | Biosynthetic gene cluster of compounds and application thereof |
| CN107868789B (en) * | 2015-12-31 | 2020-11-17 | 沈阳福洋医药科技有限公司 | Colimycin biosynthesis gene cluster |
| EP3607952B1 (en) | 2017-04-06 | 2022-07-06 | Shenyang Fuyang Pharmaceutical Technology Co., Ltd. | Use of carrimycin or pharmaceutically acceptable salt thereof for manufacture of medicament for treatment and/or prevention of tumors |
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| CN100519757C (en) * | 2002-07-29 | 2009-07-29 | 浩鼎生技公司 | Tiacumicin production |
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| CN102993251A (en) * | 2012-12-17 | 2013-03-27 | 苏州纳微生物科技有限公司 | Method for purifying tiacumicin B by high performance liquid chromatography |
| CN102993251B (en) * | 2012-12-17 | 2016-03-16 | 苏州纳微科技有限公司 | A kind of method of high-efficient liquid phase chromatogram purification TCM B |
| WO2014111254A1 (en) | 2013-01-15 | 2014-07-24 | Astellas Pharma Europe Ltd | Composition of tiacumicin compounds |
| CN103275152B (en) * | 2013-05-29 | 2015-11-18 | 华北制药集团新药研究开发有限责任公司 | A kind of preparation method of high-purity fidaxomicin |
| CN103275152A (en) * | 2013-05-29 | 2013-09-04 | 华北制药集团新药研究开发有限责任公司 | Preparation method of high-purity fidaxomicin |
| CN104418925A (en) * | 2013-09-05 | 2015-03-18 | 重庆乾泰生物医药有限公司 | Method for preparing high-purity fidaxomicin |
| CN104418925B (en) * | 2013-09-05 | 2018-09-28 | 重庆乾泰生物医药有限公司 | A method of preparing high-purity fidaxomicin |
| CN104513286A (en) * | 2013-09-27 | 2015-04-15 | 博瑞生物医药技术(苏州)有限公司 | Method for separating and purifying fidaxomicin |
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| WO2015169451A1 (en) | 2014-05-09 | 2015-11-12 | Astellas Pharma Europe Ltd | Treatment regimen tiacumicin compound |
| US10137141B2 (en) | 2014-05-09 | 2018-11-27 | Astellas Pharma Europe Ltd. | Treatment regimen tiacumicin compound |
| CN107236686B (en) * | 2017-06-21 | 2019-10-15 | 杭州华东医药集团新药研究院有限公司 | A kind of cyst fungus citrus finger and its application in regulating the microbial metabolite fidaxomicin |
| CN117024611A (en) * | 2023-03-01 | 2023-11-10 | 中国科学院南海海洋研究所 | Construction and activity application of oligosaccharide antibiotic everninomicin high-yield strain |
| CN117024611B (en) * | 2023-03-01 | 2024-05-17 | 中国科学院南海海洋研究所 | Construction of a high-yield strain of oligosaccharide antibiotic everninomicin and its application |
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|---|---|
| CN102115757A (en) | 2011-07-06 |
| CN102219815B (en) | 2013-12-18 |
| CN102115757B (en) | 2015-10-28 |
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