Idiomycin A derivatives and their preparation methods and applications

Abstract

The present invention relates to the field of organic compound production, and discloses an edemycin A derivative, its preparation method, and application. The structural formula of the edemycin A derivative is shown in formula (I); the preparation method of the edemycin A derivative comprises: adding a spermidine analogue during the fermentation and cultivation of the transformed strain; wherein the structural formula of the edemycin A derivative is shown in formula (I), and the structural formula of the spermidine analogue is shown in formula (II). The present invention also provides a fungicide containing the above-mentioned edemycin A derivative, and the application of the edemycin A derivative and the fungicide in preventing and controlling plant diseases. The edemycin A derivative has a strong antibacterial ability against tobacco bacterial wilt and a high production efficiency, and can be used as a green, low-toxic, and highly effective plant fungicide.

Description

Technical Field

[0001] The present invention relates to the field of organic compound production, and in particular to an edemectin A derivative and a preparation method and application thereof. Background Art

[0002] Tobacco bacterial wilt is a high-temperature, high-humidity soil-borne bacterial disease caused by Ralstonia solanacearum. The pathogen has a wide range of hosts, strong pathogenicity, and greatly varying physiological characteristics. It is one of the most important plant pathogens. It generally infects plants through root wounds, root tips, or secondary roots, damaging the plant's xylem, ultimately causing the entire plant to turn yellow, black, wither, and eventually die, resulting in a serious reduction in tobacco yields and significant economic losses to tobacco farmers.

[0003] Tobacco bacterial wilt has been reported in every provincial-level administrative region in my country, with the most severe damage occurring in major tobacco-producing regions such as Guangxi, Guangdong, Fujian, Hunan, Guizhou, Sichuan, and Chongqing, with signs of spreading to northern tobacco-producing areas. During outbreaks, tobacco leaf yields can drop by over 50%, severely impacting tobacco production.

[0004] Currently, in actual production, tobacco bacterial wilt prevention and control still relies on chemical agents, primarily organic copper (thiophanate-copper, nonathiacobalamin), organic sulfur (chloroquine, ethoxychlor), organic chlorine (chloride), inorganic copper (copper hydroxide), and inorganic sulfur (lime sulfur). However, the long-term use of these chemical agents alone can lead to the development of drug resistance in pathogens, imbalance in soil microbial flora, and reduced soil resistance to pathogens. Furthermore, it can also lead to excessive pesticide residues in the soil, seriously polluting the environment. Therefore, the development of new, highly effective, and low-toxic agricultural fungicides is urgent.

[0005] Ephedrine A is a linear non-ribosomal antimicrobial peptide produced by Brevibacillus brevis X23. + , G - ), fungi (yeast, mold) and mycoplasma, but its production is low and it has high cytotoxicity to model animals (mice). Therefore, the application of edemectin A in the field of pesticides is subject to certain restrictions. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art and provide a derivative of edemycin A and its preparation method and application. The edemycin A derivative has strong antibacterial ability against tobacco bacterial wilt and high production efficiency, and can be used as a green, low-toxic and high-efficiency plant fungicide.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides an edemectin A derivative, the structural formula of the edemectin A derivative is shown in formula (I),

[0008]

[0009] The second aspect of the present invention provides the use of a spermidine analogue in the preparation of the edemacin A derivative according to claim 1, wherein the structural formula of the edemacin A derivative is shown in formula (I), and the structural formula of the spermidine analogue is shown in formula (II).

[0010]

[0011] Preferably, the method for preparing the spermidine analogue comprises: subjecting tert-butyl N-(3-aminopropyl)carbamate, 4-chloro-1-butanol and potassium carbonate to a contact reaction.

[0012] Preferably, the molar ratio of tert-butyl N-(3-aminopropyl)carbamate, 4-chloro-1-butanol and potassium carbonate is 1:1.8-2.2:1.8-2.2.

[0013] Preferably, the contact reaction conditions include at least: a temperature of 80-100° C., a stirring rate of 300-600 rpm, and a time of 4-8 h.

[0014] Preferably, the solvent for the contact reaction is n-butane.

[0015] Preferably, the method for preparing the spermidine analogue further comprises: purifying and crystallizing the reaction solution obtained from the contact reaction.

[0016] Preferably, the purification process comprises: filtering the reaction solution and concentrating I to obtain a concentrate, mixing the concentrate with an acid-containing methanol solution for stirring reaction, and concentrating II to obtain a crude product.

[0017] Preferably, the crystallization process comprises: mixing the crude product with ethyl acetate for crystallization.

[0018] Preferably, the acid concentration in the acid-containing methanol solution is 5-9 M, and the stirring reaction conditions at least include: a temperature of 5-40° C., a stirring rate of 300-600 rpm, and a time of 8-14 h.

[0019] The third aspect of the present invention provides a method for preparing an edemectin A derivative, the method comprising: adding a spermidine analogue during the fermentation culture of a transformed strain; wherein the structural formula of the edemectin A derivative is as shown in formula (I), the structural formula of the spermidine analogue is as shown in formula (II),

[0020]

[0021]

[0022] Preferably, the transformed strain is Brevibacillus brevis X23ΔspeE.

[0023] Preferably, the fermentation medium used in the fermentation culture contains tryptone, yeast extract and inorganic salts; more preferably, it contains 8-12 g / L tryptone, 3-8 g / L yeast extract and 8-12 g / L sodium chloride.

[0024] Preferably, the fermentation culture conditions include at least: a temperature of 25-35° C., a rotation speed of 150-200 rpm, and a time of 40-60 h.

[0025] Preferably, the spermidine analogue is added in an amount of 1-3 mM, and is added 10-15 hours after the start of fermentation.

[0026] Preferably, the method further comprises: performing solid-liquid separation on the fermentation liquid obtained by the fermentation culture to obtain a fermentation supernatant, and subjecting the fermentation supernatant to adsorption separation and concentration III.

[0027] Preferably, the adsorption separation uses a cation exchange resin.

[0028] The fourth aspect of the present invention provides a fungicide containing the above-mentioned edemectin A derivative and / or the edemectin A derivative prepared by the above-mentioned method.

[0029] The fifth aspect of the present invention provides the use of the above-mentioned edemectin A derivatives, the edemectin A derivatives prepared by the above-mentioned method, and the above-mentioned fungicides in preventing and controlling plant diseases.

[0030] Preferably, the plant disease is tobacco bacterial wilt.

[0031] Through the above technical scheme, the beneficial effects of the present invention are as follows: the edemacin A derivatives provided by the present invention have strong antibacterial ability against tobacco bacterial wilt pathogens, and are prepared by precursor feeding of transformed strains, with a simple preparation method and high production efficiency, and the precursor raw material structure is simple, easy to obtain, and low in cost, which is conducive to the industrial production of edemacin A derivatives; therefore, the edemacin A derivatives provided by the present invention can not only be used as green, low-toxic, and highly effective plant fungicides, effectively alleviating the problems of drug resistance, a sharp decline in soil microbial regulation ability, and environmental pollution existing in existing tobacco bacterial wilt chemical fungicides, but also the preparation method thereof has great prospects for industrial production application and is worthy of further research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the hydrogen NMR spectrum of the spermidine analog in Example 1;

[0033] Figure 2 is the carbon NMR spectrum of the spermidine analog in Example 1;

[0034] Figure 3 is the mass spectrum of the spermidine analog in Example 1;

[0035] Figure 4 LC-MS analysis of the crude product, wild-type Brevibacillus brevis X23 fermentation supernatant, and Brevibacillus brevis X23ΔspeE fermentation supernatant, and high-resolution mass spectra of edemectin A derivatives in Example 1;

[0036] Figure 5 This is a graph showing the biological activities of the fermentation supernatant obtained in Example 1 in Test Example 2, the fermentation supernatant of wild-type Brevibacillus brevis X23, the fermentation supernatant of Brevibacillus brevis X23ΔspeE, and the spermidine analog Ahao (2 mM). DETAILED DESCRIPTION

[0037] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0038] The first aspect of the present invention provides an edemectin A derivative, the structural formula of the edemectin A derivative is shown in formula (I),

[0039]

[0040] During the research process, the inventors of the present invention discovered that the edemacin A derivative with a structural formula as shown in formula (I) exhibits good inhibitory activity against the Ralstonia solanacearum GMI1000 strain, the pathogen of tobacco bacterial wilt, and can be used as a new, highly effective and low-toxic plant fungicide, effectively alleviating the problems of drug resistance, a sharp decline in the regulatory ability of soil microorganisms, and environmental pollution existing in existing chemical fungicides for tobacco bacterial wilt.

[0041] The second aspect of the present invention provides the use of a spermidine analogue in the preparation of the edemacin A derivative according to claim 1, wherein the structural formula of the edemacin A derivative is shown in formula (I), and the structural formula of the spermidine analogue is shown in formula (II).

[0042]

[0043]

[0044] The present invention uses a spermidine analogue with a structural formula as shown in formula (II) as a precursor to prepare an edemectin A derivative through conversion, so that the preparation method of the edemectin A derivative is simple and the production efficiency is high. The raw material structure is simple, easy to obtain, and low in cost, which is conducive to the industrial production and development of the edemectin A derivative.

[0045] According to the present invention, preferably, the method for preparing the spermidine analog comprises: subjecting tert-butyl N-(3-aminopropyl)carbamate, 4-chloro-1-butanol, and potassium carbonate to a contact reaction. The inventors have discovered that, in this preferred embodiment, using tert-butyl N-(3-aminopropyl)carbamate as a substrate is advantageous for increasing the yield of the spermidine analog and reducing the preparation cost.

[0046] According to the present invention, preferably, the molar ratio of tert-butyl N-(3-aminopropyl)carbamate, 4-chloro-1-butanol, and potassium carbonate is 1:1.8-2.2:1.8-2.2. The inventors have found that under this preferred embodiment, the yield of spermidine analogs is further improved.

[0047] According to the present invention, preferably, the contact reaction conditions include at least: a temperature of 80-100°C, specifically 80°C, 85°C, 90°C, 95°C, 100°C, or any value between the foregoing two values; a stirring rate of 300-600 rpm, specifically 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, or any value between the foregoing two values; and a reaction time of 4-8 hours, specifically 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or any value between the foregoing two values. The inventors have found that under this preferred embodiment, the production efficiency of spermidine analogs is further improved.

[0048] According to the present invention, a contact reaction system of tert-butyl N-(3-aminopropyl)carbamate, 4-chloro-1-butanol, and potassium carbonate is carried out in an organic solvent. Specifically, tert-butyl N-(3-aminopropyl)carbamate and an organic solvent are mixed to form a solution, and then 4-chloro-1-butanol and potassium carbonate are added. Preferably, the solvent for the contact reaction is n-butane. The inventors have discovered that this preferred embodiment is conducive to further improving the production efficiency of spermidine analogs.

[0049] According to the present invention, preferably, the method for preparing the spermidine analogue further comprises: purifying and crystallizing the reaction solution obtained from the contact reaction to obtain a pure product of the spermidine analogue.

[0050] According to the present invention, the purification process preferably comprises filtering the reaction solution and concentrating it (I) to obtain a concentrate, mixing the concentrate with an acid-containing methanol solution, stirring and reacting the mixture, and concentrating it (II) to obtain a crude product. The inventors have discovered that this preferred embodiment is beneficial for improving the purity and yield of the spermidine analog.

[0051] In the present invention, filtration can be performed using methods commonly used in the art, such as membrane filtration or filter paper filtration. Concentration I and Concentration II can be performed using conventional solution concentration methods, such as vacuum rotary evaporation or nitrogen blowing. The acid in the acid-containing methanol solution can be at least one of hydrochloric acid, sulfuric acid, and nitric acid.

[0052] According to the present invention, preferably, the concentration of the acid in the acid-containing methanol solution is 5-9M, specifically 5M, 6M, 7M, 8M, 9M, or any value between the two aforementioned values; the stirring reaction conditions include at least: a temperature of 5-40°C, specifically 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or any value between the two aforementioned values; a stirring rate of 300-600rpm, specifically 300rpm, 350rpm, 400rpm, 450rpm, 500rpm, 550rpm, 600rpm, or any value between the two aforementioned values; a time of 8-14h, specifically 8h, 10h, 12h, 14h, or any value between the two aforementioned values. The inventors have found that under this preferred embodiment, the purity and yield of the spermidine analogue are further improved.

[0053] According to the present invention, the crystallization can be carried out by any compound crystallization method. Preferably, the crystallization process includes: mixing the crude product with ethyl acetate for crystallization. The inventors have found that under this preferred embodiment, the yield of spermidine analogs is further improved.

[0054] The third aspect of the present invention provides a method for preparing an edemectin A derivative, the method comprising: adding a spermidine analogue during the fermentation culture of a transformed strain; wherein the structural formula of the edemectin A derivative is as shown in formula (I), the structural formula of the spermidine analogue is as shown in formula (II),

[0055]

[0056]

[0057] The preparation method of edemectin A derivatives provided by the present invention utilizes a transformation strain to efficiently convert a spermidine analog precursor into a target product, edemectin A derivatives. The preparation method is green and environmentally friendly, and has high production efficiency.

[0058] According to the present invention, the transformant strain can be any microbial strain capable of converting the spermidine analog represented by formula (II) into the edemectin A derivative represented by formula (I). The strain can be obtained by self-screening or modification, or purchased. Preferably, the transformant strain is Brevibacillus brevis X23ΔspeE, the construction method of which is described in Example 2 of CN115925552A. The inventors have discovered that this preferred embodiment is conducive to increasing the conversion rate of the spermidine analog precursor, thereby increasing the yield of the edemectin A derivative.

[0059] According to the present invention, the fermentation culture can employ a culture medium capable of providing nutrients for the growth and reproduction of the transformed strain. Preferably, the fermentation culture employs a fermentation medium containing tryptone, yeast extract, and inorganic salts; more preferably, it comprises 8-12 g / L tryptone, 3-8 g / L yeast extract, and 8-12 g / L sodium chloride. Exemplarily, the fermentation culture employs LB medium.

[0060] In the present invention, before the transformed strain is inoculated into the fermentation medium, it can be activated to enhance the growth ability of the strain and improve the conversion efficiency of the spermidine analog. For example, the transformed strain is inoculated into LB solid medium and cultured at a temperature of 25-35°C for 20-30 hours to obtain a single colony. The single colony is then picked and inoculated into LB liquid medium, which is shaken and cultured on a shaker at a temperature of 25-35°C and a rotation speed of 150 rpm-200 rpm for 8-14 hours. The activated bacterial liquid is then transferred to the fermentation medium at a ratio of 1% for fermentation culture.

[0061] According to the present invention, preferably, the fermentation culture conditions include at least: a temperature of 25-35°C, specifically 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, or any value between the two aforementioned values; a rotation speed of 150-200 rpm, specifically 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, or any value between the two aforementioned values; and a time of 40-60 h, specifically 40 h, 45 h, 50 h, 55 h, 60 h, or any value between the two aforementioned values. The inventors have found that under this preferred embodiment, the production efficiency of edemectin A derivatives is further improved.

[0062] According to the present invention, preferably, the amount of the spermidine analog added (i.e., the final concentration) is 1-3 mM, specifically 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, or any value between the two aforementioned values; the addition time is 10-15 hours after the start of fermentation, specifically 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or any time between the two aforementioned times. The inventors have found that under this preferred embodiment, the conversion rate of the spermidine analog precursor is beneficial. The spermidine analog must be sterilized by filtration through a 0.22 μm filter before addition.

[0063] According to the present invention, conventional methods can be used to separate edemacin A derivatives from the fermentation broth obtained by fermentation culture. Preferably, the method further comprises: performing solid-liquid separation on the fermentation broth obtained by fermentation culture to obtain a fermentation supernatant, and subjecting the fermentation supernatant to adsorption separation and concentration III to improve the separation efficiency of the edemacin A derivatives.

[0064] According to the present invention, solid-liquid separation can be performed by conventional methods in the art, such as centrifugation, filtration, etc. Adsorption separation preferably uses a cation exchange resin to improve the separation efficiency of edemectin A derivatives.

[0065] The fourth aspect of the present invention provides a fungicide containing the above-mentioned edemectin A derivative and / or the edemectin A derivative prepared by the above-mentioned method.

[0066] In the present invention, the edemacin A derivative can be used as a fungicide, the fermentation broth or fermentation clear liquid obtained by adding a spermidine analogue during the fermentation culture of the above-mentioned transformed strain can be used as a fungicide, or the edemacin A derivative can be used as an active ingredient and prepared with other excipients as a fungicide.

[0067] Based on the fact that edemectin A derivatives have the activity of inhibiting the pathogen Ralstonia solanacearum GMI1000 strain of tobacco bacterial wilt, the fifth aspect of the present invention provides the use of the above-mentioned edemectin A derivatives, the edemectin A derivatives prepared by the above-mentioned method, and the above-mentioned fungicide in preventing and controlling plant diseases.

[0068] According to the present invention, the plant disease can be any disease caused by plant pathogens. Preferably, the plant disease is tobacco bacterial wilt.

[0069] According to a particularly preferred embodiment of the present invention, the preparation method of edemectin A derivative comprises:

[0070] (1) Using n-hexane as a reaction solvent, tert-butyl N-(3-aminopropyl)carbamate, 4-chloro-1-butanol, and potassium carbonate are mixed in a molar ratio of 1:1.8-2.2:1.8-2.2, and reacted at a temperature of 5-40° C. and a stirring rate of 300-600 rpm for 4-8 hours to obtain a reaction solution, filtering the reaction solution and concentrating it (I) to obtain a concentrate, mixing the concentrate with an acid-containing methanol solution (the acid concentration is 5-9 M), stirring and reacting it at a temperature of 5-40° C. and a stirring rate of 300-600 rpm for 8-14 hours, and then concentrating it (II) to obtain a crude product; mixing the crude product with ethyl acetate for crystallization to obtain a spermidine analog represented by formula (II);

[0071] (2) The transformed strain Brevibacillus brevis X23ΔspeE was inoculated into LB solid medium, cultured at a temperature of 25-35°C for 20-30 hours to obtain a single colony, and the single colony was selected and inoculated into LB liquid medium, cultured at a temperature of 25-35°C and a rotation speed of 150-200 rpm for 10-15 hours, and the spermidine analogue represented by formula (II) was added to a final concentration of 1-3 mM, and the culture was continued for 40-60 hours to obtain a fermentation broth;

[0072] (3) The fermentation liquid is centrifuged to obtain a fermentation supernatant, which is adsorbed by a cation exchange resin and then eluted with 15-25 mM ammonia water. The obtained eluate is concentrated to obtain the edemacin A derivative shown in formula (I).

[0073] The present invention will be described in detail below through examples.

[0074] In the following examples, the tobacco bacterial wilt pathogen Ralstonia solanacearum GMI1000 was provided by the Vegetable and Floriculture Research Institute of the Chinese Academy of Agricultural Sciences; the cation exchange resin was WorkBeads 100S model, purchased from Bio-Works, USA; raw materials and reagents such as tert-butyl N-(3-aminopropyl)carbamate (CAS No. 75178-96-0), 4-chloro-1-butanol, and potassium carbonate were all conventional commercially available products; and Brevibacillus brevis X23ΔspeE was obtained by modifying the wild-type Brevibacillus brevis X23 (provided by Hunan Agricultural University) using the method provided in Example 2 of CN115925552A as the starting strain.

[0075] In the following examples, the formula of LB liquid medium is: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride;

[0076] The formula of LB solid medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar.

[0077] In the following examples, nuclear magnetic resonance was performed using a nuclear magnetic resonance spectrometer (Bruker 500), and LC-MS was performed using an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer.

[0078] Example 1

[0079] (1) At 90°C, 4-chloro-1-butanol (2.16 g, 20.0 mM) and potassium carbonate (2.76 g, 20.0 mM) were added to a n-butane solution containing tert-butyl N-(3-aminopropyl)carbamate (1.74 g, 10.0 mM), and the mixture was stirred at a rate of 120 rpm for 6 h to obtain a reaction solution. The filtrate after filtering the reaction solution was concentrated under reduced pressure to obtain a concentrate. The concentrate was transferred to a methanol solution containing 7 M HCl, stirred at a rate of 500 rpm for overnight reaction (about 12 h) at 25°C, and then the methanol was removed by concentration under reduced pressure to obtain a crude product. The crude product was mixed with ethyl acetate for crystallization to obtain 1.75 g of a white powdery substance (total yield 79.9%), i.e., a pure product of a spermidine analogue. The white powdery substance was prepared into a mother liquor with a concentration of 500 mM using sterile ultrapure water as a solvent, and filtered for sterilization.

[0080] The white powdery substance was taken as a sample for NMR and LC-MS analysis. The hydrogen spectrum results were as follows: Figure 1 As shown, the carbon spectrum results are as follows Figure 2 The mass spectrometry results are shown in Figure 3As shown, the structural formula of spermidine analogue was identified as Abbreviated as Ahao;

[0081] (2) The strain Brevibacillus brevis X23ΔspeE was inoculated into LB solid culture medium, activated and cultured at 30°C for 24 hours to obtain a single colony, and a single colony was picked and inoculated into a 50 mL Erlenmeyer flask containing 10 mL LB liquid culture medium, and placed in a 30°C shaker at 180 rpm for 12 hours. The activated bacterial solution was then transferred to a 5000 mL Erlenmeyer flask containing 1000 mL LB liquid culture medium, and placed in a 30°C shaker at 180 rpm for 12 hours. The Ahao mother solution obtained in the appropriate step (1) was then added to the culture solution to make the final concentration of Ahao reach 2 mM, and the culture was continued for 36 hours to obtain a fermentation broth;

[0082] (3) After the fermentation is completed, take an appropriate amount of fermentation liquid and measure the OD of the bacterial liquid using an enzyme marker 600 The remaining fermentation liquid was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered and sterilized to obtain the fermentation liquid. A trace amount of the fermentation liquid was used as a sample for LC-MS analysis.

[0083] The fermentation supernatant was adsorbed by a cation exchange resin and then eluted with 20 mM ammonia water. The eluate was concentrated and dried under reduced pressure to obtain 40.74 mg of a crude product.

[0084] Example 2

[0085] (1) At 80°C, 4-chloro-1-butanol (1.94 g, 18.0 mM) and potassium carbonate (2.48 g, 18.0 mM) were added to a n-butane solution containing tert-butyl N-(3-aminopropyl)carbamate (1.74 g, 10.0 mM), and the mixture was stirred at a rate of 100 rpm for 8 h to obtain a reaction solution. The filtrate after filtering the reaction solution was concentrated under reduced pressure to obtain a concentrate. The concentrate was transferred to a methanol solution containing 5 M HCl, stirred at 40°C at a rate of 400 rpm for 14 h, and then concentrated under reduced pressure to remove methanol to obtain a crude product. The crude product was mixed with ethyl acetate for crystallization to obtain 1.56 g of a white powdery substance (total yield 71.2%), i.e., a pure product of the spermidine analogue represented by formula (II). The white powdery substance was prepared into a mother liquor with a concentration of 500 mM using sterile ultrapure water as a solvent, and filtered and sterilized for later use.

[0086] (2) Brevibacillus brevis X23ΔspeE strain was inoculated into LB solid culture medium, activated and cultured at 25°C for 30 h to obtain a single colony, and a single colony was picked and inoculated into a 50 mL Erlenmeyer flask containing 10 mL LB liquid culture medium, and placed in a 30°C shaker at 180 rpm for 12 h. The activated bacterial solution was then transferred to a 5000 mL Erlenmeyer flask containing 1000 mL LB liquid culture medium, and placed in a 25°C shaker at 200 rpm for 10 h. The Ahao mother solution obtained in the appropriate step (1) was then added to the culture solution to make the final Ahao concentration reach 1 mM, and the culture was continued for 30 h to obtain a fermentation broth;

[0087] (3) After the fermentation is completed, take an appropriate amount of fermentation liquid and measure the OD of the bacterial liquid using an enzyme marker 600 The remaining fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered and sterilized to obtain a fermentation supernatant, which was adsorbed by a cation exchange resin and then eluted with 15 mM ammonia water. The eluate was concentrated and dried under reduced pressure to obtain 37.17 mg of a crude product.

[0088] Example 3

[0089] (1) At 100° C., 4-chloro-1-butanol (2.38 g, 22.0 mM) and potassium carbonate (3.04 g, 22.0 mM) were added to a n-butane solution containing tert-butyl N-(3-aminopropyl)carbamate (1.74 g, 10.0 mM), and the mixture was stirred at a rate of 150 rpm for 4 h to obtain a reaction solution. The filtrate after filtering the reaction solution was concentrated under reduced pressure to obtain a concentrate. The concentrate was transferred to a methanol solution containing 9 M HCl, stirred at a rate of 600 rpm for 8 h at 5° C., and then concentrated under reduced pressure to remove methanol to obtain a crude product. The crude product was mixed with ethyl acetate for crystallization to obtain 1.38 g of a white powdery substance (total yield 63.0%), i.e., a pure product of the spermidine analog represented by formula (II). The white powdery substance was prepared into a mother liquor with a concentration of 500 mM using sterile ultrapure water as a solvent, and filtered and sterilized for later use.

[0090] (2) Brevibacillus brevis X23ΔspeE strain was inoculated into LB solid culture medium, activated and cultured at 35°C for 20 h to obtain a single colony, and a single colony was picked and inoculated into a 50 mL Erlenmeyer flask containing 10 mL LB liquid culture medium, and placed in a 30°C shaker at 180 rpm for 12 h. The activated bacterial solution was then transferred to a 5000 mL Erlenmeyer flask containing 1000 mL LB liquid culture medium, and placed in a 35°C shaker at 150 rpm for 15 h. The Ahao mother solution obtained in step (1) was then added to the culture solution to make the final Ahao concentration reach 3 mM, and the culture was continued for 45 h to obtain a fermentation broth;

[0091] (3) After the fermentation is completed, take an appropriate amount of fermentation liquid and measure the OD of the bacterial liquid using an enzyme marker 600 The remaining fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered and sterilized to obtain a fermentation supernatant, which was adsorbed by a cation exchange resin and then eluted with 25 mM ammonia water. The eluate was concentrated and dried under reduced pressure to obtain 37.71 mg of a crude product.

[0092] Example 4

[0093] The crude product of the edemacin A derivative shown in (I) was prepared according to the method of Example 1, except that step (2) was replaced by:

[0094] (2) The Brevibacillus brevis X23ΔspeE strain was inoculated into LB solid culture medium and activated and cultured at 30°C for 24 h to obtain a single colony. A single colony was picked and inoculated into a 50 mL Erlenmeyer flask containing 10 mL LB liquid culture medium, and the culture was shaken at 180 rpm at 30°C for 12 h. The activated bacterial solution was then transferred to a 5000 mL Erlenmeyer flask containing 1000 mL LB liquid culture medium, and the culture was shaken at 180 rpm at 30°C for 5 h. The Ahao mother solution obtained in the appropriate step (1) was then added to the culture solution to make the final concentration of Ahao reach 2 mM. The culture was continued for 36 h to obtain a fermentation broth.

[0095] Step (3) yielded 26.35 mg of crude product.

[0096] Example 5

[0097] The crude product of the edemacin A derivative shown in (I) was prepared according to the method of Example 1, except that the final concentration of Ahao in step (2) was replaced from 2 mM to 4 mM.

[0098] Step (3) yielded 32.61 mg of crude product.

[0099] Example 6

[0100] The crude product of the edemacin A derivative shown in (I) was prepared according to the method of Example 1, except that step (1) was replaced by:

[0101] (1) To a n-butane solution of tert-butyl N-(3-aminopropyl)carbamate (1.74 g, 10.0 mM) was added 4-chloro-1-butanol (2.16 g, 20.0 mM) and potassium carbonate (2.76 g, 20.0 mM) at 90°C, and the mixture was stirred at 500 rpm for 6 h to obtain a reaction solution. The filtrate after filtering the reaction solution was concentrated under reduced pressure to obtain a concentrate. The concentrate was mixed with ethyl acetate for crystallization to obtain 0.14 g of a white powdery substance (total yield 6.4%), i.e., a pure product of the spermidine analogue represented by formula (II). The white powdery substance was prepared into a mother liquor with a concentration of 500 mM using sterile ultrapure water as a solvent, and the mother liquor was filtered and sterilized for later use.

[0102] Step (3) gave 25.70 mg of crude product.

[0103] Comparative Example 1

[0104] (1) The wild-type Brevibacillus brevis X23 strain was inoculated into LB solid medium and activated at 30°C for 24 h to obtain a single colony. A single colony was picked and inoculated into a 50 mL Erlenmeyer flask containing 10 mL of LB liquid medium. The culture was shaken at 30°C at 180 rpm for 12 h. The activated bacterial solution was then transferred to a 5000 mL Erlenmeyer flask containing 1000 mL of LB liquid medium and shaken at 30°C at 180 rpm for 48 h to obtain a fermentation broth.

[0105] (2) After the fermentation is completed, take an appropriate amount of fermentation liquid and measure the OD of the bacterial liquid using an enzyme marker 600 The remaining fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered and sterilized to obtain the fermentation supernatant. A trace amount of the fermentation supernatant was used as a sample for LC-MS analysis.

[0106] Comparative Example 2

[0107] (1) Brevibacillus brevis X23ΔspeE strain was inoculated into LB solid medium and activated at 30°C for 24 h to obtain a single colony. A single colony was picked and inoculated into a 50 mL Erlenmeyer flask containing 10 mL of LB liquid medium. The culture was shaken at 30°C at 180 rpm for 12 h. The activated bacterial solution was then transferred to a 5000 mL Erlenmeyer flask containing 1000 mL of LB liquid medium and shaken at 30°C at 180 rpm for 48 h to obtain a fermentation broth.

[0108] (2) After the fermentation is completed, take an appropriate amount of fermentation liquid and measure the OD of the bacterial liquid using an enzyme marker 600 The remaining fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered and sterilized to obtain the fermentation supernatant. A trace amount of the fermentation supernatant was used as a sample for LC-MS analysis.

[0109] Test Example 1

[0110] The crude products obtained in Examples 1-6 were dissolved in sterile water and then subjected to LC-MS analysis. The wild-type Brevibacillus brevis X23 cells obtained in Comparative Example 1 were used as control 1, and the Brevibacillus brevisX23ΔspeE cells obtained in Comparative Example 2 were used as control 2. The results are shown in FIG. Figure 4 As shown in Figure 2, m / z = 756.4232 ([M+H] + )'s target molecule - edemectin A derivative, and the structural formula of edemectin A derivative was identified as;

[0111]

[0112] The content of edemacin A derivatives in the fermentation broths obtained in Examples 1-6 and Comparative Examples 1-2 was detected, and the yield of edemacin A derivatives was calculated using the following formula. The results are shown in Table 1.

[0113] Yield of edemectin A derivatives = weight of crude edemectin A derivatives ÷ (content of edemectin A derivatives in fermentation broth × fermentation volume) × 100%.

[0114] Table 1

[0115]

[0116] Test Example 2 Detection of the inhibitory activity of edemectin A derivatives against tobacco bacterial wilt

[0117] Ralstonia solanacearum GMI1000, the pathogen of tobacco bacterial wilt, was used as the target bacteria for bioactivity determination.

[0118] First, the GMI1000 strain was activated with LB solid medium and cultured at 30°C for 24 h. Then, a single clone was picked and inoculated into an EP tube containing 1 mL of LB liquid medium, placed on a thermomixer, and cultured overnight at 30°C and 900 rpm. 10 μL (10 8 cfu / mL) was mixed with 200 mL of NB solid medium (10 g / L peptone, 3 g / L beef powder, 5 g / L sodium chloride, pH 7.2) pre-cooled to 45-50°C, and poured into plates (17.5 mL / plate); after the plates were naturally cooled and solidified, holes were punched on the plates with a sterilized puncher (8.0 mm in diameter), and the fermentation supernatant obtained in step (3) of Example 1 was added to the holes in an amount of 100 μL / hole. The cells were cultured at 30°C for 24 h, and the diameter of the inhibition zone was measured. The test was repeated three times, and the activity was expressed by the inhibition diameter. The wild-type Brevibacillus brevis X23 strain (abbreviated as X23) was used as control 1, the Brevibacillus brevis X23ΔspeE strain (abbreviated as ΔspeE) was used as control 2, and the spermidine analog Ahao was used as control 3. The results are shown in FIG. Figure 5 shown.

[0119] from Figure 5 As can be seen, the diameter of the inhibition zone for wild-type Brevibacillus brevis X23 was 18 mm, while Brevibacillus brevis X23ΔspeE exhibited no inhibition zone. However, the fermentation supernatant obtained by feeding the precursor spermidine analog Ahao to the fermentation broth of Brevibacillus brevis X23ΔspeE (abbreviated as ΔspeE+Ahao) restored antibacterial activity, producing an inhibition zone diameter of 14 mm, a 22.2% decrease compared to wild-type Brevibacillus brevis X23 (considering that the bacterial concentration decreased by 13.1% after deleting the speE gene, the activity of this derivative was only slightly lost). Control wells containing 2 mM spermidine analog Ahao showed no antibacterial activity. The restoration of the antibacterial activity of ΔspeE+Ahao indicates that the expected target molecule, the edemacin A derivative, has been generated and exhibits good antibacterial activity.

[0120] In summary, the present invention chemically synthesizes the spermidine analog Ahao and then biosynthesizes the edemectin A derivative, which is convenient to prepare and low in cost, and exhibits good inhibitory activity against tobacco bacterial wilt, and is worthy of further research and development.

[0121] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. An edemectin A derivative, characterized in that: The structural formula of the edemacin A derivative is shown in formula (I), (I)。 2. Application of spermidine analogs in the preparation of edemacin A derivatives, wherein: The structural formula of the edemectin A derivative is shown in formula (I), and the structural formula of the spermidine analog is shown in formula (II). (AND), (II)。 3. The use according to claim 2, characterized in that The preparation method of the spermidine analogue comprises: N -tert-Butyl (3-aminopropyl) carbamate, 4-chloro-1-butanol and potassium carbonate are contacted to react.

4. The use according to claim 3, characterized in that described N -The molar ratio of tert-butyl (3-aminopropyl) carbamate, 4-chloro-1-butanol and potassium carbonate is 1:1.8-2.2:1.8-2.2; The contact reaction conditions include at least: a temperature of 80-100°C, a stirring rate of 300-600 rpm, and a time of 4-8 hours; The solvent for the contact reaction is n-butane.

5. The use according to claim 3, characterized in that The method for preparing the spermidine analogue further comprises: purifying and crystallizing the reaction solution obtained from the contact reaction.

6. The use according to claim 5, characterized in that The purification process includes: filtering the reaction solution and concentrating I to obtain a concentrate, mixing the concentrate with an acid-containing methanol solution for stirring and reacting, and concentrating II to obtain a crude product; The crystallization process comprises: mixing the crude product with ethyl acetate for crystallization; The acid concentration in the acid-containing methanol solution is 5-9 M, and the stirring reaction conditions at least include: a temperature of 5-40° C., a stirring rate of 300-600 rpm, and a time of 8-14 h.

7. A method for preparing an edemectin A derivative, characterized in that: The method comprises: adding a spermidine analogue during the fermentation culture process of the transformed strain; Wherein, the structural formula of the edemacin A derivative is shown as formula (I), and the structural formula of the spermidine analog is shown as formula (II). (AND), (II); The transformed strain is Brevibacillus Brevibacillus brevis X23 ΔspeE .

8. The method according to claim 7, characterized in that The fermentation culture medium used in the fermentation culture contains tryptone, yeast extract and inorganic salts; The fermentation culture conditions include at least: a temperature of 25-35°C, a rotation speed of 150-200 rpm, and a time of 40-60 hours; The amount of the spermidine analogue added is 1-3 mM, and the addition time is 10-15 hours after the start of fermentation.

9. The method according to claim 8, characterized in that The fermentation culture medium used in the fermentation culture contains 8-12 g / L tryptone, 3-8 g / L yeast extract and 8-12 g / L sodium chloride.

10. The method according to any one of claims 7 to 9, characterized in that The method further comprises: performing solid-liquid separation on the fermentation liquid obtained by the fermentation culture to obtain a fermentation supernatant, and performing adsorption separation and concentration on the fermentation supernatant III; The adsorption separation adopts cation exchange resin.

11. A fungicide, characterized in that: Containing the edemectin A derivative according to claim 1 and / or the edemectin A derivative prepared according to the method of any one of claims 7 to 10.

12. Use of the edemectin A derivative according to claim 1, the edemectin A derivative obtained by the method according to any one of claims 7 to 10, and the fungicide according to claim 11 in preventing and controlling plant diseases.

13. The use according to claim 12, characterized in that The plant disease is tobacco bacterial wilt.

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