CN114380764B - Thiazoline siderophore compound and preparation method and application thereof - Google Patents
Thiazoline siderophore compound and preparation method and application thereof Download PDFInfo
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- CN114380764B CN114380764B CN202111128139.0A CN202111128139A CN114380764B CN 114380764 B CN114380764 B CN 114380764B CN 202111128139 A CN202111128139 A CN 202111128139A CN 114380764 B CN114380764 B CN 114380764B
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- ethyl acetate
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
- C07D277/08—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
- C07D277/10—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
- A01N43/74—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,3
- A01N43/78—1,3-Thiazoles; Hydrogenated 1,3-thiazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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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
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a thiazoline iron carrier compound and a preparation method and application thereof, which are characterized in that the structural formula of the thiazoline iron carrier compound is shown as I, the preparation method comprises the steps of obtaining a fermentation product of the thiazoline iron carrier compound by fermenting and culturing actinomycetes with the preservation number of CCTCC No: M2020953, then soaking the fermentation product with ethyl acetate to extract a crude extract, and separating and purifying the crude extract by normal phase silica gel column chromatography, reverse medium pressure column chromatography and reverse phase semi-preparative high performance liquid chromatography on the basis.
Description
Technical Field
The invention relates to a siderophore compound, in particular to a thiazoline siderophore compound extracted from marine actinomycetes and a preparation method and application thereof.
Background
Iron is an essential element for the survival of bacteria and limits their growth during vertebrate infection when the iron concentration is low. Under iron starvation conditions, the microorganisms biosynthesize and secrete siderophores by upregulating the production of relevant enzymes, thereby facilitating the uptake of iron by cells from the external microenvironment. For Fe 3+ Is a key functional attribute of siderophores. In addition, siderophores play an important role in maintaining virulence of pathogenic bacteria. Generally, micromolar concentrations of iron are required for the survival of marine microorganisms, however, the iron content of surface water in the ocean is only 0.01-2nM. Thus, marine microorganisms have evolved strategies for absorbing iron from the surrounding environment, and one of the most common and effective strategies for iron accumulation is the production of iron carriers.
The inventor finds a thiazoline siderophore type new natural product and names the thiazoline siderophore type new natural product as the result of chemical investigation of an ethyl acetate extract of Streptomyces diaastaticus NBU2966 under rice fermentation. In addition, through screening of antibacterial activity, the antibacterial peptide has a certain degree of biological activity against staphylococcus aureus, and therefore, can be used for development and utilization of antibacterial lead medicaments for preventing, controlling and treating infection and diseases caused by staphylococcus aureus. At present, the chemical structure and the anti-staphylococcus aureus activity of the compound are not reported, so that the related medicines are not seen in the market.
Disclosure of Invention
The invention aims to solve the technical problem of providing a thiazoline siderophore compound with an inhibiting effect on staphylococcus aureus, and a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
1. a thiazoline siderophore compound, the structural formula of which is shown in (I);
2. the preparation method of the thiazoline siderophore compound is characterized by comprising the following steps:
(1) Fermentation production
The preservation number is CCTCC NO: streaking M2020953 marine actinomycetes (Streptomyces sp.4-7) on a plate of a Gao's No. 1 solid culture medium, culturing and activating for 7 days in a 28 ℃ incubator, selecting a single colony, inoculating the single colony in a Gao's No. 1 liquid culture medium, culturing for 3 days at a temperature of 28 ℃ and a rotation speed of 180rpm/min on a shaker, collecting seed liquid, inoculating the seed liquid into a rice culture medium according to an inoculation amount of 10% in volume ratio, and culturing for 30 days at a temperature of 28 ℃ to obtain a fermented product;
(2) Extract extraction
Adding equal amount of ethyl acetate into the fermentation product obtained in the step (1), repeatedly soaking for 3 times, then decompressing and evaporating ethyl acetate leaching liquor to dryness, and adding a mixture of ethyl acetate and ethyl acetate in a volume ratio of 1:1, repeatedly extracting the mixed solution consisting of ethyl acetate and water for 3 times, combining ethyl acetate phases obtained by three times of extraction, and obtaining a crude extract after concentrating the ethyl acetate extract under reduced pressure and evaporating to dryness;
(3) Isolation preparation of Compounds
Firstly, the volume ratio of 1:1, dissolving the mixed solvent of dichloromethane and methanol, adding 200-300-mesh silica gel for stirring sample, performing normal-phase medium-pressure column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with a volume ratio of (100; performing reversed-phase medium-pressure column chromatography gradient elution on the collected 7 th component, collecting elution fractions by using acetonitrile-water with the volume ratio of 20-100% as an eluent, arranging the elution fractions from large to small according to the fraction polarity, and combining to obtain 18 components; separating and purifying the 14 th component by using semi-preparative reverse phase high performance liquid chromatography, wherein a mobile phase is formed by mixing acetonitrile and water according to a volume ratio of 50:
the preparation method of the rice culture medium in the step (1) comprises the following steps: is prepared by dissolving 80g of rice and 4.2g of sea salt in 120mL of seawater.
The flow rate for the isolation preparation of the compound by semi-preparative reverse phase high performance liquid chromatography described in step (3) was 2.0mL/min.
3. The thiazoline siderophore compound is used for preparing a staphylococcus aureus inhibitor.
Compared with the prior art, the invention has the advantages that: the thiazoline iron carrier compound is separated, fermentation products of the thiazoline iron carrier are obtained through microbial fermentation culture, then the fermentation products are soaked and extracted by ethyl acetate to obtain crude extract, and the crude extract is separated and purified through normal-phase medium-pressure silica gel column chromatography, reverse-phase medium-pressure silica gel column chromatography and reverse-phase semi-preparative high performance liquid chromatography.
The marine actinomycete (Streptomyces sp.4-7) is NBU2966 strain with the preservation number of CCTCC NO: m2020953, deposited in China center for type culture Collection in 12.21.2020 at the storage address of Wuhan university in Wuhan, china.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
The structural formula of the thiazoline siderophore compound is shown as the following formula (I):
example 2
The method for separating and preparing the thiazoline siderophore compound shown in the structural formula (I) in the embodiment 1 specifically comprises the following steps:
(1) Fermentation production
The preservation number is CCTCC NO: streaking M2020953 marine actinomycetes (Streptomyces sp.4-7) on a plate of a Gao's 1 solid culture medium, culturing and activating for 7 days in a 28 ℃ incubator, selecting a single colony, inoculating the single colony in a Gao's 1 liquid culture medium, culturing the single colony on a shaker at the temperature of 28 ℃ and the rotating speed of 180rpm/min for increasing bacteria, collecting seed liquid after culturing for 3 days, then inoculating the seed liquid into a rice culture medium according to the inoculation amount of 10% by volume, and culturing for 30 days at the temperature of 28 ℃ to obtain a fermented product;
(2) Extract extraction
Adding equal amount of ethyl acetate into the fermentation product obtained in the step (1), repeatedly soaking for 3 times, then decompressing and evaporating ethyl acetate leaching liquor to dryness, and adding a mixture of ethyl acetate and ethyl acetate in a volume ratio of 1:1, repeatedly extracting for 3 times by using a mixed solution consisting of ethyl acetate and water, combining ethyl acetate phases obtained by three times of extraction, and concentrating an ethyl acetate extraction liquid under reduced pressure and evaporating to dryness to obtain a crude extract;
(3) Isolation preparation of compounds
Firstly, the volume ratio of 1:1, dissolving the mixed solvent of dichloromethane and methanol, adding 200-300-mesh silica gel for stirring sample, performing normal-phase medium-pressure column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with a volume ratio of (100; performing reversed-phase medium-pressure column chromatography gradient elution on the collected 7 th component, collecting elution fractions by using acetonitrile-water with the volume ratio of 20-100% as an eluent, arranging the elution fractions from large to small according to the fraction polarity, and combining to obtain 18 components; separating and purifying the 14 th component by using semi-preparative reverse phase high performance liquid chromatography, wherein a mobile phase is formed by mixing acetonitrile and water according to a volume ratio of 50:
compound I of the present invention is a white powder, and its excimer peak m/z 338.0997[ 2 ] M + H is given by high resolution mass spectrum (HR-ESI-MS) in positive ion mode] + . Bonding of 13 C NMR spectrum to determine its molecular formula as C 15 H 19 N 3 O 2 S 2 Of the compound 1 H and 13 the C NMR spectral data are shown in Table 1:
TABLE 1D NMR data (DMSO-D) for Compound I 6 )
| No. | δH.multi.(J in Hz) | δc，tYpe |
| 1 | - | 115.7，C |
| 2 | - | 158.3，C |
| 3 | 6.96，d(8.1) | 116.8，CH |
| 4 | 7.43，t(7.7) | 133.4，CH |
| 5 | 6.95，t(7.7) | 119.3，CH |
| 6 | 7.41，d(8.0) | 130.4，CH |
| 2’ | - | 171.6，C |
| 4’ | 5.30，td(9.2，3.5) | 78.0，CH |
| 5’ | 3.62，t(10.4)：3.50，t(10.4) | 31.2，CH 2 |
| 2” | 4.64，d(3.5) | 72.8，CH |
| 4” | - | 73.5，C |
| 5” | 3.04，d(10.5)；2.72，d(10.5) | 40.2，CH 2 |
| 6” | - | 174.8，C |
| 7” | 2.34，s | 34.9，CH 3 |
| 8” | 1.26，s | 13.1，CH 3 |
| NH 3 | 7.15，s | - |
| NH h | 7.27，s | - |
| 2-OH | 1234，s | - |
Note 1: s-singlet, d-doublet, t-triplet, m-multiplet.
Note 2: 1 h was obtained at 600 MHzNMR; 13 c was obtained at 150 MHzNMR.
Example 3
Activity and application of thiazolines described in example 1
(1) Experimental sample
Preparing a solution of a sample to be detected: the test sample is the purified compound I isolated and purified in the above example 1, and a proper amount of the sample is precisely weighed and prepared into a solution with a required concentration by DMSO for testing the antibacterial activity. The indicator bacterium used in this experiment was staphylococcus aureus (ATCC 6538).
(2) Experimental methods
The 96-well plate antibacterial test method comprises the following steps: in a 96-well plate, mueller-Hinton broth with a final volume of 100. Mu.L/well was used as the minimal medium, wherein the concentration of the compound was diluted to a concentration of 256 to 1. Mu.g/mL in a two-fold broth dilution method and 5X 10 5 Bacterial cultures were added at turbidity of CFU/mL. The plates were incubated overnight at 37 ℃ and then observed, and the concentration of the compound corresponding to a well in which bacteria could not be observed with the naked eye was determined as the MIC of the lowest inhibitory concentration of the compound. Meanwhile, gentamicin is used as a positive control.
(3) Results of the experiment
In the 96-well plate Vibrio resistance test, the MIC of Compound I against Staphylococcus aureus was determined to be 64. Mu.g/ml.
The above description is not intended to limit the invention, nor is the invention limited to the examples set forth above. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (5)
1. A thiazoline siderophore compound is characterized in that the structural formula of the thiazoline siderophore compound is shown as (I);
2. a method for preparing the thiazoline siderophore compound according to claim 1, comprising the steps of:
(1) Fermentation production
The preservation number is CCTCC NO: m2020953 marine actinomycetes (Steptomyces sp.4-7) are streaked on a plate of a Gaulter 1 solid culture medium, cultured and activated in a 28 ℃ incubator for 7 days, then single colony is selected and inoculated in a Gaulter 1 liquid culture medium, the strain is cultured on a rocking bed at the temperature of 28 ℃ and the rotating speed of 180rpm/min for 3 days, seed liquid is collected, then the seed liquid is inoculated in a rice culture medium according to the inoculation amount of 10 percent of the volume ratio, and cultured for 30 days at the temperature of 28 ℃ to obtain a fermented product, wherein the preparation method of the rice culture medium is as follows: dissolving 80g of rice and 4.2g of sea salt in 120mL of seawater to prepare the rice-sea salt solution;
(2) Extract extraction
Adding equal amount of ethyl acetate into the fermentation product obtained in the step (1), repeatedly soaking for 3 times, then decompressing and evaporating ethyl acetate leaching liquor to dryness, and adding a mixture of ethyl acetate and ethyl acetate in a volume ratio of 1:1, repeatedly extracting the mixed solution consisting of ethyl acetate and water for 3 times, combining ethyl acetate phases obtained by three times of extraction, and obtaining a crude extract after concentrating the ethyl acetate extract under reduced pressure and evaporating to dryness;
(3) Isolation preparation of Compounds
Firstly, the volume ratio of 1:1, dissolving the mixed solvent of dichloromethane and methanol, adding 200-300-mesh silica gel for stirring sample, performing normal-phase medium-pressure column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with a volume ratio of (100; performing reversed-phase medium-pressure column chromatography gradient elution on the collected 7 th component, collecting elution fractions by using acetonitrile-water with the volume ratio of 20-100% as an eluent, arranging the elution fractions from large to small according to the fraction polarity, and combining to obtain 18 components; separating and purifying the 14 th component by using semi-preparative reverse phase high performance liquid chromatography, wherein a mobile phase is formed by mixing acetonitrile and water according to a volume ratio of 50:
3. the method of claim 2, wherein the gradient elution of the reversed-phase MPLC of step (3) is performed at a time of 120min from 20 to 100% acetonitrile.
4. The method for preparing a thiazoline siderophore compound according to claim 2, wherein the method comprises the following steps: the flow rate for the isolation preparation of the compound by semi-preparative reverse phase high performance liquid chromatography described in step (3) was 2.0mL/min.
5. Use of the thiazoline siderophore compound of claim 1 in the preparation of a staphylococcus aureus inhibitor.
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