Method for preparing acetyl glucosamine derivative through biotransformation of marine microbacterium and application of acetyl glucosamine derivative
By preparing acetaminophen derivatives through biotransformation using marine microbacteria, the problem of poor structural stability of acetaminophen has been solved, resulting in novel compounds with anti-inflammatory effects and meeting the diverse needs of drug development.
Patent Information
- Application Number
- CN202510953917.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-11-21
AI Technical Summary
Glucosamine has poor structural stability and is prone to hydrolysis and polymerization reactions in chemical environments or biological bodies, which affects its stability and activity in long-term applications. In addition, its structure is relatively simple, making it difficult to meet the diverse needs of drug development.
Acetaminoglycine derivatives were prepared by biotransformation using marine microbacterium sp. HN-3-14. Through specific culture medium and fermentation process, combined with chromatographic separation technology, four novel acetylglucosamine derivatives were obtained.
The prepared acetylglucosamine derivatives exhibited significant anti-inflammatory effects under low cytotoxicity, with anti-inflammatory effects at concentrations of 10-100 μM, thus expanding their functional diversity.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method and application for preparing acetaminophen derivatives using marine microbacteria through biotransformation. Background Technology
[0002] Glucosamine acetylglucosamine (GlcNAc), also known as acetylglucosamine, is a monosaccharide derivative widely found in organisms. Its structure is formed by replacing the 2-hydroxyl group of glucose with an acetylamino group. Glucosamine acetylglucosamine is an important building block of many glycoproteins, glycolipids, and polysaccharides, participating in various physiological processes such as cell recognition and signal transduction. In terms of biological activity, acetylglucosamine has antioxidant and anti-inflammatory effects. Recent studies have also found that it has potential in regulating immunity. However, research on acetylglucosamine still has many shortcomings. Structurally, acetylglucosamine has poor structural stability and is prone to hydrolysis and polymerization reactions under certain chemical environments or specific conditions in vivo, affecting its stability and activity in long-term applications. Secondly, its relatively simple structure limits the diversity of its functions, making it difficult to meet the needs of drug development. Therefore, future research needs to delve deeper into the relationship between its structure and function and explore ways to improve its structural stability and expand its functions through chemical modification and other methods. Summary of the Invention
[0003] The purpose of this invention is to provide a method and application for preparing acetaminophen derivatives using marine microbacteria through biotransformation.
[0004] To achieve the above objectives, the present invention adopts the following technical solution: A class of acetylglucosamine derivatives, the structural formula of which is shown below: .
[0005] A method for preparing the acetylglucosamine derivative includes the following steps: (1) Microbacteria ( Microbacterium HN-3-14 sp. was inoculated into SDA medium and cultured at 8℃ and 150 r / min for 7 days to obtain seed culture. The microbacterium was deposited at Guangdong Provincial Microbial Culture Collection Center on March 3, 2025, with accession number GDMCC No. 65964. (2) The seed liquid obtained in step (1) is inoculated into a special culture medium and fermented at 28°C for 30 days to obtain the fermentation liquid; (3) The fermentation broth obtained in step (2) is first filtered, and the filtrate is extracted three times with ethyl acetate. The extracts are combined and concentrated under reduced pressure to obtain an extract. (4) After dissolving the extract, the sample is loaded onto the chromatographic column for preliminary separation to obtain the components to be separated; (5) The components obtained in step (4) are separated by HPLC to obtain acetylglucosamine derivatives.
[0006] Furthermore, in step (2), the inoculation amount of seed solution is 0.4% v / v.
[0007] Further, the formula of the special culture medium mentioned in step (2) is as follows: 2-10 g of soluble starch, 3-8.5 g of N-acetyl-D-glucosamine, 0.5-3.0 g of (NH4)2SO4, 1-3 g of yeast extract, and 5-15 g of sea salt, dissolved in 1L of deionized water.
[0008] Furthermore, in step (3), the volume ratio of the filtrate to ethyl acetate is 1:1.
[0009] Furthermore, the HPLC preparation conditions in step (5) are as follows: Ultimate XB-C column. 18 The column was prepared using a methanol-water mobile phase with gradient elution for 60 minutes, methanol concentration ranging from 10% to 80%, and a flow rate of 10 mL / min. The detection wavelength was 210 nm, the column temperature was 28 °C, and the injection volume was 500 μL.
[0010] The acetylglucosamine derivatives mentioned above can be used to prepare anti-inflammatory drugs.
[0011] The beneficial effects of this invention are as follows: This invention utilizes microbial bio-fermentation to obtain four novel acetylglucosamine derivatives. The resulting compounds exhibit low cytotoxicity and certain anti-inflammatory effects at concentrations of 10-100 μM. Attached Figure Description
[0012] Figure 1 This is the high-resolution mass spectrum of compound 1.
[0013] Figure 2 This is the 1H NMR spectrum of compound 1.
[0014] Figure 3 This is the carbon NMR spectrum of compound 1.
[0015] Figure 4 For compound 1 1 H- 1 H COSY diagram.
[0016] Figure 5 Here is the HMQC diagram for compound 1.
[0017] Figure 6 Here is the HMBC diagram for compound 1.
[0018] Figure 7 The NOESY diagram is for compound 1.
[0019] Figure 8 This is the high-resolution mass spectrum of compound 2.
[0020] Figure 9 This is the 1H NMR spectrum of compound 2.
[0021] Figure 10 The image shows the carbon NMR spectrum of compound 2.
[0022] Figure 11 For compound 2 1 H- 1 H COSY diagram.
[0023] Figure 12 Here is the HMQC diagram for compound 2.
[0024] Figure 13 Here is the HMBC diagram of compound 2.
[0025] Figure 14 This is the high-resolution mass spectrum of compound 3.
[0026] Figure 15 This is the 1H NMR spectrum of compound 3.
[0027] Figure 16 This is the carbon NMR spectrum of compound 3.
[0028] Figure 17 For compound 3 1 H- 1 H COSY diagram.
[0029] Figure 18 Here is the HMQC diagram for compound 3.
[0030] Figure 19 Here is the HMBC diagram for compound 3.
[0031] Figure 20 This is the high-resolution mass spectrum of compound 4.
[0032] Figure 21 This is the 1H NMR spectrum of compound 4.
[0033] Figure 22 This is the carbon NMR spectrum of compound 4.
[0034] Figure 23 For compound 4 1 H- 1 H COSY diagram.
[0035] Figure 24 Here is the HMQC diagram for compound 4.
[0036] Figure 25 Here is the HMBC diagram for compound 4.
[0037] Figure 26 Biosynthesis of compounds 1-4.
[0038] Figure 27 This indicates the anti-inflammatory activity of compound 4. Detailed Implementation
[0039] To make the content of this invention easier to understand, the technical solutions of this invention will be further described below in conjunction with specific embodiments, but this does not limit the scope of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials in the following embodiments can be purchased from the market.
[0040] Example 1: Fermentation of the strain and obtaining monomers The strain used in the screening of this invention is a microbacterium ( Microbacterium sp.) HN-3-14 was deposited on March 3, 2025 at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 65964, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. The screening and identification of the strain are described in the paper "Isolation and Structural Identification of Secondary Metabolites of Marine Actinomycete HN-3-14".
[0041] 1. Seed liquid preparation Under aseptic conditions, take out the activated Microbacterium sp. HN-3-14 strain SDA plate, pick out an agar block containing a single colony and put it into 100 mL of SDA liquid medium (Guangdong Huankai Microbial Technology Co., Ltd., batch number: 230413A20), place it in a constant temperature shaker incubator, and incubate at 28℃ and 150 r / min for 7 days to obtain seed culture.
[0042] 2. Large-scale fermentation of microbial strains Prepare 50L of dedicated liquid culture medium and divide it into 25 fermentation flasks (5L fermentation flasks, each containing 2L of culture medium). After autoclaving, place them in the inoculation room to cool. In a clean bench, use a 10mL pipette to add 8mL of seed culture to each fermentation flask and seal the flasks. Place the fermentation flasks in a 28℃ constant temperature incubator and shake for 30 days to obtain the 3-1-14 fermentation broth.
[0043] The formula for the special liquid culture medium (1L) is as follows: soluble starch (5 g), N-acetyl-D-glucosamine (7 g), (NH4)2SO4 (2.5 g), yeast extract (2.0 g), sea salt (15 g), and purified water to 1L.
[0044] 3. Extract preparation Take the total fermentation broth of HN-3-14 obtained in step (2), filter it with silk, extract the filtrate three times with an equal amount of ethyl acetate, combine the extracts, and concentrate it under reduced pressure at 40°C using a rotary evaporator to obtain a brown extract (15.5g).
[0045] 4. Isolation of compounds The brown extract obtained in step (3) was dissolved in 50% methanol aqueous solution and loaded onto a Sephadex LH-20 column (8*150cm). Elution was performed with pure methanol, and the mobile phase flow rate was adjusted to 1 drop / 2s. The receiving volume for each sample was 20mL. Each component was concentrated and analyzed by high-performance liquid chromatography (HPLC). The detection conditions were an Agilent 1260 Infinity analytical HPLC system with Ultimate XB-C. 18 Analytical column (10×250 mm); mobile phase: methanol-water, gradient elution, elution time: 60 min, methanol concentration: 10% to 80%, flow rate: 1 mL / min; detection wavelength: 210 nm; column temperature: 28 ℃; injection volume: 10 μL, components were combined based on chromatogram similarity.
[0046] The fraction containing the compound to be separated was collected, and compound preparation was performed using preparative HPLC. HPLC preparation conditions: Agilent 1260 Infinity preparative liquid chromatograph; Ultimate XB-C column. 18 A preparative column (30×250 mm) was used; the mobile phase was a methanol-water system with gradient elution for 60 min, with methanol concentration ranging from 10% to 80% and a flow rate of 10 mL / min; the detection wavelength was 210 nm; the column temperature was 28 ℃; and the injection volume was 500 uL. Compound 1 (15 mg, 51.0 min), compound 2 (10 mg, 42.3 min), compound 3 (8 mg, 29.2 min), and compound 4 (20 mg, 32.8 min) were prepared. The prepared samples were evaporated to dryness and stored for later use.
[0047] Example 2: Structural analysis of the compound 1. Compound 1 like Figure 1 As shown, the high-resolution mass spectrometry data for compound 1 is 554.2017 (precise value 554.1991 [MH]). - The molecular formula is C.24 H 33 N3O 12 It has 10 degrees of unsaturation. The proton NMR spectrum of compound 1 shows three acetylmethyl signals, suggesting the presence of three glucosamine units in the molecule. Figure 2 Compound 1's carbon spectrum shows six highly overlapping groups of carbon atom signals (δ). C (154.8 / 154.7, 141.8 / 141.6, 122.9 / 122.8, 107.3 / 107.2, 89.6 / 89.5, 85.7 / 85.6), each group has two carbon atoms, where δ C The signals 154.8 / 154.7 (C-4 / 4'), 141.8 / 141.6 (C-1 / 1'), 122.9 / 122.8 (C-2 / 2'), and 107.3 / 107.2 (C-3 / 3') are presumed to be carbon atom signals of substituted furan fragments, indicating that the carbon atoms at positions 1 and 4 of glucosamine are connected by an oxygen bridge, and dehydration forms a stable furan structure. δ C 89.6 / 89.5 and 85.7 / 85.6 are presumed to be the 5th and 6th carbon atoms of glucosamine furanylformate. After subtracting the two furanyl structures; carbon spectroscopy. δ C The signals at 126.7 and 135.4 are trisubstituted double bond signals. Based on the degree of molecular unsaturation, it is inferred that the last glucosamine should be an open-ring structure containing unsaturated double bonds. Figure 3 ). use 1 H- 1 The H COSY and HMBC experiments verified the hypothesis. The COSY spectrum showed a spin-coupled fragment =CHCH(OH)CH(OH)CH2-. The HMBC spectrum showed correlation signals between H-1´´ and C-2´´, C-3´´, indicating that the glucosamine fragment underwent dehydration at positions 2 and 3 to form double bonds. The 1´´ carbon signal in this fragment was located at a higher field, indicating that its corresponding carbon was not connected to a heteroatom. The HMBC spectrum showed a strong correlation signal between H-1´´ and the C-1, C1´ of the two furanose glucosamines, indicating that the carbon atom at position 1´´ acted as a carbon bridge connecting the two terminal carbons of the furanose glucosamines (…). Figures 4-6 ).
[0048] The configuration of the compound was determined, its biosynthetic structure was predicted, and it was confirmed by the NOESY experiment. The NOESY spectrum (...) Figure 7 H-1´´ and H-3´´ have correlated signals, therefore the double bond geometry is: Z Based on the biosynthetic pathway, the hydroxyl groups attached to C-5, C-5', C-4'', and C-5'' in the compound did not participate in the synthetic reaction. Therefore, it is inferred that its configuration is the same as that of the reaction initiator, glucosamine, and is as follows: R , R , S, R .
[0049] Compound 1 1 H- 1 The following are the relevant signals for H COSY, major HMBC, and key NOESY: ; In summary, the structure of compound 1 was identified as N-(2-((4) S 5 R , Z )-2-acetamido-1-(3-acetamido-5-(( R )-1,2-dihydroxyethyl)furan-2-yl)-4,5,6-trihydroxyhex-2-en-1-yl)-5-(( S )-1,2-dihydroxyethyl)furan-3-yl)acetamide, identified as a new compound in the Scifinder database.
[0050] The structure of compound 1 is shown below: .
[0051] 2. Compound 2 The high-resolution mass spectrometry data of compound 2 are as follows: m / z 452.1680 [MH] - The molecular formula is C. 20 H 27 N3O9 has 9 degrees of unsaturation ( Figure 8 Compound 2 showed three acetylmethyl signals in its 1H NMR spectrum, but only 20 carbon signals in its 1C NMR spectrum. The 1C NMR spectrum of compound 2 also showed six highly overlapping groups of carbon atoms, each group containing two carbon atoms, suggesting the presence of two furanyl-fragmented glucosamine structures in the molecule. Figures 9-10 After subtracting the three acetylmethyl carbon signals, the compound also contained an AX2 spin-coupling system, and the fragments were linked using HMBC experiments. In the HMBC spectrum ( Figure 13 Compound 2 shows a strong correlation between H-1´´ and C-2´´, H-2´´ and C-3´´, and H-1´ and the C-1 and C1´ of the two furanose groups, indicating that the carbon atom at position 1´´ acts as a carbon bridge connecting the two furans. 1 H- 1 The H COSY and key HMBC related signals are shown below: .
[0052] The configuration of compound 2 was determined primarily using a biosynthetic deduction method. Based on the biosynthetic pathway, the hydroxyl groups attached to C-5 and C-5' in the compound did not participate in the synthetic reaction. Therefore, it was deduced that its configuration is the same as that of the reaction initiator, glucosamine, and is as follows: R , R .
[0053] In summary, the structure of compound 2 was identified as N,N'-((2-acetamidoethane-1,1-diyl)bis(5-(( R )-1,2-dihydroxyethyl)furan-2,3-diyl))diacetamide, retrieved from the SciFinder database as a new compound.
[0054] The molecular structure of the compound is as follows: .
[0055] 3. Compound 3 The high-resolution mass spectrometry data for compound 3 is 369.1305 ([MH)). - The molecular formula is C. 16 H 22 N2O8 has 7 degrees of unsaturation ( Figure 14 Compound 3 showed two acetylmethyl signals in its 1H NMR spectrum, suggesting it is a diacetylaminoglycan derivative, but its 1C NMR spectrum showed 16 carbon signals. Figures 15-16 ). Compounds 1 H- 1 H COSY spectrum ( Figure 17 The image shows two CH2CH spin segments, HMBC spectrum ( Figure 19 On the HMBC spectrum, H-3' correlates with C-1' and C-5', suggesting the presence of furan-substituted glucosamine in the compound. H-3 also correlates with C-1 and C-5, while H-1 correlates with C-1'. Compounds H-1 and H-4 are also correlated on the HMBC spectrum. 1 H- 1 The HCOSY spectrum shows a W-type coupling signal, indicating that the relative configuration of H-1 and H-4 in compound 3 is cis. 1 H- 1 The H COSY and key HMBC related signals are shown below: .
[0056] Based on the biosynthetic pathway, the C-5 hydroxyl group in the compound did not participate in the synthetic reaction, suggesting that its configuration is the same as that of the reaction initiator, glucosamine. R .
[0057] In summary, the structure of compound 3 was identified as N,N'-((2S *,5 R *)-5,5'-bis(( R ()-1,2-dihydroxyethyl)-2,5-dihydro-[2,2'-bifuran]-3,3'-diyl)diacetamide, a novel compound was identified in the SciFinder database. The structural formula of compound 3 is shown below: .
[0058] 4. Compound 4 The high-resolution mass spectrometry data for compound 4 is 309.1096 ([MH)). - The molecular formula is C. 14 H 18 N2O6 has 7 degrees of unsaturation ( Figure 20 Compound 4 showed two acetylmethyl signals in its 1H NMR spectrum, suggesting it is a diacetyl glucosamine derivative, but its 1C NMR spectrum only showed 14 carbon signals. Figures 21-22 ). Compounds 1 H- 1 H COSY spectrum ( Figure 23 The double bond correlation signal was shown, combined with the HMBC experiment ( Figure 25 It is inferred that the glucosamine fragment of the compound has been degraded, leaving only the furan fragment. The HMBC spectrum shows correlation signals between H-1' and C-1, indicating that the carbon-lowering acetylglucosamine fragment is linked at position 1 to the 3-position of another glucosamine. Compounds H-1 and H-4... 1 H- 1 The H COSY spectrum shows a W-type coupling signal, indicating that the relative configuration of H-1 and H-4 in compound 4 is cis. 1 H- 1 The following are the relevant signals for HCOSY and key HMBC: .
[0059] The configuration of compound 4 was determined primarily using a biosynthetic deduction method. Based on the biosynthetic pathway, the C-5 hydroxyl group in the compound did not participate in the synthetic reaction, thus deducing that its configuration is the same as that of the reaction initiator, glucosamine. R .
[0060] In summary, the structure of compound 4 was identified as N,N'-((2 S *,5 R *)-5-(( R The compound )-1,2-dihydroxyethyl)-2,5-dihydro-[2,2'-bifuran]-3,3'-diyl)diacetamide was identified as a new compound in the SciFinder database.
[0061] The structure of compound 4 is shown below: .
[0062] The NMR data of compounds 1-4 are summarized in Table 1.
[0063] Table 1. NMR numbers of compounds 1-4 Note: a The solvent is CD3OD; b The solvent is DMSO-d6.
[0064] To clarify the origin of compounds 1-4 and determine the absolute configuration of the relevant chiral carbons, their biosynthetic pathways were deduced. Based on the structures of compounds 1-4, it is speculated that they all originate from N-acetyl-D-glucosamine (a) in the culture medium. First, the hydroxyl group at position 4 of N-acetyl-D-glucosamine (a) undergoes a hemiacetal reaction with the carbonyl group at position 1 to generate a cyclic glucosamine (b). Further dehydration generates a furanyl glucosamine derivative (c). The unsaturated hydrogen at position 1 of this compound undergoes a nucleophilic addition reaction with the carbonyl group of another molecule of chain glucosamine (a) to generate a diglucosamine derivative (d). Then, it undergoes an intermolecular nucleophilic substitution reaction with c to generate a triglucosamine derivative (e), which further undergoes a dehydration reaction to generate compound 1. The sugar chain of the triglucosamine derivative (e) undergoes a continuous oxidation-decarboxylation reaction to generate compound 2. The furanyl glucosamine derivative (c) undergoes a nucleophilic substitution reaction with the cyclic glucosamine (b) to generate compound 3, which further undergoes oxidation-decarboxylation to generate compound 4. The reaction flow is as follows: Figure 26 As shown.
[0065] Example 3 Evaluation of the anti-inflammatory activity of the compound The anti-inflammatory activity of compounds 1-4 was evaluated in RAW 264.7 mouse mononuclear macrophages. First, the cytotoxicity of the compounds was determined using the CCK-8 assay. The results showed that compounds 1-4 did not exhibit significant cytotoxicity at concentrations of 1-200 μM (with DMSO as solvent) (cell viability greater than 85%).
[0066] The NO content in cell supernatant was determined using the Griess method. Cells in the logarithmic growth phase were treated with a concentration of 5 × 10⁻⁶. 5200 μL of cell suspension per cell / mL was seeded into 96-well cell culture plates, and 200 μL of 1×PBS (pH 7.2) was added. Cells were cultured at 37℃ and 5% CO2 to allow adherence. The supernatant was discarded. The blank control group was treated with 100 μL of DMEM medium, the model group with an equal volume of DMEM medium containing 1 µg / mL LPS (containing the same volume of DMSO as the sample solution), and the experimental groups with equal volumes of DMSO solutions containing 1 µg / mL LPS at different concentrations for compounds 1-4. Each group had three replicates. After 24 h of culture, the supernatant was collected, and the OD value of each well at 540 nm was measured and recorded using a kit to calculate the degree of inhibition of NO release in each group. Taking compound 4 as an example, the results showed that compound 4 had a certain anti-inflammatory effect at concentrations of 10-100 μM. Figure 27 ).
[0067] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.
Claims
1. A class of acetylglucosamine derivatives, characterized in that: The structural formula of the acetylglucosamine derivative is shown below: 。 2. A method for preparing the acetylglucosamine derivative as described in claim 1, characterized in that: Includes the following steps: (1) Microbacteria ( Microbacterium HN-3-14 sp. was inoculated into SDA medium and cultured at 8℃ and 150 r / min for 7 days to obtain seed culture. The microbacterium was deposited at Guangdong Provincial Microbial Culture Collection Center on March 3, 2025, with accession number GDMCC No. 65964. (2) The seed liquid obtained in step (1) is inoculated into a special culture medium and fermented at 28°C for 30 days to obtain the fermentation liquid; (3) The fermentation broth obtained in step (2) is first filtered, and the filtrate is extracted three times with ethyl acetate. The extracts are combined and concentrated under reduced pressure to obtain an extract. (4) After dissolving the extract, the sample is loaded onto the chromatographic column for preliminary separation to obtain the components to be separated; (5) The components obtained in step (4) are separated by HPLC to obtain acetylglucosamine derivatives.
3. The method according to claim 2, characterized in that: In step (2), the inoculation amount of seed liquid is 0.4% v / v.
4. The method according to claim 2, characterized in that: The formula for the special culture medium mentioned in step (2) is as follows: 2-10 g of soluble starch, 3-8.5 g of N-acetyl-D-glucosamine, 0.5-3.0 g of (NH4)2SO4, 1-3 g of yeast extract, and 5-15 g of sea salt, dissolved in 1L of deionized water.
5. The method according to claim 2, characterized in that: In step (3), the volume ratio of filtrate to ethyl acetate is 1:
1.
6. The method according to claim 2, characterized in that: HPLC preparation conditions in step (5): Ultimate XB-C column 18 Preparative column; mobile phase: methanol-water, gradient elution, elution time: 60 min, methanol concentration: 10% to 80%, flow rate: 10 mL / min; detection wavelength: 210 nm; column temperature: 28 ℃; injection volume: 500 uL.
7. The use of the acetylglucosamine derivative as described in claim 1 in the preparation of anti-inflammatory drugs.