CN114380814B - Oxazole siderophore compound and preparation method and application thereof - Google Patents
Oxazole siderophore compound and preparation method and application thereof Download PDFInfo
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- CN114380814B CN114380814B CN202111127701.8A CN202111127701A CN114380814B CN 114380814 B CN114380814 B CN 114380814B CN 202111127701 A CN202111127701 A CN 202111127701A CN 114380814 B CN114380814 B CN 114380814B
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Abstract
The invention discloses an oxazole iron carrier compound and a preparation method and application thereof, which are characterized in that the structural formula of the compound is shown as I, the preparation method comprises the steps of obtaining a fermentation product of the oxazole 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 of the crude extract.
Description
Technical Field
The invention relates to a siderophore compound, in particular to an oxazole 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 to absorb 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 new oxazole siderophore type natural product and names the product as streptachelin A in chemical investigation of ethyl acetate extract of actinomyces maritima Diastaticaticus NBU2966 under rice fermentation. In addition, through screening of antibacterial activity, the antibacterial peptide has a certain degree of biological activity of resisting 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 found in the market.
Disclosure of Invention
The invention aims to solve the technical problem of providing a phenol/thiazoline siderophore compound with an inhibitory 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. an oxazole siderophore compound, the structural formula of which is shown as (I);
2. a preparation method of an oxazole siderophore compound comprises the following steps:
(1) Fermentation production
The preservation number is CCTCC NO: marking marine actinomycetes (Steptomyces sp.4-7) of M2020953 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 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% of the volume ratio, and culturing for 30 days at the temperature of 28 ℃ to obtain a fermentation 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, mixing the raw materials in a volume ratio of 1:1, adding 200-300 mesh silica gel for mixing samples after dissolving, performing normal-phase medium-pressure column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with a volume ratio of (100; subjecting the collected 8 th component to reversed-phase medium-pressure column chromatography gradient elution, collecting elution fractions by using acetonitrile-water with a 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 10 components; and (3) separating and purifying the obtained 6 th component by using a solution of acetonitrile and water in a volume ratio of 17 to 83 as a mobile phase through semi-preparative reverse phase high performance liquid chromatography to obtain a compound, wherein the structure of the compound is shown as (I):
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.
And (3) performing reversed-phase MPLC gradient elution with acetonitrile of 20-100% for 120min.
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 application of the oxazole compound in preparing a staphylococcus aureus inhibitor.
Compared with the prior art, the invention has the advantages that: the oxazole iron carrier compound is separated out, a fermentation product of oxazole iron carriers is obtained through microbial fermentation culture, then the fermentation product is soaked and extracted by ethyl acetate to obtain a 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 (Steptomyces sp.4-7) is NBU2966 strain with the preservation number of CCTCC NO: m2020953, deposited in China center for type culture Collection (CGMCC) at 12/21/2020, with the deposition address of Wuhan university, wuhan, china.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
The structural formula of the oxazole siderophore compound is shown as the following (I):
example 2
The preparation method of the oxazole siderophore compound shown in the structural formula (I) in the embodiment 1 comprises the following steps:
(1) Fermentation production
The preservation number is CCTCC NO: marking marine actinomycetes (Steptomyces sp.4-7) of M2020953 on a plate of a Gao's No. 1 solid culture medium, culturing and activating in a 28 ℃ incubator for 7 days, selecting a single colony, inoculating in a Gao's No. 1 liquid culture medium, culturing and increasing bacteria on a shaker at the temperature of 28 ℃ and the rotating speed of 180rpm/min, 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 at the temperature of 28 ℃ for 30 days 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, and adding a solution prepared from the following components 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, adding 200-300 mesh silica gel for mixing samples after dissolving, performing normal-phase medium-pressure column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with a volume ratio of (100; subjecting the collected 8 th component to reversed-phase medium-pressure column chromatography gradient elution, collecting elution fractions by using acetonitrile-water with a 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 10 components; and (3) separating and purifying the obtained 6 th component by using a solution of acetonitrile and water in a volume ratio of 17 to 83 as a mobile phase through semi-preparative reverse phase high performance liquid chromatography to obtain a compound, wherein the structure of the compound is shown as (I):
the compound I of the invention is white powder, and the high resolution mass spectrum (HR-ESI-MS) under the positive ion mode gives the excimer peak m/z 290.0603[ M ] +H ] thereof] + . Bonding with 13 C NMR spectrum to determine its molecular formula as C 13 H 11 N 3 O 3 S, of the compound 1 H and 13 the C NMR spectral data are shown in Table 1:
TABLE 1D NMR data for Compound I (DMSO-D6)
| No. | δ H .multi.(Jin Hz) | δ C .type |
| 1 | - | 110.0.C |
| 2 | - | 156.3.C |
| 3 | 7.08.d(8.4) | 117.1.CH |
| 4 | 7.45.t(7.6) | 133.1.CH |
| 5 | 7.02.t(7.6) | 119.8.CH |
| 6 | 7.84.d(7.8) | 127.5.CH |
| 2’ | - | 160.8.C |
| 4’ | - | 134.4.C |
| 5’ | 8.88.s | 140.4.C |
| 2” | - | 159.6.C |
| 4” | 5.18.t(91) | 79.2.CH |
| 5” | 3.63.m | 34.3.CH 2 |
| 6” | - | 171.7.C |
| NH a | 7.40.s | - |
| NH b | 7.43.s | - |
| 2-OH | 10.56.s | - |
Note 1: s-singlet, d-doublet, t-triplet, m-multiplet; 1 h is in 600MHz NMR; 13 c was obtained by 150MHz NMR.
Example 3
Activity and application of oxazole Compound 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 by 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 assay, 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 present invention, and the present invention is not limited to the above examples. 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. An oxazole siderophore compound, which is characterized in that the structural formula of the oxazole siderophore compound is shown as (I);
2. a method for preparing an oxazole siderophore compound as claimed in claim 1, characterized by comprising the steps of:
(1) Fermentation production
The preservation number is CCTCC NO: marking marine actinomycetes (Steptomyces sp.4-7) of M2020953 on a plate of a Gao's No. 1 solid culture medium, culturing and activating in a 28 ℃ incubator for 7 days, selecting a single colony, inoculating in a Gao's No. 1 liquid culture medium, culturing and increasing bacteria on a shaker at the temperature of 28 ℃ and the rotating speed of 180rpm/min, 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 percent by volume, and culturing at the temperature of 28 ℃ for 30 days 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 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, adding 200-300 mesh silica gel for mixing samples after dissolving, performing normal-phase medium-pressure column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with a volume ratio of (100; subjecting the collected 8 th component to reversed-phase medium-pressure column chromatography gradient elution, collecting elution fractions by using acetonitrile-water with a 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 10 components; and (3) separating and purifying the obtained 6 th component by using a solution of acetonitrile and water in a volume ratio of 17 to 83 as a mobile phase through semi-preparative reverse phase high performance liquid chromatography to obtain a compound, wherein the structure of the compound is shown as (I):
3. the method for preparing an oxazole siderophore compound according to claim 2, characterized in that: and (3) performing reversed-phase MPLC gradient elution with acetonitrile of 20-100% for 120min.
4. The method for preparing an oxazole siderophore compound according to claim 2, characterized in that: 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 oxazole compound as defined in claim 1 for preparing a staphylococcus aureus inhibitor.
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