CN102219815B - 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
The invention discloses six tiacumicins compounds, a preparation method and a use of the six tiacumicins compounds in preparing antibacterial drugs. With the present invention, dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 is adopted as original bacteria; tiaS5-O-methyl transferase gene of the dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 is knocked out to obtain a tiaS5-O-methyl transferase gene knockout mutant strain (TCM 70); a plurality of column chromatography technologies such as positive silica gel, reverse phase silica gel, gel and the like, preparation of thin-layer chromatography technique and the like are adopted for crude extracts of the mutant strain fermentation products to obtain the compounds 1-6 from the mutant strain TCM 70. The six tiacumicins compounds provide good inhibitory activity against staphylococcus aureus, bacillus thuringiensis, enterococcus faecalis and the like. Compared to a parent compound of the tiacumicin B, the compound 1 and the compound 6 provide a lower MIC value against the enterococcus faecalis and the staphylococcus aureus, showing that the compound 1 and the compound 6 provide the better inhibitory activity, in particular the inhibitory activity of the compound 6 against the staphylococcus aureus is raised by 8 times than the parent compound of the tiacumicin B. Therefore, the compound 6 is expected to be a new antibacterial drug through research and development.
Description
Technical field:
The present invention relates to six kinds of platforms and hook 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.It is the general name of a series of 18 ring macrolide antibiotics that platform hooks mycin (Tiacumicins).TCM B (Tiacumicin B) is a primary product in the bacterial strain of producing platform hook mycin (Tiacumicins), it has 18 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 hook mould chlorins compound.
Summary of the invention:
The purpose of this invention is to provide six kinds of platforms and hook 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 for Tiacumicin B oxygen methyltransgerase, obtain this oxygen methyl transferase gene deletion mutantion strain, be separated to six kinds of new platforms and hooked mould chlorins compound from the culture of this mutant strain, 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 for the preparation of antibacterials, thereby realized purpose of the present invention.
Six kinds of platforms of the present invention hook mould chlorins compound, and its structure is suc as formula shown in (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 usingd finger sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 as original bacterium, the tiaS5-oxygen methyl transferase gene of this bacterium is carried out and knocked out, obtain tiaS5-oxygen methyl transferase gene knockout mutant strain (TCM70).Mutant strain tunning crude extract is adopted to the column chromatography chromatogram technology such as purification on normal-phase silica gel, reverse phase silica gel, gel and Preparative TLC thin layer chromatography technology etc., from mutant strain TCM70, obtain compound 1-6.
Compound 1-6 of the present invention separates and obtains from the fermented liquid of tiaS5-oxygen methyl transferase gene knockout mutant strain (TCM70).Further preferably, in the fermentation culture process of mutant strain, add macroporous resin as sorbent material, enrichment adsorption compound 1-6 then separates and obtains compound 1-6 from this sorbent material.The preferred preparation method of compound 1-6 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 fermented liquid, through ethanol elution, ethanol extraction is after concentration and recovery ethanol, be extracted with ethyl acetate, ethyl acetate layer obtains the meta-bolites crude extract after concentrated, by this crude extract through silica gel column chromatography, using volume ratio from the chloroform-methanol of 100: 0～5: 1 as the eluent gradient elution, collecting the chloroform-methanol volume ratio is 100: 1-20: the cut eluted under 1 gradient, again through gel filtration chromatography, using the volume ratio chloroform-methanol of 1: 1 as the eluent wash-out, and then with the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, acetonitrile/water gradient elution 180min with volume fraction from 10%-85%, obtain 6 cuts, what the volume fraction of wherein take was got off from the acetonitrile/water gradient elution of 55%-60% is cut 1, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 60%-65% is cut 2, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 65%-70% is cut 3, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 70%-75% is cut 4, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 75%-80% is cut 5, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 80%-85% is cut 6, cut 1 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, acetonitrile/water gradient elution 60min with 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, the chloroform-methanol that the volume ratio of take is 8: 1 is the eluent wash-out, collect cut, obtain compound 5 (26.5mg), cut 2 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, acetonitrile/water gradient elution 160min with volume fraction from 10%-68%, the cut that the acetonitrile/water gradient elution of collected volume mark 65-68% gets off, use again the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 8: 1 launches as developping agent, collect the cut that the RF value is 0.45, obtain compound 2 (171.5mg).Cut 3 is through the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 8: 1 launches as developping agent, collects the cut that the RF value is 0.5, and by gel filtration chromatography, using the volume ratio chloroform-methanol of 1: 1 as the eluent wash-out, obtain compound 3 (36.4mg).Cut 4 is through the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collect the cut that the RF value is 0.5, by half preparative high-performance liquid chromatographic, the acetonitrile/water wash-out that the volume ratio of take is 65%, flow velocity 3ml/min, the cut that the collection retention time is 40.5min, finally by the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collect the cut that the RF value is 0.55, obtain compound 4 (7.8mg).Cut 5 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, the acetonitrile/water gradient elution 180min with volume fraction from 10%-75%, the cut that the acetonitrile/water gradient elution of collected volume mark 70-75% gets off, use again the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collects the cut that the RF value is 0.6, obtains compound 1 (285.0mg).Cut 6 is through the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collect the cut that the RF value is 0.65, by the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, acetonitrile/water gradient elution 60min with volume fraction from 10%-78%, the cut that the acetonitrile/water gradient elution of collected volume mark 75-78% gets off, then use silica gel column chromatography, ethyl acetate-methyl alcohol that the volume ratio of usining is 50: 1 is as the moving phase wash-out, obtain compound 6 (39.7mg).
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 comprises flow A phase and mobile B phase, mobile phase A phase: the trifluoroacetic acid of the acetonitrile of 10% (volume fraction)+0.08% (volume fraction), solvent is water, the B phase flows: the acetonitrile of 90% (volume fraction), and solvent is water; Sample introduction program: 0-20min, mobile phase ratio is A phase/B phase (volume ratio): 95: 5-0: 100,20-21min, mobile phase ratio is A phase/B phase (volume ratio): 0: 100, and 21-22min, mobile phase ratio is A phase/B phase (volume ratio): 0: 100-95: 5,22-30min, mobile 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: valid data do not detected.
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, know the structure of compound 1 by inference suc as formula shown in (1), wherein R
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, know the structure of compound 2 by inference suc as formula shown in (1), wherein R
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, know the structure of compound 3 by inference suc as formula shown in (1), wherein R
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, know the structure of compound 4 by inference suc as formula shown in (1), wherein R
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, know the structure of compound 5 by inference suc as formula shown in (1), wherein R
1=Z, R
2=OH, R
3=Me, 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, know the structure of compound 6 by inference suc as formula shown in (1), wherein R
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 hooks mould chlorins compound, is the new compound that platform hooks 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: the minimal inhibitory concentration (μ g/ml) of each compound to three kinds of bacteriums
The clear and definite demonstration of result in table 2, compound 1-6 all has bacteriostatic activity preferably, the MIC value is between 1-64 μ g/ml, with the parent compound TCM B, compare, 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 parent compound of specific activity for streptococcus aureus TCM B 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 hooked mould chlorins compound from the mutant strain that refers to sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085, has enriched platform and has hooked 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 the activity of inhibition, and being expected to becomes new antibiotic new drug for research and development.
For finger sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL 18085 of the present invention, in the application, openly be recorded in the past United States Patent (USP), in the patent that its patent No. is 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.
The accompanying drawing explanation:
Fig. 1 is that the standard substance TCM B contrasts the peak position that goes out of figure and compound 1-6 with the HPLC of the secondary metabolite of mutant strain TCM70, the HPLC condition: chromatographic column is phenomex 150 * 4.6mm (SphereClone SAX), moving phase comprises flow A phase and mobile B phase, mobile phase A phase: the trifluoroacetic acid of the acetonitrile of 10% (volume fraction)+0.08% (volume fraction), solvent is water, the B phase flows: the acetonitrile of 90% (volume fraction), and solvent is water; Sample introduction program: 0-20min, mobile phase ratio is A phase/B phase (volume ratio): 95: 5-0: 100,20-21min, mobile phase ratio is A phase/B phase (volume ratio): 0: 100, and 21-22min, mobile phase ratio is A phase/B phase (volume ratio): 0: 100-95: 5,22-30min, mobile phase ratio is A phase/B phase (volume ratio): 95: 5, detect wavelength 254nm, flow velocity 1ml/min.Wherein 1 means that compound 1,2 means that compound 2,3 means that compound 3,4 means that compound 4,5 means that compound 5,6 means compound 6.
Fig. 2 is the structure schematic 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 schematic 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 to double exchange mutant strain TCM70; Shown the position of primer and clip size and the PCR stripe size of displacement in 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 to further illustrate 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.Hook mycin synthetic gene bunch sequence according to the platform obtained, 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 hook 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.Then knocking out plasmid outward with reference to the method construct of PCR-targeting then is transferred in conjunction with in the donor bacterium shifted.Concrete steps are as follows: (1) proceeds to cosmid plasmid pCSG10 in intestinal bacteria E.coliBW25113/pIJ790 and obtains E.coli BW25113/pIJ790/pCSG10, with the L-arabinose of 10mmol/L, induce λ/red recombination system to express, and its preparation is become to electricity, and to turn competent cell stand-by.(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, using this as pcr template, with 70PTs and 70PTa primer, go out the PCR product of 1.4kb, the PCR reaction system of 50 μ l: high-fidelity DNA polymerase 3U by pcr amplification, 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, add water to 50 μ l.The PCR reaction conditions is: 94 ℃ of 5min of denaturation; 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 of 1.4kb is reclaimed to purifying stand-by.(3) PCR product electricity being proceeded to the competent cell prepared in (1) step recombinates it, dull and stereotyped (containing 100 μ g/ml penbritins with the LB screening, 50 μ g/ml kantlex, 50 μ g/ml apramycins) upper 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 built is forwarded in 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 cultivate 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 4ml YMS substratum, as the recipient bacterium of conjugal transfer.Donor bacterium E.coliET12567/pUZ8002/pCSG70 containing 25 μ g/ml kantlex, in 37 ℃ grows to OD in the LB liquid nutrient medium of 25 μ g/ml paraxin and 50 μ g/ml apramycins at 50ml
600value is about at 0.8 o'clock, and centrifugal collection thalline (4000r/min, 10min) cleans thalline 3 times with LB, is suspended in 300 μ l LB substratum, as the donor bacterium of conjugal transfer.Above-mentioned recipient bacterium and donor bacterium are respectively got to 100 μ l and mix and coat not containing on any antibiotic 2CMY solid medium, after drying up, in 30 ℃, cultivate 16h.Then cover with the 3ml sterilized water after flat board being taken out, with sterilizing, be coated with the light strike-off stick of rod surface, sucking-off wipe off containing bacteria liquid.After cleaning, with containing antibiotic water, cover flat board, its final concentration is 35 μ g/ml apramycins and 50 μ g/ml trimethoprims, after drying up, is placed in 30 ℃ of incubators, cultivates and observes afterwards in 7 days.
Grow small colonies on the conjugal transfer flat board after, 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 containing in same antibiotic 2.5ml YMS liquid nutrient medium, cultivates 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 positive clone (seeing Fig. 2-B) of 1735bp size fragment by above-mentioned PCR primer amplification, 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 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 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, cultivate 3d, then by 5% inoculum size, be inoculated in the 12L fermented liquid, 200ml * 60 bottle, 28 ℃, 200rpm, cultivate 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, after the ethanol extraction decompression recycling ethanol, with 4L ethyl acetate room temperature extraction 4 times, the ethyl acetate layer concentrating under reduced pressure obtains the meta-bolites crude extract of mutant strain TCM70.The meta-bolites crude extract detects through HPLC, and its result as shown in Figure 1.
Embodiment 3: the separation of compound 1-6
Get the meta-bolites crude extract of embodiment 2 middle finger sporangiocyst bacterium mutant strain TCM70 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: under 1 gradient, the cut of wash-out contains target compound 1-6, collect this cut, this cut is through gel sephadex LH-20 column chromatography chromatogram (30g), by volume ratio, be that 1: 1 chloroform-methanol is as the moving phase wash-out, eluate is after concentrated, and then with the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, acetonitrile/water gradient elution 180min with volume fraction from 10%-85%, obtain 6 cuts.
What the volume fraction of take was got off from the acetonitrile/water gradient elution of 55%-60% is cut 1, through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, acetonitrile/water gradient elution 60min with 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), the chloroform-methanol that the volume ratio of take is 8: 1 is the eluent wash-out, collect cut, obtain compound 5 (26.5mg).
What the volume fraction of take was got off from the acetonitrile/water gradient elution of 60%-65% is cut 2, through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, acetonitrile/water gradient elution 160min with volume fraction from 10%-68%, the cut that the acetonitrile/water gradient elution of collected volume mark 65～68% gets off, use again Preparative TLC chromatography (silica gel G, 20 * 20cm), the chloroform-methanol that the volume ratio of usining is 8: 1 launches as developping agent, collect the cut that the RF value is 0.45, obtain compound 2 (171.5mg).
What the volume fraction of take was got off from the acetonitrile/water gradient elution of 65%-70% is cut 3, through Preparative TLC chromatography (silica gel G, 20 * 20cm), the chloroform-methanol that the volume ratio of usining is 8: 1 launches as developping agent, collect the cut that the RF value is 0.5, and, by gel filtration chromatography (Sephadex LH-20), using the volume ratio chloroform-methanol of 1: 1 as the eluent wash-out, collect cut, obtain compound 3 (36.4mg).
What the volume fraction of take was got off from the acetonitrile/water gradient elution of 70%-75% is cut 4, through Preparative TLC chromatography (silica gel G, 20 * 20cm), the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collect the cut that the RF value is 0.5, by half preparative high-performance liquid chromatographic (YMC*GEL ODS-A, 120A S-5 μ m, 250 * 10mm), the acetonitrile/water wash-out that the volume ratio of take is 65%, flow velocity 3ml/min, the cut that the collection retention time is 40.5min, finally by Preparative TLC chromatography (silica gel G, 20 * 20cm), the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collect the cut that the RF value is 0.55, obtain compound 4 (7.8mg).
What the volume fraction of take was got off from the acetonitrile/water gradient elution of 75%-80% is cut 5, through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, acetonitrile/water gradient elution 180min with volume fraction from 10%-75%, the cut that the acetonitrile/water gradient elution of collected volume mark 70～75% gets off, use again Preparative TLC chromatography (silica gel G, 20 * 20cm), the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collect the cut that the RF value is 0.6, obtain compound 1 (285.0mg).
What the volume fraction of take was got off from the acetonitrile/water gradient elution of 80%-85% is cut 6, through Preparative TLC chromatography (silica gel G, 20 * 20cm), the chloroform-methanol that the volume ratio of usining is 10: 1 launches as developping agent, collect the cut that the RF value is 0.65, by the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, acetonitrile/water gradient elution 60min with volume fraction from 10%-78%, the cut that the acetonitrile/water gradient elution of collected volume mark 75～78% gets off, then use silica gel column chromatography (300-400 order), ethyl acetate-methyl alcohol that the volume ratio of usining 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, through Structural Identification, its structure is suc as formula shown in (1), wherein compound 1:R
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 Antibacterial Activity of compound 1-6
Adopt streptococcus aureus Staphylococcus aureus ATCC 29213, bacillus thuringiensis Bacillusthuringensis, 29,212 three kinds of bacteriums of enterococcus faecalis Enterococcus faecalis ATCC, as indicator, carry out the MIC pH-value determination pH.
Strain golden look staphylococcus Staphylococcus aureus ATCC 29213 and bacillus thuringiensis Bacillus thuringensis bacterial strain are cultivated and carry out MIC mensuration in Mueller-Hinton (MH) broth cultures as indicator, and the enterococcus faecalis Enterococcus faecalis ATCC 29212 bacterial strains heart leach liquor substratum (Brain heart infusion broth) that makes to require mental skill is cultivated.The mensuration of MIC, with reference to the National Committee for Clinical Laboratory Standards method, is used micro-broth dilution method (broth microdilution method) to measure the MIC (antimicrobial spectrum mensuration) of compound to each indicator.
Antibacterials stock solution preparation: more than each sample precision takes 5mg, use the DMSO through 0.22 μ m filtering with microporous membrane to make solvent, be configured to 5.12mg/ml.The antibacterials stock solution prepared should be preserved in 4 ℃ of refrigerators.
The preparation of MIC plate: aseptic technique, the compound solution of different concns after 2 doubling dilutions is added to respectively in 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 judgement: to detect by an unaided eye, in aperture, the lowest concentration of drug of bacteria growing inhibiting is MIC fully, the results are shown in Table 2.The demonstration compound 1-6 that its result is clear and definite has still retained antibacterial activity, and the MIC value is between 1-64ug/ml.Compound 1-6 has better bacteriostatic activity as can be seen here, and being expected to research and development becomes antibiotic new drug.
Claims (2)
1. six kinds of platforms as the formula (1) hook the preparation method of mould chlorins compound, it is characterized in that:
In the substratum of tiaS5-oxygen methyl transferase gene knockout mutant strain TCM70 after adding tiaS5-oxygen methyl transferase gene to referring to sporangiocyst bacterium Dactylosporangium aurantiacum subsp.hamdenensis NRRL18085 to be knocked out macroporous resin, after fermentation culture, isolate the macroporous resin in fermented liquid, through ethanol elution, ethanol extraction is after concentration and recovery ethanol, be extracted with ethyl acetate, ethyl acetate layer obtains the meta-bolites crude extract after concentrated, by this crude extract through silica gel column chromatography, using volume ratio from the chloroform-methanol of 100:0～5:1 as the eluent gradient elution, collecting the chloroform-methanol volume ratio is the cut eluted under the 100:1-20:1 gradient, again through gel filtration chromatography, using the chloroform-methanol of volume ratio 1:1 as the eluent wash-out, and then with the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, acetonitrile/water gradient elution with volume fraction from 10%-85%, what the volume fraction of wherein take was got off from the acetonitrile/water gradient elution of 55%-60% is cut 1, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 60%-65% is cut 2, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 65%-70% is cut 3, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 70%-75% is cut 4, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 75%-80% is cut 5, what the volume fraction of take was got off from the acetonitrile/water gradient elution of 80%-85% is cut 6,
Cut 1 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, 120A aperture, 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, the acetonitrile/water gradient elution with 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, the chloroform-methanol that the volume ratio of take is 8:1 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-shaped filling material, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, the acetonitrile/water gradient elution with volume fraction from 10%-68%, the cut that the acetonitrile/water gradient elution of collected volume mark 65-68% gets off, use again the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 8:1 launches as developping agent, collects the cut that the RF value is 0.45, obtains compound 2; Cut 3 is through the Preparative TLC chromatography, and the chloroform-methanol that the volume ratio of usining is 8:1 launches as developping agent, collects the cut that the RF value is 0.5, and by gel filtration chromatography, usings the chloroform-methanol of volume ratio 1:1 as the eluent wash-out, obtains compound 3; Cut 4 is through the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10:1 launches as developping agent, collect the cut that the RF value is 0.5, by half preparative high-performance liquid chromatographic, the acetonitrile/water wash-out that the volume ratio of take is 65%, flow velocity 3ml/min, the cut that the collection retention time is 40.5min, finally by the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10:1 launches as developping agent, collect the cut that the RF value is 0.55, obtain compound 4; Cut 5 is through the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, 120A aperture, 50um particle diameter, 40 * 2.5cm I.D., flow velocity is 20ml/min, the acetonitrile/water gradient elution with volume fraction from 10%-75%, the cut that the acetonitrile/water gradient elution of collected volume mark 70-75% gets off, use again the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10:1 launches as developping agent, collects the cut that the RF value is 0.6, obtains compound 1; Cut 6 is through the Preparative TLC chromatography, the chloroform-methanol that the volume ratio of usining is 10:1 launches as developping agent, collect the cut that the RF value is 0.65, by the anti-phase medium pressure liquid chromatography of ODS, YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 20 * 2.5cm I.D., flow velocity is 15ml/min, the acetonitrile/water gradient elution with volume fraction from 10%-78%, the cut that the acetonitrile/water gradient elution of collected volume mark 75-78% gets off, then use silica gel column chromatography, ethyl acetate-methyl alcohol that the volume ratio of usining is 50:1, as the moving phase wash-out, obtains compound 6;
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. in claim 1, six kinds of platforms as the formula (1) hook the application of mould chlorins compound in the preparation antibacterials.
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| 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 |
| JO3464B1 (en) | 2013-01-15 | 2020-07-05 | Astellas Pharma Europe Ltd | Compositions of Tiacumicin Compounds |
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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 |
| CN105505954B (en) * | 2015-12-31 | 2019-01-22 | 沈阳福洋医药科技有限公司 | Rokitamycin biological synthesis gene cluster |
| RU2746047C1 (en) | 2017-04-06 | 2021-04-06 | Шенянг Фуянг Фармасьютекал Технолоджи Ко., Лтд. | The use of carrimycin and its pharmaceutically acceptable salts for the production of medicines for treatment and / or tumor prevention |
| 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 dactylosporangium aurantiacum and its application in regulating microorganism metabolism object feldamycin |
| CN117024611B (en) * | 2023-03-01 | 2024-05-17 | 中国科学院南海海洋研究所 | Construction and activity application of oligosaccharide antibiotics everninomicin high-yield strain |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4918174A (en) * | 1986-09-26 | 1990-04-17 | Abbott Laboratories | Tiacumicin compounds |
| CN1149220C (en) * | 1996-07-12 | 2004-05-12 | 艾博特公司 | Bromotiacumicin compounds |
| CN102115757A (en) * | 2010-12-14 | 2011-07-06 | 中国科学院南海海洋研究所 | Biosynthetic gene cluster of tiacumicins and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100972542B1 (en) * | 2002-07-29 | 2010-07-28 | 옵티머 파마슈티칼즈, 인코포레이티드 | Tiacumicin production |
-
2010
- 2010-12-16 CN CN201010592416.9A patent/CN102115757B/en active Active
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- 2011-04-25 CN CN 201110104051 patent/CN102219815B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4918174A (en) * | 1986-09-26 | 1990-04-17 | Abbott Laboratories | Tiacumicin compounds |
| CN1149220C (en) * | 1996-07-12 | 2004-05-12 | 艾博特公司 | Bromotiacumicin compounds |
| CN102115757A (en) * | 2010-12-14 | 2011-07-06 | 中国科学院南海海洋研究所 | Biosynthetic gene cluster of tiacumicins and application thereof |
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| CN102219815A (en) | 2011-10-19 |
| CN102115757A (en) | 2011-07-06 |
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