CN102952179A - Preparation method for high-purity micafungin precursor compound - Google Patents
Preparation method for high-purity micafungin precursor compound Download PDFInfo
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- CN102952179A CN102952179A CN2011102443053A CN201110244305A CN102952179A CN 102952179 A CN102952179 A CN 102952179A CN 2011102443053 A CN2011102443053 A CN 2011102443053A CN 201110244305 A CN201110244305 A CN 201110244305A CN 102952179 A CN102952179 A CN 102952179A
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Abstract
The invention discloses a preparation method for a micafungin precursor compound by using a Coleophoma sp. fermentation culture. The method comprises the following steps: carrying out filtration, digestion, resin adsorption, desorption, condensation and the like on the Coleophoma sp. fermentation culture so as to obtain a crude extract of FR901379; and carrying out further purification on the crude extract of the FR901379 by using silica gel chromatography so as to obtain a micafungin precursor compound FR901379 product with high purity. The invention has the following advantages: resin adsorption and silica gel chromatography are used to extract and separate the FR901379 in the method to prepare the micafungin precursor compound FR901379 with high purity, and the method is simple and feasible and is suitable for industrial production.
Description
Technical field
The invention belongs to the industrial microbial technology field, be specifically related to the preparation method of a kind of high purity MFG precursor compound FR901379.
Background technology
In recent years, along with spreading of extensive application, chemotherapy of tumors, organ transplantation and the acquired immune deficiency syndrome (AIDS) of extensive pedigree antibiotic and immunosuppressor etc., the crowd who causes immunity system to be suppressed is on the increase, the sickness rate of deep fungal infection begins to present obvious ascendant trend, and the utilization along with antifungal drug, the resistance of fungi is also more and more stronger, so that the application of antifungal drug has the trend of swift and violent increase.Therefore, the medicine of anti-deep fungal infection has become one of study hotspot of anti-infective, day by day causes people's concern.
Echinocandin is the novel antifungal drug of a class, and is obviously different from the mechanism of action of present clinical antifungal drug commonly used, is β-1, and the inhibitor of 3-glucan synthase acts on the cell walls of fungi.Cell walls is difference maximum between fungi and the mammalian cell, so the interference cell wall is synthetic, the integrity that affect cell walls will cause fungal cell's death and to the people without injury, so side effect is less, security is higher.
The echinocandin class medicine has had 3 kind listings, i.e. Caspofungin, MFG and anidulafungins at present.Wherein MFG is that Japanese rattan pool company develops, and 2005 is the 2nd kind of echinocandin antifungal agent thing of FDA approval after Caspofungin by the U.S. FDA authentication.The deep fungal infection that Candida, Eurotium are caused has extensive anti-microbial effect; Candida albicans, Candida glabrata, candida krusei and other candidiasis to anti-azole drug all have good anti-microbial activity.Simultaneously, MFG is without renal toxicity, and with other drug almost without interacting, therefore good potential applicability in clinical practice is arranged.
MFG is the synthetic echinocandin antifungal agent thing of a kind of water-soluble semi, by precursor compound FR901379 synthetic obtaining after the deacylation enzymic hydrolysis.MFG precursor compound FR901379 is the fermentating metabolism product of sheath Phoma (Coleophoma sp.), is the main raw material of synthetic MFG, and its structural formula is as follows:
At present, few about the bibliographical information of MFG precursor compound FR901379.Journal of bioscience and bioengineering, The journal of antibiotics, Bioorganic﹠amp; Three periodicals of medicinal chemistry letters have the document that relates on a small quantity FR901379, are the report of microorganism culturing and structural modification aspect, have no report about extracting refining aspect.
Summary of the invention
The object of the invention is to develop a kind of easy and simple to handle, solvent for use toxicity is low, is suitable for the method for suitability for industrialized production MFG precursor compound FR901379.The present invention adopts polymeric adsorbent to extract separation MFG precursor compound FR901379, and is further purified by silica gel column chromatography, to prepare highly purified MFG precursor compound FR901379 product.Simple for process, be suitable for suitability for industrialized production, for MFG synthetic provides strong raw material basis.
The below specifically describes the present invention:
In the methods of the invention, through to MFG precursor compound FR901379 filtering fermentation liquor, lixiviate, resin absorption, desorb, the step such as concentrated, obtain the crude extract of FR901379, through silica gel column chromatography the FR901379 crude extract is further purified again, obtains highly purified MFG precursor compound FR901379 product.
Particularly, the present invention relates to the preparation method of a kind of high purity MFG precursor compound FR901379, comprise the steps:
1) MFG precursor compound FR901379 fermented liquid is carried out solid-liquid separation, obtain the solid fermenting culture;
2) add the polar solvent stirring and leaching in the solid fermenting culture, carry out solid-liquid separation after lixiviate is finished, obtain MFG precursor compound FR901379 vat liquor;
3) in MFG precursor compound FR901379 vat liquor, add water, adjust the polar solvent ratio, import polymeric adsorbent absorption;
4) the polymeric adsorbent mixed solution gradient desorption of polar solvent and water;
5) with the concentrated solvent of sloughing of MFG precursor compound FR901379 stripping liquid, filtration, drying get MFG precursor compound FR901379 crude extract;
6) crude extract with MFG precursor compound FR901379 carries out the silica gel column chromatography separation, uses the mixed solvent wash-out;
7) the silica gel elutriant concentrated after, use solvent crystallization, after filtration, drying, obtain highly purified MFG precursor compound FR901379.
Step 1 wherein) described solid-liquid separation can be filter press, vacuum filtration, centrifuging.
Step 2) described lixiviate, polar solvent is methyl alcohol or ethanol, the volume ratio of add-on and fermented liquid is 1: 2-1: 5, the single extraction time is 30-90min, extracting times is 1-3 time.
Step 3) described vat liquor adds water and adjusts the polar solvent ratio, and the polar solvent ratio after the adjustment is 20-40%, employed polymeric adsorbent be pore radius 20～
Polymeric adsorbent, can be HZ816, D312 macroporous adsorbent resin.
Step 4) described desorb polar solvent is methyl alcohol or ethanol, and after gradient concentration was 1 times of column volume of 40-60% concentration wash-out, it is complete that 70-90% concentration is eluted to the whole wash-outs of MFG precursor compound FR901379.
Step 5) 40-45 ℃ of lower concentrating under reduced pressure of described stripping liquid simmer down to is concentrated into polar solvent concentration less than 2%, filters, and 40-45 ℃ of lower vacuum-drying obtains MFG precursor compound FR901379 crude extract solid.
Step 6) described silica gel is 100-300 order silica gel G, preferred 200-300 order, and mixed solvent is ethyl acetate-sherwood oil of 7: 3 of volume ratio or acetone-sherwood oil of 6: 4 of volume ratio.
Step 7) described crystallization be with the silica gel elutriant after HPLC detects, merge area percentage greater than 95% part, 40-45 ℃ of concentrating under reduced pressure, add sherwood oil or isopropyl ether in enriched material, add-on is 3-8 times of enriched material weight, after fully disperseing, be cooled to 0-10 ℃, filter, vacuum-drying gets MFG precursor compound FR901379 fine powder.
Products obtained therefrom of the present invention can be for the synthesis material of MFG, and the weight content of MFG precursor compound FR901379 can reach more than 90%, and the area normalization percentage composition reaches more than 95%, and sample recovery rate is greater than 75%.
The present invention has the following advantages: 1. all adopt conventional separating medium and conventional separation means, the purifying cost is low.2. technique is simple, and is quality controllable.3. sample purity is high, can reach more than 90%, and the rate of recovery is high, and omnidistance total recovery reaches on 75%.
Description of drawings
FR901379 solid fermenting culture vat liquor HPLC collection of illustrative plates among Fig. 1 embodiment 1
FR901379 fine powder HPLC collection of illustrative plates among Fig. 2 embodiment 1
Embodiment
Following embodiment only is used for setting forth realizing method of the present invention, should not be construed as limitation of the present invention.
MFG precursor compound FR901379 fermented liquid used in the present invention is that research and development centre of magnificent medicine stock company obtains with the microorganism culturing means.The HZ-816 macroporous adsorbent resin, Shanghai Huazhen Science and Technology Co., Ltd.; The D312 macroporous adsorbent resin, the large chemical industrial company in east, Shandong.The reagent such as methyl alcohol, ethanol, acetone, sherwood oil are commercially available.The high performance liquid chromatograph that the present invention uses is 996 type detectors, 515 pumps (Waters company).
Embodiment 1
Get MFG precursor compound FR901379 fermented liquid 5.0L, fermentation unit 1320 μ g/mL, suction filtration obtains 1.1kg solid fermenting culture, adds 95% ethanol 1.25L, stirring at normal temperature lixiviate 60min, suction filtration is collected filtrate, and adding 1L concentration is 95% ethanol in the filter cake, stirring at normal temperature 30min final vacuum suction filtration, merge filtered liquid (seeing accompanying drawing 1), adding water adjustment alcohol concn is 30%, uses the 800mLD312 resin absorption, 50% concentration ethanol washing 2400mL, with 80% concentration ethanol wash-out, complete to the FR901379 wash-out, 80% ethanol eluate is evaporated to alcohol concn less than 2% under 40 ℃, be cooled to 20 ℃, suction filtration, 40 ℃ of vacuum-dryings get FR901379 crude extract 7.16g, dry method is splined on the 500mL silicagel column, with ethyl acetate-sherwood oil 7; 3 wash-outs, HPLC detects, and merges area percentage 95% above part, and 40 ℃ of concentrating under reduced pressure add sherwood oil 20mL, are cooled to 0 ℃, filter, and vacuum-drying gets MFG precursor compound FR901379 fine powder 5.46g, content 92.1% (seeing accompanying drawing 2).
Get MFG precursor compound FR901379 fermented liquid 10.0L, fermentation unit 1140 μ g/mL, filter, obtain 2.1kg solid fermented liquid culture, add 95% ethanol 3.0L, stirring at normal temperature lixiviate 90min, suction filtration, collect filtrate (similar to accompanying drawing 1 peak shape), adding 2.0L concentration is 95% ethanol in the filter cake, and stirring at normal temperature 60min final vacuum suction filtration merges filtered liquid, adding water adjustment alcohol concn is 35%, use the 1200mLHZ-816 resin absorption, 50% concentration methanol wash 1200mL is with 85% concentration methanol-eluted fractions, complete to the FR901379 wash-out, under 40 ℃ 85% meoh eluate is evaporated to methanol concentration less than 2%, is cooled to 20 ℃, suction filtration, 40 ℃ of vacuum-dryings get MFG precursor compound FR901379 crude extract 12.63g.Dry method is splined on the 700mL silicagel column, with 7: 3 wash-outs of ethyl acetate-sherwood oil, HPLC detects, and merges area percentage 95% above part, 40 ℃ of concentrating under reduced pressure, add sherwood oil 30mL, be cooled to 5 ℃, filter vacuum-drying, get MFG precursor compound FR901379 fine powder 9.45g, content 92.8% (similar to accompanying drawing 2 peak shapes).
Embodiment 3
Get MFG precursor compound FR901379 fermented liquid 8.0L, fermentation unit 1260 μ g/mL, suction filtration, obtain 1.4kg solid fermented liquid culture, add methyl alcohol 4L, stirring at normal temperature lixiviate 30min, suction filtration, collect filtrate, add 2L methyl alcohol in the filter cake, stirring at normal temperature 30min final vacuum suction filtration merges filtered liquid (similar to accompanying drawing 1 peak shape), adding water adjustment methanol concentration is 40%, use the 1200mLD312 resin absorption, 50% concentration methanol wash 2000mL is with 80% concentration methanol-eluted fractions, complete to the FR901379 wash-out, under 40 ℃ 80% meoh eluate is evaporated to methanol concentration less than 2%, is cooled to 20 ℃, suction filtration, 40 ℃ of vacuum-dryings get FR901379 crude extract 9.45g.Dry method is splined on the 500mL silicagel column, with acetone-sherwood oil 6; 4 wash-outs, HPLC detects, and merges area percentage 95% above part, 40 ℃ of concentrating under reduced pressure, add isopropyl ether 30mL, be cooled to 10 ℃, filter, vacuum-drying gets MFG precursor compound FR901379 fine powder 8.22g, content 93.5% (similar to accompanying drawing 2 peak shapes).
Claims (9)
1. the preparation method of a high purity MFG precursor compound FR901379 comprises the steps:
1) MFG precursor compound FR901379 fermented liquid is carried out solid-liquid separation, obtain the solid fermenting culture;
2) add the polar solvent stirring and leaching in the solid fermenting culture, carry out solid-liquid separation after lixiviate is finished, obtain MFG precursor compound FR901379 vat liquor;
3) turn down MFG precursor compound FR901379 vat liquor Semi-polarity solvent strength, import polymeric adsorbent absorption;
4) polymeric adsorbent is with containing the stripping liquid of polar solvent with the gradient concentration desorb;
5) with the concentrated polar solvent of sloughing of MFG precursor compound FR901379 stripping liquid, filter, get MFG precursor compound FR901379 crude extract;
6) crude extract with MFG precursor compound FR901379 carries out the silica gel column chromatography separation, uses the mixed solvent wash-out;
7) the silica gel column chromatography elutriant concentrated after, use solvent crystallization, after filtration, drying, obtain highly purified MFG precursor compound FR901379.
2. method according to claim 1, wherein step 2), 4) described polar solvent is methyl alcohol or ethanol.
3. method according to claim 1, wherein step 2) volume ratio of described polar solvent add-on and fermented liquid is 1: 2-1: 5.
4. method according to claim 1, wherein step 3) described polar solvent concentration is reduced to 20-40%.
5. method according to claim 1, wherein step 3) described polymeric adsorbent is pore radius 20-
Polymeric adsorbent, be preferably HZ-816 or D312 macroporous adsorbent resin.
6. method according to claim 1, step 4 wherein) described stripping liquid is with the gradient concentration desorb, after process was 1-3 times of column volume of stripping liquid wash-out of 40-60% concentration, it is complete that the stripping liquid of 70-90% concentration is eluted to the whole wash-outs of MFG precursor compound FR901379.
7. method according to claim 1, wherein step 6) described mixed solvent is ethyl acetate-sherwood oil of 7: 3 of volume ratio or acetone-sherwood oil of 6: 4 of volume ratio.
8. method according to claim 1, wherein step 7) solvent that uses of described crystallization is sherwood oil or isopropyl ether, add-on be enriched material weight 3-8 doubly.
9. method according to claim 1, wherein step 7) described Tc is 0-10 ℃.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861044A (en) * | 2014-05-29 | 2015-08-26 | 上海天伟生物制药有限公司 | Solvate for cyclic peptide compound, as well as preparation method and use thereof |
| CN104877012A (en) * | 2014-05-29 | 2015-09-02 | 上海天伟生物制药有限公司 | Crystal of cyclic peptide compound and preparation method and application of crystal |
| CN105968173A (en) * | 2016-06-28 | 2016-09-28 | 成都雅途生物技术有限公司 | Method for purifying micafungin precursors FR901379 |
| CN106399431A (en) * | 2016-11-28 | 2017-02-15 | 无锡福祈制药有限公司 | Preparation method of micafungin precursor |
| CN108250274A (en) * | 2016-12-28 | 2018-07-06 | 浙江华谱新创科技有限公司 | Mikafen high efficiency separation and purification method |
| CN111187339A (en) * | 2018-11-15 | 2020-05-22 | 江苏豪森药业集团有限公司 | Method for extracting FR901379 from fermentation liquor |
| CN111909244A (en) * | 2020-08-14 | 2020-11-10 | 卓和药业集团有限公司 | Preparation method of high-purity micafungin precursor FR901379 |
| CN114874919A (en) * | 2022-05-09 | 2022-08-09 | 中国科学院青岛生物能源与过程研究所 | Micafungin precursor FR901379 high-yield strain and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861044A (en) * | 2014-05-29 | 2015-08-26 | 上海天伟生物制药有限公司 | Solvate for cyclic peptide compound, as well as preparation method and use thereof |
| CN104877012A (en) * | 2014-05-29 | 2015-09-02 | 上海天伟生物制药有限公司 | Crystal of cyclic peptide compound and preparation method and application of crystal |
| CN104877012B (en) * | 2014-05-29 | 2019-03-01 | 上海天伟生物制药有限公司 | Crystal of cyclic peptide compound and its preparation method and application |
| CN104861044B (en) * | 2014-05-29 | 2019-03-01 | 上海天伟生物制药有限公司 | A kind of solvate of cyclic peptide compound and its preparation method and application |
| CN105968173A (en) * | 2016-06-28 | 2016-09-28 | 成都雅途生物技术有限公司 | Method for purifying micafungin precursors FR901379 |
| CN105968173B (en) * | 2016-06-28 | 2019-12-03 | 成都雅途生物技术有限公司 | The purification process of mikafen precursor FR901379 |
| CN106399431A (en) * | 2016-11-28 | 2017-02-15 | 无锡福祈制药有限公司 | Preparation method of micafungin precursor |
| CN108250274A (en) * | 2016-12-28 | 2018-07-06 | 浙江华谱新创科技有限公司 | Mikafen high efficiency separation and purification method |
| CN111187339A (en) * | 2018-11-15 | 2020-05-22 | 江苏豪森药业集团有限公司 | Method for extracting FR901379 from fermentation liquor |
| CN111187339B (en) * | 2018-11-15 | 2023-12-01 | 江苏豪森药业集团有限公司 | Method for extracting FR901379 from fermentation broth |
| CN111909244A (en) * | 2020-08-14 | 2020-11-10 | 卓和药业集团有限公司 | Preparation method of high-purity micafungin precursor FR901379 |
| CN114874919A (en) * | 2022-05-09 | 2022-08-09 | 中国科学院青岛生物能源与过程研究所 | Micafungin precursor FR901379 high-yield strain and application thereof |
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