CN113308407B

CN113308407B - Deep sea streptomycete, tianyamycin series compound and application thereof

Info

Publication number: CN113308407B
Application number: CN202110668740.2A
Authority: CN
Inventors: 韩壮; 王佳伟; 翟吉安; 高兆明
Original assignee: Institute of Deep Sea Science and Engineering of CAS
Current assignee: Institute of Deep Sea Science and Engineering of CAS
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2023-08-18
Anticipated expiration: 2041-06-16
Also published as: CN113308407A

Abstract

The application relates to the field of biological medicine, in particular to streptomyces abyssal and tiaryamycin series compounds and application thereof. The application separates 4 compounds (compounds 1-4) from a fermentation culture of a strain Streptomyces sp.LDBS7850 with anti-pathogenic bacteria activity, which is derived from Ma Liya nano-sea ditch sediment, and the structural identification of the compounds 1-4 is carried out to obtain novel tetramic acid compounds which are named as Tianyamycin A-D. These four compounds showed strong inhibitory MRSA activity. In addition, these four compounds have varying degrees of inhibitory effect on methicillin-sensitive staphylococcus aureus, enterococcus faecalis.

Description

Deep sea streptomycete, tianyamycin series compound and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to streptomyces abyssal and tiaryamycin series compounds and application thereof.

Background

Since the first discovery of the antibiotic penicillin, the chinese, by the uk scientist alexander fleming (alexander. Fleming), in 1928, antibiotics have played a tremendous role in the infectious disease in humans. However, the overabundance of antibiotics and overuse has led to an increasing variety of resistant and multi-resistant bacteria. Drug-resistant bacterial infection has become a non-negligible public health problem, and poses a great threat to human health. The World Health Organization (WHO) has called for and encouraged the development of new antibiotics many times, and thus, society has urgent needs for some new antibiotics with unique mechanisms.

With the full exploitation and utilization of the landed microorganism resources, the discovery of novel natural products presents a bottleneck, which makes the development of novel antibiotics more difficult. "ocean going to the essential drugs" is a new trend. Marine environments have microbial living environments that are incomparable to land environments, and this extreme environment determines the species diversity, genetic diversity, and metabolic diversity of marine microorganisms. The Ma Liya nanometer sea ditch is known to be the deepest part of the ocean, has high water pressure, complete darkness, low temperature and low oxygen content, is one of the most severe areas on the earth, develops microbial resources of the areas, and is more hopeful to discover active metabolites with novel structures.

Disclosure of Invention

In view of this, the present application provides Streptomyces abyssal, tianyamycin series compounds and uses thereof. The application separates 4 compounds (compounds 1-4) from a fermentation culture of a strain Streptomyces sp.LDBS7850 with anti-MRSA activity, which is derived from Ma Liya nano-sea ditch sediment, and the structural identification of the compounds 1-4 is carried out to obtain novel tetramic acid compounds, which are named as Tianyamycin A-D. These four compounds showed strong inhibitory MRSA activity. In addition, these four compounds have varying degrees of inhibitory effect on methicillin-sensitive staphylococcus aureus, enterococcus faecalis.

In order to achieve the above object, the present application provides the following technical solutions:

the application provides Streptomyces sp.LDBS7850 with a preservation number of CCTCC NO: m2021363.

The application also provides application of the Streptomyces sp.LDBS7850 in fermentation preparation of tetramic acid compounds.

In some embodiments of the application, the tetramic acid-type compound has a structural formula as shown in formula I or formula II:

wherein:

R ₁ selected from:

R ₂ selected from:

R ₃ selected from:

H CH ₃ CH ₂ CH ₃ ；

R ₄ or R is ₆ Independently selected from:

H CH ₃ CH ₂ CH ₃ OH；

R ₅ selected from:

H OH ＝O CH ₃ ；

R ₇ selected from:

the application also provides a compound or an isomer or salt thereof, which is a tetramic acid compound, and the structural formula of the tetramic acid compound is shown as formula I or formula II:

wherein:

R ₁ selected from:

R ₂ selected from:

R ₃ selected from:

H CH ₃ CH ₂ CH ₃ ；

R ₄ or R is ₆ Independently selected from:

H CH ₃ CH ₂ CH ₃ OH；

R ₅ selected from:

H OH ＝O CH ₃ ；

R ₇ selected from:

in some embodiments of the application, the compound or isomer or salt thereof has a structural formula as shown in any one of formulas III-VI:

the application also provides a preparation method of the compound or the isomer or the salt thereof, and the Streptomyces deep sea sp.LDBS7850 is adopted for fermentation culture to obtain a fermentation culture; extracting, and separating to obtain the compound.

In some embodiments of the application, the fermentation medium employed for the fermentation comprises, per L:

10g of soluble starch, 5g of yeast powder, 3g of tryptone, 21g of sea salt and 1L of ddH ₂ O, pH 7.2-7.6; and/or

The extraction comprises the following steps:

step 1: separating the fermentation culture to obtain thalli and fermentation liquor;

step 2: taking the fermentation broth, and carrying out equal volume of ethyl acetate: extracting with n-hexane (volume ratio of 1:1) for 3 times, collecting extractive solution, evaporating to dryness to obtain fermentation broth extract, and preserving at-20deg.C;

step 3: adding 2-4 times of the volume of the thallus into a methanol-acetone (volume ratio is 1:4) mixed solution, carrying out ultrasonic treatment for 20min, and filtering and collecting a leaching solution; repeating for 3 times; concentrating the leaching solution in vacuum at 34 ℃ and 150-300hPa until the solid-liquid ratio in a rotary steaming bottle is 1:2, and adding 50mL of ultrapure water for dissolution; adding an equal volume of ethyl acetate-n-hexane (1:1) mixed solution for extraction for 3 times, and evaporating the extract at 34 ℃ and 100-300hPa to prepare a bacterial extract;

step 4: combining the thallus extract and the fermentation broth extract to obtain a crude extract; and/or

The separation comprises the following steps:

step 1: separating the crude extract with C18 reverse chromatographic column, sequentially eluting with gradient concentration methanol-water system (50%, 70%,90%, 100%), and collecting the eluates of each gradient to obtain crude fraction;

step 2: performing TLC thin layer chromatography on the crude fractions respectively, developing with chloroform and methanol, soaking in 10% sulfuric acid ethanol solution, and baking at 170deg.C for color development;

step 3: eluting with absolute ethyl alcohol by adopting SephadexLH20 of a sephadex column;

step 4: semi-preparative high performance liquid chromatography with 90-100% ACN/H ₂ O,0.05% TFA as mobile phase, to afford the compound.

More importantly, the application also provides application of the deep sea Streptomyces sp.LDBS7850 or the compound or the isomer or salt thereof in preparing bacteriostatic agent and/or drug-resistant bacteria resistant drugs.

In some embodiments of the application, the bacteria include enterococcus faecalis H119, methicillin-resistant staphylococcus aureus H57 (MRSA), methicillin-sensitive staphylococcus aureus G280 (MSSA).

More practically, the application also provides a bacteriostatic agent and/or a drug for resisting drug-resistant bacteria, which comprises a fermentation culture of Streptomyces sp.LDBS7850 and/or the compound or an isomer or salt thereof, and pharmaceutically acceptable auxiliary materials.

The inventor separates and purifies a compound Tianyamycin series compound with a new structure from a microbial strain which is preserved in a laboratory and is derived from a Mariana sea ditch. Subsequent researches show that the Tianyamycin series compounds have strong anti-Methicillin-resistant staphylococcus aureus (Methicillin-resistantStaphylococcus aureus, MRSA) growth inhibition activity and can be used for developing medicines for treating MRSA infection. In addition, the Tianyamycin compound has different degrees of inhibition effects on methicillin-sensitive staphylococcus aureus and enterococcus faecalis.

Description of biological preservation

Biological material: streptomyces LDBS7850, classified and named: streptomyces sp.LDBS7850 was deposited at China Center for Type Culture Collection (CCTCC) with the accession number CCTCC NO: m2021363.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows an HPLC profile of a fermented crude extract of Streptomyces sp.

FIG. 2 shows the chemical structures of compounds 1-4;

FIG. 3 shows the chemical structure of Compound 1;

FIG. 4 shows the principle of Compound 1 ¹ H- ¹ H COSY, HMBC and NOESY related signals;

FIG. 5 shows the chemical structure of Compound 2;

FIG. 6 shows the principle of Compound 2 ¹ H- ¹ H COSY, HMBC and NOESY related signals;

FIG. 7 shows the chemical structure of Compound 3;

FIG. 8 shows the chemical structure of Compound 4;

FIG. 9 shows MIC test results for compounds 1-4;

FIG. 10 shows mass spectra of Compound 1;

FIG. 11 shows mass spectra of Compound 2;

FIG. 12 shows mass spectra of Compound 3;

FIG. 13 shows mass spectra of Compound 4;

FIG. 14 shows compounds 1 and 3 ¹ H NMR(500MHZ，DMSO-d ₆ ) A spectrogram;

FIG. 15 shows compounds 1 and 3 ¹³ C NMR(201MHZ，Chloroform-d ₆ ) A spectrogram;

FIG. 16 shows 135 and 90 degree DEPT profiles for compounds 1 and 3;

FIG. 17 shows HSQC patterns for Compounds 1 and 3;

FIG. 18 shows compounds 1 and 3 ¹ H- ¹ H COSY pattern;

FIG. 19 shows NOESY patterns of Compounds 1 and 3;

FIG. 20 shows HMBC patterns of Compounds 1 and 3;

FIG. 21 shows compounds 2 and 4 ¹ H NMR(500 MHz,DMSO-d ₆ ) A spectrogram;

FIG. 22 shows compounds 2 and 4 ¹³ C NMR(201 MHz,Chloroform-d ₆ ) A spectrogram;

FIG. 23 shows 135 and 90 degree DEPT profiles for compounds 2 and 4;

FIG. 24 shows HSQC patterns for Compounds 2 and 4;

FIG. 25 shows compounds 2 and 4 ¹ H- ¹ H COSY pattern;

FIG. 26 shows NOESY patterns of Compounds 2 and 4;

fig. 27 shows HMBC profiles for compounds 2 and 4.

Detailed Description

The application discloses a streptomyces abyssal and tiaryamycin series compound and application thereof, and a person skilled in the art can refer to the content of the compound and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present application. While the methods and applications of this application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this application, without departing from the spirit or scope of the application.

The first purpose of the application is that: a tetramic acid compound having an activity against pathogenic bacteria growth is provided.

The structural formula of the tetramic acid compound is shown as formula I:

the second object of the application is: provides Streptomyces sp.LDBS7850 which is a deep sea mold capable of producing the tetramic acid compound.

The third object of the present application: provides a preparation method of the compounds 1-4 shown in the formula I. The method is characterized by comprising the following steps.

a. Preparing a fermentation culture of Streptomyces sp.LDBS7850;

b. and separating the fermentation culture to obtain thalli and fermentation liquor. Equal volume of ethyl acetate in the broth: extracting with n-hexane (1:1) mixture for 3 times, collecting extractive solution, evaporating to dryness, and storing in a refrigerator at-20deg.C. Adding 2-4 times of thallus volume of methanol-acetone (1:4) mixed solution into thallus, performing ultrasonic treatment for 20min, and filtering to collect leaching solution; the thallus extraction is repeated for 3 times; pouring the collected 1500mL leaching liquor into a rotary steaming bottle, vacuum concentrating at 34 ℃ and 150-300hPa until the solid-liquid ratio in the rotary steaming bottle is 1:2, and adding ultrapure water for full dissolution; then, adding an equal volume of ethyl acetate-n-hexane (1:1) mixture for extraction for 3 times, adding the extract into a rotary steaming bottle, steaming at 34 ℃ and 100-300hPa, and weighing. And combining the thallus extract and the fermentation liquor extract to obtain a crude extract.

c. Separating the crude extract by C18 reverse chromatographic column, sequentially eluting with gradient concentration methanol-water system (50%, 70%,90%, 100%), and eluting with 600mL each gradient; performing TLC thin layer chromatography on the crude fractions respectively, developing with chloroform and methanol, soaking in 10% sulfuric acid ethanol solution, and baking at 170deg.C for color development; then Sephadex LH20 (absolute ethyl alcohol elution) is adopted for the next separation; finally, the mixture was prepared by semi-preparative high performance liquid chromatography (90-100% ACN/H ₂ O,0.05% tfa) to give pure compound.

Preferably, the Streptomyces deep seaThe fermentation culture of the sp.LDBS7850 is obtained by inoculating Streptomyces abyssal sp.LDBS7850 into a fermentation medium for fermentation, wherein each liter of the fermentation medium contains: 10g of soluble starch, 5g of yeast powder, 3g of tryptone, 21g of sea salt and 1L of ddH ₂ O；pH 7.2-7.6。

A fourth object of the application is to provide the use of Streptomyces sp.

The fifth object of the application is to provide the application of any one of the compounds 1-4 shown in the formula I or the pharmaceutically acceptable salt thereof in preparing the drug-resistant bacteria.

Preferably, the antibacterial agents described herein are anti-methicillin-resistant staphylococcus aureus, methicillin-sensitive staphylococcus aureus, enterococcus faecalis agents.

A fifth object of the present application is to provide an anti-three pathogenic bacteria agent as described above, characterized by comprising an effective amount of any one of the compounds 1 to 4 represented by the formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier;

4 compounds (compounds 1-4) were isolated from a fermentation culture of the strain Streptomyces sp.LDBS7850 having anti-MRSA activity from Ma Liya nanotrench sediment, and the structure of the compounds 1-4 was identified as tetramic acid new compounds, named TianyamycinA-D (FIG. 2), which showed strong MRSA inhibition activity. In addition, these four compounds have varying degrees of inhibitory effect on methicillin-sensitive staphylococcus aureus, enterococcus faecalis. We tested the Minimum Inhibitory Concentration (MIC) on tiaryamycina-D using a double dilution method, and the data indicate that tiaryamycina-D has mic=0.125 μg/mL for MRSA, mic=0.125 μg/mL for methicillin-sensitive staphylococcus aureus, and mic=2 μg/mL for positive control vancomycin, demonstrating that the compound has stronger anti-MRSA activity than vancomycin, with the potential to be further developed into a novel antimicrobial.

In addition, the mic=0.625 μg/mL for TianyamycinA-D against enterococcus faecalis, the MIC >32 μg/mL for imipenem-resistant pseudomonas aeruginosa, imipenem-resistant acinetobacter baumannii, ESBLs-positive escherichia coli, ESBLs-negative escherichia coli, enterococcus faecium, imipenem-sensitive acinetobacter baumannii, imipenem-sensitive pseudomonas aeruginosa, ESBLs-positive klebsiella pneumoniae, ESBLs-negative klebsiella pneumoniae.

The deep sea streptomycete, the Tianyamycin series compounds and the application thereof provided by the application can be obtained from the market by using raw materials and reagents.

The application is further illustrated by the following examples:

example 1: streptomyces sp.LDBS7850 culture and 16s rRNA gene identification

1. Isolation and cultivation of Streptomyces sp.LDBS7850 strain.

Solid culture of 1.Streptomyces sp.LDBS7850:

the 1.1Streptomyces sp.LDBS7850 strain was isolated from sediment at 7850m depth in Ma Liya nm, and was maintained in Marine Broth2216 glycerol medium.

2. 16s rRNA gene sequence analysis of Streptomyces sp.

2.1 Extraction of Streptomyces sp.LDBS7850 Strain genomic DNA, PCR amplification of 16s rRNA Gene, sequencing and species identification. The genome of Streptomyces sp.LDBS7850 was extracted using a DNA extraction kit (TIANGEN) and a part was used as a template, and PCR amplification was performed using a 16S universal primer and Taq enzyme from TAKARA under conditions such as annealing temperature of 55 ℃. And sending the amplified PCR product to China big gene company for bidirectional sequencing. The sequences obtained were then spliced and analyzed by an EzBiocloud online database BLAST alignment.

2.1.1 16s rRNA gene PCR amplification system and amplification procedure:

amplification system:

deionized water: 16.75 μl, water template DNA: 1. Mu.L of each of the upstream and downstream primers, 0.5. Mu.L of dNTP:1 μL,10 xBuffer: 2.5 μL, mgCl2:1.5 μL, taq:0.25 μL; a total of 25. Mu.L of system.

Primer:

27F：AGAGTTTGATCCTGGCTCAG

1492R：TACGGCTACCTTGTTACGACTT

PCR amplification procedure:

95℃*5min+(95℃*30sec+55℃*30sec+72℃*60sec)*30cycle+72℃*5min。

2.1.2 Sequencing of PCR products

And (3) sending the amplified PCR product to a Hua big gene sequencing company for bidirectional sequencing, and performing sequence splicing and online BLAST comparison analysis on a sequence result by using an online sequence splicing program of the Hua big gene company, wherein the sequence of the 16srRNA is shown in a sequence table.

The 16s rRNA gene (the nucleotide sequence of which is shown in the sequence table) is sequenced and morphologically (the growth morphology of which on a Marine Broth2216 medium is shown in figure 1) is analyzed and identified, and the similarity of the 16s rRNA sequence and Streptomyces tendae streptomyces is 100%, and the sequence is named: streptomyces sp.LDBS7850.

Example 2: the use of Streptomyces sp.LDBS7850 for the production of the series of compounds of the Tianyamycin family.

1. Fermentation, extraction of Streptomyces sp.LDBS7850 and extraction and separation of Tianyamycin.

(1) Fermentation: the strain Streptomyces sp.LDBS7850 glycerol was taken out from the refrigerator at-80℃and inoculated onto a Marine Broth2216 plate by the inoculating loop streaking method, incubated in a constant temperature incubator at 30℃for 4-5 days until a large amount of white spores grew, and a small amount of spores were picked up to 50mL of SPY medium (10 g of soluble starch; 5g of yeast extract; 3g of tryptone; 21g of sea salt; 1LddH2O; pH 7.2-7.6) by the inoculating loop and shake-incubated for 3 days (180 rpm,34 ℃) to obtain a seed solution. 50mL of the seed solution was inoculated into 1L of SPY fermentation medium, and cultured at constant temperature for 7 days (180 rpm,34 ℃).

(2) Extraction: 6L of fermentation broth LDBS7850 was subjected to suction filtration with a Buchner funnel for 7 days, and the fermentation broth and the cells were separated. After separation of the cells from the broth, the broth was treated with an equal volume of ethyl acetate: extracting with n-hexane (1:1) mixture for 3 times, collecting extractive solution, evaporating to dryness, and storing in a refrigerator at-20deg.C. Adding 2-4 times of thallus volume of methanol-acetone (1:4) mixed solution into thallus, performing ultrasonic treatment for 20min, and filtering to collect leaching solution; the thallus extraction is repeated for 3 times; pouring the collected 1500mL of leaching liquor into a rotary steaming bottle, concentrating in vacuum at 34 ℃ and 150-300hPa until the solid-liquid ratio in the rotary steaming bottle is 1:2, and adding 50mL of ultrapure water for full dissolution; then adding an equal volume of ethyl acetate-n-hexane (1:1) mixed solution for extraction for 3 times, evaporating the extract at 34 ℃ and under the condition of 100-300hPa, and weighing to obtain a crude extract.

(3) Separating the crude extract: the extract was first subjected to preliminary separation using a C18 reverse chromatography column, and 600mL was eluted sequentially with a gradient concentration methanol-water system (50%, 70%,90%, 100%) and 100% acetonitrile, and dried by evaporation to give 5 fractions fr.1 (2.335 g), fr.2 (0.1903 g), fr.3 (0.2007 g), fr.4 (0.4498 g), fr.5 (0.1967 g). The 5 fractions were each analyzed by TLC thin layer chromatography, fr.2 developed with chloroform: methanol (9:1); fr.3 using chloroform: acetone (11:1) development; fr.4 using chloroform: developing with methanol (3:1), soaking in 10% sulfuric acid ethanol solution, and heating at 170deg.C for developing. Mixing Fr.4 and Fr.5 obtained in the previous step, evaporating to dryness, separating in the second step with SephadexLH20 column, and preparing the separated mixture by semi-preparative high performance liquid chromatography (90-100% ACN/H) ₂ O,0.05% TFA) 38min,3mL/min gave compound 1 (11.0 mg), compound 2 (13.0 mg), compound 3 (2.9 mg), compound 4 (3.8 mg).

2. Structural analysis and identification of compounds

The structure of the compounds 1-4 (figure 2) is determined by one-dimensional, two-dimensional nuclear magnetic resonance (1D, 2D-NMR) and liquid chromatography-mass spectrometry (LC-MS), which are Tetramic acid derivatives, and 1 and 3,2 and 4 are keto-enol tautomers.

TianyamycinA (Compound 1)

Brown solid of formula C ₂₄ H ₃₉ NO ₅ ，ESI-HRMS m/z 422.2985[M+H] ⁺ ，444.2806[M+Na] ⁺ (calculated 422.2901[ M+H ]] ⁺ ，444.2721[M+Na] ⁺ ). The unsaturation of the compound was calculated to be 6 according to the formula. The nuclear magnetic data of the compounds are shown in Table 1. Nuclear magnetic resonance carbon spectrum ¹³ C NMR data indicated a low field with three carbons delta _C 198.15,184.11,164.43; three carbon signals delta _C 134.03,114.31,103.75 is presumed to be an alkenyl carbon, with one hydroxy-substituted olefinic carbon also being present in the lower three carbons; the above groups contributed 4 unsaturations, the remaining 2 unsaturations, indicating that the compound has 2 more cyclic structures. The UV absorption spectrum of the compound has strong absorption at 220nm and 271nm, which indicates that amide, ketone and enol structures exist. HMBC spectra show H-7 (delta) _H 2.20,3H) and C-5 (delta) _C 134.03 And C-6 (delta) _C 114.31 A correlation exists between; h-6 (delta) _H 5.53, d, 1H) and C-5 and C-4 (delta) _C 184.11 Is related even to C-3 (delta) _C 103.75 A weak W-shape exists ⁴ J CH correlation, the above correlation signals indicate that compound 1 has a 3-acyltetramic acid five-membered ring structure and that the acyl group is ortho to C-5 (delta) _C 134.03 Through olefinic bond C-6 (delta) _C 114.31 To methyl C-7 (. Delta.C12.80). ¹ H- ¹ H-13 (delta H1.47,1.57,2H) and H-12 (delta) in the H COSY spectra _H 3.92,1H) and H-14 (delta) _H 3.46,1H) are related, H-10 (delta) _H 3.51,1H) and H-9 (delta) _H 3.96,1H) and H-11 (delta) _H 1.44, overlapped); h-23 in HMBC (delta) _H 1.13, overlapped) with C-12 (δC68.59) and C-13 (δC44.57); according to ¹³ C-10, C-12 and C-14 (. Delta.) in C NMR _C 79.44 Chemical shift, the three methines are attached to oxygen atoms; in summary, it is assumed that there is a 2,3,4, 6-tetrasubstituted tetrahydropyran ring, wherein C-11 (delta) _C 36.59 C-12 are each methyl C-23 (. Delta.) and C-12 _C 23.26 And hydroxy substitution. ¹ H- ¹ In the H COSY spectrum, H-24 (delta) _H 1.12,3H) is associated with H-9; HMBC shows H-24 and C-8 (delta) _C 198.15 C-9 and C-10, H-9 being associated with C-3, C-8, C-10, C-24 and C-11; the above association identifies that the tetra-substituted tetrahydropyran ring and the 3-acyltetramic acid five membered ring are linked by C-8 and C-9, wherein C-8 is a hydroxy substituted olefinic carbon. ¹³ The remaining 2 symmetrical methyl groups, 5 methylene groups and 1 methine group signals in C NMR, ¹ chemical shifts δ1.10-1.60 in H NMR are highly overlapping fat regions. Indicating that the compound also has one isooctane fatty chain. ¹ H- ¹ H-14 in H COSYH-15(δ _H 1.26,1.53, overlapped) indicating that the branched fatty chain is linked to the tetrahydropyran ring through C-14. The planar structure of compound 1 was finally presumed to be shown in FIG. 3, which is a novel compound designated as TianyamycinA.

From the NOESY spectrum data of the compounds, the partial relative configuration of the compounds can be determined. H-10 and H-11 of the six-membered tetrahydropyran ring moiety are related, demonstrating that they are on the same side; h-12 is associated with H-14 and H-23 on the same side. The H-25 and H-6 correlations demonstrate that Δ5,6 is in the E configuration. Whereas the relative configuration of C-8 and C-9 could not be measured by NOESY. It was found in experiments that this compound was left at room temperature for 1-2 days, partially changed to compound 3, keto-enol tautomer, and that a pair of compound signals was present in NMR tested in this paper, with a ratio of about (1:3=10:7) in deuterated chloroform.

Tianyamycin B (Compound 2)

Brown oil of formula C ₂₄ H ₃₉ NO ₅ ，ESI-HRMS m/z 422.2975[M+H] ⁺ ，444.2796[M+Na] ⁺ . The nuclear magnetic data of the compounds are shown in Table 2. methyl-CH based on 135 degree and 90 degree DEPT spectra information ₃ 25.14,23.24,14.25,12.80,7.79 methylene-CH ₂ 44.45,32.53,32.09,30.59,29.49,28.93,26.44,23.24, methyne-CH 114.33,82.93,79.44,68.62,40.23,36.62, tetrasubstituted carbon-C198.17,184.09,164.44,134.02,103.75. Compound 2 has the same molecular weight and ultraviolet absorption as compound 1, has the same molecular formula and skeleton structure, and compound 2 in DEPT diagram has one methyl delta less than compound 1 _C 22.87 (C-21) and one methine delta C28.21 (C-20), 2 methylene delta _C 30.09 (C-20), δC22.80 (C-21), it is presumed that the isooctane of the side chain is changed to linear octane. ¹ H- ¹ H COSY and HMBC demonstrate our speculation (FIG. 2). Thus, compound 2 has a planar structure as shown in FIG. 5, and is a novel compound designated as Tianyamycin B.

Tianyamycin C (Compound 3)

Brown solid of formula C ₂₄ H ₃₉ NO ₅ ，ESI-HRMS m/z 422.3014[M+H] ⁺ ，444.2843[M+Na] ⁺ The compound is the same as the compound 1Tautomers largely become compound 1 after 1-2 days of standing at room temperature. Of compounds ¹ H NMR ¹³ C NMR is shown in Table 1. The chemical shifts of C-2, C-8 and C-9 were found to vary greatly by comparison with the data for Compound 1 (comparison of data for 1 and 3). C-8 of compound 1 changes from enol to keto, and keto C-2 changes from enol to enol in compound 3. The final structure is shown in FIG. 7, designated as Tianyamycin C, as a novel compound.

Tianyamycin D (Compound 4)

Brown solid of formula C ₂₄ H ₃₉ NO ₅ ，ESI-HRMS m/z 422.2975[M+H] ⁺ ，444.2977[M+Na] ⁺ The compound is tautomeric with compound 2 and becomes mostly compound 2 after 1-2 days of standing at room temperature. Of compounds ¹ H NMR ¹³ C NMR is shown in Table 2. The chemical shifts of C-2, C-8 and C-9 were found to vary greatly by comparison with the data for compound 2 (comparison of data for 2 and 4). C-8 of compound 2 changes from enol to keto, and keto C-2 changes from enol to enol in compound 4. The final structure is shown in FIG. 8, designated as Tianyamycin D, as a novel compound.

TABLE 1 Compounds 1 and 3 ¹³ C NMR(200MHz,Chloroform-d ₆ ) And ¹ H NMR(500MHz,

DMSO-d ₆ ) Data

TABLE 2 Compounds 2 and 4 ¹³ C NMR(200MHz,Chloroform-d ₆ ) And ¹ H NMR(500MHz,DMSO-d ₆ ) Data

Table 3 mass spectrometry data for compounds 1-4

Example 3: test analysis of antibacterial Activity of Compounds 1-4 shown in formula I

1. Antibacterial activity detection indicator

The antibacterial activity detection indicator used in this section is as follows:

TABLE 4 Table 4

2. Biological Activity test

(1) Antibacterial Activity experiment

1) Activation of indicator bacteria: the experimental pathogenic bacteria are inoculated from a gravity maintaining tube to an LB liquid culture medium for activation for 24 hours, then inoculated on an LB flat plate by a streaking method, and placed in a constant temperature incubator for culture at 30 ℃ for overnight. And (3) selecting single colonies, inoculating the single colonies to an LB liquid culture medium, and placing a constant-temperature shaking table at 37 ℃ for culture at 220rpm overnight for later use.

2) The compound sample to be tested is dissolved and diluted by methanol to prepare a solution with the concentration of 1 mg/mL. The positive control vancomycin is prepared into 1mg/mL solution and filtered for later use.

3) Culturing the indicator bacteria at 37 ℃ to OD ₆₀₀ =1.0 to 1.4, the bacterial solution was diluted 1000-fold, 200 μl of the diluted solution was pipetted into LB solid plates with a pipette, and uniformly coated with a coating bar.

4) Taking a corresponding number of sterilized filter paper sheets on a sterile empty flat plate, respectively dripping 5 mu L of methanol (negative control), vancomycin (positive control) and a sample to be tested on the filter paper sheets, and repeating dripping once after the solution dripped for the first time is completely dried.

5) The dried filter paper sheet was taken with forceps and placed on an LB plate, and the filter paper sheet was gently pressed to fix it on the medium while the label was written on the back. Placing in a constant temperature incubator at 37 ℃ for culturing for 12 hours.

(2) Minimum inhibitory concentration experiment (minimuminhibitory concentration, MIC)

1) Marking and activating pathogenic bacteria by using LB, placing the pathogenic bacteria into a constant temperature incubator at 37 ℃ for culturing for 24 hours, picking 2-3 single colonies, and culturing for 24 hours by using MH culture medium at 37 ℃.

2) Sucking the bacterial liquid, diluting the bacterial liquid to 0.5 McAb turbidity by using sterile water, and diluting 1000 times by using MH culture medium to ensure that the concentration of the bacterial liquid reaches 10 ⁴ -10 ⁵ CFU/mL. Preincubation was carried out for 2 hours at 37 ℃.

3) The TianyamycinA-D was diluted in advance with a two-fold gradient of DMSO for 12 gradients. The initial concentration was 6.4. Mu.g/. Mu.L.

4) 199. Mu.L of pre-incubation bacteria solution was added to ninety-six well plates, and 1. Mu.L of a gradient diluted TianyamycinA-D DMSO solution was added. The final liquid volume per well was 200 μl. Two rows of parallel controls were made.

3. Results of biological Activity

(1) Antibacterial activity

The bacteriostatic activity experiment used in the application adopts a filter paper sheet method to detect the inhibition effect on the following 12 pathogenic bacteria: ESBLs positive Escherichia coli G106, ESBLs negative Escherichia coli K8, enterococcus faecalis H119, ampicillin sensitive enterococcus faecium G258, methicillin resistant staphylococcus aureus H57, methicillin sensitive staphylococcus aureus G280, imipenem sensitive Acinetobacter baumannii H422, imipenem resistant Acinetobacter baumannii E292, imipenem sensitive Pseudomonas aeruginosa E248, imipenem resistant Pseudomonas aeruginosa F197, ESBL positive Klebsiella pneumoniae F161, ESBL negative Klebsiella pneumoniae O244. Methanol was used as a negative control and vancomycin was used as a positive control, respectively.

The results show that 10 μg of Tianyamycin (compounds 1-4) has various degrees of inhibitory effects on enterococcus faecalis H119, methicillin-resistant Staphylococcus aureus H57 (MRSA), methicillin-sensitive Staphylococcus aureus G280 (MSSA). There was no significant inhibitory effect on the remaining ESBLs-positive escherichia coli G106, ESBLs-negative escherichia coli K8, enterococcus faecium G258, imipenem-sensitive acinetobacter baumannii H422, imipenem-sensitive pseudomonas aeruginosa E248, ESBLs-positive pneumogram Lei Bajun F161, ESBLs-negative klebsiella pneumoniae O244, imipenem-resistant acinetobacter baumannii E292, imipenem-resistant pseudomonas aeruginosa F197. The center point was 10 μg vancomycin as a positive control.

(2) Minimum Inhibitory Concentration (MIC)

We tested the Minimum Inhibitory Concentration (MIC) on tiaryamycina-D using a double dilution method, and the data indicate that tiaryamycina-D has mic=0.125 μg/mL for MRSA, mic=0.125 μg/mL for MSSA, and mic=2 μg/mL for the positive control vancomycin, demonstrating that the compound has stronger anti-MRSA activity than vancomycin, with the potential to be further developed into a novel antimicrobial. In addition, the MIC of TianyamycinA-D against enterococcus faecalis H119=0.625 μg/mL.

The TianyamyccinA-D is against imipenem drug-resistant pseudomonas aeruginosa F197, imipenem drug-resistant Acinetobacter baumannii E292, ESBLs positive escherichia coli G106, ESBLs negative escherichia coli K8, enterococcus faecium G258, imipenem sensitive Acinetobacter baumannii H422, imipenem sensitive pseudomonas aeruginosa E248, ESBls positive klebsiella pneumoniae F161, ESBLs negative klebsiella pneumoniae O244 MIC >32 mug/mL. MIC test results are shown in fig. 9.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Sequence listing

<110> institute of deep sea science and engineering of China academy of sciences

<120> Streptomyces deep sea, tianyamycin series compounds and uses thereof

<130> MP21003555

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1557

<212> DNA

<213> Streptomyces tendae LDBS7850

<400> 1

acggagagtt tgatcctggc tcaggacgaa cgctggcggc gtgcttaaca catgcaagtc 60

gaacgatgaa ccacttcggt ggggattagt ggcgaacggg tgagtaacac gtgggcaatc 120

tgccctgcac tctgggacaa gccctggaaa cggggtctaa taccggatac tgaccctcgc 180

aggcatctgc gaggttcgaa agctccggcg gtgcaggatg agcccgcggc ctatcagctt 240

gttggtgagg taatggctca ccaaggcgac gacgggtagc cggcctgaga gggcgaccgg 300

ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc 360

acaatgggcg aaagcctgat gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta 420

aacctctttc agcagggaag aagcgaaagt gacggtacct gcagaagaag cgccggctaa 480

ctacgtgcca gcagccgcgg taatacgtag ggcgcaagcg ttgtccggaa ttattgggcg 540

taaagagctc gtaggcggct tgtcacgtcg gttgtgaaag cccggggctt aaccccgggt 600

ctgcagtcga tacgggcagg ctagagttcg gtaggggaga tcggaattcc tggtgtagcg 660

gtgaaatgcg cagatatcag gaggaacacc ggtggcgaag gcggatctct gggccgatac 720

tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 780

cgtaaacggt gggcactagg tgtgggcaac attccacgtt gtccgtgccg cagctaacgc 840

attaagtgcc ccgcctgggg agtacggccg caaggctaaa actcaaagga attgacgggg 900

gcccgcacaa gcggcggagc atgtggctta attcgacgca acgcgaagaa ccttaccaag 960

gcttgacata caccggaaag catcagagat ggtgcccccc ttgtggtcgg tgtacaggtg 1020

gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080

acccttgtcc cgtgttgcca gcaggccctt gtggtgctgg ggactcacgg gagaccgccg 1140

gggtcaactc ggaggaaggt ggggacgacg tcaagtcatc atgcccctta tgtcttgggc 1200

tgcacacgtg ctacaatggc cggtacaatg agctgcgata ccgcaaggtg gagcgaatct 1260

caaaaagccg gtctcagttc ggattggggt ctgcaactcg accccatgaa gtcggagtcg 1320

ctagtaatcg cagatcagca ttgctgcggt gaatacgttc ccgggccttg tacacaccgc 1380

ccgtcacgtc acgaaagtcg gtaacacccg aagccggtgg cccaacccct tgtgggaggg 1440

agctgtcgaa ggtgggactg gcgattggga cgaagtcgta acaaggtagc cgtaccggaa 1500

ggtgcggctg gatcacctcc tttctaagga gcacatagcc gactgcaagc aaatgtc 1557

Claims

1. Application of Streptomyces sp LDBS7850 in fermentation preparation of tetramic acid compounds;

the preservation number of the Streptomyces abyssal (Streptomyces sp.) LDBS7850 is CCTCC NO: m2021363;

the structural formula of the tetramic acid compound is shown in any one of the formulas III to VI:

2. the compound is characterized in that the structural formula is shown in any one of the formulas III to VI:

3. the method for producing a compound according to claim 2, wherein a fermentation culture is obtained by fermenting and culturing Streptomyces sp.LDBS 7850; extracting, separating to obtain compound;

the preservation number of the Streptomyces abyssal (Streptomyces sp.) LDBS7850 is CCTCC NO: m2021363.

4. A method of preparing according to claim 3, wherein the fermentation medium employed for the fermentation comprises, per L:

10g of soluble starch, 5g of yeast powder, 3g of tryptone, 21g of sea salt and 1LddH ₂ O, pH 7.2-7.6; and/or

The extraction comprises the following steps:

step 2: taking the fermentation broth, and carrying out equal volume of ethyl acetate: extracting with n-hexane at volume ratio of 1:1 for 3 times, collecting extractive solution, evaporating to dryness to obtain fermentation broth extract, and preserving at-20deg.C;

step 3: adding 2-4 times of thallus volume of methanol-acetone into the thallus, mixing the liquid at a volume ratio of 1:4, performing ultrasonic treatment for 20min, and filtering to collect leaching solution; repeating for 3 times; concentrating the leaching solution in vacuum at 34 ℃ and 150-300hPa until the solid-liquid ratio in a rotary steaming bottle is 1:2, and adding 50mL of ultrapure water for dissolution; adding equal volume of ethyl acetate-n-hexane, extracting with mixed solution for 3 times at 34 deg.C and 100-300hPa, and evaporating to dryness to obtain thallus extract;

The separation comprises the following steps:

step 1: separating the crude extract with C18 reverse chromatographic column, sequentially eluting with methanol-water system with gradient concentration of 50%,70%,90%, and 100%, and collecting the eluates with each gradient to obtain crude fraction;

step 3: eluting with absolute ethanol by using Sephadex LH 20;

5. The use of a compound as claimed in claim 2 in the preparation of a bacteriostatic agent.

6. The use according to claim 5, wherein the bacteria comprise enterococcus faecalis, methicillin-resistant staphylococcus aureus MRSA or methicillin-sensitive staphylococcus aureus MSSA.

7. A bacteriostatic agent comprising a compound according to claim 2 and a pharmaceutically acceptable adjuvant.