Nocardia-derived siderophore compound and separation and identification method
By isolating and identifying 11 new structures of iron carrier compounds from Nocardia, the problem of limited research on Nocardia ferrocarrier compounds was solved, and the structural diversity of iron carrier compounds was achieved. It provides important materials for the discovery of drug-leading compounds.
Patent Information
- Application Number
- CN202411577252.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-06
- Publication Date
- 2025-02-14
AI Technical Summary
The research on Nocardia iron carrier compounds in the prior art is relatively limited, especially the novel structure and active iron carriers have not been fully explored.
Coublibactin A (1) – Coublibactin K (11) was isolated and identified from Nocardia coubleae DSM 44960, and an isolation and identification method was developed, including seed culture and fermentation. Culture, extraction and separation, column chromatography and purification.
11 new structures of iron carrier compounds were successfully extracted and identified, enriching the structural diversity of iron carrier compounds and being of great significance to the discovery of drug lead compounds.
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Figure CN119431492A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of natural product chemistry, and in particular relates to a type of iron carrier compounds derived from Nocardia and a separation and identification method thereof. Background Art
[0002] Nocardia belongs to the phylum Actinobacteria, class Actinobacteria, suborder Corynebacterineae, family Nocardiaceae. It is a type of aerobic, non-motile, catalase-positive actinomycetes, with N. asteroides as the model species, and is widely distributed in aquatic and terrestrial habitats. Nocardia can produce a variety of complex natural products such as peptides, alkaloids, lactones, quinones, terpenes, steroids, etc. with biological activities such as antibacterial, antitumor, antioxidant, and protein inhibitors during secondary metabolism, so it can be used as a potential microbial resource for new drug discovery. During its growth process, Nocardia can produce siderophore compounds. Siderophores are a class of low molecular weight (200-2000 Da) organic compounds that can efficiently chelate Fe (III). According to the chemical properties of the siderophores and the iron coordination site, they can be divided into hydroxamic acid type, catechol type, carboxylic acid type and mixed type. Siderophore compounds are of great significance in biological functions and applications. When the iron ion concentration in microorganisms is low, the organism will secrete siderophores to efficiently absorb iron ions in the environment to meet its own growth needs. In addition, siderophores are important virulence factors for some pathogenic microorganisms to infect hosts, and are also signal molecules between microbial communities, which can help microorganisms cope with harmful oxidative stress. Siderophores have a wide range of application values in agriculture and medicine: in agriculture, siderophores can be used to inhibit the infection of plant pathogenic fungi, and because they can bind heavy metal ions, they are also used in biodegradation; siderophores have high affinity with bacterial surface receptor proteins and can be used to develop biosensors, and can even be used as carriers to deliver antimicrobial drugs to pathogenic microorganisms to achieve targeted inhibition; siderophores secreted by pathogenic bacteria can also be used as potential drug targets. The research on Nocardia siderophores, especially those with novel structures and activities, has important scientific value and development potential. Summary of the invention
[0003] The first object of the present invention is to provide a class of siderophore compounds derived from Nocardia.
[0004] The second object of the present invention is to provide a method for separating and identifying the siderophore compounds derived from Nocardia.
[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a type of siderophore compound derived from Nocardia, the structural formula of which is shown below:
[0006] The iron carrier compound is obtained by separation from Nocardia coubleae DSM 44960 through fermentation culture.
[0007] The second aspect of the present invention provides a method for separating and identifying the siderophore compounds derived from Nocardia, comprising the following steps: (1) Prepare seed culture medium, inoculate Nocardia coubleae DSM 44960 into the seed culture medium, and culture at 30°C and 200 rpm for 4 days to obtain seed solution; (2) The seed solution obtained in step (1) was inoculated into the fermentation medium, and cultured at 30°C and 200 rpm with shaking for 5 days to obtain a fermentation product; (3) separating the fermentation broth and the bacterial cells in the fermentation product obtained in step (2), extracting the fermentation broth with ethyl acetate four times, extracting the bacterial cells with ethyl acetate four times, combining the ethyl acetate phases and concentrating under reduced pressure to obtain a crude extract; (4) The crude extract obtained in step (3) was chromatographed on a normal phase silica gel column using a petroleum ether-ethyl acetate gradient elution (v / v 100:0, 50:1, 20:1, 10:1, 5:1), followed by a dichloromethane-methanol gradient elution (v / v 100:0, 50:1, 20:1, 10:1, 5:1, 0:1) to obtain 7 components N1-N7, of which component N5 was chromatographed on an ODS column using a methanol-water gradient elution (v / v 90:10, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to obtain 24 subcomponents. Components N5-20 and N5-21 were purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min, and 80% acetonitrile aqueous solution was used for isocratic elution to obtain compounds Coublibactin A (1), Coublibactin B (2), Coublibactin C (3), Coublibactin D (4), and Coublibactin G (7). Component N5-22 was first chromatographed on a Sephadex LH-20 gel column with methanol as the eluent, and then purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min and eluted isocratically with 89% acetonitrile aqueous solution to obtain compounds Coublibactin E (5), Coublibactin F (6), and Coublibactin G (7). H (8); component N6 was purified by ODS column chromatography using a methanol-water gradient elution (v / v 100:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to obtain 19 subcomponents. Components N6-17 were first purified by Sephadex LH-20 gel column chromatography using methanol as the eluent, and then purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min and isocratically eluted with 81% acetonitrile aqueous solution to obtain compounds Coublibactin I (9), Coublibactin J (10), and Coublibactin K (11); (5) The compounds Coublibactin A (1) -Coublibactin K (11) obtained in step (4) were dissolved in methanol, respectively, and an aqueous solution of gallium sulfate was added. The mixture was stirred at 25 °C for 1 h. The reaction solution was freeze-dried and purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min. The mixture was isocratically eluted with 84% acetonitrile aqueous solution to obtain the gallium complex of the compounds Coublibactin A (1) -Coublibactin K (11).
[0008] Preferably, the preparation method of the seed culture medium described in step (1) comprises the following steps: 37 g of brain heartinfusion powder, 20 g of glucose and 1 L of pure water, and sterilization at 121°C for 30 min.
[0009] Preferably, the method for preparing the fermentation medium in step (2) comprises the following steps: 7.5 g of soy peptone, 30 g of glucose, 1 g of dipotassium hydrogen phosphate, 5.5 g of calcium carbonate, 5 g of ammonium sulfate and 1 L of pure water, and sterilizing at 121°C for 30 min.
[0010] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects: The present invention provides 11 new-structure iron carrier compounds, Coublibactin A (1)-Coublibactin K (11). The extraction method is mature, the process is simple, the yield of the obtained products is high, and the structures are correct after nuclear magnetic resonance and mass spectrometry detection, which enriches the structural diversity of iron carrier compounds and is of great significance for the discovery of drug lead compounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 The structural formula of the compounds Coublibactin A (1) - Coublibactin K (11) of the present invention.
[0012] Figure 2 The (+)-HR-ESI-MS spectrum of the gallium complex of Coublibactin A (1), a compound of the present invention.
[0013] Figure 3 The gallium complex of the compound Coublibactin A (1) of the present invention is dissolved in CDCl3 1 H-NMR spectrum.
[0014] Figure 4 The gallium complex of the compound Coublibactin A (1) of the present invention is dissolved in CDCl3 13C-NMR spectrum.
[0015] Figure 5 The DEPT-135 spectrum of the gallium complex of Coublibactin A (1), a compound of the present invention, dissolved in CDCl3.
[0016] Figure 6 The gallium complex of the compound Coublibactin A (1) of the present invention is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0017] Figure 7 The HSQC spectrum of the gallium complex of Coublibactin A (1), a compound of the present invention, dissolved in CDCl3.
[0018] Figure 8 The HMBC spectrum of the gallium complex of Coublibactin A (1), a compound of the present invention, dissolved in CDCl3.
[0019] Fig. 9 The NOESY spectrum of the gallium complex of Coublibactin A (1), a compound of the present invention, dissolved in CDCl3.
[0020] Fig.10 The HSQC-TOCSY spectrum of the gallium complex of Coublibactin A (1), a compound of the present invention, dissolved in CDCl3.
[0021] Fig.11 The (+)-HR-ESI-MS spectrum of the compound Coublibactin B (2) gallium complex of the present invention.
[0022] Fig.12 The gallium complex of the compound Coublibactin B (2) of the present invention is dissolved in CDCl3 1 H-NMR spectrum.
[0023] Fig.13 The gallium complex of the compound Coublibactin B (2) of the present invention is dissolved in CDCl3 13 C-NMR spectrum.
[0024] Fig.14 The DEPT-135 spectrum of the gallium complex of Coublibactin B (2), a compound of the present invention, dissolved in CDCl3.
[0025] Fig.15 The gallium complex of the compound Coublibactin B (2) of the present invention is dissolved in CDCl3 1H– 1 H COSY spectrum.
[0026] Fig.16 The HSQC spectrum of the gallium complex of Coublibactin B (2), a compound of the present invention, dissolved in CDCl3.
[0027] Fig.17 The HMBC spectrum of the gallium complex of Coublibactin B (2), a compound of the present invention, dissolved in CDCl3.
[0028] Fig.18 The NOESY spectrum of the gallium complex of Coublibactin B (2), a compound of the present invention, dissolved in CDCl3.
[0029] Fig.19 The HSQC-TOCSY spectrum of the gallium complex of Coublibactin B (2), a compound of the present invention, dissolved in CDCl3.
[0030] Fig. 20 The (+)-HR-ESI-MS spectrum of the compound Coublibactin C (3) gallium complex of the present invention.
[0031] Fig.21 The compound of the present invention, Coublibactin C (3) gallium complex, is dissolved in CDCl3 1 H-NMR spectrum.
[0032] Fig. 22 The compound of the present invention, Coublibactin C (3) gallium complex, is dissolved in CDCl3 13 C-NMR spectrum.
[0033] Fig.23 This is the DEPT-135 spectrum of the compound Coublibactin C (3) gallium complex of the present invention dissolved in CDCl3.
[0034] Fig.24 The compound of the present invention, Coublibactin C (3) gallium complex, is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0035] Fig.25 The HSQC spectrum of the compound Coublibactin C (3) gallium complex of the present invention dissolved in CDCl3.
[0036] Fig.26This is the HMBC spectrum of the compound Coublibactin C (3) gallium complex of the present invention dissolved in CDCl3.
[0037] Fig. 27 The NOESY spectrum of the compound Coublibactin C (3) gallium complex of the present invention dissolved in CDCl3.
[0038] Fig.28 The HSQC-TOCSY spectrum of the compound Coublibactin C (3) gallium complex of the present invention dissolved in CDCl3.
[0039] Fig.29 The (+)-HR-ESI-MS spectrum of the compound Coublibactin D (4) gallium complex of the present invention.
[0040] Fig.30 The gallium complex of the compound Coublibactin D (4) of the present invention is dissolved in CDCl3 1 H-NMR spectrum.
[0041] Fig.31 The gallium complex of the compound Coublibactin D (4) of the present invention is dissolved in CDCl3 13 C-NMR spectrum.
[0042] Fig.32 The DEPT-135 spectrum of the compound Coublibactin D (4) gallium complex of the present invention dissolved in CDCl3.
[0043] Fig.33 The gallium complex of the compound Coublibactin D (4) of the present invention is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0044] Fig.34 The HSQC spectrum of the compound Coublibactin D (4) gallium complex of the present invention dissolved in CDCl3.
[0045] Fig.35 The HMBC spectrum of the compound Coublibactin D (4) gallium complex of the present invention dissolved in CDCl3.
[0046] Fig.36 The NOESY spectrum of the compound Coublibactin D (4) gallium complex of the present invention dissolved in CDCl3.
[0047] Fig.37The HSQC-TOCSY spectrum of the compound Coublibactin D (4) gallium complex of the present invention dissolved in CDCl3.
[0048] Fig.38 The (+)-HR-ESI-MS spectrum of the compound Coublibactin E (5) gallium complex of the present invention.
[0049] Fig.39 The gallium complex of the compound Coublibactin E (5) of the present invention is dissolved in CDCl3 1 H-NMR spectrum.
[0050] Fig.40 The gallium complex of the compound Coublibactin E (5) of the present invention is dissolved in CDCl3 13 C-NMR spectrum.
[0051] Fig.41 The DEPT-135 spectrum of the gallium complex of Coublibactin E (5), a compound of the present invention, dissolved in CDCl3.
[0052] Fig.42 The gallium complex of the compound Coublibactin E (5) of the present invention is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0053] Fig.43 The HSQC spectrum of the gallium complex of Coublibactin E (5), a compound of the present invention, dissolved in CDCl3.
[0054] Fig.44 The HMBC spectrum of the compound Coublibactin E (5) gallium complex of the present invention dissolved in CDCl3.
[0055] Fig.45 The NOESY spectrum of the compound Coublibactin E (5) gallium complex of the present invention dissolved in CDCl3.
[0056] Fig.46 The HSQC-TOCSY spectrum of the compound Coublibactin E (5) gallium complex of the present invention dissolved in CDCl3.
[0057] Fig.47 The (+)-HR-ESI-MS spectrum of the compound Coublibactin F (6) gallium complex of the present invention.
[0058] Fig.48The compound of the present invention, Coublibactin F (6) gallium complex, is dissolved in CDCl3 1 H-NMR spectrum.
[0059] Fig.49 The compound of the present invention, Coublibactin F (6) gallium complex, is dissolved in CDCl3 13 C-NMR spectrum.
[0060] Fig.50 The DEPT-135 spectrum of the compound Coublibactin F (6) gallium complex of the present invention dissolved in CDCl3.
[0061] Fig.51 The compound of the present invention, Coublibactin F (6) gallium complex, is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0062] Fig.52 The HSQC spectrum of the compound Coublibactin F (6) gallium complex of the present invention dissolved in CDCl3.
[0063] Fig.53 The HMBC spectrum of the compound Coublibactin F (6) gallium complex of the present invention dissolved in CDCl3.
[0064] Fig.54 The NOESY spectrum of the compound Coublibactin F (6) gallium complex of the present invention dissolved in CDCl3.
[0065] Fig.55 The HSQC-TOCSY spectrum of the compound Coublibactin F (6) gallium complex of the present invention dissolved in CDCl3.
[0066] Fig.56 The (+)-HR-ESI-MS spectrum of the compound Coublibactin G (7) gallium complex of the present invention.
[0067] Fig.57 The gallium complex of the compound Coublibactin G (7) of the present invention is dissolved in CDCl3 1 H-NMR spectrum.
[0068] Fig.58 The gallium complex of the compound Coublibactin G (7) of the present invention is dissolved in CDCl3 13 C-NMR spectrum.
[0069] Fig.59 This is the DEPT-135 spectrum of the compound Coublibactin G (7) gallium complex of the present invention dissolved in CDCl3.
[0070] Fig.60 The gallium complex of the compound Coublibactin G (7) of the present invention is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0071] Fig.61 The HSQC spectrum of the compound Coublibactin G (7) gallium complex of the present invention dissolved in CDCl3.
[0072] Fig.62 The HMBC spectrum of the compound Coublibactin G (7) gallium complex of the present invention dissolved in CDCl3.
[0073] Fig.63 The NOESY spectrum of the compound Coublibactin G (7) gallium complex of the present invention dissolved in CDCl3.
[0074] Fig.64 The HSQC-TOCSY spectrum of the gallium complex of Coublibactin G (7), a compound of the present invention, dissolved in CDCl3.
[0075] Fig.65 The (+)-HR-ESI-MS spectrum of the compound Coublibactin H (8) gallium complex of the present invention.
[0076] Fig.66 The compound of the present invention, Coublibactin H (8) gallium complex, is dissolved in CDCl3 1 H-NMR spectrum.
[0077] Fig.67 The compound of the present invention, Coublibactin H (8) gallium complex, is dissolved in CDCl3 13 C-NMR spectrum.
[0078] Fig.68 The DEPT-135 spectrum of the compound Coublibactin H (8) gallium complex of the present invention dissolved in CDCl3.
[0079] Fig.69 The compound of the present invention, Coublibactin H (8) gallium complex, is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0080] Fig.70 The HSQC spectrum of the compound Coublibactin H (8) gallium complex of the present invention dissolved in CDCl3.
[0081] Fig.71 This is the HMBC spectrum of the compound Coublibactin H (8) gallium complex dissolved in CDCl3.
[0082] Fig.72 The NOESY spectrum of the compound Coublibactin H (8) gallium complex of the present invention dissolved in CDCl3.
[0083] Fig.73 The HSQC-TOCSY spectrum of the compound Coublibactin H (8) gallium complex of the present invention dissolved in CDCl3.
[0084] Fig.74 The (+)-HR-ESI-MS spectrum of the compound Coublibactin I (9) gallium complex of the present invention.
[0085] Fig.75 The gallium complex of the compound Coublibactin I (9) of the present invention is dissolved in CDCl3 1 H-NMR spectrum.
[0086] Fig.76 The gallium complex of the compound Coublibactin I (9) of the present invention is dissolved in CDCl3 13 C-NMR spectrum.
[0087] Fig.77 The DEPT-135 spectrum of the compound Coublibactin I (9) gallium complex of the present invention dissolved in CDCl3.
[0088] Fig.78 The gallium complex of the compound Coublibactin I (9) of the present invention is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0089] Fig.79 This is the HSQC spectrum of the compound Coublibactin I (9) gallium complex of the present invention dissolved in CDCl3.
[0090] Fig.80 The HMBC spectrum of the compound Coublibactin I (9) gallium complex of the present invention dissolved in CDCl3.
[0091] Fig.81 The NOESY spectrum of the compound Coublibactin I (9) gallium complex of the present invention dissolved in CDCl3.
[0092] Fig.82 The HSQC-TOCSY spectrum of the compound Coublibactin I (9) gallium complex of the present invention dissolved in CDCl3.
[0093] Fig.83 The (+)-HR-ESI-MS spectrum of the compound Coublibactin J (10) gallium complex of the present invention.
[0094] Fig.84 The gallium complex of the compound Coublibactin J (10) of the present invention is dissolved in CDCl3 1 H-NMR spectrum.
[0095] Fig.85 The gallium complex of the compound Coublibactin J (10) of the present invention is dissolved in CDCl3 13 C-NMR spectrum.
[0096] Fig.86 The DEPT-135 spectrum of the gallium complex of Coublibactin J (10), a compound of the present invention, dissolved in CDCl3.
[0097] Fig.87 The gallium complex of the compound Coublibactin J (10) of the present invention is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0098] Fig.88 The HSQC spectrum of the gallium complex of Coublibactin J (10), a compound of the present invention, dissolved in CDCl3.
[0099] Fig.89 The HMBC spectrum of the gallium complex of Coublibactin J (10), a compound of the present invention, dissolved in CDCl3.
[0100] Fig.90 The NOESY spectrum of the gallium complex of Coublibactin J (10), a compound of the present invention, dissolved in CDCl3.
[0101] Fig.91 The HSQC-TOCSY spectrum of the gallium complex of Coublibactin J (10), a compound of the present invention, dissolved in CDCl3.
[0102] Fig.92 The (+)-HR-ESI-MS spectrum of the compound Coublibactin K (11) gallium complex of the present invention.
[0103] Fig.93 The compound of the present invention, Coublibactin K (11) gallium complex, is dissolved in CDCl3 1 H-NMR spectrum.
[0104] Fig.94 The compound of the present invention, Coublibactin K (11) gallium complex, is dissolved in CDCl3 13 C-NMR spectrum.
[0105] Fig.95 This is the DEPT-135 spectrum of the compound Coublibactin K (11) gallium complex of the present invention dissolved in CDCl3.
[0106] Fig.96 The compound of the present invention, Coublibactin K (11) gallium complex, is dissolved in CDCl3 1 H– 1 H COSY spectrum.
[0107] Fig.97 The HSQC spectrum of the compound Coublibactin K (11) gallium complex of the present invention dissolved in CDCl3.
[0108] Fig.98 The HMBC spectrum of the compound Coublibactin K (11) gallium complex of the present invention dissolved in CDCl3.
[0109] Fig.99 The NOESY spectrum of the compound Coublibactin K (11) gallium complex of the present invention dissolved in CDCl3.
[0110] Fig.100 The HSQC-TOCSY spectrum of the compound Coublibactin K (11) gallium complex of the present invention dissolved in CDCl3. DETAILED DESCRIPTION
[0111] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.
[0112] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0113] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0114] Example 1 A method for separating and identifying siderophore compounds derived from Nocardia comprises the following steps: 1. Seed cultivation 50 μL of spores of Nocardia coubleae DSM 44960 were inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured at 30 °C and 200 rpm for 4 days to obtain seed solution. The preparation method of the seed culture medium comprises the following steps: 37 g of brain heart infusion powder, 20 g of glucose and 1 L of pure water, and sterilization at 121° C. for 30 min.
[0115] 2. Liquid fermentation culture The seed solution obtained in step 1 was inoculated into 200 mL of fermentation medium (1000 mL Erlenmeyer flask) at an inoculum ratio of 1:50, and cultured at 30°C and 200 rpm with shaking for 5 days to obtain a fermentation product; The preparation method of the fermentation medium comprises the following steps: 7.5 g of soy peptone, 30 g of glucose, 1 g of dipotassium hydrogen phosphate, 5.5 g of calcium carbonate, 5 g of ammonium sulfate and 1 L of pure water, and sterilizing at 121°C for 30 min.
[0116] 3. Extraction and separation of compounds Coublibactin A (1) – Coublibactin K (11) The fermentation product obtained in step 2 was centrifuged at 6000 rpm for 5 min, and the supernatant and bacteria were collected separately. The supernatant was extracted with ethyl acetate for 4 times and then concentrated and evaporated to obtain a crude extract of the supernatant. The bacteria were pre-frozen at -80 °C overnight, dried in a freeze dryer, and extracted with ethyl acetate for 20 min for 4 times. The extract was concentrated and evaporated to obtain a crude extract of the bacteria. The supernatant and the crude extract of the bacteria were combined and weighed to obtain a crude extract. The extract was chromatographed on a normal phase silica gel column using a petroleum ether-ethyl acetate gradient elution (v / v 100:0, 50:1, 20:1, 10:1, 5:1), followed by a dichloromethane-methanol gradient elution (v / v 100:0, 50:1, 20:1, 10:1, 5:1, 0:1) to obtain 7 components N1-N7, of which component N5 was chromatographed on an ODS column using a methanol-water gradient elution (v / v The components N5-20 and N5-21 were purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min and eluted isocratically with 80% acetonitrile aqueous solution to obtain compounds Coublibactin A (1), Coublibactin B (2), Coublibactin C (3), Coublibactin D (4), and Coublibactin G (7). The component N5-22 was first chromatographed on a Sephadex LH-20 gel column with methanol as the eluent, and then purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min and eluted isocratically with 89% acetonitrile aqueous solution to obtain compound Coublibactin E (5), Coublibactin F (6), and Coublibactin H (8); component N6 was chromatographed on an ODS column using a methanol-water gradient elution (v / v 100:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to obtain 19 subcomponents. Components N6-17 were first chromatographed on a Sephadex LH-20 gel column using methanol as the eluent, and then purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min. Compounds Coublibactin I (9), Coublibactin J (10), and Coublibactin K (11) were obtained by isocratic elution with 81% acetonitrile aqueous solution. The structural formulas of the compounds CoublibactinA (1)-Coublibactin K (11) are shown in the figure. Figure 1 shown.
[0117] 4. Gallium sulfate derivatization of compounds Coublibactin A (1)–Coublibactin K (11) The compounds Coublibactin A (1) -Coublibactin K (11) (1.8 μmol) obtained in step 3 were dissolved in 1 mL of methanol, and 1 mL of an aqueous solution of gallium sulfate (7.0 μmol) was added. The mixture was stirred and reacted at 25 °C for 1 h. The reaction solution was freeze-dried and purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min. The gallium complex of the compound Coublibactin A (1) -Coublibactin K (11) was obtained by isocratic elution with 84% acetonitrile aqueous solution. Its structure was determined by HR-ESI-MS, 1D NMR and 2D NMR spectroscopic techniques. The structural confirmation data are as follows: Gallium complex of compound Coublibactin A (1): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +789 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 211 (4.62), 228 (4.62), 272 (4.23) and 340 (4.01) nm; CD (MeOH) λ max (Δε): 234 (+50.18), 270 (+20.39) and 336 (+8.00) nm; IR (KBr) ν max : 3403, 2922, 2852, 1737, 1669, 1610, 1591, 1559, 1532, 1477 and 1469 cm -1 ; Positive ion mode HRESIMS m / z 772.4517 [M+H] + (The calculated value is C 40 H 62 N5O 10 , 772.4491) and m / z 838.3541 [M–3+Ga+H] + (The calculated value is C 40 H 59 GaN5O 10 , 838.3512); Gallium complex of compound Coublibactin B (2): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +939 (c 0.1, MeOH); UV (MeOH) λ max(log ε): 210 (4.52), 227 (4.52), 272 (4.12) and 340 (3.89) nm; CD (MeOH) λ max (Δε): 234 (+45.25), 268 (+20.84) and 336 (+7.98) nm; IR (KBr) ν max : 3403, 2923, 2852, 1736, 1671, 1610, 1588, 1559, 1532, 1476 and 1468 cm -1 ; Positive ion mode HRESIMS m / z 786.4671 [M+H] + (The calculated value is C 41 H 64 N5O 10 , 786.4648) and m / z 852.3691 [M–3+Ga+H] + (The calculated value is C 41 H 61 GaN5O 10 , 852.3669); Gallium complex of compound Coublibactin C (3): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +789 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 212 (4.58), 227 (4.58), 272 (4.18) and 339 (3.95) nm; CD (MeOH) λ max (Δε): 234 (+29.86), 268 (+12.25) and 335 (+4.68) nm; IR (KBr) ν max : 3405, 2924, 2853, 1737, 1669, 1610, 1593, 1559, 1532, 1477 and 1468 cm -1 ; Positive ion mode HRESIMS m / z 744.4206 [M+H] + (The calculated value is C 38 H 58 N5O 10 , 744.4178), m / z 810.3222 [M–3+Ga+H] + (The calculated value is C 38 H 55 GaN5O 10 , 810.3199); Gallium complex of compound Coublibactin D (4): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +843 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 211 (4.68), 227 (4.67), 272 (4.28) and 339 (4.05) nm; CD (MeOH) λ max (Δε): 234 (+43.24), 271 (+17.04) and 338 (+6.69) nm; IR (KBr) ν max : 3402, 2923, 2852, 1736, 1670, 1610, 1590, 1559, 1532 and 1477 cm -1 ; Positive ion mode HRESIMS m / z 758.4364 [M+H] + (The calculated value is C 39 H 60 N5O 10 , 758.4335) and m / z 824.3357 [M–3+Ga+H] + (The calculated value is C 39 H 57 GaN5O 10 , 824.3356); Gallium complex of compound Coublibactin E (5): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +531 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 210 (4.42), 227 (4.41), 272 (4.02) and 339 (3.78) nm; CD (MeOH) λ max (Δε): 234 (+24.11), 270 (+11.84) and 333 (+4.31) nm; IR (KBr) ν max : 3402, 2923, 2852, 1738, 1669, 1610, 1593, 1558, 1532, 1477 and 1468 cm -1 ; Positive ion mode HRESIMS m / z 800.4814 [M+H] + (The calculated value is C 42 H 66 N5O 10 , 800.4804) and m / z 866.3852 [M–3+Ga+H] + (The calculated value is C 42 H 63 GaN5O10 , 866.3825); Gallium complex of compound Coublibactin F (6): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +663 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 211 (4.77), 227 (4.76), 272 (4.37) and 341 (4.14) nm; CD (MeOH) λ max (Δε): 235 (+55.88), 269 (+26.28) and 335 (+10.15) nm; IR (KBr) ν max : 3403, 2924, 2852, 1737, 1671, 1610, 1590, 1559, 1532, 1477 and 1467 cm -1 ; Positive ion mode HRESIMS m / z 828.5133 [M+H] + (The calculated value is C 44 H 70 N5O 10 , 828.5117), m / z 894.4133 [M–3+Ga+H] + (The calculated value is C 44 H 67 GaN5O 10 , 894.4138); Gallium complex of compound Coublibactin G (7): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +825 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 213 (4.78), 228 (4.78), 272 (4.39) and 339 (4.15) nm; CD (MeOH) λ max (Δε): 235 (+54.48), 267 (+24.84) and 335 (+9.81) nm; IR (KBr) ν max : 3403, 2925, 2853, 1737, 1670, 1610, 1588, 1558, 1532, 1476 and 1468 cm -1 ; Positive ion mode HRESIMS m / z 758.4362 [M+H] + (The calculated value is C 39 H 60 N5O 10 , 758.4335) and m / z 824.3381 [M–3+Ga+H] + (The calculated value is C39 H 57 GaN5O 10 , 824.3356); Gallium complex of compound Coublibactin H (8): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +690 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 211 (4.67), 228 (4.66), 272 (4.27) and 340 (4.03) nm; CD (MeOH) λ max (Δε): 234 (+34.65), 268 (+14.21) and 333 (+5.81) nm; IR (KBr) ν max : 3403, 2923, 2852, 1737, 1670, 1610, 1588, 1558, 1532, 1476 and 1468 cm -1 ; Positive ion mode HRESIMS m / z 814.4989 [M+H] + (The calculated value is C 43 H 68 N5O 10 , 814.4961) and m / z 880.4007 [M–3+Ga+H] + (The calculated value is C 43 H 65 GaN5O 10 , 880.3982); Gallium complex of compound Coublibactin I (9): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +852 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 210 (4.55), 224 (4.53), 272 (4.14) and 339 (3.90) nm; CD (MeOH) λ max (Δε): 234 (+32.81), 270 (+13.84) and 335 (+5.30) nm; IR (KBr) ν max : 3402, 2923, 2852, 1737, 1671, 1608, 1559, 1532, 1477 and 1468 cm -1 ; Positive ion mode HRESIMS m / z 758.4362 [M+H] + (The calculated value is C 39 H 60 N5O 10, 758.4335) and m / z 824.3382 [M–3+Ga+H] + (The calculated value is C 39 H 57 GaN5O 10 , 824.3356); Gallium complex of compound Coublibactin J (10): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +807 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 210 (4.57), 227 (4.56), 272 (4.17) and 339 (3.94) nm; CD (MeOH) λ max (Δε): 233 (+30.36), 269 (+14.85) and 338 (+6.26) nm; IR (KBr) ν max : 3401, 2922, 2852, 1736, 1670, 1608, 1559, 1533, 1477, 1469 cm -1 ; Positive ion mode HRESIMS m / z 730.4049 [M+H] + (The calculated value is C 37 H 56 N5O 10 , 730.4022) and m / z 796.3070 [M–3+Ga+H] + (The calculated value is C 37 H 53 GaN5O 10 , 796.3043); Gallium complex of compound Coublibactin K (11): light orange-red amorphous solid, easily soluble in methanol and chloroform; [α]25 D +597 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 211 (4.67), 227 (4.65), 272 (4.26) and 339 (4.02) nm; CD (MeOH) λ max (Δε): 233 (+40.38), 270 (+16.84) and 340 (+6.42) nm; IR (KBr) ν max : 3402, 2924, 2853, 1738, 1670, 1607, 1558, 1532, 1477 and 1467 cm -1 ; Positive ion mode HRESIMS m / z 786.4677 [M+H] + (The calculated value is C 41 H64 N5O 10 , 786.4648) and m / z 852.3693 [M–3+Ga+H] + (The calculated value is C 41 H 61 GaN5O 10 , 852.3669); Table 1 Gallium complex of Coublibactin A (1) 1 H- and 13 C-NMR spectrum peaks
[0118] o represents overlapped signals.
[0119] Table 2 Gallium complex of Coublibactin B (2) 1 H- and 13 C-NMR spectrum peaks
[0120] o represents overlapped signals.
[0121] Table 3 Gallium complex of Coublibactin C (3) 1 H- and 13 C-NMR spectrum peaks
[0122] o represents overlapped signals.
[0123] Table 4 Gallium complex of compound Coublibactin D (4) 1 H- and 13 C-NMR spectrum peaks
[0124] o represents overlapped signals.
[0125] Table 5 Gallium complex of compound Coublibactin E (5) 1 H- and 13 C-NMR spectrum peaks
[0126] o represents overlapped signals.
[0127] Table 6 Compounds of Coublibactin F (6) Gallium complexes 1 H- and 13 C-NMR spectrum peaks
[0128] o represents overlapped signals.
[0129] Table 7 Gallium complex of Coublibactin G (7) 1 H- and 13 C-NMR spectrum peaks
[0130] o represents overlapped signals.
[0131] Table 8 Gallium complex of Coublibactin H (8) 1 H- and 13 C-NMR spectrum peaks
[0132] o represents overlapped signals.
[0133] Table 9 Gallium complex of compound Coublibactin I (9) 1 H- and 13 C-NMR spectrum peaks
[0134] o represents overlapped signals.
[0135] Table 10 Gallium complex of compound Coublibactin J (10) 1 H- and 13 C-NMR spectrum peaks
[0136] o represents overlapped signals.
[0137] Table 11 Gallium complex of compound Coublibactin K (11) 1 H- and 13 C-NMR spectrum peaks
[0138] o represents overlapped signals.
[0139] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.
Claims
1. A type of siderophore compound derived from Nocardia, characterized in that: The structural formula is as follows: 。 2. A method for separating and identifying siderophore compounds derived from Nocardia as claimed in claim 1, characterized in that: The following steps are involved: (1) Prepare a seed culture medium, inoculate Nocardia coubleae DSM 44960 into the seed culture medium, and culture at 30°C and 200 rpm for 4 days to obtain a seed solution; (2) The seed solution obtained in step (1) was inoculated into the fermentation medium, and cultured at 30°C and 200 rpm with shaking for 5 days to obtain a fermentation product; (3) separating the fermentation broth and the bacterial cells in the fermentation product obtained in step (2), extracting the fermentation broth with ethyl acetate four times, extracting the bacterial cells with ethyl acetate four times, combining the ethyl acetate phases and concentrating under reduced pressure to obtain a crude extract; (4) The crude extract obtained in step (3) was subjected to normal phase silica gel column chromatography, and gradient elution was performed with petroleum ether-ethyl acetate, and the gradient elution volume ratio v / v was 100:0, 50:1, 20:1, 10:1, 5:1, followed by gradient elution with dichloromethane-methanol, and the gradient elution volume ratio v / v was 100:0, 50:1, 20:1, 10:1, 5:1, 0:1, to obtain 7 components N1-N7, among which component N5 was subjected to ODS column chromatography, and gradient elution was performed with methanol-water, and the gradient elution volume ratio v / v was 90:10, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, to obtain 24 subcomponents. Components N5-20 and N5-21 were purified by semi-preparative RP-HPLC using YMC-Pack The components N5-22 were purified by ODS-A column at a flow rate of 2 mL / min and isocratically eluted with 80% acetonitrile aqueous solution to obtain compounds Coublibactin A (1), Coublibactin B (2), Coublibactin C (3), Coublibactin D (4), and Coublibactin G (7). Component N5-22 was first chromatographed on a Sephadex LH-20 gel column with methanol as the eluent, and then purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min and isocratically eluted with 89% acetonitrile aqueous solution to obtain compounds Coublibactin E (5), Coublibactin F (6), and Coublibactin G (7). H (8); component N6 was purified by ODS column chromatography using a methanol-water gradient elution with a gradient elution volume ratio of 100:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0 to obtain 19 subcomponents. Components N6-17 were first purified by Sephadex LH-20 gel column chromatography using methanol as the eluent, and then purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min and isocratically eluted with 81% acetonitrile aqueous solution to obtain compounds Coublibactin I (9), Coublibactin J (10), and Coublibactin K (11); (5) The compounds Coublibactin A (1) -Coublibactin K (11) obtained in step (4) were dissolved in methanol, respectively, and an aqueous solution of gallium sulfate was added. The mixture was stirred at 25 °C for 1 h. The reaction solution was freeze-dried and purified by semi-preparative RP-HPLC using a YMC-Pack ODS-A column at a flow rate of 2 mL / min. The mixture was isocratically eluted with 84% acetonitrile aqueous solution to obtain the gallium complex of the compounds Coublibactin A (1) -Coublibactin K (11).
3. The method for separating and identifying siderophore compounds derived from Nocardia according to claim 2, characterized in that: The preparation method of the seed culture medium described in step (1) comprises the following steps: 37 g of brain heart infusion powder, 20 g of glucose and 1 L of pure water, and sterilization at 121°C for 30 min.
4. The method for separating and identifying siderophore compounds derived from Nocardia according to claim 2, characterized in that: The method for preparing the fermentation medium described in step (2) comprises the following steps: 7.5 g of soy peptone, 30 g of glucose, 1 g of dipotassium hydrogen phosphate, 5.5 g of calcium carbonate, 5 g of ammonium sulfate and 1 L of pure water, and sterilizing at 121°C for 30 min.