A carbazole alkaloid, its preparation method and application

Abstract

The application belongs to the technical field of natural medicine, and particularly relates to a carbazole alkaloid, and further discloses a preparation method and application thereof. The application firstly discovers carbazole alkaloids carbazomycin I and carbazomycin J from liquid fermentation products of an actinomycete Saccharopolyspora phattalungensis DSM 45584 strain, identifies a biosynthesis gene cluster cabz thereof through bioinformatics and other means, and identifies a biosynthesis process thereof. The discovery can provide a model for synthetic biology creation of new antibiotics, and serve as a lead drug of new antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to a carbazole alkaloid, and further discloses its preparation method and application. Background Technology

[0002] Natural products are a crucial pillar of modern drug discovery and development, especially natural products and their derivatives derived from plants and microorganisms, which are the primary sources of lead compounds and clinical drugs in new drug development. However, the field of natural product science has long faced two major challenges: firstly, the discovery rate of new natural lead molecules is declining year by year, with some even suggesting that microbial resources as natural products are nearing depletion; secondly, the supply of drug sources is fragile, as most newly discovered natural lead compounds are (ultra)trace components, making it difficult to obtain sufficient samples for preclinical drug research. Encouragingly, genomic analysis shows that almost all microbial strains have a large number of unknown secondary metabolite genes (clusters) yet to be discovered, and the natural products discovered to date represent only a small fraction of their theoretical output. Therefore, there is room for improvement in the yield and structural diversity of microbial secondary metabolites, but sufficient understanding and control of their biosynthetic pathways are necessary. Thus, discovering new natural lead compounds and identifying their biosynthetic gene clusters, and using synthetic biology and other methods to elucidate their biosynthetic processes and regulate their metabolism, represents the forefront of research in natural product science.

[0003] Carbazole is a broad-spectrum antibiotic with antifungal, antibacterial, and anti-yeast properties. There are eight homologues: A, B, C, D, E, F, G, and H. It has important applications in the pharmaceutical and plant biocontrol fields, with a large market demand and broad market prospects. The field anticipates developing more carbazole analogues as lead drugs for novel antibiotics. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a novel carbazomycin alkaloid, which includes carbazomycin I and carbazomycin J, and the carbazomycin alkaloid can be used as a lead drug for a novel antibiotic.

[0005] The second technical problem to be solved by the present invention is to provide a method for preparing and applying the above-mentioned carbazole alkaloids.

[0006] To solve the above-mentioned technical problems, the present invention provides a carbazole alkaloid having the structure shown in formula (P): ; in, R is methyl, and the carbazomycin alkaloid is designated as carbazomycin I, having the structure shown in formula (Ⅰ); ; or, The R is The carbazo alkaloid is designated as carbazomycin J and has the structure shown in formula (Ⅰ); .

[0007] This invention also discloses a method for preparing the carbazole alkaloid by fermentation, comprising: fermenting actinomycetes... Saccharopolyspora phattalungensis The steps for inoculating DSM 45584 into a suitable fermentation medium for fermentation culture; The actinomycetes Saccharopolyspora phattalungensis DSM strain 45584 is deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany, with accession number DSM No: 45584, and deposit date of February 9, 2011.

[0008] Specifically, in the method for preparing the carbazole alkaloid by fermentation, the fermentation medium comprises: 1-3 wt% soluble starch, 0.5-2 wt% glucose, 0.3-0.8 wt% peptone, 0.3-0.8 wt% yeast extract, 0.3-0.5 wt% NaCl, 0.03-0.08 wt% K₂HPO₄, 0.03-0.08 wt% MgSO₄·7H₂O, 0.1-0.3 wt% CaCO₃, and natural pH; Preferably, the fermentation medium comprises: 2 wt% soluble starch, 1 wt% glucose, 0.5 wt% peptone, 0.5 wt% yeast extract, 0.4 wt% NaCl, 0.05 wt% K2HPO4, 0.05 wt% MgSO4·7H2O, 0.2 wt% CaCO3, and natural pH. Preferably, the parameters of the fermentation culture step include: culture temperature 28-32℃, shaking culture for 5-8 days, and rotation speed 200-250 rpm.

[0009] Specifically, the method for preparing the carbazole alkaloid by fermentation further includes the use of the actinomycetes. Saccharopolyspora phattalungensis The steps for seed culture of DSM 45584 inoculated into seed culture medium; Preferably, the seed culture medium comprises tryptic soy peptone liquid medium; Preferably, the tryptic soy peptone liquid culture medium comprises the following components: 15-20 g / L tryptic peptone, 2-4 g / L soy peptone, 2-3 g / L glucose, 4-6 g / L NaCl, 2-4 g / L K2HPO4, and pH 7.2-7.4. Preferably, the tryptic soy peptone liquid culture medium comprises the following components: 17 g / L tryptic peptone, 3 g / L soy peptone, 2.5 g / L glucose, 5 g / L NaCl, 2.5 g / L K2HPO4, pH 7.3; Preferably, the parameters for the seed culture step include: culture temperature 28-32℃, shaking culture for 36-60h, and rotation speed 200-250rpm.

[0010] Specifically, the method for preparing the carbazole alkaloid by fermentation further includes the use of the actinomycetes. Saccharopolyspora phattalungensis The steps for inoculating DSM 45584 into solid culture medium for activation culture; Preferably, the solid culture medium includes ISP4 solid culture medium; As a basic culture medium in this field, the ISP4 solid culture medium comprises: 8-12 g / L soluble starch, 0.5-2 g / L K2HPO4, 0.5-1.5 g / L MgSO4·7H2O, 1-3 g / L CaCO3, 20 g / L agar, and pH 7.0-7.4. Preferably, the ISP4 solid culture medium comprises: 10 g / L soluble starch, 1 g / L K2HPO4, 1 g / L MgSO4·7H2O, 2 g / L CaCO3, 20 g / L agar, and pH 7.2. Preferably, the parameters for the activation and culture step include: culture temperature 26-30℃, culture time 2-4 days.

[0011] Specifically, the method for preparing the carbazole alkaloid by fermentation further includes the steps of extracting and purifying the carbazole alkaloid, specifically including: Extraction: The fermentation broth was collected and extracted with organic solvents to obtain a crude extract, which was then subjected to reverse silica gel column chromatography to obtain the eluted fraction. Purification: The eluted fraction was collected and purified by gel column chromatography and semi-preparative HPLC to obtain the carbazole alkaloid.

[0012] Specifically, in the method for preparing the carbazole alkaloid by fermentation, the parameters of the reverse silica gel column chromatography in the extraction step include: the bonded phase is octadecyl, the matrix is ​​amorphous silica gel, the particle size is 200-300 mesh, the pore size is 8.0-10.0 nm, and the pore volume is 0.8-1.0 ml / g.

[0013] Specifically, in the method for preparing the carbazole alkaloid through fermentation, the purification step includes: The parameters for the gel column chromatography include: using Sephadex LH-20 / dextran gel with a particle size of 100-200 mesh; The HPLC separation and purification steps include: Semi-preparative reversed-phase high-performance liquid chromatography was used with a mobile phase of methanol-water (30%:70% v / v) and isocratic elution at a flow rate of 2 mL / min for 15 min. Carbazomycin I and carbazomycin J were eluted at different times, and the corresponding fractions were collected. or, A reversed-phase column was used, with mobile phase A being water and mobile phase B being acetonitrile. The isocratic elution program was: mobile phase A: mobile phase B = 70 wt%: 30 wt%, for 15 min. Carbazomycin I and carbazomycin J were eluted at different times, thereby collecting the corresponding fractions.

[0014] The present invention also discloses the use of the carbazomycin alkaloids or the carbazomycin alkaloids extracted by the method for the preparation of novel antibiotics. Specifically, the carbazomycin alkaloids carbazomycin I and carbazomycin J can serve as source molecules for novel antibiotics.

[0015] This invention also discloses an actinomycete. Saccharopolyspora phattalungensis The use of DSM 45584 for the fermentation preparation of the above-mentioned carbazole alkaloids; The actinomycetes Saccharopolyspora phattalungensis DSM strain 45584 is deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany, with accession number DSM No: 45584, and deposit date of February 9, 2011.

[0016] This invention is the first to obtain from actinomycetes Saccharopolyspora phattalungensis Two carbazomycin alkaloids, carbazomycin I and carbazomycin J, were found in the rice fermentation products of DSM 45584 strain. Their biosynthetic gene clusters were identified using bioinformatics and other methods. cabz The study identified its biosynthetic process, a finding that could provide a paradigm for the synthetic biology creation of novel antibiotics, serving as a lead drug for new antibiotics. Attached Figure Description

[0017] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a 1H NMR spectrum of the compound carbazomycin I of this invention; Figure 2 This is a carbon NMR spectrum of the compound carbazomycin I of this invention; Figure 3 This is the DEPT135 NMR spectrum of the compound carbazomycin I of this invention; Figure 4 This is the HSQC NMR spectrum of the compound carbazomycin I of this invention; Figure 5 NMR of the compound carbazomycin I of this invention 1 H- 1 HCOSY spectral data plot; Figure 6 This is the HMBC NMR spectrum of the compound carbazomycin I of this invention; Figure 7 This is the NOESY NMR spectrum data of the compound carbazomycin I of this invention; Figure 8 This is a mass spectrometry data of the compound carbazomycin I of this invention; Figure 9 This is a 1H NMR spectrum of the compound carbazomycin J of this invention; Figure 10 This is a carbon NMR spectrum of the compound carbazomycin J of this invention; Figure 11 This is the DEPT135 NMR spectrum of the compound carbazomycin J of this invention; Figure 12 This is the HSQC NMR spectrum of the compound carbazomycin J of this invention; Figure 13 NMR of the compound carbazomycin J of this invention 1 H- 1 HCOSY spectral data plot; Figure 14 This is the HMBC NMR spectrum of the compound carbazomycin J of this invention; Figure 15This is the NOESY NMR spectrum of the compound carbazomycin J of this invention; Figure 16 This is a mass spectrometry data of the compound carbazomycin J of this invention; Figure 17 This is a biosynthetic route diagram for the carbazomycin I and carbazomycin J of this invention. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0019] In the following embodiments of the present invention, actinomycetes are used. Saccharopolyspora phattalungensis Fermentation was performed using strain DSM45584, and the desired carbazole alkaloid structure was obtained by extraction and purification of the fermentation broth. This strain is an actinomycete. Saccharopolyspora phattalungensis DSM strain 45584, preservation name is Saccharopolyspora phattalungensis DSM 45584; deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany; deposit date: February 9, 2011; accession number: DSM No: 45584.

[0020] In the following embodiments of the present invention, the actinomycetes... Saccharopolyspora phattalungensis In the activation of strain DSM45584, any actinomycete activation medium widely accepted in the art can be used. As an example, in the following embodiments of the present invention, ISP4 solid medium is used to activate the strain. Specifically, as a basic culture medium in the art, in the following embodiments of the present invention, the components of the ISP4 solid medium include (in 1L): 10g soluble starch, 1g MgSO4·7H2O, 2g CaCO3, 1g K2HPO4, 20g agar, 1L distilled water, pH 7.2.

[0021] In the following embodiments of the present invention, the actinomycetes Saccharopolyspora phattalungensisWhen culturing the DSM45584 strain, any actinomycete seed culture medium generally accepted in the art can be used. As an example, in the following embodiments of the present invention, tryptic soy peptone liquid medium is used for seed culture to obtain the desired seed culture. Specifically, as an achievable method, in the following embodiments of the present invention, the tryptic soy peptone liquid medium comprises the following components: 17 g / L tryptic peptone, 3 g / L soy peptone, 2.5 g / L glucose, 5 g / L NaCl, 2.5 g / L K₂HPO₄, pH 7.3.

[0022] Example 1 In this embodiment, actinomycetes Saccharopolyspora phattalungensis The cryopreserved dry powder of DSM 45584 strain was spread on ISP4 agar plates and incubated at 30°C to revive the actinomycete.

[0023] On ISP4 plates, the aerial mycelium initially appeared white during the early stages of cultivation, easily forming single colonies. After one week, a large number of white mycelia began to appear. Morphological and ITS sequence identification confirmed that this bacterium is... Saccharopolyspora phattalungensis Genus. The name of this fungus is... Saccharopolyspora phattalungensis DSM 45584.

[0024] Example 2 The preparation method of carbazole alkaloids described in this embodiment includes the following steps: (1) Actinomycetes Saccharopolyspora phattalungensis DSM 45584 strain was activated and cultured on ISP4 solid plates and then incubated at 30°C to revive the actinomycete. (2) Collect the spores produced after activation in step (1) above, inoculate them into tryptic soy peptone liquid medium, and culture them at 30°C with shaking for 48 hours at 220 rpm to obtain seed liquid; (3) The obtained seed liquid was transferred to a 1 L Erlenmeyer flask containing 200 mL of fermentation medium at a 5% inoculation rate, and cultured at 30℃ with shaking for 7 days at a speed of 220 rpm. The obtained fermentation liquid was then collected. The fermentation medium comprises the following components (per 1L): 20g soluble starch, 10g glucose, 5g peptone, 5g yeast extract, 4g NaCl, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 2g CaCO3, with a natural pH of 7.3.

[0025] Example 3 In this embodiment, the fermentation broth obtained in Example 2 was collected, extracted three times with ethyl acetate, and concentrated to obtain a crude extract. The crude extract was further subjected to reverse silica gel column chromatography (bonded phase: octadecyl, matrix: amorphous silica gel, particle size: 200-300 mesh, pore size: 8.0-10.0 nm, pore volume: 0.8-1.0 ml / g), and a total of 10 fractions were obtained. The tenth elution fraction was collected to obtain the desired elution fraction.

[0026] The eluted fraction obtained above was initially purified by gel column chromatography (using Sephadex LH-20 / dextran gel with a particle size of 100-200 mesh), and then separated and purified by semi-preparative HPLC to obtain the desired carbazomycin I and carbazomycin J.

[0027] In this embodiment, the HPLC separation and purification process is as follows: semi-preparative reversed-phase high-performance liquid chromatography: ODS-2 Hypersil column, 5µm, 250mm×10mm; the mobile phase is a methanol-water system with a volume ratio of 30%:70%, and isocratic elution is performed at a flow rate of 2mL / min for 15min; the pump model is Hitachi pump L-7100, and the UV lamp model is UVdetector L-7400.

[0028] In this embodiment, fractions containing carbazomycin I and carbazomycin J were collected at 13.5 min and 14.7 min, respectively, and carbazomycin I (5.6 mg) and carbazomycin J (1.6 mg) were collected, respectively.

[0029] Example 4 In this embodiment, the structures of the carbazomycin I and carbazomycin J collected above were analyzed and identified.

[0030] In this embodiment, the carbazomycin I and carbazomycin J were determined to have... 1 H and 13 The C NMR data are assigned as shown in Table 1 below: Table 1 Carbazole alkaloids 1 H and 13 C NMR data attribution

[0031] Note: a Recorded at 400MHz, bRecorded at 100 MHz In this embodiment, various identification spectra of the carbazomycin I alkaloid are shown in the appendix. Figures 1-8 As shown, various identification spectra of the carbazomycin J are attached. Figures 9-16 As shown, the specific structures of the two carbazole alkaloids mentioned above were thus determined.

[0032] In summary, based on mass spectrometry, nuclear magnetic resonance spectroscopy and other data, this embodiment confirms that the structure of the carbazomycin I is as shown in formula (Ⅰ), and the structure of the carbazomycin J is as shown in formula (Ⅱ).

[0033]

[0034] Example 5 This embodiment describes the biosynthetic gene clusters of the above-mentioned carbazole alkaloids carbazomycin I and carbazomycin J. cabz The biosynthetic process was identified.

[0035] In this embodiment, the biosynthetic gene clusters and biosynthetic processes of the carbazomycin I and carbazomycin J are as follows: their whole genome data were uploaded to the antiSMASH database, and the gene clusters of the carbazomycins were found through literature comparison. cabz The BLAST tool was used to identify the function of each open reading frame (ORF) on the gene cluster, and the biosynthesis process of carbazole alkaloids was determined by combining the structural characteristics of carbazole alkaloids.

[0036] In this embodiment, the experimental materials involved include: Saccharopolyspora phattalungensis DSM45584 whole genome data, antiSMASH database, BLAST, 2ndFind and other bioinformatics tools.

[0037] Will Saccharopolyspora phattalungensis The whole genome data of DSM 45584 was uploaded to the antiSMASH database, and analysis yielded data on all its secondary metabolic gene clusters. Using the biosynthetic gene cluster and key functional genes of the carbazole alkaloid carquinostatin A (CQS) reported in the literature, the biosynthetic gene cluster cabz responsible for the synthesis of carbazomycin I and carbazomycin J was identified.

[0038] Subsequently, bioinformatics analysis tools such as BLAST and 2ndFind were used to identify gene clusters. cabz The functions of the seven encoded proteins are as follows: Cabz1, Cabz2, and Cabz7 are labeled as SAM-dependent methyltransferases; Cabz3 is labeled as a TDP-L-rhamnosyl-dependent transferase; Cabz4 is labeled as a thiamine-dependent synthase; Cabz5 is labeled as a hypothetical protein; and Cabz6 is labeled as a β-ketoacyl-ACP synthase. Accordingly, the biosynthetic process of the carbazomycin I and carbazomycin J of the present invention is as follows: Figure 17 As shown, (A) represents the biosynthetic gene cluster. cabz The identification results (B) are the results of the biosynthesis process identification.

[0039] Example 6 In this embodiment, the preliminary screening results of antibacterial activity showed that the compounds carbazomycin I and carbazomycin J of the present invention have good inhibitory activity against Micrococcus luteus, and the positive control drug is abramycin sulfate.

[0040] In summary, this invention is the first to utilize actinomycetes... Saccharopolyspora phattalungensis The natural compounds carbazomycin I and carbazomycin J were discovered in the rice fermentation products of DSM 45584 strain, and their biosynthetic gene clusters were identified using bioinformatics and other methods. cabz The study identified its biosynthetic process, a finding that could provide a paradigm for the synthetic biology creation of novel antibiotics, serving as a lead drug for new antibiotics.

[0041] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A carbazole alkaloid, characterized in that, The carbazole alkaloid has the structure shown in formula (P): ； in, R is methyl, and the carbazomycin alkaloid is designated as carbazomycin I, having the structure shown in formula (Ⅰ); ； or, The R is The carbazo alkaloid is designated as carbazomycin J and has the structure shown in formula (Ⅰ); 。 2. A method for preparing the carbazole alkaloid of claim 1 by fermentation, characterized in that, Including actinomycetes Saccharopolyspora phattalungensis The steps for inoculating DSM 45584 into a suitable fermentation medium for fermentation culture; The method further includes the steps of extracting and purifying the carbazole alkaloid, specifically including: Extraction: The fermentation broth was collected and extracted with organic solvents to obtain a crude extract, which was then subjected to reverse silica gel column chromatography to obtain the eluted fraction. Purification: The eluted fraction was collected and purified by gel column chromatography and HPLC to obtain the carbazole alkaloid; in the extraction step, the parameters of the reverse silica gel column chromatography included: the bonded phase was octadecyl, the matrix was amorphous silica gel, the particle size was 200-300 mesh, the pore size was 8.0-10.0 nm, and the pore volume was 0.8-1.0 ml / g; In the purification step, the parameters of the gel column chromatography include: using Sephadex LH-20 / dextran gel with a particle size of 100-200 mesh; The HPLC separation and purification steps include: using semi-preparative reversed-phase high-performance liquid chromatography with a mobile phase of methanol-water (volume ratio 30%:70%), isocratic elution at a flow rate of 2 mL / min, wherein carbazomycin I and carbazomycin J are eluted at different times, thereby receiving the corresponding fractions; or, The HPLC separation and purification steps include: using a reversed-phase column, with mobile phase A being water and mobile phase B being acetonitrile, and an isocratic elution program of mobile phase A: mobile phase B = 70wt%: 30wt%, wherein carbazomycin I and carbazomycin J are eluted at different times, thereby receiving the corresponding fractions; The actinomycetes Saccharopolyspora phattalungensis DSM strain 45584 is deposited at the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany, with accession number DSM No: 45584, and deposit date of February 9, 2011.

3. The method for preparing the carbazole alkaloid by fermentation according to claim 2, characterized in that, The parameters for the fermentation and culture steps include: culture temperature 28-32℃, shaking culture for 5-8 days, and rotation speed 200-250 rpm.

4. The method for preparing the carbazole alkaloid by fermentation according to claim 2, characterized in that, The fermentation medium comprises: 1-3 wt% soluble starch, 0.5-2 wt% glucose, 0.3-0.8 wt% peptone, 0.3-0.8 wt% yeast extract, 0.3-0.5 wt% NaCl, 0.03-0.08 wt% K₂HPO₄, 0.03-0.08 wt% MgSO₄·7H₂O, 0.1-0.3 wt% CaCO₃, and natural pH.

5. The method for preparing the carbazole alkaloid by fermentation according to claim 2, characterized in that, The method further includes processing the actinomycetes. Saccharopolyspora phattalungensis The steps for seed culture of DSM 45584 inoculated into seed culture medium.

6. The method for preparing the carbazole alkaloid by fermentation according to claim 5, characterized in that, The seed culture medium includes tryptic soy peptone liquid medium.

7. The method for preparing the carbazole alkaloid by fermentation according to claim 6, characterized in that, The tryptic soy peptone liquid culture medium comprises the following components: 15-20 g / L tryptic peptone, 2-4 g / L soy peptone, 2-3 g / L glucose, 4-6 g / L NaCl, 2-4 g / L K2HPO4, and pH 7.2-7.

4.

8. The method for preparing the carbazole alkaloid by fermentation according to claim 5, characterized in that, The parameters for the seed culture step include: culture temperature 28-32℃, shaking culture for 36-60h, and rotation speed 200-250rpm.

9. The method for preparing the carbazole alkaloid by fermentation according to claim 2, characterized in that, The method further includes processing the actinomycetes. Saccharopolyspora phattalungensis The steps for inoculating DSM 45584 into solid culture medium for activation culture.

10. The method for preparing the carbazole alkaloid by fermentation according to claim 9, characterized in that, The solid culture medium includes ISP4 solid culture medium.

11. The method for preparing the carbazole alkaloid by fermentation according to claim 10, characterized in that, The ISP4 solid culture medium comprises the following components: 8-12 g / L soluble starch, 0.5-2 g / L K2HPO4, 0.5-1.5 g / L MgSO4·7H2O, 1-3 g / L CaCO3, 20 g / L agar, and pH 7.0-7.

4.

12. The method for preparing the carbazole alkaloid by fermentation according to claim 9, characterized in that, The parameters for the activation and culture step include: culture temperature 26-30℃, culture time 2-4 days.

13. Use of the carbazole alkaloid of claim 1 or the carbazole alkaloid extracted by the method of any one of claims 2-12 in the preparation of an antibiotic that inhibits Micrococcus luteus.

14. An actinomycete Saccharopolyspora phattalungensis DSM 45584 is used for the fermentation preparation of the carbazole alkaloid of claim 1; The actinomycetes Saccharopolyspora phattalungensis DSM strain 45584 is deposited at the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany, with accession number DSM No: 45584, and deposit date of February 9, 2011.

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