Method for preparing picopyranapine from marine fungi GHQ-208

Abstract

The invention belongs to the field of compound preparation, and particularly relates to a method for preparing pyridinopyranine by using marine fungi GHQ-208. The invention discloses a preparation method for producing mixed-source terpenoids on the basis of fungi Penicillium sp. GHQ-208 (the preservation number is CCTCC NO: M 2025229) derived from mangrove forest bottom mud in Hainan province, and application of the mixed-source terpenoids as a green insecticide. The preparation process of the compound comprises the following steps: firstly, carrying out fermentation culture on a preserved strain GHQ-208 to obtain a metabolite, and then carrying out multi-step separation and purification processes, including silica gel column chromatography, reverse ODS silica gel column chromatography, LH-20 gel column chromatography and chromatographic separation technologies such as analysis and semi-preparative HPLC (High Performance Liquid Chromatography). A novel biological source insecticidal compound which is novel in structure, unique in action mechanism and environmentally friendly is developed from secondary metabolites of marine fungi, and a new candidate substance is provided for research and development of green pesticides.

Description

Technical Field

[0001] This invention belongs to the field of compound preparation, specifically relating to a method for preparing diazonopyr using marine fungus GHQ-208. Background Technology

[0002] Pesticides play a vital role in agricultural production and public health, but with increasing pest resistance and rising environmental safety requirements, there is an urgent need to develop novel, highly effective, and low-toxicity pesticides. Currently, global agriculture faces a growing threat from pests, particularly piercing-sucking insects such as aphids, which severely damage various crops including vegetables, fruits, grains, and tea, leading to significant declines in yield and quality. However, long-term reliance on traditional pesticides (such as organophosphates, carbamates, pyrethroids, and neonicotinoids) has resulted in increasingly prominent pesticide resistance problems. Some aphid populations have even developed resistance hundreds to thousands of times to existing pesticides, severely reducing control effectiveness. Furthermore, the high toxicity of traditional pesticides to non-target organisms such as bees and aquatic life raises serious ecological safety concerns. Therefore, developing novel, highly effective, and environmentally friendly pesticides with novel mechanisms of action has become a key research focus, crucial for ensuring sustainable agricultural development and reducing ecological risks.

[0003] Pyripyropene A (PP-A) is a meroterpenoid compound derived from Aspergillus fumigatus FO-1289, which has significant insecticidal activity and pharmacological effects.

[0004] In agriculture, PP-A is a green and highly effective insecticide with a mechanism of action different from traditional neurotoxic or respiratory-inhibiting insecticides. Studies have shown that PP-A specifically targets proteins related to the insect nervous system, causing pests to exhibit abnormal excitement and excessive wandering behavior, ultimately leading to death. This compound exhibits excellent insecticidal activity against major agricultural pests such as aphids and whiteflies, with an LC50 as low as 0.98 mg / L against juvenile aphids and only 2.5 mg / L against whiteflies. Furthermore, it causes no phytotoxicity to crops and possesses outstanding environmentally friendly characteristics.

[0005] Furthermore, PP-A is one of the most potent known inhibitors of cholesterol-O-acyltransferase (SOAT), with an IC50 of up to 58 nM for its inhibitory activity against SOAT, and it exhibits high selectivity for the SOAT-2 subtype in human hepatocytes. This characteristic makes it a promising candidate for pharmaceutical development in the treatment of dyslipidemia-related diseases such as atherosclerosis and steatosis.

[0006] Based on the unique insecticidal mechanism and pharmacological activity of PP-A, the development of its novel derivatives or formulations is of great significance for solving the problem of resistance to existing insecticides and expanding their application in the pharmaceutical field. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the various problems, defects, and deficiencies in the prior art, and to provide a method for preparing the mixed-source terpenoid compound difenocyanate based on the fungus *Penicillium sp. GHQ-208* (accession number CCTCC NO: M2025229) from Hainan mangrove sediment, and its application as a green insecticide. The chemical formula of the terpenoid compound is C2. 31 H 37 NO 10 The structure is shown in Formula 1—pyripyropene A.

[0008] The mixed-source terpenoid compound, difenosulfuron, was isolated from the liquid fermentation products of the fungus *Penicillium sp. GHQ-208* in the mangrove sediment of Hainan. Studies have shown that this compound exhibits strong insecticidal activity and is environmentally friendly, indicating its potential for further development into novel, environmentally friendly insecticides.

[0009] This invention relates to the use of the fungus Penicillium sp. GHQ-208, accession number: CCTCC NO: M 2025229, accession date: February 17, 2025; depositary institution: China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0010] The purpose of this invention is to provide a compound with green and environmentally friendly insecticidal properties. Its structural formula is shown in Formula I. .

[0011] The compound was prepared based on the fungus Penicillium sp. GHQ-208, derived from the sediment of Hainan mangroves (accession number: CCTCC NO: M 2025229).

[0012] Another object of the present invention is to provide a method for preparing the compound.

[0013] Another object of the present invention is to provide the use of the compound in the preparation of insecticides.

[0014] This invention is achieved through the following technical solution: The mixed-source terpenoids were isolated from the liquid fermentation products of the fungus *Penicillium sp. GHQ-208* in the mangrove sediment of Hainan. Studies have shown that these compounds possess strong insecticidal activity and are environmentally friendly, indicating their potential for further development into novel, environmentally friendly insecticides.

[0015] This invention relates to the use of the fungus Penicillium sp. GHQ-208, accession number: CCTCC NO: M 2025229, accession date: February 17, 2025; depositary institution: China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0016] The compounds of Formula I of the present invention can be obtained by microbial fermentation culture to obtain fermentation products containing such compounds, and then the crude fermentation extract can be separated and purified by silica gel column chromatography, reversed ODS silica gel column chromatography, LH-20 gel column chromatography and semi-preparative HPLC.

[0017] The method for preparing the compound includes the following steps: (1) Activation of strain: Penicillium sp. GHQ-208 with preservation number CCTCC NO: M 2025229 was inoculated into PDA solid slant medium and cultured at 28℃ for 3 days; (2) Liquid fermentation: Take the activated cells from step (1) and inoculate them into the No. 2 liquid culture medium for fungi, and ferment them at 28℃ and 220 rpm for 13 days; (3) Processing of fermentation products: (a) The fermentation broth was filtered and separated to obtain the supernatant and mycelium; (b) Extract the supernatant three times with an equal volume of ethyl acetate, and combine the extracts; (c) The mycelium was extracted with 80% acetone solution. The resulting extract was concentrated under reduced pressure to remove acetone, and then extracted three times with an equal volume of ethyl acetate. (4) Combine the ethyl acetate extracts obtained in steps (b) and (c), concentrate under reduced pressure, and obtain crude extract; (5) ODS reversed-phase silica gel column chromatography: The crude extract was separated using an ODS reversed-phase silica gel column with a gradient elution of methanol-water (1:1, v / v) as the mobile phase, and the target components were collected. (6) Semi-preparative high performance liquid chromatography purification: The components obtained in step (5) are further purified by semi-preparative high performance liquid chromatography with a preparation gradient of acetonitrile:water = 45:55; (7) LH-20 gel column chromatography: The product obtained in step (6) was finally purified by LH-20 dextran gel column with methanol as the mobile phase to obtain the compound shown in Formula I.

[0018] The fermentation medium in step (1) contains the following components (w / v): mannitol 2%, maltose 2%, glucose 1%, monosodium glutamate 1%, KH2PO4 0.05%, MgSO4·7H2O 0.03%, yeast extract 0.3%, corn steep liquor 0.1%, and the remainder is aged seawater. The pH value of the medium is 6.5.

[0019] The present invention also includes the use of the method for preparing the compound in the preparation of insecticides.

[0020] The beneficial effects of this invention are as follows: This invention provides a method for preparing mixed-source terpenoid compounds based on the fungus *Penicillium sp. GHQ-208* (accession number: CCTCC NO: M 2025229) from the sediment of Hainan mangroves, and its application as a green insecticide. This invention develops a novel bio-based insecticide compound with a novel structure, unique mechanism of action, and environmental friendliness from the secondary metabolites of marine fungi, providing a new candidate substance for the development of green pesticides. Attached Figure Description

[0021] Figure 1 The compound described in this invention 1 1H NMR (400MHz, DMSO-d6) plot; Figure 2 The compound described in this invention 13 C NMR (100 MHz, DMSO-d6) plot; Figure 3 The compound DEPT-135 described in this invention ( 13 Figure C, 100MHz, DMSO-d6); Figure 4 The compound HMBC described in this invention ( 1 H- 13 Figure C, 400MHz, DMSO-d6); Figure 5 The diagram shows the compound COSY (400 MHz, DMSO-d6) described in this invention. Figure 6 The compound HMQC described in this invention ( 1 H- 13 Figure C, 400MHz, DMSO-d6. Detailed Implementation

[0022] The chemical structure of compound I referred to in the following examples: .

[0023] Example 1: Fermentation production and purification of compound I 1. Fermentation production Fermentation culture of the production strain: Following the conventional method for culturing microorganisms, take an appropriate amount of the fungus Penicillium sp. GHQ-208, inoculate it onto PDA solid slant medium, and incubate it in a 28℃ incubator for 4 days.

[0024] Take an appropriate amount of spores of the fungus Penicillium sp. GHQ-208, which has been cultured on an slant for 4 days, and inoculate them into a 1L Erlenmeyer flask containing 150mL of culture medium [culture medium composition (g / L): mannitol 20, maltose 20, glucose 10, monosodium glutamate 10, KH2PO4 0.5, MgSO4·7H2O 0.3, yeast extract 0.3, corn steep liquor 1]. Incubate at 28℃ and 220rpm for 13 days to obtain the fermentation product.

[0025] 2. Obtaining the extract The fermentation broth was separated from the supernatant and mycelium using gauze. The mycelium was treated three times with methanol and concentrated under reduced pressure until no methanol remained. The resulting aqueous phase was extracted three times with an equal volume of ethyl acetate. The fermentation broth was directly extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to obtain a crude extract.

[0026] 3. Isolation and purification of compounds After the extract was dissolved in methanol, reversed ODS column chromatography was performed using methanol-water as the elution system. Fractions of the same type were combined to obtain five fractions. Fraction 3 was first separated and purified by semi-preparative high-performance liquid chromatography (acetonitrile:water = 45:55), and then purified using an LH-20 dextran gel column with gradient elution using methanol as the mobile phase to obtain compound I.

[0027] Compound I is a yellow powder with the molecular formula C. 31 H 37 NO 10 The cation mass spectrometry (ESI-MS) result was 584.3 [M+H], with an unsaturation degree of 14. 1 H and 13 The C NMR values ​​are shown in Table 1.

[0028] Table 1 Compound I 1 H and 13 C NMR data (400 and 100 MHz, in DMSO-d6)

[0029] a) The signal attribution in this table is based on the results of DEPT, HMQC, and HMBC spectral analysis. The multiplicity of the carbon signal was determined using the DEPT method and represented by s (singleton), d (doubleton), t (tripleton), q (quartet), and m (multipleton), respectively.

[0030] b) The numbers and codes in this column represent the... 1 H- 1 In the H COSY spectrum and the corresponding rows 1 H gives the coupling correlation signal 1 H nucleus.

[0031] c) The numbers and codes in this column represent the corresponding lines in the HMBC spectrum. 1 H gives the coupling correlation signal 13 C core.

[0032] Figure 1-6 The NMR spectrum of this compound. Figure 1 The compound described in this invention 1 1H NMR (400MHz, DMSO-d6) plot; Figure 2 The compound described in this invention 13 C NMR (100 MHz, DMSO-d6) plot; Figure 3 The compound DEPT-135 described in this invention ( 13 Figure C, 100MHz, DMSO-d6); Figure 4 The compound HMBC described in this invention ( 1 H- 13 Figure C, 400MHz, DMSO-d6); Figure 5 The diagram shows the compound COSY (400 MHz, DMSO-d6) described in this invention. Figure 6 The compound HMQC described in this invention ( 1 H- 13 Figure C, 400MHz, DMSO-d6.

Claims

1. A method for preparing pyranoline using marine fungus GHQ-208, the structural formula of pyranoline is shown as formula I, characterized in that: Pyranofuran is prepared based on a Hainan mangrove mud-derived fungus Penicillium sp. GHQ-208, preservation number: CCTCC NO: M 2025229, ​ 。 2. A process for the preparation of Pyranol using marine fungus GHQ-208 as claimed in claim 1 wherein, comprising the following steps: (1) strain activation: inoculate Penicillium sp. GHQ-208 with a preservation number of CCTCC NO: M 2025229 into PDA solid slant medium, and culture at 28°C for 3 days, the fungus Penicillium sp. GHQ-208, preservation number: CCTCC NO: M 2025229, preservation date: February 17, 2025, preservation unit: China Center for Type Culture Collection, address: Wuhan, China; (2) liquid fermentation: inoculate the strain activated in step (1) into fungal No. 2 liquid medium, and ferment and culture at 28°C, 220 rpm on a shaking table for 13 days; (3) fermentation product processing: (a). filter the fermentation broth to obtain supernatant and mycelium; (b). extract the supernatant with an equal volume of ethyl acetate 3 times, and combine the extract; (c). immerse the mycelium in 80% acetone solution, and after the extract is concentrated under reduced pressure to remove acetone, extract with an equal volume of ethyl acetate 3 times; (4) combine the ethyl acetate extracts obtained in steps (b) and (c), and concentrate under reduced pressure to obtain a crude extract; (5) ODS reverse phase silica gel column chromatography: separate the crude extract using ODS reverse phase silica gel column, and perform gradient elution with methanol-water, v / v 1:1, as the mobile phase, and collect the target components; (6) semi-preparative high performance liquid chromatography purification: further purify the components obtained in step (5) by semi-preparative high performance liquid chromatography, and the preparation gradient is acetonitrile: water = 45:55; (7) LH-20 gel column chromatography: finally purify the product obtained in step (6) using LH-20 dextran gel column, and obtain compound pyranofuran using methanol as the mobile phase.

3. The method for preparing difenazone using marine fungus GHQ-208 as described in claim 3, characterized in that: The fermentation medium in step (1) comprises the following components, w / v: mannitol 2%, maltose 2%, glucose 1%, monosodium glutamate 1%, KH2PO4 0.05%, MgSO4·7H2O 0.03%, yeast extract powder 0.3%, corn syrup 0.1%, and the balance is old seawater, and the pH value of the medium is 6.

5.

4. Use of the method for preparing pyranofuran from marine fungus GHQ-208 according to claim 1 in the preparation of an insecticide.

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