A novel sixteen-membered macrocyclic lactone compound, its preparation method and application

Abstract

This invention relates to the field of agricultural biological control technology, specifically disclosing a novel bafilomycin analogue with antifungal activity, its preparation method, and its application. The bafilomycin analogue is a novel natural compound obtained by fermentation culture of Streptomyces sp. TH21, component activity tracing, and subsequent separation and extraction. This type of compound exhibits good activity against plant pathogenic fungi and has the potential to be developed into a novel, highly efficient, and environmentally friendly biological fungicide.

Description

Technical Field

[0001] This invention relates to the field of agricultural biological control technology, specifically to a novel sixteen-membered macrocyclic lactone compound with activity against plant pathogenic fungi, its preparation method, and its application. Background Technology

[0002] Biological control aligns better with the development trend of green prevention and control of plant diseases. Biocontrol bacteria, especially actinomycetes, are diverse and produce complex and varied secondary metabolites, making them an important type of biological control and fungicide (He Yawen et al., 2004). Macrolides are the most typical secondary metabolites of actinomycetes (especially Streptomyces). Their rich chemical diversity and broad biological activity have attracted much attention in the pharmaceutical and agricultural fields, resulting in the most successful development. Based on the size of the lactone ring, the active macrolide compounds that have been discovered and applied include twelve-membered (such as succinylcholine), fourteen-membered (such as erythromycin), fifteen-membered (such as azithromycin), sixteen-membered (such as tylosin, avermectin, spiramycin, milbemycin, epothilone, bafloxacin, etc.), and eighteen-membered, twenty-two-membered, twenty-three-membered, and twenty-four-membered macrolides (such as azathromycin, nystatin, novomycin, amphotericin B, anisomniacin derivatives, rapamycin, sirolimus, fedamycin, etc.), as well as spinosad, etc. These macrolide compounds are widely used in agriculture (insecticides, antibacterial agents, etc.) and medicine (immunosuppressants, antitumor agents, etc.).

[0003] Bafilomycin is a class of characteristic sixteen-membered macrocyclic lactones produced by Streptomyces, best known for its mechanism of action as a specific inhibitor of vacuolar ATPase (V-ATPase). These compounds exhibit a variety of biological activities, including antitumor, antiviral, and antifungal activity. Bafilomycin A1 inhibits the growth of Candida albicans, while bafilomycin C1 exerts its antifungal effect by disrupting the ergosterol biosynthesis pathway. However, current research primarily focuses on the medical potential of these compounds, and their application potential in the control of agricultural plant diseases requires further investigation. The applicant's research group is dedicated to the discovery of active actinomycetes and their secondary metabolites in samples from special ecological environments (ocean, lake, base and crop base). A strain of Streptomyces sp. TH21 was isolated from the bottom mud of soil in a large vegetable planting base. Through activity tracing, a new class of sixteen-membered macrocyclic lactone compounds, bafilomycin H (1) and 19-O-methylhygrolidin (2), were isolated from its secondary metabolites. They have significant anti-plant pathogenic fungal activity and have the potential to be developed into biological fungicides. Summary of the Invention

[0004] The purpose of this invention is to provide a novel sixteen-membered macrocyclic lactone compound that can be isolated from the fermentation products of Streptomyces sp. TH21 and has a certain inhibitory effect on various pathogenic fungi.

[0005] This invention provides the following technical solution: A sixteen-membered macrocyclic lactone compound, with the following chemical structural formula: ; R represents segment 1 or segment 2 in the diagram; The aforementioned compound 1 (R represents fragment 1) and compound 2 (R represents fragment 2) were obtained by fermenting and culturing Streptomyces sp. TH21 and then extracting and separating the fermentation broth. Both compounds exhibited certain antifungal activity and showed promising application prospects.

[0006] This invention also provides a Streptomyces strain capable of producing the aforementioned sixteen-membered macrocyclic lactone compounds. This Streptomyces strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33060 and is classified as *Streptomyces sp.*. This Streptomyces strain was isolated by the inventors' team from soil in a vegetable planting base in Huzhou City, Zhejiang Province. Its internal marker is TH21, and it was deposited at CGMCC on December 13, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The 16S rRNA of this strain is registered in GenBank under the number PX944784. Compounds 1 and 2, isolated from its secondary metabolites, exhibit antifungal activity against plant pathogenic fungi.

[0007] This invention also provides a method for preparing the above-mentioned sixteen-membered macrocyclic lactone compound, comprising the following steps: (1) Inoculate Streptomyces sp. TH21 into a liquid culture medium containing carbon and nitrogen sources and carry out fermentation culture. After fermentation, collect the fermentation broth, remove the mycelium, and retain the clear liquid. (2) The clear liquid obtained in step (1) is sequentially adsorbed through macroporous adsorption resin, then eluted with organic solvent to obtain eluent, and then the eluent is concentrated under reduced pressure to obtain crude extract of fermentation broth. (3) The crude extract of fermentation broth in step (2) was subjected to silica gel column chromatography and gel column chromatography to obtain a fraction containing sixteen-membered macrocyclic lactone compounds. (4) The fractionated sample from step (3) was purified by reverse-phase preparative column chromatography to separate the sixteen-membered macrocyclic lactone compound bafilomycin H (1) and compound 19-O-methylhygrolidin (2); As a preferred embodiment of the method of the present invention, Streptomyces sp. TH21 is fermented in a liquid culture medium. The carbon source in step (1) is selected from one or a combination of glucose, soluble starch, dextrin, sucrose, corn starch, maltose, industrial molasses, glycerol, sorbitol, mannitol, lactose, xylan, or more of the above substances, preferably a combination of glucose and soluble starch, or a combination of glucose and corn starch; the nitrogen source is selected from one or a combination of yeast powder, yeast extract, soybean meal, soybean flour, peanut meal, malt extract, peptone, beef extract, yeast extract, corn steep liquor, wheat bran, gluten powder, urea, ammonium salt, or two or more of the above substances, preferably yeast powder, malt extract, or a combination thereof.

[0008] In the above preparation method, the clarified liquid is obtained by centrifuging the fermentation broth to separate the mycelium. In step (2), the clarified liquid is adsorbed by a macroporous adsorption resin and then eluted with an organic solvent to obtain an eluent. The eluent is then concentrated under reduced pressure to obtain a crude extract of the fermentation broth. Preferably, the macroporous adsorption resin is selected from DIAION HP20 resin or LX-60 resin. The organic solvent is preferably selected from lower ketones, C1-C3 alcohols, or mixtures thereof, and preferably from acetone, methanol, ethanol, or a combination of two or more of these.

[0009] In the above preparation method, as a preferred embodiment of the method of the present invention, the silica gel particle size used in step (3) for silica gel column chromatography is selected from 100-200 mesh, the elution solvent used for silica gel column chromatography is chloroalkanes, or a mixed solvent system of chloroalkanes and C1-C3 alcohols, the elution method used for silica gel column chromatography is preferably gradient elution, the chloroalkanes are preferably from chloroform, dichloromethane or mixtures thereof, and the C1-C3 alcohols are preferably from methanol, ethanol or mixtures thereof. The elution method is chloroform:methanol = 100:0-70:30 (V / V) gradient elution, or dichloromethane:methanol = 100:0-70:30 (V / V) gradient elution; preferably, Sephadex LH-20 gel column is used for gel column chromatography, the chloroalkanes are preferably from chloroform, dichloromethane or mixtures thereof, and the C1-C3 alcohols are preferably from methanol, ethanol or mixtures thereof. The elution solvent used is a chloroform:methanol system (90:10-10:90, V / V) or a dichloromethane:methanol system (90:10-10:90, V / V), preferably chloroform:methanol = 50:50 (V / V) or dichloromethane:methanol = 50:50 (V / V).

[0010] In the above preparation method, as a preferred embodiment of the method of the present invention, the reversed-phase column chromatography in step (4) uses C18 reversed-phase packing material. The elution solvent used in the reversed-phase column chromatography is selected from acetonitrile / water solution, methanol / water solution, or an aqueous solution of a mixed solvent of methanol and acetonitrile. The elution solvent is preferably an acetonitrile to water volume ratio of 30:70-100:0, or a methanol to water volume ratio of 50:50-100:0, more preferably an acetonitrile to water volume ratio of 70:30-85:15 in an acetonitrile aqueous solution, or a methanol to water volume ratio of 85:15-95:5 in a methanol aqueous solution.

[0011] This invention also provides the application of the above-mentioned sixteen-membered macrocyclic lactone compounds in the treatment of plant pathogenic fungi. The fungi mentioned are preferably *Botryosphaeria dothidea*, *Ceratocystis*, *Alternaria solani*, *Alternaria alternata*, *Valsa mali* (apple tree rot fungus), *Colletotrichum camelliae*, *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, and *Colletotrichum fructicola*.

[0012] The beneficial effects of this invention are as follows: The sixteen-membered macrocyclic lactone bafilomycin compounds, namely compounds 1 and 2, provided by this invention were isolated from the secondary metabolites of Streptomyces sp. TH21 and exhibited activity against plant pathogenic fungi, thus expanding the activity spectrum of such compounds and showing promise for development into bio-based fungicides. Attached Figure Description

[0013] Figure 1 This is the hydrogen spectrum of the compound bafilomycin H(1).

[0014] Figure 2 This is the carbon spectrum of the compound bafilomycin H(1).

[0015] Figure 3 This is the DEPT135° spectrum of the compound bafilomycin H(1).

[0016] Figure 4 This is the high-resolution mass spectrum of the compound bafilomycin H(1).

[0017] Figure 5This is the hydrogen spectrum of compound 19-O-methylhygrolidin (2).

[0018] Figure 6 This is the carbon spectrum of compound 19-O-methylhygrolidin (2).

[0019] Figure 7 This is the DEPT 135° spectrum of compound 19-O-methylhygrolidin (2).

[0020] Figure 8 This is the high-resolution mass spectrum of compound 19-O-methylhygrolidin (2).

[0021] Figure 9 These are the NMR data (H: 400 MHz; C: 100 MHz) of the new sixteen-membered macrocyclic lactone compounds 1 and 2 in Acetone-d6.

[0022] Figure 10 The compounds bafilomycin H (1) and 19-O-methylhygrolidin (2) exhibit antifungal activity.

[0023] Figure 11 Streptomyces The morphology of sp.TH21 in ISP2 medium.

[0024] Figure 12. Determination of the effect of the compound on Alternaria alternifolia using the toxic plate method. Alternaria alternata active.

[0025] Figure 13. Determination of the activity of the compound against Alternaria solani using the poisoned plate method.

[0026] Figure 14 shows the determination of the compound's activity against Valsa mali, the apple tree rot pathogen, using the filter paper disc method. Detailed Implementation

[0027] The specific embodiments of the present invention will be further described below.

[0028] The fermentation culture of the new sixteen-membered macrolide-producing bacterium Streptomyces sp. TH21, as well as the extraction, isolation, and activity experiments of the compounds, are detailed in the specific examples. The following examples are further illustrations of the invention but do not limit the scope of the invention.

[0029] This invention provides a 16-membered macrocyclic lactone bafilomycin-like compound with the following chemical structural formula: ; R represents segment 1 or segment 2 in the diagram; This invention also provides a Streptomyces sp. strain capable of producing the aforementioned 16-membered macrocyclic lactone bafilomycin. This Streptomyces strain was isolated by the inventors' team from soil from a large vegetable planting base in Huzhou, Zhejiang Province. Its internal marker is TH21. It was deposited on December 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.33060. It is classified as Streptomyces sp., and its 16S rRNA is registered in GenBank under the number PX944784. Figure 1 As shown, this strain forms white aerial mycelia on ISP2 medium, and the early spores are white with abundant spores.

[0030] The technical solution of the present invention will be described below through specific implementation examples.

[0031] Example 1: Fermentation culture of Streptomyces sp. TH21 (1) Fermentation strain: The fermentation strain is Streptomyces sp. TH21.

[0032] (2) Slant culture: Solid culture medium (g / L) (composition: yeast extract 4.0, malt extract 10.0, glucose 4.0, CoCl2·6H2O 0.005, agar 18.0, distilled water, pH 7.2-7.4) was sterilized at 121℃ for 20 min, and then cultured at 28℃ for 6-8 days after inoculation.

[0033] (3) Seed culture: The seed culture medium (g / L) (glucose 4.0, malt extract 10.0, yeast extract 4.0, CaCO3 2.0, distilled water, pH 7.2-7.4) was sterilized at 121℃ for 20 min. 250 ml of the medium was dispensed into each 1000 ml Erlenmeyer flask. The Streptomyces spores on the slant were then washed off with sterile water to prepare a spore suspension with a concentration of 1×10⁻⁶. 7 -1×10 8 spores / ml. Add 2 ml of spore suspension to each bottle, place on a shaker, rotate at 250 r / min, and incubate at 28℃ for 48 h to obtain seed culture.

[0034] (4) Fermentation: The fermentation medium (g / L) (4g yeast powder, 40g soluble starch, 10g glucose, 10g malt extract, 2g CaCO3, 1g FeSO4·7H2O, 1g ZnSO4·7H2O, 1g MnCl2·4H2O, prepared with distilled water, pH 7.2-7.4) was sterilized at 121℃ for 20 min. The seed culture was inoculated into a 50L fermenter (30L capacity) at a 5% (volume percentage) inoculation rate and cultured at 28℃ with a stirring speed of 200-300 r / min and an aeration rate of 1000 L / h for 7 days.

[0035] Example 2: Extraction and separation of bafilomycin-like compounds (hexadecyl macrocyclic lactones)

[0036] Fermentation was carried out according to the method in Example 1 to obtain 30 L of fermentation broth. The broth was centrifuged at 4000 rpm for 10 min to obtain a clear liquid. The clear liquid was adsorbed onto DIAION HP20 resin and eluted with 5 L of ethanol to obtain an ethanol eluent. The eluent was concentrated under reduced pressure at 45 °C to remove the organic phase to 1 L. Then, it was extracted three times with 2 times the volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated to dryness under reduced pressure at 45 °C to obtain 340 g of viscous crude extract.

[0037] The obtained viscous crude extract was mixed with silica gel, dried, and subjected to silica gel (particle size 100-200 mesh) column chromatography. Gradient elution was performed using chloroform:methanol (100:0, 95:5, 90:10, 85:15, 80:20, and 70:30, V / V), and the relevant components were collected. Target component 1 was obtained by TLC detection and antibacterial activity tracing. Target component 1 was separated by gel column chromatography (chloroform:methanol = 50:50, V / V), and then further purified using reversed-phase column chromatography to obtain the target product.

[0038] The reversed-phase column chromatography conditions are as follows: Liquid chromatography system: Shimadzu LC-8A preparative high-performance liquid chromatograph; Column: ZORBAX-C18 (250 mm * 21.2 mm); Flow rate: 20 mL / min; Detection wavelength: λ = 220 nm / 254 nm; The eluent was methanol to water in a volume ratio of 85:15 to 95:5 (25 min). The compound with a retention time of 22 min was collected to obtain bafilomycin H (1) 12 mg and the compound with a retention time of 17 min was collected to obtain 19-O-methylhygrolidin (2) 15 mg.

[0039] Example 3: Structural Identification of New Sixteen-Membered Macrocyclic Lactone Compounds New sixteen-membered macrocyclic lactone compounds 1 and 2 were identified by 1D and 2D NMR, MS and other spectroscopic analyses, including bafilomycin H (1) and compound 19-O-methylhygrolidin (2). 1 H and 13 C NMR (Acetone-d6) data can be found Figure 9 : Specific spectral data can be found in the instruction manual. Figure 1-14 As shown.

[0040] The structural formulas of the new sixteen-membered macrocyclic lactones are determined as follows: ; Compound bafilomycin H (1), R is fragment 1; compound 19-O-methylhygrolidin (2), R is fragment 2.

[0041] The physicochemical properties of the new sixteen-membered macrocyclic lactone compounds 1 and 2 are as follows: bafilomycin H (1): Appearance: White amorphous powder; Solubility: Easily soluble in dichloromethane, methanol, ethanol, and ethyl acetate; slightly soluble in water. Melting point: 140-142 ℃; Molecular formula: C 31 H 44 O5; Specific curl: = − 29.2 (c 0.08, MeOH); High-resolution mass spectrometry (HRESI-MS): 519.3083 [M + Na] + (C 31 H 44 The calculated value of O5Na is 519.3086. Ultraviolet absorption spectrum (UV absorption spectrum) λ max (Methanol) nm (logε): 248, 292; Infrared absorption spectrum (IR absorption spectrum) V max cm -1: 3444, 2969, 1703, 1639, 1384, 1357, 1261, 1148, 1090, 1028, 803.

[0042] 19-O-methylhygrolidin (2); Appearance: White amorphous powder; Solubility: Easily soluble in dichloromethane, methanol, ethanol, and ethyl acetate; slightly soluble in water. Melting point: 141-144 ℃; Molecular formula: C 40 H 62 O 11 ; Specific curl: = + 42.6 (c 0.08, MeOH); High-resolution mass spectrometry (HRESI-MS): 741.4191 [M + Na] + (C 40 H 62 O 11 The calculated value of Na is 741.4190. Ultraviolet absorption spectrum (UV absorption spectrum) λ max (Methanol) nm (logε): 251, 292; Infrared absorption spectrum (IR absorption spectrum) V max cm -1 : 3445, 2966, 1701, 1385, 1261, 1091, 1030, 801.

[0043] Example 4: Antibacterial activity test The antibacterial activity of compounds bafilomycin H (1) and 19-O-methylhygrolidin (2) was tested. The fungicide testing technique (Pesticide Testing Techniques and Evaluation Methods, Huang Guoyang, China Agricultural Press, 2000, 24-30) was used to test the inhibitory activity of the compounds against *Botryosphaeria dothidea*, *Ceratocystis*, *Alternaria solani*, *Alternaria alternata*, *Valsa mali* (the causal agent of apple tree rot), *Colletotrichum camelliae*, *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, and *Colletotrichum fructicola*, among other pathogens. The commercially available pesticide tebuconazole was used as a control. The results are shown in […]. Figure 10 Compounds bafilomycin H (1) and 19-O-methylhygrolidin (2) both showed inhibitory effects on the tested pathogens. Among them, they showed better activity against Alternaria solani, Alternaria alternata, and Valsa mali, the causal agent of apple tree rot, with MBC (mg / L) ranging from 8 to 15 mg / L. Their activity against Alternaria solani was better than that against tebuconazole.

[0044] The antifungal activities of compounds bafilomycin H (1) and 19-O-methylhygrolidin (2) against *Alternaria solani*, *Alternaria alternata*, and *Valsa mali*, the causal agent of apple tree rot, were tested using the plate method and filter paper method. The plate method employed two concentrations of the fungus: 1, 2, and 5 mg / L. The concentrations on the filter paper were 50 μg and 25 μg / filter paper, with each treatment repeated three times. The mixtures were incubated at 28 °C for 4–7 days. A control group without the fungus was included. The mycelial inhibition rate of the pathogens was measured.

[0045] The antibacterial activities of compounds bafilomycin H (1) and 19-O-methylhygrolidin (2) were determined by the plate assay. The results showed that at concentrations of 1, 2, and 5 mg / L, bafilomycin H (1) exhibited antibacterial activities of 52%, 66%, and 84% against *Alternaria alternata*, and 52%, 57%, and 71% against *Alternaria solani*, respectively. 19-O-methylhygrolidin (2) showed antibacterial activities of 38%, 59%, and 75% against *Alternaria alternata*, and 37%, 58%, and 68% against *Alternaria solani*, respectively. Figure 12-13 The antibacterial test results using the filter paper method showed that, at 50 μg / filter paper, the compounds bafilomycin H (1) and 19-O-methylhygrolidin (2) exhibited antibacterial activities of 87% and 78% respectively against the apple tree rot pathogen Valsa mali. Figure 14 ).

Claims

1. A sixteen-membered macrocyclic lactone compound, characterized in that, Its chemical structural formula is as follows: R represents segment 1 or segment 2 in the diagram.

2. A Streptomyces strain that produces the sixteen-membered macrocyclic lactone compound as described in claim 1, characterized in that, The strain in question is Streptomyces sp. TH21, with accession number CGMCC No. 33060.

3. A method for preparing the sixteen-membered macrocyclic lactone compound of claim 1, the method comprising the following steps: (1) Streptomyces sp. TH21, whose preservation number is (CGMCC No.33060). Fermentation was carried out in a liquid culture medium containing carbon and nitrogen sources, and the fermentation broth was collected. (2) The fermentation broth collected in step (1) is centrifuged to remove mycelium and retain the clear liquid. The clear liquid is then passed through macroporous adsorption resin for adsorption, and then eluted with organic solvent to obtain eluent. After that, the eluent is concentrated under reduced pressure, silica gel column chromatography and gel column chromatography are performed to obtain a fraction containing sixteen-membered macrocyclic lactones. The sixteen-membered macrocyclic lactones are then purified and separated by reverse-phase preparative column chromatography.

4. The method according to claim 3, characterized in that... The carbon source in step (1) is selected from one or a combination of glucose, soluble starch, dextrin, sucrose, corn starch, maltose, industrial molasses, glycerol, sorbitol, mannitol, lactose, xylan, or more of the above substances, preferably a combination of glucose and soluble starch, or a combination of glucose and corn starch; the nitrogen source is selected from one or a combination of yeast powder, yeast extract, soybean meal, soybean flour, peanut meal, malt extract, peptone, beef extract, yeast extract, corn steep liquor, wheat bran, gluten powder, urea, ammonium salt, or two or more of the above substances, preferably yeast powder, malt extract, or a combination thereof.

5. The method according to claim 3, characterized in that... The resin mentioned in step (2) is a macroporous adsorption resin, preferably DIAION HP20 or LX-60, and the organic solvent is selected from lower ketones, C1-C3 alcohols or mixtures thereof, preferably acetone, methanol, ethanol or a combination of two or more thereof.

6. The method according to claim 3, characterized in that... The silica gel used in step (2) for silica gel column chromatography has a particle size of 100-200 mesh, and the elution solvent used for silica gel column chromatography is a chloroalkane, or a mixed solvent system of chloroalkane and C1-C3 alcohol. The chloroalkane is preferably chloroform, dichloromethane, or a mixture thereof, and the C1-C3 alcohol is preferably methanol, ethanol, or a mixture thereof. The preferred elution method for the silica gel column chromatography is gradient elution; the elution method is chloroform:methanol = 100:0-70:30 (V / V) gradient elution, or dichloromethane:methanol = 100:0-70:30 (V / V) gradient elution; the gel type used for the gel column chromatography is Sephadex LH-20 dextran gel, and the elution solvent used for the gel column chromatography is a mixed solvent system of chloroalkanes and C1-C3 alcohols; the preferred elution method used for the gel column chromatography is isocratic elution; the chloroalkanes are preferably selected from chloroform, dichloromethane, or mixtures thereof, and the C1-C3 alcohols are preferably selected from methanol, ethanol, or mixtures thereof. The elution solvent used is a chloroform:methanol system (90:10-10:90, V / V) or a dichloromethane:methanol system (90:10-10:90, V / V), preferably chloroform:methanol = 50:50 (V / V) or dichloromethane:methanol = 50:50 (V / V).

7. The method according to claim 3, characterized in that... The reversed-phase column used in step (2) is a reversed-phase C18 column. The elution solvent used in the reversed-phase column chromatography is selected from acetonitrile / water solution, methanol / water solution, or an aqueous solution of a mixture of methanol and acetonitrile. The elution solvent is preferably an acetonitrile to water volume ratio of 30:70-100:0, or a methanol to water volume ratio of 50:50-100:

0. More preferably, the acetonitrile to water volume ratio in the acetonitrile aqueous solution is 70:30-85:15, or the methanol to water volume ratio in the methanol aqueous solution is 85:15-95:

5.

8. The sixteen-membered macrocyclic lactone compound according to claim 1 is used for resistance to plant pathogenic fungi.

9. The application according to claim 8, characterized in that, The plant pathogenic fungi include *Botryosphaeria dothidea*, *Ceratocystis*, *Alternaria solani*, *Alternaria alternata*, *Valsa mali* (the causal agent of apple tree rot), *Colletotrichum cameelliae*, *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, and *Colletotrichum fructicola*.

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