CN106905342B - The structure and its preparation of two kinds of active metabolites of rose yellow streptomycete - Google Patents
The structure and its preparation of two kinds of active metabolites of rose yellow streptomycete Download PDFInfo
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
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Abstract
The invention discloses rose yellow streptomycete (Streptomyces roseoflavus) two kinds of antibacterial activity metabolites-pentaene macrolides metabolite Roflamycoin and Menmyco-A structure and preparation method thereof.The two compounds with bacteriostatic activity are obtained after isolating and purifying in 0116 fermentation liquid of rose yellow streptomycete bacterial strain, and the development and application of efficient agricultural antibiotic is expected to be used for.
Description
Technical field
In the two kinds of big rings of pentaene generated the present invention relates to rose yellow streptomycete (Streptomyces roseoflaver)
The structure and preparation method thereof of esters antibacterial activity metabolite Roflamycoin and Menmyco-A.
Background technique
Rose yellow streptomycete (Streptomyces roseoflaver) bacterium that culture presevation number is CGMCC N0.1471
Strain 0116 is the screening of biocontrol agents method that this laboratory uses autonomous innovation, is sieved from potato scab dieback soil
One plant of Antagonistic Fungi.The bacterial strain and its fermentation liquid are a variety of to verticillium dahliae, cucurbits powdery mildew bacterium, potato scab bacterium etc.
Important phytopathogen has very strong inhibiting effect, there is good biological and ecological methods to prevent plant disease, pests, and erosion application potential.Two during the last ten years, we are to this
Bacterial strain and its fermentation liquid antimicrobial spectrum, Substance extraction and identification, antibiotin synthetic gene cluster, genetic conversion system are built
Vertical, chitinase gene clone, biocontrol microorganisms and its activated product dosage form etc. have conducted extensive research, and apply for country related to this
Patent of invention 6, wherein being obtained respectively in terms of rose yellow streptomycete negative regulator gene, fermentation material aqua, fermentation material bacteria residue
3 national inventing patent (negative regulator genes for rose yellow streptomycete and its preparation method and application
2012100837464；Application 2005101174228 of the 0116 fermentation liquid bacteria residue of rose yellow streptomycete mutation as fertilizer；It is a kind of
Biological bactericide and preparation method thereof is 200510117421.3).Other three patents still in application have: " a kind of biology kills
Microbial inoculum wettable powder, number of patent application 200910074685.3 ", a kind of " wettable of rose yellow streptomycete biological bactericide
Pulvis, number of patent application 201110375945.8 " and " a kind of biocontrol microorganisms auxotype tablet, number of patent application
201410156782.8".What the above patent application was directed to is all the different preparations of rose yellow streptomycete viable bacteria or fermentation material research and development,
There is no any patent applications for being directed to its active metabolite compound structure.
Our researchs to 0116 active metabolite structure of rose yellow streptomycete bacterial strain in the past, for a long time without it is breakthrough into
Exhibition, until the second half year in 2016, by being separated, extracted to the antibacterial active constituents in rose yellow streptomycete metabolite and
Structural Identification (data are not delivered), with methanol extraction fermentation liquid precipitate, rotary evaporation obtains yellow powder substance, carries out liquid phase
Simultaneously purification and recovery is analyzed, and carries out Structural Identification through Shanghai Qin Zhen Biotechnology Co., Ltd, finally obtains active component
Roflamycoin (molecular formula C40H66O12, molecular weight 738) and Menmyco-A (molecular formula C41H68O12, molecular weight 752), the two
Structure is polyene macrolides substance.Wherein Roflamycoin is retrieved, for external report known compound, 1971
It is reported for the first time by Schlegel et al., and its determining chemical structure in 1981, at home and abroad also there is no conducts for the compound
Medicine, animal drugs and other goods registrations, or enter any information of clinical research, only in foreign countries, there are two be used as antibacterial
The patent of medicine and animal drugs, WO9964040A1 and WO2015164289A1.And Menmyco-A has not been reported yet, we
It is named as Menmyco-A.Therefore, this patent application is related to rose yellow streptomycete metabolite Roflamycoin and
Structure of Menmyco-A and preparation method thereof has novelty.
Summary of the invention
It is an object of the present invention to: provide two kinds of pentaene macrolide antibiotic Roflamycoin and
Menmyco-A, shown in structural formula such as formula (1), (2):
(1)Roflamycoin
(2)Menmyco-A
Roflamycoin (former name flavomycoin) of the present invention, is molecular weight 738, and chemical formula is
C40H66O12, which belongs to 36 yuan of pentaene macrolide antibiotics, earliest from 5068 mycelium of rose yellow streptomycete JA
It is isolated, prime report in 1971, determination its chemical structure in 1981, not yet find at present CHINESE REGION report and answer
With.Menmyco-A of the present invention, molecular weight 752, molecular formula C41H68O12, it is similar to Roflamycoin, in structure
Upper similar to Roflamycoin, the substituent group of No. 35 carbon is become outside isobutyl group that other are identical from isopropyl on division ring, at present
The structure not yet finds to report.
It is a further object of the invention to provide natural above-mentioned Roflamycoin of the invention and
The preparation method of Menmyco-A, includes the following steps:
Fermented and cultured saves the rose yellow streptomycete Streptomyces that number is CGMCC No.1471 first
Roseoflver bacterial strain 0116；Then from fermentation liquid two kinds of antibiotic Roflamycoin described in extracting and developing and
Menmyco-A。
The fermented and cultured step are as follows: connect saving to number for the rose yellow streptomycete bacterial strain 0116 of CGMCC No.1471
Kind cultivates mature acquisition fermentation liquid in fermentation medium.
Wherein fermentation medium is glucose 2.4%, soluble starch 0.8%, groundnut meal 1.5%, corn pulp
0.8%, sodium chloride 0.4%, calcium carbonate 0.3%, potassium dihydrogen phosphate 0.02%.
Wherein fermentation culture conditions are 30 DEG C, 200rpm, are cultivated 5 days.
The extracting and developing step includes: about 3 times of volumes methanols of precipitating after fermentation liquid 4500rpm is centrifuged 20min
30min is impregnated and is ultrasonically treated, filter paper filtering obtains yellow methanol solution, 50 DEG C methanol is evaporated under reduced pressure in Rotary Evaporators
To doing, appropriate ethyl acetate is added, sonic oscillation mixes them thoroughly, it is seen that there is yellow powder substance to be gradually precipitated, it is quiet
It after setting 2h, filters, obtains yellow powder substance, as containing there are two types of the antibiosis of compound R oflamycoin and Menmyco A
Plain crude product.It using HPLC preparation chromatography prepared by two compounds, preparation condition is chromatographic column: Waters X
5 μm of 19 × 150mm of OBDTM of Prep C18, mobile phase acetonitrile: water=45:55, flow velocity 10mL/min, detector wavelength
363nm.Using continuous sample introduction mode, in the corresponding retention time at two peaks, the mobile phase of detector outlet is picked up.It is being protected from light
Under the conditions of, 50 DEG C in Rotary Evaporators, 75r/min falls the maximum rotary evaporation of acetonitrile, then -40 in freeze drier
DEG C, the sample containing water and a small amount of acetonitrile is freeze-dried in sample bottle, obtains two pure compounds.
The general system that above-mentioned fermented and cultured and extraction separating step are substance Roflamycoin and Menmyco-A of the present invention
Preparation Method, the conventional means being well known to those skilled in the art, should not be construed as limiting the invention, without departing substantially from this hair
In the case where bright spirit and essence, to modifications or substitutions made by the method for the present invention, step or condition, model of the invention is belonged to
It encloses.
The antibiotic Roflamycoin and Menmyco-A that the present invention narrates, which are derived from, is isolated from Washington state horse
The rose yellow streptomycete Streptomyces roseoflaver bacterial strain 0116 of bell potato shot hole dieback soil.The generation
Bacterium has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as on September 30th, 2005
CGMCC, address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100080), point
Class is named as rose yellow streptomycete Streptomyces roseoflaver, deposit number are as follows: CGMCCNo.1471.Therefore, originally
Invention provides a kind of for producing the rose yellow streptomycete Streptomyces of Roflamycoin and Menmyco-A
Roseoflaver bacterial strain 0116.
The Structural Identification of Roflamycoin and Menmyco-A: according to HR-ESI-MS,13C-NMR、1H-NMR、1H-1H
The analysis (see photo) of COSY, HMBC, HSQC, NOESY, it is determined that the chemical structure of Roflamycoin and Menmyco A, two
Person is in CD3NMR data in OD is as shown in Table 1 and Table 2.
1 Roflamycoin of table is in CD3OD's1H-NMR(600MHz)、13The structural assignment of C-NMR (150MHz) spectral peak
2 Menmyco-A of table is in CD3In OD1H-NMR(600MHz)、13The structural assignment of C-NMR (150MHz) spectral peak
Detailed description of the invention
The structure of Fig. 1 rose yellow streptomycete two kinds of active metabolites Roflamycoin and Menmyco-A.
The high resolution mass spectrum (HR-ESI-MS) of Fig. 2 compound R oflamycoin.
Fig. 3 compound R oflamycoin's1H NMR figure.
Fig. 4 compound R oflamycoin's13C NMR (DEPT 135) figure.
Fig. 5 compound R oflamycoin's1H-1H COSY figure.
The HMBC of Fig. 6 compound R oflamycoin schemes.
The HSQC of Fig. 7 compound R oflamycoin schemes.
The NOESY of Fig. 8 compound R oflamycoin schemes.
The high resolution mass spectrum (HR-ESI-MS) of Fig. 9 compound Menmyco-A.
Figure 10 compound Menmyco-A's1H NMR figure.
Figure 11 compound Menmyco-A's13C NMR (DEPT 135) figure.
Figure 12 compound Menmyco-A's1H-1H COSY figure.
The HMBC of Figure 13 compound Menmyco-A schemes.
The HSQC of Figure 14 compound Menmyco-A schemes.
The NOESY of Figure 15 compound Menmyco-A schemes.
Claims (5)
1. a kind of preparation method of Menmyco-A, which is characterized in that the structure of the Menmyco-A is as follows:
The preparation method includes the following steps: the rose yellow that the number of fermented and cultured culture presevation first is CGMCC No.1471
Streptomycete bacterial strain 0116；Then the Menmyco-A described in extracting and developing from fermentation liquid.
2. preparation method as described in claim 1, which is characterized in that fermentation culture method are as follows: be by culture presevation number
The rose yellow streptomycete bacterial strain 0116 of CGMCC No.1471, which is inoculated in fermentation medium, cultivates mature acquisition fermentation liquid.
3. preparation method as claimed in claim 1 or 2, which is characterized in that fermentation medium components are glucose 2.4%, can
Soluble starch 0.8%, groundnut meal 1.5%, corn pulp 0.8%, sodium chloride 0.4%, calcium carbonate 0.3%, potassium dihydrogen phosphate
0.02%.
4. preparation method as claimed in claim 1 or 2, which is characterized in that fermentation culture conditions are 30 DEG C, 200rpm, culture 5
It.
5. preparation method as described in claim 1, which is characterized in that the extraction separating step includes: by fermentation liquid 4500rpm
After being centrifuged 20mins, 30mins is impregnated and be ultrasonically treated to about 3 times of volumes methanols of precipitating, and filter paper filtering obtains yellow methanol solution,
50 DEG C methanol is evaporated to dryness under reduced pressure in Rotary Evaporators, adds appropriate ethyl acetate, sonic oscillation keeps it sufficiently mixed
It closes, it is seen that there is yellow powder substance to be gradually precipitated, after standing 2h, filter, obtain yellow powder substance, as contain and have the right
It is required that the crude product of Menmyco-A described in 1；It using HPLC preparation chromatography prepared by the Menmyco-A, preparation condition is
Chromatographic column: Waters5 μm of 19 × 150mm of OBDTM of Prep C18, mobile phase acetonitrile: water=45:55, flow velocity
10mL/min, detector wavelength 363nm；Using continuous sample introduction mode, in the corresponding retention time of the Menmyco-A chromatographic peak
It is interior, pick up the mobile phase of detector outlet；Under the conditions of being protected from light, 50 DEG C in Rotary Evaporators, 75r/min by acetonitrile utmostly
Rotary evaporation fall, then -40 DEG C in freeze drier, by the sample containing water and a small amount of acetonitrile, freezing is dry in sample bottle
It is dry, obtain the Menmyco-A sterling.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999064040A1 (en) * | 1998-06-08 | 1999-12-16 | Advanced Medicine, Inc. | Novel polyene macrolide compounds and uses |
| CN103125527A (en) * | 2011-11-22 | 2013-06-05 | 河北农业大学 | Wettable powder of streptomyces roseoflavus biological bactericide |
| WO2015164289A1 (en) * | 2014-04-21 | 2015-10-29 | Cidara Therapeutics, Inc. | Compositions and methods for the treatment of fungal infections |
-
2017
- 2017-02-21 CN CN201710091330.XA patent/CN106905342B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999064040A1 (en) * | 1998-06-08 | 1999-12-16 | Advanced Medicine, Inc. | Novel polyene macrolide compounds and uses |
| CN103125527A (en) * | 2011-11-22 | 2013-06-05 | 河北农业大学 | Wettable powder of streptomyces roseoflavus biological bactericide |
| WO2015164289A1 (en) * | 2014-04-21 | 2015-10-29 | Cidara Therapeutics, Inc. | Compositions and methods for the treatment of fungal infections |
Non-Patent Citations (6)
| Title |
|---|
| Pavel Grigorjev et al..Roflamycoin -- a new channel-forming antibiotic.《Biochimica et Biopttvsica Acta》.1985,第821卷第297-304页. * |
| Roflamycoin -- a new channel-forming antibiotic;Pavel Grigorjev et al.;《Biochimica et Biopttvsica Acta》;19851231;第821卷;第297-304页 * |
| S R Naik et al..Structural studies on a oxohexaene polyene antibiotic (HA-1-92) produced by Streptomyces CDRIL-312.《India Journal of Chemistry》.2001,第40B卷第1183-1186页. * |
| Structural studies on a oxohexaene polyene antibiotic (HA-1-92) produced by Streptomyces CDRIL-312;S R Naik et al.;《India Journal of Chemistry》;20011231;第40B卷;第1183-1186页 * |
| 玫瑰黄链霉菌Men-myco-93-63 有效成分的初步提取与纯化;赵志泉等;《河北农业大学学报》;20070131;第30卷(第1期);第76-78、104页 * |
| 玫瑰黄链霉菌对根结线虫的防效及对土壤微生物区系的影响;乔丹娜等;《中国蔬菜》;20141231;第9卷;第30-36页 * |
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