CN106905342A - The structure of two kinds of active metabolites of rose yellow streptomycete and its preparation - Google Patents
The structure of two kinds of active metabolites of rose yellow streptomycete and its preparation Download PDFInfo
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- CN106905342A CN106905342A CN201710091330.XA CN201710091330A CN106905342A CN 106905342 A CN106905342 A CN 106905342A CN 201710091330 A CN201710091330 A CN 201710091330A CN 106905342 A CN106905342 A CN 106905342A
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- roflamycoin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/08—Bridged systems
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
Abstract
The invention discloses rose yellow streptomycete（Streptomyces roseoflavus）The structure of big ring Inner esters metabolite Roflamycoin and the Menmyco A of two kinds of antibacterial activity metabolite-pentaenes and preparation method thereof.The two have bacteriostatic activity compound isolated and purified from the zymotic fluid of rose yellow streptomycete bacterial strain 0116 after obtain, be expected to be used for the development and application of efficient agricultural antibiotic.
Description
Technical field
The present invention relates to the big ring Inner of two kinds of pentaenes that rose yellow streptomycete (Streptomyces roseoflaver) is produced
Structure of esters antibacterial activity metabolite Roflamycoin and Menmyco-A and preparation method thereof.
Background technology
Culture presevation numbering is rose yellow streptomycete (Streptomyces roseoflaver) bacterium of CGMCC N0.1471
Strain 0116 is screening of biocontrol agents method of this laboratory using autonomous innovation, is sieved from potato scab dieback soil
One plant of Antagonistic Fungi.The bacterial strain and its zymotic fluid are various to verticillium dahliae, cucurbits powdery mildew bacterium, potato scab bacterium etc.
Important phytopathogen has very strong inhibitory action, there is good biological and ecological methods to prevent plant disease, pests, and erosion application potential.Two during the last ten years, and we are to this
Bacterial strain and its zymotic fluid antimicrobial spectrum, Substance are extracted and identification, antibiotin synthetic gene cluster, genetic conversion system are built
Vertical, chitinase gene clone, biocontrol microorganisms and its activated product formulation etc. have carried out numerous studies, apply for country related to this
Patent of invention 6, wherein in terms of rose yellow streptomycete negative regulator gene, fermentate aqua, fermentate bacteria residue, obtaining respectively
3 national inventing patents (negative regulator genes for rose yellow streptomycete and its preparation method and application
2012100837464；The zymotic fluid bacteria residue of rose yellow streptomycete mutation 0116 as fertilizer application 2005101174228；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 is killed
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 above patent application was directed to is all the different preparations of rose yellow streptomycete viable bacteria or fermentate research and development,
Not for any patent application of its active metabolite compound structure.
Our researchs to the active metabolite structure of rose yellow streptomycete bacterial strain 0116 in the past, for a long time it is not breakthrough enter
Exhibition, until the second half year in 2016, separated by the antibacterial active constituents in rose yellow streptomycete metabolite, extract and
Structural Identification (data are not delivered), with methanol extraction fermentation liquid precipitate, rotary evaporation obtains yellow powder material, carries out liquid phase
Recovery is analyzed and purified, and Structural Identification is carried out through Shanghai Qin Zhen bio tech ltd, finally give active component
Roflamycoin (molecular formula C40H66O12, molecular weight 738) and Menmyco-A (molecular formula C41H68O12, molecular weight 752), the two
Structure is polyene macrocyclic Inner Esters.Wherein Roflamycoin, through retrieval, is external report known compound, 1971
Reported first by Schlegel et al., and determined its chemical constitution in 1981, the compound does not have conduct also at home and abroad
Medicine, animal drugs and other goods registrations, or enter any information of clinical research, only there are two abroad as antibacterial
The patent of medicine and animal drugs, WO9964040A1 and WO2015164289A1.And Menmyco-A has not yet to see report, we
It is Menmyco-A to be named.Therefore, this patent application is related to rose yellow streptomycete metabolite Roflamycoin and
Structure of Menmyco-A and preparation method thereof, with novelty.
The content of the invention
An object of the present invention is：There is provided two kinds of big ring Inner esters antibiotic Roflamycoin of pentaene 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, the compound belongs to 36 yuan of pentaene macrolide antibiotics, earliest from the mycelium of rose yellow streptomycete JA 5068
Isolated, prime report in 1971 determines its chemical constitution in 1981, not yet find at present CHINESE REGION report and should
With.Menmyco-A of the present invention, molecular weight is 752, and molecular formula is C41H68O12, it is similar to Roflamycoin, in structure
Upper similar to Roflamycoin, No. 35 substitution bases of carbon are changed into isobutyl group other are identical outward from isopropyl on division ring, at present
The structure not yet finds report.
It is a further object of the invention to provide natural origin of the invention above-mentioned Roflamycoin and
The preparation method of Menmyco-A, comprises the following steps：
Fermented and cultured preserves the rose yellow streptomycete Streptomyces that numbering is CGMCC No.1471 first
Roseoflver bacterial strains 0116；Then two kinds of antibiotic Roflamycoin from zymotic fluid described in extracting and developing and
Menmyco-A。
The fermented and cultured step is：The rose yellow streptomycete bacterial strain 0116 that numbering is CGMCC No.1471 will be preserved to connect
Plant and ripe acquisition zymotic fluid is cultivated 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, and 200rpm is cultivated 5 days.
The extracting and developing step includes：After zymotic fluid 4500rpm is centrifuged into 20min, about 3 times of volumes methanols of precipitation
Soak and ultrasonically treated 30min, filter paper filtering obtains yellow methanol solution, and 50 DEG C methyl alcohol is evaporated under reduced pressure in Rotary Evaporators
To dry, appropriate ethyl acetate is added, sonic oscillation is sufficiently mixed it, it is seen that have yellow powder material gradually to separate out, quiet
After putting 2h, suction filtration obtains yellow powder material, as contains two kinds of antibiosis of compound R oflamycoin and Menmyco A
Plain crude product.Two compounds are prepared using HPLC preparative chromatographies, 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 two corresponding retention times at peak, the mobile phase of detector outlet is accessed.In lucifuge
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, by the sample containing water and a small amount of acetonitrile mounted in freeze-drying in sample bottle, obtain two pure compounds.
Above-mentioned fermented and cultured and the general system that extraction separating step is material Roflamycoin and Menmyco-A of the present invention
Preparation Method, the conventional meanses 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 of bright spirit and essence, the modification or replacement made to the inventive method, step or condition belong to model of the invention
Enclose.
The antibiotic Roflamycoin and Menmyco-A that the present invention is narrated are derived from being isolated from Washington state horse
The rose yellow streptomycete Streptomyces roseoflaver bacterial strains 0116 of bell potato shot hole dieback soil.The generation
Bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 30th (referred to as in September in 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, and deposit number is：CGMCCNo.1471.Therefore, originally
Invention provides a kind of rose yellow streptomycete Streptomyces for producing Roflamycoin and Menmyco-A
Roseoflaver bacterial strains 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 constitution of Roflamycoin and Menmyco A, two
Person is in CD3NMR data in OD is as shown in Table 1 and Table 2.
The Roflamycoin of table 1 is in CD3OD's1H-NMR(600MHz)、13The structural assignment of C-NMR (150MHz) spectral peak
The Menmyco-A of table 2 is in CD3In OD1H-NMR(600MHz)、13The structural assignment of C-NMR (150MHz) spectral peak
Brief description of the drawings
Two kinds of structures of active metabolite Roflamycoin and Menmyco-A of Fig. 1 rose yellow streptomycetes.
The high resolution mass spectrum (HR-ESI-MS) of Fig. 2 compound Rs oflamycoin.
Fig. 3 compound Rs oflamycoin's1H NMR scheme.
Fig. 4 compound Rs oflamycoin's13C NMR (DEPT 135) figure.
Fig. 5 compound Rs oflamycoin's1H-1H COSY scheme.
The HMBC figures of Fig. 6 compound Rs oflamycoin.
The HSQC figures of Fig. 7 compound Rs oflamycoin.
The NOESY figures of Fig. 8 compound Rs oflamycoin.
The high resolution mass spectrum (HR-ESI-MS) of Fig. 9 compounds Menmyco-A.
Figure 10 compounds Menmyco-A's1H NMR scheme.
Figure 11 compounds Menmyco-A's13C NMR (DEPT 135) figure.
Figure 12 compounds Menmyco-A's1H-1H COSY scheme.
The HMBC figures of Figure 13 compounds Menmyco-A.
The HSQC figures of Figure 14 compounds Menmyco-A.
The NOESY figures of Figure 15 compounds Menmyco-A.
Claims (7)
1.Roflamycoin and Menmyco-A structures such as formula（1）、（2）It is shown：
（1）Roflamycoin
（2）Menmyco-A.
2. a kind of two kinds of preparation methods of antibiotic Roflamycoin and Menmyco-A described in claim 1, its feature exists
In comprising the following steps：Fermented and cultured culture presevation numbering is the rose yellow streptomycete bacterial strain of CGMCC No. 1471 first
0116；Then two kinds of antibiotic Roflamycoin and Menmyco-A from zymotic fluid described in extracting and developing.
3. preparation method as claimed in claim 2, it is characterised in that the fermentation culture method is：Culture presevation is numbered
Ripe acquisition zymotic fluid is cultivated for the rose yellow streptomycete bacterial strain 0116 of CGMCC No. 1471 is inoculated in fermentation medium.
4. preparation method as claimed in claim 2 or claim 3, it is characterised in that fermentation medium components used are 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%.
5. the preparation method as described in right 2 or 3, it is characterised in that fermentation culture conditions are 30 DEG C, 200 rpm are cultivated 5 days.
6. preparation method as claimed in claim 2, it is characterised in that the extraction separating step includes：By zymotic fluid 4500
After rpm is centrifuged 20 mins, precipitation is soaked and ultrasonically treated 30 mins with about 3 times of volumes methanols, filter paper filtering, obtains yellow methyl alcohol
Solution, 50 DEG C are evaporated to dryness under reduced pressure methyl alcohol in Rotary Evaporators, add appropriate ethyl acetate, and sonic oscillation fills it
Divide mixing, it is seen that there is yellow powder material gradually to separate out, after standing 2 h, suction filtration obtains yellow powder material, as contains
Have the right two kinds of antibiotic crude products of compound in requirement 1；Two compounds are prepared using HPLC preparative chromatographies, are prepared
Condition is chromatographic column：Waters X Bridge® Prep C18 5 μm OBDTM19 × 150 mm, mobile phase acetonitrile：Water=
45:55, the mL/min of flow velocity 10, the nm of detector wavelength 363；Using continuous sample introduction mode, in two corresponding retention times at peak
It is interior, access the mobile phase of detector outlet；Under the conditions of lucifuge, 50 DEG C in Rotary Evaporators, 75 r/min are by acetonitrile maximum journey
The rotary evaporation of degree falls, then -40 DEG C in freeze drier, the sample containing water and a small amount of acetonitrile is freezed in sample bottle
Dry, obtain two pure compounds.
7. preparation method as claimed in claim 4, it is characterised in that Extraction solvent used is methyl alcohol.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710091330.XA CN106905342B (en) | 2017-02-21 | 2017-02-21 | The structure and its preparation of two kinds of active metabolites of rose yellow streptomycete |
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| CN201710091330.XA CN106905342B (en) | 2017-02-21 | 2017-02-21 | The structure and its preparation of two kinds of active metabolites of rose yellow streptomycete |
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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 (4)
| Title |
|---|
| PAVEL GRIGORJEV ET AL.: "Roflamycoin -- a new channel-forming antibiotic", 《BIOCHIMICA ET BIOPTTVSICA ACTA》 * |
| S R NAIK ET AL.: "Structural studies on a oxohexaene polyene antibiotic (HA-1-92) produced by Streptomyces CDRIL-312", 《INDIA JOURNAL OF CHEMISTRY》 * |
| 乔丹娜等: "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", 《中国蔬菜》 * |
| 赵志泉等: "玫瑰黄链霉菌Men-myco-93-63 有效成分的初步提取与纯化", 《河北农业大学学报》 * |
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