CN102952179B - A kind of preparation method of high-purity micafungin precursor compound - Google Patents
A kind of preparation method of high-purity micafungin precursor compound Download PDFInfo
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- CN102952179B CN102952179B CN201110244305.3A CN201110244305A CN102952179B CN 102952179 B CN102952179 B CN 102952179B CN 201110244305 A CN201110244305 A CN 201110244305A CN 102952179 B CN102952179 B CN 102952179B
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Abstract
The invention discloses one and utilize sheath Phoma (Coleophoma? sp.) fermenting culture prepares the method for micafungin precursor compound.The method comprise sheath Phoma fermenting culture is filtered, lixiviate, resin absorption, desorb, the step such as concentrated, obtain the crude extract of FR901379; Through silica gel column chromatography, FR901379 crude extract is further purified again, obtains highly purified micafungin precursor compound FR901379 product.The invention has the advantages that and adopt resin absorption, silica gel column chromatography extraction and isolation FR901379, highly purified micafungin precursor compound FR901379 can be prepared, simple for process, be suitable for suitability for industrialized production.
Description
Technical field
The invention belongs to industrial microbial technology field, be specifically related to the preparation method of a kind of high-purity micafungin precursor compound FR901379.
Background technology
In recent years, along with the extensive application of extensive pedigree antibiotic and immunosuppressor, chemotherapy of tumors, the spreading of organ transplantation and acquired immune deficiency syndrome (AIDS), the crowd causing immunity system to be suppressed is on the increase, the sickness rate of deep fungal infection starts to present obvious ascendant trend, and along with the utilization of antifungal drug, the resistance of fungi is also more and more stronger, makes the application of antifungal drug have the swift and violent trend increased.Therefore, medicine one of study hotspot becoming anti-infective of anti-deep fungal infection, causes the concern of people day by day.
Echinocandin is the novel antifungal drug of a class, obviously different from the mechanism of action of current clinical conventional antifungal drug, is the inhibitor of β-1,3-glucan synthase, acts on the cell walls of fungi.Cell walls is difference maximum between fungi and mammalian cell, so the synthesis of interference cell wall, the integrity affecting cell walls will cause that fungal cell is dead and to people's fanout free region, therefore side effect is less, and security is higher.
Current echinocandin class medicine has had 3 kind listings, i.e. Caspofungin, MFG and anidulafungin.Wherein MFG be Japanese Teng Ze company develop, 2005 by U.S. FDA certification, be after Caspofungin FDA approval the 2nd kind of echinocandin antifungal agent thing.Extensive anti-microbial effect is had to the deep fungal infection that Candida, Eurotium cause; Good anti-microbial activity is all had to the Candida albicans of resistance to azole drug, Candida glabrata, candida krusei and other candidiasis.Meanwhile, MFG without renal toxicity, and with other drug almost without interacting, therefore has good potential applicability in clinical practice.
MFG is a kind of water-soluble semi synthesis echinocandin antifungal agent thing, is synthesized obtain by precursor compound FR901379 after deacylation enzymic hydrolysis.Micafungin precursor compound FR901379 is the fermentating metabolism product of sheath Phoma (Coleophomasp.), and be the main raw material of synthesis MFG, its structural formula is as follows:
At present, the bibliographical information about micafungin precursor compound FR901379 is few.Journalofbioscienceandbioengineering, Thejournalofantibiotics, Bioorganic & medicinalchemistryletters tri-periodicals have the document relating to FR901379 on a small quantity, be the report of microorganism culturing and structural modification aspect, in Hydrolysis kinetics, have no report.
Summary of the invention
The object of the invention is to exploitation a kind of easy and simple to handle, solvent for use toxicity is low, is suitable for the method for suitability for industrialized production micafungin precursor compound FR901379.The present invention adopts polymeric adsorbent extraction and isolation micafungin precursor compound FR901379, and is further purified by silica gel column chromatography, to prepare highly purified micafungin precursor compound FR901379 product.Simple for process, be suitable for suitability for industrialized production, the synthesis for MFG provides strong raw material basis.
Below the present invention is specifically described:
In the methods of the invention, through to micafungin 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, FR901379 crude extract is further purified again, obtains highly purified micafungin precursor compound FR901379 product.
Particularly, the present invention relates to the preparation method of a kind of high-purity micafungin precursor compound FR901379, comprise the steps:
1) solid-liquid separation is carried out to micafungin precursor compound FR901379 fermented liquid, obtain solid fermenting culture;
2) add polar solvent stirring and leaching in solid fermenting culture, after lixiviate completes, carry out solid-liquid separation, obtain micafungin precursor compound FR901379 vat liquor;
3) add water in micafungin precursor compound FR901379 vat liquor, adjustment polar solvent ratio, imports polymeric adsorbent absorption;
4) the mixed solution gradient desorption of polymeric adsorbent polar solvent and water;
5) slough solvent by concentrated for micafungin precursor compound FR901379 stripping liquid, filtration, drying, obtain micafungin precursor compound FR901379 crude extract;
6) crude extract of micafungin precursor compound FR901379 is carried out silica gel column chromatography separation, use mixed solvent wash-out;
7) silica gel elutriant concentrated after, with solvent crystallization, after filtration, dry, obtain highly purified micafungin precursor compound FR901379.
Wherein step 1) described in solid-liquid separation can be filter press, vacuum filtration, centrifuging.
Step 2) described in lixiviate, polar solvent is methyl alcohol or ethanol, and the volume ratio of add-on and fermented liquid is 1: 2-1: 5, and single extraction time is 30-90min, and extracting times is 1-3 time.
Step 3) described in vat liquor to add water adjustment polar solvent ratio, the polar solvent ratio after adjustment is 20-40%, the polymeric adsorbent used for pore radius 20 ~
polymeric adsorbent, can be HZ816, D312 macroporous adsorbent resin.
Step 4) described in desorb polar solvent be methyl alcohol or ethanol, gradient concentration is that after 40-60% concentration wash-out 1 times of column volume, it is complete that 70-90% concentration is eluted to the whole wash-out of micafungin precursor compound FR901379.
Step 5) described in stripping liquid simmer down to 40-45 DEG C at concentrating under reduced pressure, be concentrated into polar solvent concentration and be less than 2%, filter, vacuum-drying at 40-45 DEG C, obtains micafungin precursor compound FR901379 crude extract solid.
Step 6) described in silica gel be 100-300 order silica gel G, preferred 200-300 order, mixed solvent is the ethyl acetate-light petrol of volume ratio 7: 3 or the acetone-petroleum ether of volume ratio 6: 4.
Step 7) described in crystallization be by silica gel elutriant through HPLC detect after, merge area percentage and be greater than 95% part, 40-45 DEG C of concentrating under reduced pressure, in enriched material, add sherwood oil or isopropyl ether, add-on is 3-8 times of enriched material weight, fully after dispersion, be cooled to 0-10 DEG C, filter, vacuum-drying, obtains micafungin precursor compound FR901379 fine powder.
Products obtained therefrom of the present invention can for the synthesis material of MFG, and the weight content of micafungin precursor compound FR901379 can reach more than 90%, and 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, purifying cost is low.2. technique is simple, quality controllable.3. sample purity is high, and can reach more than 90%, the rate of recovery is high, and omnidistance total recovery reaches on 75%.
Accompanying drawing explanation
FR901379 solid fermenting culture vat liquor HPLC collection of illustrative plates in Fig. 1 embodiment 1
FR901379 fine powder HPLC collection of illustrative plates in Fig. 2 embodiment 1
Embodiment
Following embodiment only realizes method of the present invention for setting forth, and should not be construed as limitation of the present invention.
Micafungin precursor compound FR901379 fermented liquid used in the present invention is that research and development centre of Hua Yao stock company microorganism culturing means obtain.HZ-816 macroporous adsorbent resin, Shanghai Huazhen Science and Technology Co., Ltd.; D312 macroporous adsorbent resin, Shandong Dong great chemical industrial company.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 micafungin precursor compound FR901379 fermented liquid 5.0L, fermentation unit 1320 μ g/mL, suction filtration, obtain 1.1kg solid fermenting culture, add 95% ethanol 1.25L, stirring at normal temperature lixiviate 60min, suction filtration, collect filtrate, the ethanol that 1L concentration is 95% is added in filter cake, stirring at normal temperature 30min final vacuum suction filtration, merge filtered liquid (see accompanying drawing 1), the adjustment alcohol concn that adds water is 30%, use 800mLD312 resin absorption, 50% concentration ethanol washing 2400mL, with 80% concentration ethanol wash-out, complete to FR901379 wash-out, at 40 DEG C, 80% ethanol eluate is evaporated to alcohol concn and is less than 2%, be cooled to 20 DEG C, suction filtration, 40 DEG C of vacuum-dryings, obtain FR901379 crude extract 7.16g, dry method is splined on 500mL silicagel column, with ethyl acetate-light petrol 7, 3 wash-outs, HPLC detects, and merges area percentage 95% with upper part, 40 DEG C of concentrating under reduced pressure, add sherwood oil 20mL, be cooled to 0 DEG C, filter, vacuum-drying, obtains micafungin precursor compound FR901379 fine powder 5.46g, content 92.1% (see accompanying drawing 2).
Embodiment 2
Get micafungin precursor compound FR901379 fermented liquid 10.0L, fermentation unit 1140 μ g/mL, filter, obtain 2.1kg solid culture fermentation broth, add 95% ethanol 3.0L, stirring at normal temperature lixiviate 90min, suction filtration, collect filtrate (similar to accompanying drawing 1 peak shape), the ethanol that 2.0L concentration is 95% is added in filter cake, stirring at normal temperature 60min final vacuum suction filtration, merge filtered liquid, the adjustment alcohol concn that adds water is 35%, use 1200mLHZ-816 resin absorption, 50% concentration methanol wash 1200mL, by 85% concentration methanol-eluted fractions, complete to FR901379 wash-out, at 40 DEG C, 85% meoh eluate is evaporated to methanol concentration and is less than 2%, be cooled to 20 DEG C, suction filtration, 40 DEG C of vacuum-dryings, obtain micafungin precursor compound FR901379 crude extract 12.63g.Dry method is splined on 700mL silicagel column, with ethyl acetate-light petrol 7: 3 wash-out, HPLC detects, and merges area percentage 95% with upper part, 40 DEG C of concentrating under reduced pressure, add sherwood oil 30mL, be cooled to 5 DEG C, filter, vacuum-drying, obtain micafungin precursor compound FR901379 fine powder 9.45g, content 92.8% (similar to accompanying drawing 2 peak shape).
Embodiment 3
Get micafungin precursor compound FR901379 fermented liquid 8.0L, fermentation unit 1260 μ g/mL, suction filtration, obtain 1.4kg solid culture fermentation broth, add methyl alcohol 4L, stirring at normal temperature lixiviate 30min, suction filtration, collect filtrate, 2L methyl alcohol is added in filter cake, stirring at normal temperature 30min final vacuum suction filtration, merge filtered liquid (similar to accompanying drawing 1 peak shape), the adjustment methanol concentration that adds water is 40%, use 1200mLD312 resin absorption, 50% concentration methanol wash 2000mL, by 80% concentration methanol-eluted fractions, complete to FR901379 wash-out, at 40 DEG C, 80% meoh eluate is evaporated to methanol concentration and is less than 2%, be cooled to 20 DEG C, suction filtration, 40 DEG C of vacuum-dryings, obtain FR901379 crude extract 9.45g.Dry method is splined on 500mL silicagel column, with acetone-petroleum ether 6; 4 wash-outs, HPLC detects, and merges area percentage 95% with upper part, 40 DEG C of concentrating under reduced pressure, add isopropyl ether 30mL, be cooled to 10 DEG C, filter, vacuum-drying, obtains micafungin precursor compound FR901379 fine powder 8.22g, content 93.5% (similar to accompanying drawing 2 peak shape).
Claims (5)
1. a preparation method of high-purity micafungin precursor compound FR901379, comprises the steps:
1) solid-liquid separation is carried out to micafungin precursor compound FR901379 fermented liquid, obtain solid fermenting culture;
2) add polar solvent stirring and leaching in solid fermenting culture, after lixiviate completes, carry out solid-liquid separation, obtain micafungin precursor compound FR901379 vat liquor;
3) turn down micafungin 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 gradient concentration desorb;
5) slough polar solvent by concentrated for micafungin precursor compound FR901379 stripping liquid, filter, obtain micafungin precursor compound FR901379 crude extract;
6) crude extract of micafungin precursor compound FR901379 is carried out silica gel column chromatography separation, use mixed solvent wash-out;
7) silica gel column chromatography elutriant concentrated after, with solvent crystallization, after filtration, dry, obtain highly purified micafungin precursor compound FR901379;
Wherein step 2), 4) described in polar solvent be methyl alcohol or ethanol; Step 3) described in polar solvent concentration be reduced to 20-40%; Step 3) described in polymeric adsorbent be HZ-816 or D312 macroporous adsorbent resin.
2. method according to claim 1, wherein step 2) described in polar solvent add-on and the volume ratio of fermented liquid be 1: 2-1: 5.
3. method according to claim 1, wherein step 4) described in stripping liquid with gradient concentration desorb, process is that after the stripping liquid washing 1-3 times of column volume of 40-60% concentration, it is complete that the stripping liquid of 70-90% concentration is eluted to the whole wash-out of micafungin precursor compound FR901379.
4. method according to claim 1, wherein step 6) described in mixed solvent be the ethyl acetate-light petrol of volume ratio 7: 3 or the acetone-petroleum ether of volume ratio 6: 4.
5. method according to claim 1, wherein step 7) described in the solvent that uses of crystallization be sherwood oil or isopropyl ether, add-on be the 3-8 of enriched material weight doubly.
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| EP3150623B1 (en) * | 2014-05-29 | 2019-04-03 | Shanghai Techwell Biopharmaceutical Co., Ltd | Solvate of cyclic peptide compound, preparation method for same, and uses thereof |
| 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 |
| 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 |
| 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 |
| CN114874919B (en) * | 2022-05-09 | 2023-06-27 | 中国科学院青岛生物能源与过程研究所 | High-yield strain of micafungin precursor FR901379 and application thereof |
Citations (1)
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| CN1051757A (en) * | 1989-11-13 | 1991-05-29 | 藤泽药品工业株式会社 | New polypeptide compound and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1051757A (en) * | 1989-11-13 | 1991-05-29 | 藤泽药品工业株式会社 | New polypeptide compound and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 棘白菌素类抗真菌药物的结构与微生物合成;滕云 等;《中国医药工业杂志》;20101231;第41卷(第10期);776-781 * |
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