A fungus-derived cyclic peptide compound and its preparation method and application

Abstract

The present invention discloses a cyclic peptide compound derived from fungi, and a preparation method and application thereof. The present invention isolates Aspergillus versicolor 2‑18 from the marine animal luxuriant membrane sponge, and obtains a new cyclic pentapeptide compound cyclo‑[N‑Me‑L‑Tyr‑L‑Val‑L‑Pro‑L‑Tyr‑L‑Phe] through fermentation, culture, extraction and separation, with a purity of 95%‑97%. The compound has a novel structure, a unique source and a simple preparation method. The cyclic peptide compound prepared by the present invention has a certain inhibitory effect on Escherichia coli and Staphylococcus aureus, provides a new lead compound for the research and development of new antibacterial drugs, and also provides a scientific basis for the development of marine fungal resources.

Description

Technical Field

[0001] The invention belongs to the technical field of marine microorganisms and natural product applications thereof, and specifically relates to a cyclic peptide compound derived from fungi and a preparation method and application thereof. Background Art

[0002] Peptide compounds have always been an important target for the development of new drugs due to their advantages such as low toxicity, strong targeting and small molecular weight. Among them, cyclic peptide compounds are cyclic compounds formed by amino acids connected end to end through peptide bonds. Due to their restricted and constrained conformation, cyclic peptide compounds have higher target selectivity, metabolic stability and membrane permeability compared with chain linear peptides, resulting in greatly improved bioavailability. Cyclic peptide compounds play an important role in the medical field as antibiotic drugs. Many cyclic peptide compounds such as vancomycin, daptomycin, polymyxin, etc. have been widely used clinically as antibacterial drugs. The discovery of new active lead compounds based on natural products has always been a fast and effective way to develop new drugs. At present, more and more cyclic peptide compounds have been discovered and identified from plants, marine organisms and microorganisms.

[0003] Marine fungi can tolerate the extreme environments of high salt, high pressure, low oxygen, and low light that are unique to the ocean. Compared with terrestrial fungi, the specificity of their living conditions leads to the diversity of marine fungi in species, genetic composition, and ecological functions. In addition, marine fungi are an important source of active natural products. According to statistics, 70-80% of marine fungal secondary metabolites exhibit various pharmacological activities and biological functions. In terms of the application value of marine fungi, marine fungal secondary metabolites are of great significance for the research and development of new cyclopeptide antibiotic drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a fungus-derived cyclic peptide compound and a preparation method and application thereof.

[0005] The structural formula of the cyclic peptide compound is as follows:

[0006] .

[0007] The fermentation preparation method of the cyclic peptide compound is as follows: the fungus Aspergillus versicolor 2-18 from marine animals is inoculated into a fungal liquid culture medium for fermentation and culture, and the cyclic peptide compound is obtained after separation and purification.

[0008] The Aspergillus versicolor 2-18 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 24, 2024. The address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41474.

[0009] The specific operation steps of the fermentation preparation method of the cyclic peptide compound are:

[0010] 1) Inoculating Aspergillus versicolor 2-18 into a fungal liquid culture medium and fermenting it for 20-50 days to obtain a fermentation product of the strain; filtering, extracting the fermentation broth with ethyl acetate, and concentrating it under reduced pressure to obtain a fermentation broth extract; extracting the mycelium with ethyl acetate, and concentrating it under reduced pressure to obtain a mycelium extract; combining the fermentation broth extract and the mycelium extract to obtain a total crude extract;

[0011] 2) subjecting the total crude extract in step 1) to normal phase silica gel column chromatography, gradient eluting with an organic solvent, collecting the eluate, and combining similar components after the eluate is detected by thin layer chromatography, wherein the organic solvent is petroleum ether-ethyl acetate or dichloromethane-methanol;

[0012] 3) The eluted components in step 2) with an eluent volume ratio of 20:1-10:1 are further separated and purified by reverse phase silica gel column chromatography and normal phase silica gel column chromatography, and the components with Rf of 0.4-0.6 are collected by thin layer chromatography to obtain cyclopeptide compounds.

[0013] The eluent for the reverse phase silica gel column chromatography in step 3) is methanol-water or ethanol-water; the eluent for the normal phase silica gel column chromatography is petroleum ether-ethyl acetate or dichloromethane-methanol.

[0014] The developing solvent for the thin layer chromatography detection in step 3) is petroleum ether-ethyl acetate or dichloromethane-methanol in a volume ratio of 30:1-1:1.

[0015] Application of the above cyclic peptide compounds in the preparation of antibacterial drugs.

[0016] The cyclic peptide compound of the present invention is a new cyclic pentapeptide compound obtained by fermentation, extraction and separation of Aspergillus versicolor 2-18 isolated from the marine animal luxuriant membrane sponge, with a purity of 95%-97%. The compound has a novel structure, a unique source and a simple preparation method. The cyclic peptide compound prepared by the present invention has a certain inhibitory effect on Escherichia coli and Staphylococcus aureus, provides a new lead compound for the research and development of new antibacterial drugs, and also provides a scientific basis for the development of marine fungal resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the colony morphology of Aspergillus versicolor 2-18.

[0018] Figure 2 The cyclic peptide compound prepared in Example 1 of the present invention 1 H NMR spectrum (500 MHz, DMSO-d6).

[0019] Figure 3 The cyclic peptide compound prepared in Example 1 of the present invention 13 CNMR spectrum (125MHz, DMSO-d6). DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the contents of the present invention are not limited thereto. Example 1

[0021] Isolation and identification of Aspergillus versicolor 2-18:

[0022] 1. Biological samples: Symbiotic fungi samples from luxuriant membrane sponges collected from the intertidal zone of Yantai City, Shandong Province.

[0023] 2. Isolation and identification: After the strain was cultured, isolated and purified, a pure symbiotic fungal strain was obtained. After morphological identification and strain ITS sequence analysis, it was identified as Aspergillus versicolor, with the strain number 2-18. The marine fungus Aspergillus versicolor 2-18 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 24, 2024, with the deposit number CGMCC No.41474.

[0024] Preparation and structural identification of cyclic peptide compounds from marine fungi:

[0025] The specific steps are as follows:

[0026] Take Aspergillus versicolor 2-18 strains that grow well on the PDA plate, cut into small pieces, inoculate into potato glucose liquid culture medium, each 1L triangular flask contains 300mL culture medium, a total of 100 flasks, ferment at room temperature for 30 days, filter and collect the mycelium and fermentation liquid separately.

[0027] The potato glucose liquid culture medium is composed of 500 mL of boiled juice of 200 g potato pieces, 20 g of glucose, 5 g of peptone and 500 mL of natural seawater per liter of culture medium.

[0028] About 30 L of fermentation broth was collected, extracted three times with ethyl acetate, and concentrated under reduced pressure to obtain a fermentation broth extract; the mycelium was dried and crushed, extracted three times with ethyl acetate, and concentrated under reduced pressure to obtain a mycelium extract; thin layer chromatography (TLC) detection (developed with a volume ratio of 20:1 dichloromethane-methanol or 1:1 petroleum ether-ethyl acetate, heated with anisaldehyde-sulfuric acid color developer or fluorescent detection under 254 nm ultraviolet light) showed that the color characteristics of the fermentation broth and mycelium extracts were similar and were combined into a total extract (122.7 g).

[0029] The crude extract was chromatographed on a 200-300 mesh silica gel column, and gradient eluted with petroleum ether-ethyl acetate in a volume ratio of 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, in sequence. The eluates were collected and examined by thin layer chromatography (TLC) (developed with dichloromethane-methanol in a volume ratio of 20:1 or petroleum ether-ethyl acetate in a volume ratio of 1:1, and developed with anisaldehyde-sulfuric acid color developer by heating or fluorescence detection under 254 nm ultraviolet light). Similar components were merged based on Rf values, and seven components (1-7) were obtained using the standard division of Rf values ​​of >0.8, 0.7-0.8, 0.5-0.7, 0.4-0.5, 0.3-0.4, 0.2-0.3, and <0.2, respectively. The compositions of the seven components were detected and analyzed by column chromatography separation method, thin layer chromatography (TLC) analysis detection method, and nuclear magnetic resonance structure identification method, and the target compound was obtained only in component 3 (Rf value was 0.5-0.7).

[0030] Component 3 (7.61 g), i.e., the eluted component with an Rf value of 0.5-0.7 (developed with dichloromethane-methanol in a volume ratio of 20:1 or petroleum ether-ethyl acetate in a volume ratio of 1:1), was separated by reverse phase silica gel column chromatography and normal phase silica gel column chromatography in turn. The eluent for reverse phase silica gel column chromatography was water-methanol in a volume ratio of 1:1-0:1, and the eluent for normal phase silica gel column chromatography was dichloromethane-methanol in a volume ratio of 50:1-10:1. The components were collected and detected by thin layer chromatography. The components with an Rf value of 0.4-0.6 were collected to obtain the compound shown in formula (I) (81.8 mg). After TLC detection (developed with dichloromethane-methanol in a volume ratio of 30:1, and heated with anisaldehyde-sulfuric acid as a color developer), a single, uniform pink spot was observed, which was confirmed to be a pure compound with a purity of 95%-97%. Its structure was identified as a new cyclopeptide compound by nuclear magnetic resonance spectroscopy analysis, and the structural formula is shown in (I).

[0031]

[0032] Formula (I)

[0033] The physicochemical properties and nuclear magnetic resonance data of the marine fungus-derived cyclopeptide compound (I) prepared by the above steps are as follows: white amorphous solid, easily soluble in acetone, methanol, ethanol, ethyl acetate, soluble in dichloromethane, and poorly soluble in water; the molecular formula is C 38

[0034] H 45 N5O7; High resolution mass spectrometry (HRESIMS) m / z: 706.3218 [M + Na] + , the calculated value is C 38 H 45 N5O7Na, 706.3217; H NMR ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) data are shown in Table 1.

[0035] Table 1: H NMR spectra of cyclic peptide compounds (I) 1 H NMR) and carbon spectroscopy ( 13 C NMR data (DMSO-d6)

[0036]

[0037] Structural deduction of cyclopeptide compound (I): The high-resolution mass spectrum of the compound indicated that the molecular weight of the compound was 683 and the molecular formula was C 38 H 45 N5O7, the degree of unsaturation is 19. It has fluorescence under 254nm ultraviolet light, indicating that there may be a benzene ring in the structure of this compound. 1 HNMR and 13 CNMR spectrum showed that the structure of the compound contained typical amino acid residue signals of peptide compounds. 13 The CNMR spectrum shows 5 carbonyl carbon signals ( d C 171.0, 170.7, 170.2, 169.5 and 168.9), suggesting that the compound is a cyclopentapeptide containing 5 amino acid residues. By analyzing the 2D NMR spectrum, it can be seen that there is an N-methyl-tyrosine (N-Me-Tyr), a phenylalanine (Phe), a tyrosine (Tyr), a proline (Pro) and a valine (Val) residue fragment in the structure of the cyclopeptide compound. The connection order of the amino acid residues can be determined based on the relevant signals of the HMBC spectrum. In the HMBC spectrum, N-CH3 ( d H 2.78) / Phe-C=O( d C 171.0), Phe-NH ( d H8.61) / Tyr-C=O( d C 170.2), Tyr-NH ( d H 6.93) / Pro-C=O( d C 170.7), Pro-H d ( d H 3.44) / Val-C=O( d C 169.5) and Val-NH ( d H 6.67) / N-Me-Tyr-C=O( d C 168.9) and other related signals, and the connection sequence of amino acid residues was determined to be N-Me-Tyr-Val-Pro-Tyr-Phe. The Δδ Cβ-Cγ The large chemical shift difference (10.3 ppm) indicates a cis conformation. The chirality of the amino acid residues was determined by comparing the molecular weight information and retention time of the standard derivatives and the amino acid derivatives of the compound of the present invention using the Marfey method, and the results showed that all the amino acid residues were in L-configuration.

[0038] In summary, the structure of the cyclic peptide compound represented by formula (I) is cyclo-[N-Me-L-Tyr-L-Val-L-Pro-L-Tyr-L-Phe], which is a new compound not reported in the literature.

[0039] Antibacterial activity experiment:

[0040] Escherichia coli and Staphylococcus aureus were used to evaluate the strength of the compound's antibacterial activity. The specific operation method was as follows: the bacterial culture medium used in the experiment was LB culture medium. The bacterial suspension was soaked with a sterile cotton swab and evenly applied to the culture medium. The test sample was dissolved in DMSO at a concentration of 5.0 mg / mL. 5 μL of the sample was added to a sterile filter paper with a diameter of 6 mm (25 μg per piece); and a filter paper with the same volume of DMSO was used as a negative control, and chloramphenicol was used as a positive control, with three parallels for each. The plate culture medium with the sample was placed at 37°C for 24 hours, and the experimental results were observed. If an inhibition zone appeared, the diameter of the inhibition zone was measured.

[0041] Experimental results: The diameters of the inhibition zones of the cyclic peptide compound (I) obtained above against Escherichia coli and Staphylococcus aureus were 12 mm and 9 mm, respectively (the diameters of the inhibition zones of the positive control chloramphenicol were 26 mm and 22 mm, respectively), and it had certain antibacterial activity.

[0042] The above description is only a preferred embodiment of the present invention. For relevant technicians in this field, several improvements and modifications made to the present invention should also be considered as the protection scope of the present invention.

Claims

1. A cyclic peptide compound, characterized in that: The structural formula of the compound is shown below: 。 2. The fermentation method for preparing cyclic peptide compounds according to claim 1, characterized in that: The fermentation preparation method comprises: Aspergillus versicolor ) 2-18 is inoculated into a fungal liquid culture medium for fermentation and culture, and the cyclic peptide compounds are obtained after separation and purification; The Aspergillus versicolor ( Aspergillus versicolor ) 2-18 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 24, 2024, with the deposit number CGMCC No.41474.

3. The fermentation method for preparing cyclic peptide compounds according to claim 2, characterized in that: The specific operation steps of the preparation method are: 1) Aspergillus versicolor ( Aspergillus versicolor ) 2-18 are inoculated into a fungal liquid culture medium and allowed to ferment for 20-50 days to obtain a fermentation product of the strain; the fermentation broth is filtered, extracted with ethyl acetate, and concentrated under reduced pressure to obtain a fermentation broth extract; the mycelium is extracted with ethyl acetate, and concentrated under reduced pressure to obtain a mycelium extract; the fermentation broth extract and the mycelium extract are combined to obtain a total crude extract; 2) subjecting the total crude extract in step 1) to normal phase silica gel column chromatography, gradient eluting with an organic solvent, collecting the eluate, and combining similar components after the eluate is detected by thin layer chromatography, wherein the organic solvent is petroleum ether-ethyl acetate or dichloromethane-methanol; 3) The eluted components in step 2) with an eluent volume ratio of 20:1-10:1 are further separated and purified by reverse phase silica gel column chromatography and normal phase silica gel column chromatography, and the components with Rf of 0.4-0.6 are collected by thin layer chromatography to obtain cyclopeptide compounds.

4. The fermentation method for preparing cyclic peptide compounds according to claim 3, characterized in that: The eluent for the reverse phase silica gel column chromatography in step 3) is methanol-water or ethanol-water; the eluent for the normal phase silica gel column chromatography is petroleum ether-ethyl acetate or dichloromethane-methanol.

5. The fermentation method for preparing cyclic peptide compounds according to claim 3, characterized in that: The developing solvent for the thin layer chromatography detection in step 3) is petroleum ether-ethyl acetate or dichloromethane-methanol in a volume ratio of 30:1-1:

1.

6. The use of the cyclic peptide compound according to claim 1 in the preparation of antibacterial drugs, characterized in that: The bacteria is Escherichia coli or Staphylococcus aureus.

Images