CN103554230B - Amphomycin-type compounds and preparation method thereof - Google Patents
Amphomycin-type compounds and preparation method thereof Download PDFInfo
- Publication number
- CN103554230B CN103554230B CN201310428077.4A CN201310428077A CN103554230B CN 103554230 B CN103554230 B CN 103554230B CN 201310428077 A CN201310428077 A CN 201310428077A CN 103554230 B CN103554230 B CN 103554230B
- Authority
- CN
- China
- Prior art keywords
- ecomytrin
- slant
- bacterial strain
- medium
- fim
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 0 CC(C)C(C(N(CCC1)C1C(NC(C)C(C(N(CCCC1)C1C(NC(C(C)*)C(NC(C*)C(NCC(NC(CC(O)=O)C(NCC(NC1C(C)[N+])=O)=O)=O)=O)=O)=[U])=[U])NC(C(C*2C[U-]C2)NC(C/C=C/CCCC*)=O)=O)=O)=O)NC1=[U] Chemical compound CC(C)C(C(N(CCC1)C1C(NC(C)C(C(N(CCCC1)C1C(NC(C(C)*)C(NC(C*)C(NCC(NC(CC(O)=O)C(NCC(NC1C(C)[N+])=O)=O)=O)=O)=O)=[U])=[U])NC(C(C*2C[U-]C2)NC(C/C=C/CCCC*)=O)=O)=O)=O)NC1=[U] 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides amphomycin-type compounds and a preparation method thereof. The method comprises: culturing monospore of a streptomycete strain in fresh aspartic acid agar slant, after monospore is cultured maturely in the strain slant, inoculating to a seed culture medium, then transferring to a fermentation culture medium for culturing and further for obtaining the fermentation broth; performing macroporous resin adsorption column chromatography on the fermentation broth to obtain crude extract; suspending the crude extract with water, extracting with equal volume of n-butanol, adding equal volume of distilled water, fully vibrating to mix uniformly, after two phases are separated, getting the water phase; concentrating the water phase, performing medium-pressure reverse-phase silica gel column chromatography to obtain secondary crude extract, and collecting partially the amphomycin-type compounds. The new amphomycin-type compounds are prepared by employing microbial fermentation, and have antibacterial property.
Description
[technical field]
The present invention relates to a kind of Ecomytrin compounds and preparation method thereof.
[background technology]
Along with a large amount of uses of antibacterials, there is multi-drug resistant in many pathogenic bacterium.Such as, by gram-positive microorganism, the infection (comprising nosocomial infection) caused as vancomycin resistant enterococci (VRE) and methicillin resistant Staphylococcus aureus (MRSA) and penicillin-resistant streptococcus pneumoniae (PRSP) etc. increases.The generation of drug resistance problems and spread the health that the mankind in serious threat, therefore screens new texture, emphasis that the antibacterials of novel mechanism become Recent study.
Cyclic lipopeptide antibiotic is that a class is to the very effective important antibiotic of resisting gram-positive resistant organism.Wherein, Daptomycin [Debono M, Barnhart M, Carrell C B, et al.A21978C, acomplex of new acidic peptide antibiotics:isolation, chemistry and mass spectralstructure elucidation [J] .J Antibiot, 1980, 42 (6): 761-777.] be first for clinical cyclic lipopeptide antibiotic, successfully went on the market in 2003 in the U.S., there is high bactericidal properties and rapid action, all anti-microbial effect is had to multiple drug-resistant bacteria, include but not limited to the resistant organisms such as VRE and MRSA.Daptomycin has unique mechanism of action, and persister occurrence probability is very low.Under calcium ion existent condition, daptomycin is attached on epicyte protein with the form of non covalent bond, upset cytolemma to amino acid whose transhipment, thus hinder the biosynthesizing of phosphorus wall (acid) lipid (LTA) of bacteria cell wall peptidoglycan, change the character of cytoplasmic membrane; In addition, it is also by destroying the cytolemma of bacterium, its content is made to leak and reach object [the Alborn WEJr of kill bacteria, Allen NE, Preston DA.Daptomycin disrupts membrane potential in growing Staphylococcus aureus [J] .Antimicrob Agent s Chemot her, 1991,35 (11): 2282].Significantly, daptomycin is applied to clinical sole chemical structure and the brand-new microbiotic of mechanism of action over nearly 40 years, the success of Daptomycin makes cyclic lipopeptide compound, becomes the focus of the screening of current new antibiotic and research and development.
Ecomytrin (Amphomycin) [Heinemann B, Kaplan M A, Muir R D, et al.Amphomycin, a new antibiotic [J] .Antibiot Chemother, 1953,3:1239 – 1242.] be found in eighties of last century fifties, research shows that it has resisting gram-positive bacteria effect widely.Subsequently, researchist find again a series of with it in physico-chemical property, cyclic lipopeptide compound that anti-microbial activity is close, as aspartocin, tsushimycin, laspartocin, friulimicin B etc. [Baltz R.H., Miao V. & Wrigley S.K. (2005) .Natural products to drugs:daptomycin and relatedlipopeptide antibiotics.Nat.Prod.Rep.22 (6), 717-741.].This compounds, by amino acid makeup ring lipopeptid structure, be amino acid classes and quantity, or pendent fatty chain is not quite similar.Amphomycin has hemolytic action because of it and can not medication, but it is compared with daptomycin, there is better therapeutic index [V.J.Leea.Anti-gram positive agents of natural product origin [J] .Comprehensive Medicinal Chemistry II .2007,7,653-671.].With amphomycin structure for skeleton, it is modified, contribute to finding how effective antibacterials.[the Wacowich-Sgarbi such as Cameron, S A, Boyd V A, Cameron D R, et al.Synthesis andStructure – Activity Relationship (SAR) Studies on Dab-9Substitutions of theLipopeptide Antibiotic Amphomycin.229th American ChemicalSociety Meeting, San Diego, CA, Mar 13 – 17,2005; Abs 367.] Dab of 9 in amphomycin structure and aliphatic chain are modified, obtain compound M-1396, its anti-microbial activity is suitable with daptomycin.Vancouver MIGENIX biopharmaceutical company have developed the semi-synthetic derivative MX-2401 of amphomycin [MIGENIX Inc.MIGENIX Reports.Effectiveness of MX-2401inPneumonia Model [R] .Vancouver, BC, Canada & San Diego, CA, USA, 2005, June 23/PRNewswire.], its structure is similar to amphomycin, and only in branched fatty acid and peptide ring, an amino acid is variant.[the Rubinchik E such as Rubinchik, Schneider T, et al.Mechanism of Action and Limited Cross-Resistance of New LipopeptideMX-2401.Antimicrob [J] .Agents Chemother, 2011, 55 (6): 2743 – 2754.] mechanism of action of MX-2401 is studied, find that it passes through to come in conjunction with 11 amylene phosphoric acid matrix (C55-P) synthesis of inhibiting peptide glycan, the synthesis of T suppression cell wall precursor lipid I and lipid II and cell walls phosphate precursor lipid III, different from daptomycin mechanism.[the DominiqueD such as Dominique, Haiyan Y, et al.Antimicrobial Properties of MX-2401, anExpanded-Spectrum Lipopeptide Active in the Presence of Lung Surfactant [J] .Antimicrob Agents Chemother, 2011,55 (8): 3720 – 3728.] have studied the anti-microbial activity of MX-2401, result shows that MX-2401 has good antibacterial ability to VRE, MRSA, MSSA, MRSE etc., and has good therapeutic action to bronchovesicular pneumonia animal model.Moreover MX-2401 and daptomycin compares, there is longer post antibiotic effect and transformation period, decrease administration number of times, thus decrease drug toxicity, be expected to the cyclic lipopeptide compound becoming another clinical application.Therefore, the research of amphomycin and analogue thereof is extremely important to new drug development.
[summary of the invention]
One of the technical problem to be solved in the present invention, is to provide a kind of Ecomytrin compounds, and it is a kind of new Ecomytrin compounds, has germ resistance.
The present invention realizes one of above-mentioned technical problem like this:
A kind of Ecomytrin compounds, the molecular structural formula of described Ecomytrin compounds is as follows:
Wherein R is CH (CH
3)
2or CH
2cH (CH
3)
2.
The technical problem to be solved in the present invention two, is the preparation method providing a kind of Ecomytrin compounds, and obtain a kind of new Ecomytrin compounds by fermentable, it has germ resistance.
The present invention realizes above-mentioned technical problem two like this:
A preparation method for Ecomytrin compounds, comprises the steps:
Step one: the monospore of dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 is carried out slant culture with fresh aspartic acid agar, seed culture medium is inoculated in after bacterial strain slant culture maturation, transfer again into fermention medium cultivation, obtain dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 fermented liquid;
Step 2: dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 fermented liquid step one obtained carries out macroporous resin adsorption column chromatography, obtains crude extract;
Step 3: crude extract aqueous suspension step 2 obtained, adjusts pH to 2-3, uses equal-volume n-butanol extraction, and n-butanol layer adjusts pH to 7-8, adds equal-volume distilled water, fully two-phase laminated flow water intaking phase after vibration mixing;
Step 4: carry out middle pressure reversed-phase silica gel column chromatography to obtain secondary crude product after aqueous phase liquid step 3 obtained is concentrated, when carrying out middle pressure reversed-phase silica gel column chromatography, first adopting volume fraction to be 10% acetonitrile containing the aqueous solution of 0.1% trifluoroacetic acid is after moving phase balances 20 minutes, then moving phase adopts the aqueous solution system gradient elution 120min of acetonitrile-90% ~ 30% containing 0.1% trifluoroacetic acid of volume ratio 10% ~ 70%, fraction collection Ecomytrin compounds, the flow velocity of moving phase is 5ml/min; Finally detect fraction collection liquid according to high performance liquid chromatography;
Described dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 21st, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.7792.
Further, described step one is specially:
The monospore of dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 is cultivated 7-10 days at fresh aspartic acid agar slant (ISP-2) 30 DEG C; Be inoculated in seed culture medium after dull gray streptomycete bacterial strain (Streptomycescanus) FIM-0916 slant culture maturation, and be placed on shaking culture 40 ~ 48h in constant-temperature table, temperature 30 DEG C, rotating speed 250r/min; Then obtained seed liquor transferred cultivate 110 ~ 130h, temperature 28 DEG C, rotating speed 250r/min by the culture transferring amount of 1.0% into the fermention medium of sterilization.
Further, component and the content thereof of described slant medium are as follows: yeast extract 4.0%, malt extract 10.0%, glucose 4.0%, agar 18.0%, distilled water 1L; And the pH of described slant medium is 7.3-7.5, the percentage ratio in described slant medium component is massfraction.
Further, the component of described seed culture medium and content as follows: sucrose 30.0%, KNO
31.0%, K
2hPO
41.0%, peptone 5.0%, yeast extract paste 2.0%, KCl 0.5%, MgSO
47H
2o0.5%, FeSO
4.7H
2o 0.01%, and described seed culture medium pH is 7.3-7.5, the percentage ratio in described seed culture medium component is massfraction.
Further, component and the content thereof of described fermention medium are as follows: analysis for soybean powder 10.0%, glucose 10.0%, NaCl 5.0%, KNO
31.0%, CaCO
31.0%, and described fermention medium pH is 7.3-7.5, the percentage ratio in described fermention medium component is massfraction.
Further, the high performance liquid chromatography testing conditions in described step 4 is: analytical column is ODSC18, and wherein C18 particle diameter is 5 μm, and it is 250 × 4.6mm that analytical column length is multiplied by internal diameter; Moving phase is the water-acetonitrile containing 0.1% trifluoroacetic acid of volume ratio 35:65, and flow velocity is 1.0ml/min; Column temperature is 40 DEG C; Determined wavelength is 210nm.
Tool of the present invention has the following advantages:
The present invention obtains a kind of new Ecomytrin compounds by fermentable, and this Ecomytrin compounds has anti-microbial activity, belongs to cyclic lipopeptide compound, extremely has application prospect.
[accompanying drawing explanation]
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the infrared absorpting light spectra of compound (1) in the present invention.
Fig. 2 is the ESI-MS collection of illustrative plates of compound (1) in the present invention
Fig. 3 is compound (1) in the present invention
1h-NMR spectrogram
Fig. 4 is compound (1) in the present invention
13c-NMR spectrogram
Fig. 5 is the HMQC spectrogram of compound (1) in the present invention.
Fig. 6 is compound (1) in the present invention
1h-
1h COSY spectrogram.
Fig. 7 is the HMBC spectrogram of compound (1) in the present invention.
Fig. 8 is the ROSY spectrogram of compound (1) in the present invention.
Fig. 9 is the infrared absorpting light spectra of compound (2) in the present invention.
Figure 10 is the ESI-MS collection of illustrative plates of compound (2) in the present invention
Figure 11 is compound (2) in the present invention
1h-NMR spectrogram
Figure 12 is compound (2) in the present invention
13c-NMR spectrogram
Figure 13 is the HMQC spectrogram of compound (2) in the present invention.
Figure 14 is compound (2) in the present invention
1h-
1h COSY spectrogram.
Figure 15 is the HMBC spectrogram of compound (2) in the present invention.
Figure 16 is the ROSY spectrogram of compound (2) in the present invention.
[embodiment]
Refer to shown in Fig. 1 ~ 16, embodiments of the invention are described in detail.
The present invention relates to a kind of Ecomytrin compounds, the molecular structural formula of described Ecomytrin compounds is as follows:
Wherein R is CH (CH
3)
2or CH
2cH (CH
3)
2.
The invention still further relates to the preparation method of above-mentioned Ecomytrin compounds, comprise the steps:
Step one: the monospore of dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 is carried out slant culture with fresh aspartic acid agar, seed culture medium is inoculated in after bacterial strain slant culture maturation, transfer again into fermention medium cultivation, obtain dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 fermented liquid;
Step 2: dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 fermented liquid step one obtained carries out macroporous resin adsorption column chromatography, obtains crude extract;
Step 3: crude extract aqueous suspension step 2 obtained, adjusts pH to 2-3, uses equal-volume n-butanol extraction, and n-butanol layer adjusts pH to 7-8, adds equal-volume distilled water, fully two-phase laminated flow water intaking phase after vibration mixing;
Step 4: carry out middle pressure reversed-phase silica gel column chromatography to obtain secondary crude product after aqueous phase liquid step 3 obtained is concentrated, when carrying out middle pressure reversed-phase silica gel column chromatography, first adopting volume fraction to be 10% acetonitrile containing the aqueous solution of 0.1% trifluoroacetic acid is after moving phase balances 20 minutes, then moving phase adopts the aqueous solution system gradient elution 120min of acetonitrile-90% ~ 30% containing 0.1% trifluoroacetic acid of volume ratio 10% ~ 70%, fraction collection Ecomytrin compounds, the flow velocity of moving phase is 5ml/min; Finally detect fraction collection liquid according to high performance liquid chromatography;
Described dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 21st, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.7792.
Preferably, described step one is specially:
The monospore of dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 is cultivated 7-10 days at fresh aspartic acid agar slant (ISP-2) 30 DEG C; Be inoculated in seed culture medium after dull gray streptomycete bacterial strain (Streptomycescanus) FIM-0916 slant culture maturation, and be placed on shaking culture 40 ~ 48h in constant-temperature table, temperature 30 DEG C, rotating speed 250r/min; Then obtained seed liquor transferred cultivate 110 ~ 130h, temperature 28 DEG C, rotating speed 250r/min by the culture transferring amount of 1.0% into the fermention medium of sterilization.
Component and the content thereof of described slant medium are as follows: yeast extract 4.0%, malt extract 10.0%, glucose 4.0%, agar 18.0%, distilled water 1L; And the pH of described slant medium is 7.3-7.5, the percentage ratio in described slant medium component is massfraction.
Component and the content thereof of described seed culture medium are as follows: sucrose 30.0%, KNO
31.0%, K
2hPO
41.0%, peptone 5.0%, yeast extract paste 2.0%, KCl 0.5%, MgSO
47H
2o 0.5%, FeSO
4.7H
2o0.01%, and described seed culture medium pH is 7.3-7.5, the percentage ratio in described seed culture medium component is massfraction.
Component and the content thereof of described fermention medium are as follows: analysis for soybean powder 10.0%, glucose 10.0%, NaCl 5.0%, KNO
31.0%, CaCO
31.0%, and described fermention medium pH is 7.3-7.5, the percentage ratio in described fermention medium component is massfraction.
High performance liquid chromatography testing conditions in described step 4 is: analytical column is ODS C18, and wherein C18 particle diameter is 5 μm, and it is 250 × 4.6mm that analytical column length is multiplied by internal diameter; Moving phase is the water-acetonitrile containing 0.1% trifluoroacetic acid of volume ratio 35:65, and flow velocity is 1.0ml/min; Column temperature is 40 DEG C; Determined wavelength is 210nm.
The separation method of described dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916. is as follows:
Step 100: get the soil gathered from Wuyi Mountain, soaks with sterile distilled water, filters; Get filtrate.
Step 200: the filtrate obtained in step 100 is diluted with sterile distilled water, spreading rod picks a small amount of diluent and directly coats aspartic acid agar plate, cultivates 7 ~ 20 days at then flat board being placed in 30 DEG C, obtains the single bacterium colony of streptomycete; The component (ISP-2) of described aspartic acid agar plate and content as follows: yeast extract 4.0%, malt extract 10.0%, glucose 4.0%, agar 18.0%, distilled water 1L; PH 7.3-7.5.And the percentage ratio in described component is massfraction.
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment:
One, the separation and purification of Ecomytrin compounds
By the dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 fermented liquid (10L) that obtained by aforesaid method through centrifugal, get supernatant, cross HP20 absorption with macroporous adsorbent resin, with 30% methyl alcohol, ethanol successively wash-out, collect elutriant, concentrate to obtain crude extract, with aqueous suspension, adjust pH is to 2-3, use equal-volume n-butanol extraction, get n-butanol layer adjust pH to 7-8, add equal-volume distilled water, fully two-phase laminated flow water intaking phase after vibration mixing.Aqueous sample solution carries out the reverse silica gel column chromatography of middle pressure, first adopting volume fraction to be 10% acetonitrile containing the aqueous solution of 0.1% trifluoroacetic acid is after moving phase balances 20 minutes, then moving phase adopts the acetonitrile of volume ratio 10% ~ 70%-containing the aqueous solution system gradient elution 120min of 0.1% trifluoroacetic acid, fraction collection, the flow velocity of moving phase is 5ml/min; Fraction collection liquid is detected according to high performance liquid chromatography.In fraction collection liquid, receive cut at 70 minutes to 80 minutes, recycling design, obtain compound (1) 120mg; Receive cut at 92 minutes to 105 minutes, recycling design, obtain compound (2) 205mg;
Two, the Structural Identification of Ecomytrin compounds
1, compound (1): faint yellow amorphous powder (TLC, propyl carbinol: acetic acid: water=3:1:1, Rf=0.39), soluble in water, the organic solvent such as methyl alcohol, ethanol of [α] D22=17.248 (c 1.09mg/ml, MeOH).
Ultraviolet absorption peak is 202.50 (0.5705).As shown in Figure 1, IR ν max KBr (cm
-1): 3312,3050,2932,1787,1720,1663,1529,1444,1397.Wherein, 3312cm
-1that the NH caused by molecule interchain hydrogen bond shrinks vibrational band, 1663cm
-1and 1529cm
-1, be respectively acid amides bands of a spectrum I and II, these characteristic absorbance show that the hydrophilic group of this surfactant molecule is a peptide chain.2932 ~ 2869cm
-1absorption be the C-H stretching vibration of aliphatic carbon chain, show that the hydrophobic group of this surfactant molecule is lipid acid half molecule.This compound carries out Electrospray ion trap mass spectrometry analysis after dissolve with methanol, provides molecular ion peak [M+H]
+m/z 1262.4.As shown in Figure 2, high resolution mass spectrum (HR-ESI-MS): [M+H]
+measured value is 1262.6277; Theoretical value is 1262.6263, and supposition molecular formula is C
56h
87n
13o
20, degree of unsaturation is 20.
As shown in Figure 3, compound (1)
1h-NMR (CD
3cN/D
2o=5.6:1,600MHz) spectrum in, show 7 groups of methyl hydrogen signals at high field region: [δ
h0.83 (6H, d), δ
h0.88 (3H, d), δ
h0.98 (3H, d), δ
h1.04 (3H, br), δ
h1.25 (3H, br), δ
h1.26 (3H, br)].Wherein 2 groups of bimodal methyl hydrogen chemistry environmental facies are with [δ
h0.83 (6H, d, J=12Hz)], in conjunction with carbon spectrum δ
c23.1ppm place comprises two carbon signals, shows that itself and tertiary carbon connect to form sec.-propyl; There are 2 groups of methyl hydrogen signal [δ
h1.25 (3H, br), δ
h1.26 (3H, br)] because of chemical shift close and overlapping; There are 2 and sp in low place
2hydrogen signal [the δ that the carbon atom of hydridization is connected
h5.45 (1H, J=11.8Hz, H-3), δ
h5.56 (1H, J=12.0Hz, H-4)], show in structure containing a cis-double bonds.
As shown in Figure 4,
13c-NMR (CD
3cN/D
2o=5.6:1,150MHz) show 56 carbon in conjunction with DEPT collection of illustrative plates, comprising 16 quaternary carbon signals [12 amide group carbon signal (δ
c170.6, δ
c170.9, δ
c172.2, δ
c172.2, δ
c172.8, δ
c172.9, δ
c173.0, δ
c173.2, δ
c174.0, δ
c174.2, δ
c174.3, δ
c
174.8) 4 carboxyl carbon (δ
c175.0, δ
c175.3, δ
c176.5, δ
c179.3)]; 16 methine carbon signal (δ
c28.8, δ
c30.7, δ
c47.3, δ
c45.6, δ
c50.4, δ
c50.9, δ
c51.4, δ
c51.6, δ
c52.6, δ
c54.9, δ
c55.4, δ
c56.6, δ
c59.6, δ
c62.1, δ
c122.7, δ
c134.8), wherein δ
c122.7, δ
c134.8 be double key carbon signal; 17 mesomethylene carbon signal (δ
c20.8, δ
c24.9, δ
c26.1, δ
c26.9, δ
c28.1, δ
c30.4, δ
c30.5, δ
c30.7, δ
c35.3, δ
c37.1, δ
c37.6, δ
c38.5, δ
c39.8, δ
c44.1, δ
c44.2, δ
c44.9, δ
c49.3); 7 methyl carbon signal (δ
c14.2, δ
c16.7, δ
c18.8, δ
c19.2, δ
c19.4, δ
c23.1, δ
c23.1).By reporting (Fangming K in comparative compound (1) and document, Kasia J, Joseph S.A, et al.Structures of theAspartocin Antibiotics §-A Consideration of Requirements for CyclopeptideStructures.Rec.Nat.Prod.4:2 (2010) 131-140.) cyclic lipopeptide compound aspartocin A
1h-NMR and
1c-NMR data, find that compound 1 is closely similar with the nuclear magnetic data of aspartocin A, parent nucleus forms by 11 amino acid, only few four methylene radical on aliphatic chain.
As shown in Figure 6,
1h-
1during H COSY composes, visible and δ
h3.01 (H-2a) all and δ
h5.56 (H-4) all and δ
h5.45 (H-3) are correlated with, δ
h2.00 (H-5)-δ
h1.31 (H-6)-δ
h1.24 (H-7)-δ
hbe correlated with in turn between 1.13 (H-8), δ
h0.83 (H-10, H-11) and δ
h1.47 (H-9) are correlated with, in conjunction with HSQC collection of illustrative plates, as shown in Figure 5, can in pushing-out structure containing, for example under carbon skeleton fragment: C
2-C
3-C
4, C
5-C
6-C
7-C
8, C
10-C
9-C
11.
As shown in Figure 7, in HMBC collection of illustrative plates, visible H-2 and C1 (δ
c174.0), H-4 (δ
h5.56) with C2 (δ
c35.3), C3 (δ
c122.7), C5 (δ
c28.1), C6 (δ
c30.4); H-6 (δ
h1.31) with C4 (δ
c134.8), C5 (δ
c28.1), C7 (δ
c30.5); H-8 (δ
h1.13) with C6 (δ
c30.4), C10 (δ
c23.1), C11 (δ
c23.1); H-9 and C5 (δ
c28.1), C8 (δ
c39.8), C10 (δ
c23.1), C11 (δ
c23.1) long-range relevant between, thus can pushing-out structure Segment A:
The above analysis, and in conjunction with HSQC and HMBC collection of illustrative plates, belongs to whole carbon signal of compound (1) and hydrogen signal, as shown in table 1.
Table 1 compound (1)
1h-NMR (600MHz) and
13c-NMR (150MHz) data
2, compound (2): faint yellow amorphous powder (TLC, propyl carbinol: acetic acid: water=3:1:1, R
f=0.43), [α]
d 22=14.053 (c 2.13mg/ml, MeOH), soluble in water, methyl alcohol, the organic solvents such as ethanol.Ultraviolet absorption peak is 203.0 (0.5794).As shown in Figure 9, IR ν max KBr (cm
-1): 3312,3049,2932,2869,1720,1664,1528,1439,1397.
This compound carries out Electrospray ion trap mass spectrometry analysis after dissolve with methanol, provides molecular ion peak [M-H]
-m/z 1274.7.As shown in Figure 10, high resolution mass spectrum (HR-ESI-MS): [M+H]
+measured value is 1276.6410; Theoretical value is 1276.6420, and supposition molecular formula is C
57h
89n
13o
20, degree of unsaturation is 20.
As shown in figure 11, compound (2)
1h-NMR (CDCN/D
2o=5.6:1,600MHz) spectrum in, show 7 groups of methyl hydrogen signals at high field region: [δ
h0.84 (6H, d), δ
h0.90 (3H, d), δ
h1.01 (3H, d), δ
h1.01 (3H, br), δ
h1.02 (3H, br), δ
h1.30 (3H, br)].Wherein 2 groups of bimodal methyl hydrogen chemistry environmental facies are with [δ
h0.84 (6H, d, J=12Hz)], in conjunction with carbon spectrum δ
c23.1ppm place comprises two carbon signals, shows that itself and tertiary carbon connect to form sec.-propyl; There are 2 groups of methyl hydrogen signal [δ
h1.01 (6H, br)] because of chemical shift close and overlapping; There are 2 and sp in low place
2hydrogen signal [the δ that the carbon atom of hydridization is connected
h5.45 (1H, J=11.8Hz, H-3), δ
h5.56 (1H, J=12.0Hz, H-4)], show in structure containing a cis-double bonds.
As shown in figure 12,
13c-NMR (CD
3cN/D
2o=5.6:1,150MHz) show 57 carbon in conjunction with DEPT collection of illustrative plates, comprising 16 quaternary carbon signals [12 amide group carbon signal (δ
c170.5, δ
c170.7, δ
c171.6, δ
c171.8, δ
c172.5, δ
c173.3, δ
c173.4, δ
c173.5, δ
c173.5, δ
c173.6, δ
c174.3, δ
c175.4), 4 carboxyl carbon (δ
c176.8, δ
c177.2, δ
c177.3, δ
c182.1)]; 16 methine carbon signal (δ
c28.6, δ
c30.4, δ
c47.2, δ
c48.0, δ
c51.4, δ
c52.0, δ
c52.2, δ
c54.6, δ
c55.5, δ
c56.0, δ
c56.2, δ
c57.1, δ
c60.5, δ
c62.1, δ
c122.6, δ
c134.6), wherein δ
c122.6, δ
c134.6 be double key carbon signal; 18 mesomethylene carbon signal (δ
c20.7, δ
c25.5, δ
c26.0, δ
c27.0, δ
c28.0, δ
c30.2, δ
c30.3, δ
c30.6, δ
c30.7, δ
c35.0, δ
c38.5, δ
c39.4, δ
c39.6, δ
c39.9, δ
c43.3, δ
c43.6, δ
c44.6, δ
c49.0); 7 methyl carbon signal (δ
c15.1, δ
c17.4, δ
c18.9, δ
c19.0, δ
c19.5, δ
c22.9, δ
c22.9).By reporting (Fangming K in comparative compound (2) and document, Kasia J, Joseph S.A, et al.Structures of theAspartocin Antibiotics §-A Consideration of Requirements for CyclopeptideStructures.Rec.Nat.Prod.4:2 (2010) 131-140.) cyclic lipopeptide compound aspartocin A
1h-NMR and
1c-NMR data, find that compound (2) is closely similar with the nuclear magnetic data of aspartocin A, parent nucleus forms by 11 amino acid, only few three methylene radical on aliphatic chain.
As shown in figure 14,
1h-
1during H COSY composes, visible and δ
h3.00 (H-2a) all and δ
h5.56 (H-4) all and δ
h5.46 (H-3) are correlated with, δ
h2.00 (H-5)-δ
h1.31 (H-6)-δ
h1.26 (H-7)-δ
h1.24 (H-8)-δ
h1.13 (H-9)-δ
hbe correlated with in turn between 1.48 (H-10), δ
h0.83 (H-11, H-12) and δ
h1.48 (H-10) are correlated with, in conjunction with HSQC collection of illustrative plates, as shown in figure 13, can in pushing-out structure containing, for example under carbon skeleton fragment: C
2-C
3-C
4, C
5-C
6-C
7-C
8-C
9, C
11-C
10-C
12.
As shown in figure 16, in HMBC collection of illustrative plates, visible H-2 and C1 (δ
c174.0), H-4 (δ
h5.56) with C2 (δ
c35.0), C3 (δ
c122.6), C5 (δ
c28.0), C6 (δ
c30.2); H-6 (δ
h1.31) with C4 (δ
c134.6), C5 (δ
c28.0), C7 (δ
c30.7); H-9 (δ
h1.13) with C7 (δ
c30.7), C8 (δ
c30.3), C10 (δ
c28.6), C11 (δ
c22.9), C12 (δ
c22.9) long-range relevant between, thus can pushing-out structure fragment B:
The above analysis, and according to HSQC and HMBC collection of illustrative plates, belongs to, in table 2 whole carbon signal of compound (2) and hydrogen signal.
Table 2 compound 2
1h-NMR (600MHz) and
13c-NMR (150MHz) data
Three, the Determination of Antibacterial Activity of Ecomytrin compounds
Accurately take the compound (1), the compound (2) that obtain in embodiment one, with MH broth culture (composition (g/L): beef powder 2.0, Zulkovsky starch 1.5, acid hydrolyzed casein 17.5, calcium ions 50 μ gml
-1, PH7.4) dissolve and be configured to 100ug/ml, and be diluted to required concentration.Adopt 96 orifice plate trace doubling dilutions to measure the anti-microbial activity of compound, 37 DEG C of constant incubators were cultivated after 12 ~ 18 hours, and observations, the results are shown in Table 3.
Table 3 compound (1), (2) anti-microbial activity
The above results shows that this Ecomytrin compounds has anti-microbial activity.
The present invention obtains a kind of new Ecomytrin compounds by fermentable, and this Ecomytrin compounds has anti-microbial activity, belongs to cyclic lipopeptide compound, extremely has application prospect.
Although the foregoing describe the specific embodiment of the present invention; but be familiar with those skilled in the art to be to be understood that; specific embodiment described by us is illustrative; instead of for the restriction to scope of the present invention; those of ordinary skill in the art, in the modification of the equivalence done according to spirit of the present invention and change, should be encompassed in scope that claim of the present invention protects.
Claims (7)
1. an Ecomytrin compounds, is characterized in that: the molecular structural formula of described Ecomytrin compounds is as follows:
Wherein R is CH (CH
3)
2or CH
2cH (CH
3)
2.
2. the preparation method of a kind of Ecomytrin compounds according to claim 1, is characterized in that: comprise the steps:
Step one: the monospore of dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 is carried out slant culture with fresh aspartic acid agar, seed culture medium is inoculated in after bacterial strain slant culture maturation, transfer again into fermention medium cultivation, obtain dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 fermented liquid;
Step 2: dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 fermented liquid step one obtained carries out macroporous resin adsorption column chromatography, obtains crude extract;
Step 3: crude extract aqueous suspension step 2 obtained, adjusts pH to 2-3, uses equal-volume n-butanol extraction, and n-butanol layer adjusts pH to 7-8, adds equal-volume distilled water, fully two-phase laminated flow water intaking phase after vibration mixing;
Step 4: carry out middle pressure reversed-phase silica gel column chromatography to obtain secondary crude product after aqueous phase liquid step 3 obtained is concentrated, when carrying out middle pressure reversed-phase silica gel column chromatography, first adopting volume fraction to be 10% acetonitrile containing the aqueous solution of 0.1% trifluoroacetic acid is after moving phase balances 20 minutes, then moving phase adopts the aqueous solution system gradient elution 120min of acetonitrile-90% ~ 30% containing 0.1% trifluoroacetic acid of volume ratio 10% ~ 70%, fraction collection Ecomytrin compounds, the flow velocity of moving phase is 5mL/min; Finally detect fraction collection liquid according to high performance liquid chromatography;
Described dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 21st, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.7792.
3. the preparation method of a kind of Ecomytrin compounds according to claim 2, is characterized in that: described step one is specially:
The monospore of dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 is cultivated 7-10 days at fresh aspartic acid agar slant 30 DEG C; Be inoculated in seed culture medium after dull gray streptomycete bacterial strain (Streptomyces canus) FIM-0916 slant culture maturation, and be placed on shaking culture 40 ~ 48h in constant-temperature table, temperature 30 DEG C, rotating speed 250r/min; Then obtained seed liquor transferred cultivate 110 ~ 130h, temperature 28 DEG C, rotating speed 250r/min by the culture transferring amount of 1.0% into the fermention medium of sterilization.
4. the preparation method of a kind of Ecomytrin compounds according to Claims 2 or 3, is characterized in that:
Component and the content thereof of described slant medium are as follows: yeast extract 4.0%, malt extract 10.0%, glucose 4.0%, agar 18.0%, distilled water 1L; And the pH of described slant medium is 7.3-7.5, the percentage ratio in described slant medium component is massfraction.
5. the preparation method of a kind of Ecomytrin compounds according to Claims 2 or 3, is characterized in that: component and the content thereof of described seed culture medium are as follows: sucrose 30.0%, KNO
31.0%, K
2hPO
41.0%, peptone 5.0%, yeast extract paste 2.0%, KCl 0.5%, MgSO
47H
2o 0.5%, FeSO
4.7H
2o 0.01%, and described seed culture medium pH is 7.3-7.5, the percentage ratio in described seed culture medium component is massfraction.
6. the preparation method of a kind of Ecomytrin compounds according to Claims 2 or 3, is characterized in that: component and the content thereof of described fermention medium are as follows: analysis for soybean powder 10.0%, glucose 10.0%, NaCl 5.0%, KNO
31.0%, CaCO
31.0%, and described fermention medium pH is 7.3-7.5, the percentage ratio in described fermention medium component is massfraction.
7. the preparation method of a kind of Ecomytrin compounds according to claim 2, it is characterized in that: the high performance liquid chromatography testing conditions in described step 4 is: analytical column is ODS C18, wherein C18 particle diameter is 5 μm, and it is 250 × 4.6mm that analytical column length is multiplied by internal diameter; Moving phase is the water-acetonitrile containing 0.1% trifluoroacetic acid of volume ratio 35:65, and flow velocity is 1.0mL/min; Column temperature is 40 DEG C; Determined wavelength is 210nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310428077.4A CN103554230B (en) | 2013-09-18 | 2013-09-18 | Amphomycin-type compounds and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310428077.4A CN103554230B (en) | 2013-09-18 | 2013-09-18 | Amphomycin-type compounds and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103554230A CN103554230A (en) | 2014-02-05 |
| CN103554230B true CN103554230B (en) | 2015-07-22 |
Family
ID=50008620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310428077.4A Expired - Fee Related CN103554230B (en) | 2013-09-18 | 2013-09-18 | Amphomycin-type compounds and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103554230B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5629288A (en) * | 1994-03-30 | 1997-05-13 | Hoechst Aktiengesellschaft | Lipopeptide derivatives, a process for their preparation and their use |
-
2013
- 2013-09-18 CN CN201310428077.4A patent/CN103554230B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5629288A (en) * | 1994-03-30 | 1997-05-13 | Hoechst Aktiengesellschaft | Lipopeptide derivatives, a process for their preparation and their use |
Non-Patent Citations (2)
| Title |
|---|
| Natural products to drugs: daptomycin and related lipopeptide antibiotics;Richard H Baltz;《N.P.R.》;20051104;第22卷;A 1437 A，725页3.3，3.4 * |
| Two novel amphomycin analogues from streptomyces canus strain FIM-0916;huang jian yang;《N.P.R.》;20140225;第28卷(第12期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103554230A (en) | 2014-02-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111454869B (en) | Marine streptomyces and application thereof | |
| CN103665071B (en) | Elaiophylin derivative and the application in antimicrobial agent and resistance m tuberculosis infection thereof | |
| CN102746376A (en) | Cyclopeptide antibiotics and preparation method thereof and application of cylopeptide antibiotics in preparation of antibacterial agents | |
| CN102977118B (en) | Novel antibiotic of Gram-positive bacteria and its preparation method and use | |
| CN104974100A (en) | Phenazine compounds originated from lysobacter antibioticus OH13 and preparation method and application thereof | |
| Wei et al. | Two piperazic acid-containing cyclic hexapeptides from Streptomyces alboflavus 313 | |
| Qader et al. | Shikimic acid production by Fusarium decemcellulare, an endophytic fungus isolated from Flacourtia inermis fruits | |
| CN103554230B (en) | Amphomycin-type compounds and preparation method thereof | |
| Veilumuthu et al. | Antimicrobial compounds produced by Streptomyces sp. VITGV01 against selected human pathogens | |
| CN115806881A (en) | Penicillium fungus and application thereof in preparation of antibacterial drugs | |
| CN114982763A (en) | New use of geldanamycin and its analogue | |
| CN109265461B (en) | Paenibacillus kribbensis metabolite and application thereof in biocontrol | |
| CN108441427B (en) | Arthriospora fungi and pyridone alkaloid compound produced by same | |
| CN106167494A (en) | Halogenation II type polyketide compound, preparation method and applications | |
| CN105017267A (en) | Antibiotic, and preparation and applications thereof | |
| US5279829A (en) | Fungicidal antibiotic from Streptomyces NCIMB 40212 | |
| AU631666B2 (en) | Antibiotics plusbacin | |
| Zhang et al. | Antibacterial activity and composition of the fermentation broth of Streptomyces parvus 33 | |
| CN114349762B (en) | 6/6/6/6/5/5 cyclic alkaloid compounds with novel frameworks and application thereof in preparation of antibacterial drugs | |
| CN115504990B (en) | Sugar-spiro-macrolide compound FW-5-39 and preparation method and application thereof | |
| El-Naggar et al. | Isolation and characterization of an antimicrobial substance produced by Streptomyces violatus | |
| RU2817695C1 (en) | Streptomyces species yvz014 strain - producer of antibiotic lysolipine x | |
| CN112899199B (en) | Micromonospora TMD166 and application thereof | |
| CN117821283A (en) | Pseudomonas tolla and application of cyclic lipopeptid secondary metabolite and anti-biofilm thereof | |
| CN109575040A (en) | A kind of compound and preparation method thereof with antibacterial activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150722 Termination date: 20210918 |