Streptomyces-sourced 4-MHA-pentapeptide lactone compound as well as preparation and application thereof

Abstract

The invention relates to a 4-MHA-pentapeptide lactone compound L1 derived from streptomyces as well as preparation and application of the 4-MHA-pentapeptide lactone compound L1, the 4-MHA-pentapeptide lactone compound L1 is derived from streptomyces CGMCC (China General Microbiological Culture Collection Center) 4.1912, and L1 is a 4-MHA-pentapeptide lactone monomer and belongs to a compound with a novel structure. The streptomyces secondary metabolite provided by the invention has relatively good antibacterial activity, and particularly, the compound L1 has a remarkable antibacterial effect on Staphylococcus aureus ATCC 29213, Enterococcus faecium ATCC 29212 and Enterococcus faecium ATCC 19434, so that the streptomyces secondary metabolite is suitable for research on antibacterial lead compounds or preparation of antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of microbial natural product isolation and purification technology and the pharmaceutical field, specifically involving a bioactive secondary metabolite 4-MHA-pentapeptide compound derived from Streptomyces and its preparation and application. Background Technology

[0002] Bioactive molecules have made significant contributions to the prevention and treatment of diseases in humans and animals. Secondary metabolites produced by Streptomyces are an important source of bioactive molecules. Numerous Streptomyces-derived secondary metabolites have been developed and applied in the pharmaceutical field, greatly promoting the development of modern drug research. Related products include antibiotics for treating bacterial or fungal infections, antitumor drugs, immunosuppressants, and biopesticides. *Streptomyces chloroticum* Streptomyces iakyrus They are a class of actinomycetes with great potential for development. Bioactive compounds such as actinomycin G, pteropterin, and γ-erythromycin have been isolated from them. Exploring their secondary metabolites can help discover drug lead compounds with good bioactivity.

[0003] Actinomycins are a class of pigment peptide lactone antibiotics comprising one phenoxazinone and two pentapeptide lactones, derived from the condensation of two 4-MHA-pentapeptide monomers. Actinomycin D, in particular, is used clinically due to its excellent biological activity. Currently, very few naturally occurring 4-MHA-pentapeptide monomers have been reported. Research on 4-MHA-pentapeptide compounds will not only deepen our understanding of the biosynthetic mechanism of actinomycins but also provide a richer molecular basis for drug development. Summary of the Invention

[0004] The first objective of this invention is to provide a green pigment Streptomyces. S. iakyrus The secondary metabolite derived from it is a 4-MHA-pentapeptide lactone compound.

[0005] The secondary metabolites provided by this invention are isolated and purified from *Streptomyces chlorophyll* ( S. iakyrus CGMCC4.1912, the chlorophyll-producing Streptomyces S. iakyrus CGMCC 4.1912 is derived from the China General Microbiological Culture Collection Center, accession number: CGMCC No. 4. 1912. Among the obtained compounds, there is a novel compound L1 and two compounds with known structures, L2 (actinomycin G5) and L3 (actinomycin G6). L1 is a 4-MHA-pentapeptide monomer, while L2 and L3 are 4-MHA-pentapeptide dimers. The structural formulas of the three compounds are shown below, where the upper part of the L1 structure is 4-methyl-3-hydroxy-an-aminobenzoic acid (4-MHA), and the lower part is a pentapeptide.

[0006] A second object of the present invention is to provide a method for preparing the compound.

[0007] The method for preparing the compound provided by the present invention includes the following steps: (1) Seed culture in shake flasks: *Streptomyces chlorophyll* ( S. iakyrus ) Spores of strain CGMCC 4.1912 were inoculated into TSB medium and cultured in a shaking incubator for 48 h to prepare the seed culture required for fermentation. The culture conditions were 180 rpm and 30 °C. TSB medium inactivation: Add 1.5 g of TSB medium and 50 mL of distilled water to a 250 mL Erlenmeyer flask and sterilize at 121°C for 20 minutes; (2) Large-scale culture: Pour the sterilized R5 medium into sterile flat plastic petri dishes, 25 mL of medium into each petri dish. After the medium solidifies, add 200 μL of seed liquid to each medium plate, spread it evenly, and place it in an incubator at 30℃ for 7 days. R5 medium composition (per liter): 103 g sucrose, 0.25 g K2SO4, 10.12 g MgCl2·6H2O, 10 g glucose, 0.1 g casein hydrolysate and 5 g yeast extract, 5.73 g TES, 2 mL trace element solution, 20 g agar, adjust pH to 7.0, autoclave at 121℃ for 20 minutes; After sterilization of R5 medium, add sterile components per liter: 10 mL of 0.5% KH2PO4 solution, 4 mL of 5M CaCl2·5H2O, and 15 mL of 20% L-proline. (3) Preparation of crude extract: Cut the fermented culture medium into small pieces, transfer them into Erlenmeyer flasks or beakers, add ethyl acetate and soak three times, each soaking time being more than 3 hours. Filter the soaking liquid to obtain fermentation extract, and concentrate the fermentation extract by rotary evaporation to obtain crude extract. (4) Crude extract separation and purification: The crude extract was initially separated and purified using a C18 open column and eluted with a methanol-water system. The elution gradients were 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% methanol-water, with each gradient eluting 3 times the column volume (400 mL). (5) High-performance liquid chromatography preparation of compound L1: Purification and preparation of compound L1: 60% methanol-water eluent was collected, concentrated by rotary evaporation, and then separated and purified by reversed-phase high-performance liquid chromatography to obtain 4-MHA-pentapeptide compound L1 from Streptomyces.

[0008] The fraction containing 4-MHA-pentapeptide compound L1 from Streptomyces is a 60% methanol-water eluent, and the fractions containing 4-MHA-pentapeptide compounds L2 and L3 from Streptomyces are an 80% methanol-water eluent.

[0009] The chromatographic column used in the reversed-phase high-performance liquid chromatography method is Thermo Scientific Betasil. TM The mobile phase is either a C18 reverse-phase preparative column (150*21.2 mm, 5 μm) or a YMC-Pack ODS-A reverse-phase semi-preparative column (10*250 mm, 5 μm), with methanol-water or acetonitrile aqueous solution (containing 0.1% formic acid) as the mobile phase and a detection wavelength of 210 nm.

[0010] A third object of the present invention is to provide the use of the compound in the preparation of antibacterial agents.

[0011] The antibacterial indicator bacteria described in this invention include Gram-positive bacteria: Staphylococcus aureus ( Staphylococcus aureus ), Enterococcus faecalis ( Enterococcus faecalis ), Enterococcus faecalis ( Enterococcus faecium Staphylococcus epidermidis ( Staphylococcus epidermidis Gram-negative bacteria: Klebsiella pneumoniae ( Klebsiella pneumoniae ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Acinetobacter baumannii ( Acinetobacter baumannii ) and Escherichia coli ( Escherichia coli ).

[0012] The antibacterial formulation provided by this invention has an active ingredient primarily consisting of L1, a 4-MHA-pentapeptide compound derived from *Streptomyces*, which is effective against *Staphylococcus aureus* strain (…). S. aureus ATCC 29213, Enterococcus faecalis ( E. faecalis ATCC 29212 and Enterococcus faecalis ( E. faecium ATCC 19434 has excellent antibacterial effects.

[0013] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: 1. The Streptomyces-derived 4-MHA-pentapeptide compounds L1, L2, and L3 provided in this invention were prepared from... S. iakyrusCGMCC 4.1912 specifies that compound L1 is a novel 4-MHA-pentapeptide monomer, while compounds L2 and L3 are known actinomycins G5 and G6, which are 4-MHA-pentapeptide dimers. The preparation method for the compounds provided by this invention is mature and simple, and their structures have been confirmed by NMR, mass spectrometry, and UV spectroscopy.

[0014] 2. The 4-MHA-pentapeptide compounds L1, L2, and L3 derived from Streptomyces obtained in this invention have antibacterial activity, especially L1 against strains. S. aureus ATCC 29213 E. faecalis ATCC 29212 and E. faecium ATCC19434 exhibits significant antibacterial effects and is suitable for research on antibacterial lead compounds or the preparation of antibacterial drugs. The antibacterial effects of the three compounds are as follows: compound L1 > compound L2 (actinomycin G5) > compound L3 (actinomycin G6). Attached Figure Description

[0015] Figure 1 This is the mass spectrum of compound L1 described in this invention.

[0016] Figure 2 This is the mass spectrum of compound L2 described in this invention.

[0017] Figure 3 This is the mass spectrum of compound L3 described in this invention.

[0018] Figure 4 The compound L1 described in this invention is dissolved in CD3OD. 1 H-NMR spectrum.

[0019] Figure 5 The compound L1 described in this invention is dissolved in CD3OD. 13 C-NMR spectrum.

[0020] Figure 6 The compound L1 described in this invention is dissolved in CD3OD. 1 H- 1 H COSY spectrum.

[0021] Figure 7 The image shows the HSQC spectrum of compound L1 as dissolved in CD3OD according to the present invention.

[0022] Figure 8 The image shows the HMBC spectrum of compound L1 as dissolved in CD3OD.

[0023] Figure 9 The compound L2 described in this invention is dissolved in CD3OD. 1 H-NMR spectrum.

[0024] Figure 10 The compound L2 described in this invention is dissolved in CD3OD. 13 C-NMR spectrum.

[0025] Figure 11 The compound L2 described in this invention is dissolved in CD3OD. 1 H- 1 H COSY spectrum.

[0026] Figure 12 The HMBC spectrum of compound L2 of the present invention dissolved in CD3OD is shown.

[0027] Figure 13 The compound L3 described in this invention is dissolved in CD3OD. 1 H-NMR spectrum.

[0028] Figure 14 The compound L3 described in this invention is dissolved in CD3OD. 13 C-NMR spectrum.

[0029] Figure 15 This is the ultraviolet spectrum of compound L1 of the present invention dissolved in CH3OH.

[0030] Figure 16 This is the ultraviolet spectrum of compound L2 of the present invention dissolved in CH3OH.

[0031] Figure 17 This is the ultraviolet spectrum of compound L3 of the present invention dissolved in CH3OH. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the specific embodiments described herein are only for illustration and explanation of the present invention and are not limited to the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Example 1: A method for preparing L1, a 4-MHA-pentapeptide compound derived from Streptomyces, comprising the following steps: (1) Seed culture in shake flasks: *Streptomyces chlorophyll* ( S. iakyrus ) Spores of strain CGMCC 4.1912 were inoculated into TSB medium and cultured in a shaking incubator for 48 h to prepare the seed culture required for fermentation. The culture conditions were 180 rpm and 30℃.

[0036] TSB medium composition (per liter): tryptone (17.0 g), soybean papain digest (3.0 g), glucose (2.5 g); dipotassium hydrogen phosphate as buffer (2.5 g); sodium chloride (5.0 g); pH (7.3 ± 0.2).

[0037] TSB medium inactivation: Add 1.5 g of TSB medium and 50 mL of distilled water to a 250 mL Erlenmeyer flask and sterilize at 121°C for 20 minutes.

[0038] (2) Large-scale culture: Pour the sterilized R5 medium into sterile flat plastic petri dishes, add 25 mL of medium to each petri dish, and after the medium solidifies, add 200 μL of seed liquid to each medium plate, spread it evenly, and place it in an incubator at 30℃ for 7 days.

[0039] Flat plastic petri dish specifications: 90 mm in diameter.

[0040] R5 medium composition (per liter): 103 g sucrose, 0.25 g K2SO4, 10.12 g MgCl2·6H2O, 10 g glucose, 0.1 g casein hydrolysate, 5 g yeast extract, 5.73 g TES, 2 mL trace element solution, 20 g agar. Adjust pH to 7.0 and autoclave at 121°C for 20 minutes.

[0041] Trace element solution composition (per liter): ZnCl2: 40 mg, FeCl3·6H2O: 200 mg, CuCl2·2H2O: 10 mg, MnCl2·4H2O: 10 mg, Na2B4O7·10H2O: 10 mg, (NH4)6Mo7O 24 ·4H2O: 10 mg.

[0042] After sterilization of R5 medium, add sterile components (per liter): 10 mL KH2PO4 (0.5% by mass) solution, 4 mL CaCl2·5H2O (5M), and 15 mL L-proline (20% by mass).

[0043] (3) Preparation of crude extract: Cut the fermented culture medium into small pieces, transfer them into Erlenmeyer flasks or beakers, add ethyl acetate and soak three times, each soaking time being more than 3 hours. Filter the soaking liquid to obtain fermentation extract, and concentrate the fermentation extract by rotary evaporation to obtain crude extract.

[0044] (4) Crude extract separation and purification: The crude extract was initially separated and purified using a C18 open column with gradient elution in a methanol-water system. The elution gradients were 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol-water, with each gradient eluting three column volumes (400 mL). HPLC analysis confirmed that compound L1 was enriched in the 60% methanol-water fraction, and compounds L2 and L3 were enriched in the 80% methanol-water fraction.

[0045] (5) High-performance liquid chromatography (HPLC) preparation of compounds: Purification and preparation of L1, a 4-MHA-pentapeptide compound derived from Streptomyces: A 60% methanol-water eluent was collected, concentrated by rotary evaporation, and then purified by reversed-phase high-performance liquid chromatography (RP-HPLC). HPLC preparation conditions were as follows: Thermo Scientific Betasil... TM A C18 reversed-phase preparative chromatographic column (150*21.2 mm, 5 μm) was used, with a mobile phase of 49% methanol-water solution (containing 0.1% formic acid), a detection wavelength of 210 nm, and a flow rate of 8 mL / min. t R = 43 min.

[0046] Purification and preparation of compounds L2 and L3: 80% methanol-water eluent was collected, concentrated by rotary evaporation, and then separated and purified by reversed-phase high-performance liquid chromatography. The first preparation conditions were: Thermo Scientific Betasil... TM A C18 reversed-phase preparative chromatographic column (150*21.2 mm, 5 μm) was used. The mobile phase was 65% methanol aqueous solution (containing 0.1% formic acid). The detection wavelength was 210 nm, and the flow rate was 8 mL / min. The chromatogram yielded a sample containing the compound L2. t R = 34 min) and L3 ( t R Two sub-fractions (28 min) were prepared. These fractions were then subjected to a second high-performance liquid chromatography (HPLC) preparation to obtain compound L2. The preparation conditions were: YMC-PackODS-A reversed-phase semi-preparative column (10*250 mm, 5 μm), mobile phase: 50% acetonitrile aqueous solution (containing 0.1% formic acid), detection wavelength: 210 nm, flow rate: 3 mL / min. t R = 16 min; The preparation conditions for compound L3 were: YMC-Pack ODS-A reversed-phase semi-preparative column (10*250 mm, 5 μm), mobile phase was 40% acetonitrile aqueous solution (containing 0.1% formic acid), detection wavelength was 210 nm, and flow rate was 3 mL / min.t R = 21 min.

[0047] (6) Structural identification of the compounds: The 4-MHA-pentapeptide compounds L1, L2, and L3 obtained by fermentation from Streptomyces were determined by mass spectrometry, nuclear magnetic resonance and ultraviolet spectroscopy, and their structural formulas were determined by combining the literature reports as follows. The hydrogen and carbon signals of their nuclear magnetic resonance are assigned as shown in Tables 1, 2 and 3; Figures 1-3 The ESI-MS mass spectra show the quasi-molecular ions of L1, L2, and L3, respectively. m / z 659.4 [MH] - 1254.8 [MH] - and 1271.9 [MH] - Their molecular weights are estimated to be 660, 1256 and 1273, respectively. Figures 4-8 It is a 4-MHA-pentapeptide compound L1 derived from Streptomyces. 1 H-NMR, 13 C-NMR, 1 H- 1 HCOSY, HSQC and HMBC spectra, NMR data combined with mass spectrometry data to determine its structure; Figures 9-12 For compound L2 1 H-NMR, 13 C-NMR, 1 H- 1 The structure was determined by combining H COSY and HMBC spectra with NMR data, mass spectrometry data, and literature comparison. Figures 13-14 For compound L3 1 H-NMR and 13 The structure was determined by combining C-NMR spectrum, NMR data, mass spectrometry data, and comparison with literature. Figures 15-17 The images show the UV spectra of 4-MHA-pentapeptide compounds L1, L2, and L3 derived from Streptomyces. The UV absorption of the phenoxazinone chromophore in L2 and L3 is consistent with that in the literature.

[0048] Table 1. Compound L1 13 H, 13 C-NMR data

[0049] Table 2. Compound L2 13 H, 13 C-NMR data

[0050] Table 3. Compound L3 13H, 13 C-NMR data

[0051] Example 2: Streptomyces chlorophyll S. iakyrus Antibacterial activity of CGMCC 4.1912 secondary metabolites L1-L3 Streptomyces chlorophyll S. iakyrus The CGMCC 4.1912 secondary metabolite minimum inhibitory concentration (MIC) assay used Gram-positive Staphylococcus aureus as an indicator bacterium. S. aureus ATCC 29213), Enterococcus faecalis ( E. faecalis ATCC 29212), Enterococcus faecalis ( E. faecium ATCC 19434), Staphylococcus epidermidis ( S. epidermidis ATCC 12228) and Gram-negative bacteria Klebsiella pneumoniae ( K. pneumoniae ATCC700603), Pseudomonas aeruginosa ( P. aeruginosa ATCC 27853), Acinetobacter baumannii ( A. baumannii ATCC19606) and Escherichia coli ( E. coli ATCC 25922). All strains were obtained from the Pathogenic Microbial Culture Collection Center of the Chinese Academy of Medical Sciences.

[0052] Assay method: Glycerol tubes of the above indicator bacteria were inoculated into LB liquid medium and incubated at 180 rpm / 37℃ for 16 h. The culture was then diluted with fresh LB liquid medium at a volume ratio of 1:10000, and the diluted solutions were added to 96-well plates. The test compounds L1-L3 and the positive control (levofloxacin) were dissolved in dimethyl sulfoxide and added to 96-well plates to achieve final concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.13, 0.06, and 0.03 μg / mL, respectively. The plates were incubated at 180 rpm / 37℃ for 18 h, and the results were recorded. All MIC tests were repeated. The lowest concentration at which the bacterial culture became clear for each indicator bacterium was the minimum inhibitory concentration (MIC) of the compound against that indicator bacterium. The antibacterial activities of compounds L1-L3 are shown in Tables 4 and 5. Compounds L1 to L3 correspond to 4-MHA-pentapeptide compounds L1 to L3 derived from Streptomyces.

[0053] Table 4. Inhibitory activities (MIC, µg / mL) of compounds L1–L3 against four Gram-positive bacteria

[0054] Table 5. Inhibitory activities of compounds L1–L3 against four Gram-negative bacteria (MIC, µg / mL)

[0055] The above experimental results show that, originating from S. iakyrus The secondary metabolites L1, L2, and L3 of CGMCC 4.1912 exhibited certain inhibitory activity against the tested bacteria, especially L1. S. aureus ATCC 29213 E. faecalis ATCC 29212 and E. faecium ATCC 19434 exhibits a more significant antibacterial effect, making it suitable for research on antibacterial lead compounds or the preparation of antibacterial drugs. The antibacterial effects of the three compounds are as follows: compound L1 > compound L2 (actinomycin G5) > compound L3 (actinomycin G6).

Claims

1. A 4-MHA-pentapeptide compound L1 or a pharmaceutically acceptable salt thereof derived from Streptomyces, characterized in that: L1 is a 4-MHA-pentapeptide monomer, and its structural formula is shown below: 。 2. The method for preparing the Streptomyces-derived 4-MHA-pentapeptide compound L1 according to claim 1, characterized in that... The steps include: (1) Seed culture in shake flasks: Sterilia ( S. iakyrus Spores of strain CGMCC 4.1912 were inoculated into TSB medium and cultured in a shaking incubator for 48 h to prepare the seed culture required for fermentation. The culture conditions were 180 rpm and 30 °C. TSB medium inactivation: Add 1.5 g of TSB medium and 50 mL of distilled water to a 250 mL Erlenmeyer flask and sterilize at 121°C for 20 minutes; (2) Large-scale culture: Pour the sterilized R5 medium into sterile flat plastic petri dishes, 25 mL of medium into each petri dish. After the medium solidifies, add 200 μL of seed liquid to each medium plate, spread it evenly, and place it in an incubator at 30℃ for 7 days. R5 medium composition (per liter): sucrose 103 g, K2SO4 0.25 g, MgCl2·6H2O 10.12 g, glucose 10 g, casein hydrolysate 0.1 g, yeast extract 5 g, TES 5.73 g, trace element solution 2 mL, agar 20 g, adjust pH to 7.0, autoclave at 121℃ for 20 minutes; After sterilization of R5 medium, add sterile components per liter: 10 mL of 0.5% KH2PO4 solution, 4 mL of 5M CaCl2·5H2O, and 15 mL of 20% L-proline. (3) Preparation of crude extract: Cut the fermented culture medium into small pieces, transfer them into Erlenmeyer flasks or beakers, add ethyl acetate and soak three times, each soaking time being more than 3 hours. Filter the soaking liquid to obtain fermentation extract, and concentrate the fermentation extract by rotary evaporation to obtain crude extract. (4) Crude extract separation and purification: The crude extract was initially separated and purified using a C18 open column and eluted with a methanol-water system. The elution gradients were 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% methanol-water, with each gradient eluting 3 times the column volume (400 mL). (5) High-performance liquid chromatography preparation of compound L1: Purification and preparation of compound L1: 60% methanol-water eluent was collected, concentrated by rotary evaporation, and then separated and purified by reversed-phase high-performance liquid chromatography to obtain 4-MHA-pentapeptide compound L1 from Streptomyces.

3. The preparation method according to claim 2, characterized in that: The preparative conditions for reversed-phase high-performance liquid chromatography (RP-HPLC) were as follows: C18 reversed-phase preparative column; mobile phase: 49% methanol-water solution containing 0.1% formic acid; detection wavelength: 210 nm; flow rate: 8 mL / min. t R = 43 min.

4. The preparation method according to claim 2, characterized in that: The composition of the trace element solution per liter is as follows: ZnCl2: 40 mg, FeCl3·6H2O: 200 mg, CuCl2·2H2O: 10 mg, MnCl2·4H2O: 10 mg, Na2B4O7·10H2O: 10 mg, (NH4)6Mo7O 24 ·4H2O: 10 mg.

5. A pharmaceutical composition comprising the Streptomyces-derived 4-MHA-pentapeptide compound L1 of claim 1 or a pharmaceutically acceptable salt thereof.

6. The use of the Streptomyces-derived 4-MHA-pentapeptide compound L1 or a pharmaceutically acceptable salt thereof as described in claim 1 in the preparation of an antimicrobial agent.

7. The use of the pharmaceutical composition of claim 5 or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial agent.

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