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CN-122011058-A - Three candidate antigen chimeric oligosaccharides of helicobacter pylori and preparation method thereof

CN122011058ACN 122011058 ACN122011058 ACN 122011058ACN-122011058-A

Abstract

The invention discloses three candidate antigen chimeric oligosaccharides of helicobacter pylori and a preparation method thereof, wherein a mannoheptanolide compound is prepared by protecting 2, 3-hydroxyl groups by triethyl orthoacetate, reducing an isocephalic position, carrying out ring opening on the isocephalic position by using acetylenic acid ester, protecting the 3-hydroxyl groups by acetyl to obtain mannoheptanoenic acid ester donor, carrying out glycosylation reaction with 1-azidopropanol, further reducing an azido group into amino groups, protecting 6-hydroxyl groups by glucose thioglycoside through photosensitive protecting groups, carrying out iodinated acrylic acid on the 1-hydroxyl groups, and coupling with 1-hexyne to obtain photosensitive protecting group-protected glucose acetylenic acid ester donor, preparing glucose oligosaccharide with alpha-1, 6 connection through photosensitive one-pot, protecting the 6-hydroxyl groups by using azidopropyl, reducing the azido group into amino groups, connecting two segments by using glutaric acid as a connecting arm, and carrying out three steps of removal by propylidene, acetyl removal and hydrogenation deprotection, thus obtaining the target chimeric oligosaccharide. The preparation method has mild conditions and is simple to operate.

Inventors

  • YANG YOU
  • FAN JIAHAO
  • CHANG DONGSHENG
  • JI XIANG
  • DAI CHANYUAN

Assignees

  • 华东理工大学

Dates

Publication Date
20260512
Application Date
20260205

Claims (10)

  1. 1. The three candidate antigen chimeric oligosaccharides of helicobacter pylori are characterized in that the candidate antigen chimeric oligosaccharides are chimeric oligosaccharide I, chimeric oligosaccharide II and chimeric oligosaccharide III, and the chemical structural general formulas are shown in the following formulas (I), (II) and (III) respectively: 。
  2. 2. The method for preparing three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 1, comprising (1) preparing chimeric oligosaccharide I, comprising the steps of: S1, under the action of triethyl orthoacetate and paratoluenesulfonic acid monohydrate, performing propylidene protection on 2, 3-hydroxyl in a compound 1 shown in a structural formula (1), reducing an isocephalic position to be hydroxyl under the action of lithium aluminum tri-tert-butoxide hydride, and condensing with alkynoic acid under the action of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to obtain a D-glycerol-D-mannoheptanoic acid ester donor, namely a compound 2 shown in a structural formula (2); s2, opening the ring of the triethyl orthoacetate at the 2,3 positions in the compound 2, and then performing acetyl protection on the exposed hydroxyl under the action of acetic anhydride and 4-dimethylaminopyridine to obtain a compound 3 with a structural formula shown in (3); S3, under the action of PPh 3 AuOTf and trifluoromethanesulfonic acid, carrying out glycosylation reaction on the compound 3 and 1-azidopropanol to obtain a compound 4 with a structural formula shown as (4); s4, performing an azide reaction on the compound 4 under the action of triphenylphosphine, and reducing an azide group into an amino group to obtain a compound 5 with a structural formula shown in (5); S5, reacting a compound 6 with a structural formula shown as (6) with o-nitrobenzyl bromide under the action of a potassium hydroxide aqueous solution and tetrabutylammonium bromide, protecting a 6-position exposed hydroxyl group by using a photosensitive protecting group (oNB), performing glycosylation reaction with iodinated acrylic acid under the action of N-iodinated succinimide and boron trifluoride diethyl ether, and reacting the obtained reaction product with 1-hexyne under the action of ditriphenylphosphoryl palladium dichloride, cuprous iodide and triethylamine to obtain a compound 7 with a structural formula shown as (7); S6, dissolving the compound 7 in an organic solvent, carrying out glycosylation reaction on the compound under the action of SPhosAuNTf 2 and N, N-dimethylformamide and a connector compound 8 with a structural general formula shown as (8), and removing a photosensitive protecting group (oNB) on a 6-position hydroxyl group under light irradiation after the complete reaction to obtain a compound 9 with the structural general formula shown as (9); s7, reacting the compound 9 with a compound 10 with a structural formula shown as (10) under the action of sodium hydrogen to obtain a compound 11 with a structural general formula shown as (11); S8, dissolving the compound 11 in a solvent, performing an azide reaction on the compound 11 under the action of triphenylphosphine, and reducing an azide group into an amino group to obtain a compound 12 with a structural general formula shown in (12); S9, carrying out condensation reaction on the compound 12 and glutaric acid under the action of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to obtain a compound 13 with a structural general formula shown in (13); S10, under the action of 4-dimethylaminopyridine and carbodiimide, performing condensation reaction on the compound 5 and the compound 13 to obtain a compound 14 with a structural general formula shown as (14); s11, dissolving a compound 14 in a solvent, removing propylene, removing acetyl groups under the action of sodium methoxide, and then removing protective groups by hydrogenation under the action of Pd (OH) 2 /C to obtain chimeric oligosaccharide I; (2) Preparation of chimeric oligosaccharide II comprising the steps of: A1, dissolving a compound 9 in an organic solvent, carrying out glycosylation reaction on the compound 9 and the compound 7 under the action of SPhosAuNTf 2 and N, N-dimethylformamide, and removing a photosensitive protecting group (oNB) on a 6-hydroxy group through light irradiation after the reaction is completed to obtain a compound 16 shown in a structural general formula (16); a2, reacting the compound 16 with the compound 10 under the action of sodium hydride to obtain a compound 17 with a structural general formula shown as (17); a3, dissolving the compound 17 in a solvent, and performing an azide reaction on the compound 17 under the action of triphenylphosphine to reduce an azide group into an amino group to obtain a compound 18 shown in a structural general formula (18); A4, carrying out condensation reaction on the compound 18 and glutaric acid under the action of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to obtain a compound 19 with a structural general formula shown in (19); A5, carrying out condensation reaction on the compound 5 and the compound 19 under the action of 4-dimethylaminopyridine and carbodiimide to obtain a compound 20 with a structural general formula shown as (20); A6, removing propylene from the compound 20, removing acetyl under the action of sodium methoxide, dissolving the compound in a solvent, and removing a protecting group through hydrogenation under the action of Pd (OH) 2 /C to obtain chimeric oligosaccharide II; (3) The preparation of the chimeric oligosaccharide III comprises the following steps: B1, under the action of SPhosAuNTf 2 and N, N-dimethylformamide, carrying out glycosylation reaction on the compound 9 and the compound 7, after the reaction is completed, removing a photosensitive protecting group (oNB) on a 6-position hydroxyl group by light irradiation, then adding the compound 7 and SPhosAuNTf 2 into a system again, carrying out glycosylation reaction, and after the reaction is completed, removing the photosensitive protecting group (oNB) on the 6-position hydroxyl group by light irradiation of the system, thereby obtaining a compound 22 shown in a structural general formula (22); b2, reacting the compound 22 with the compound 10 under the action of sodium hydrogen to obtain a compound 23 with a structural general formula shown as (23); b3, performing an azide reaction on the compound 23 under the action of triphenylphosphine, and reducing an azide group into an amino group to obtain a compound 24 with a structural general formula shown in (24); B4, dissolving the compound 24 in a solvent, and carrying out condensation reaction on the compound 24 and glutaric acid under the action of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to obtain a compound 25 with a structural general formula shown in (25); b5, carrying out condensation reaction on the compound 5 and the compound 25 under the action of 4-dimethylaminopyridine and carbodiimide to obtain a compound 26 with a structural formula shown as (26); B6, dissolving the compound 26 in a solvent, removing propylene, removing acetyl groups under the action of sodium methoxide, and then removing protective groups by hydrogenation under the action of Pd (OH) 2 /C to obtain the chimeric oligosaccharide III.
  3. 3. The method for preparing three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 2, characterized in that in step S1, the molar ratio of the compound 1, triethyl orthoacetate, paratoluenesulfonic acid monohydrate, lithium aluminum tri-tert-butoxide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and alkynoic acid is (1-1.2): 8-12): 0.1-0.3): 2-4): 1-2): 0.1-0.2: 1-2, the temperature of the propylidene protection reaction of the triethyl 2, 3-hydroxy orthoacetate is room temperature, the reaction temperature of the lithium aluminum tri-tert-butoxide in reducing the iso-head position is 0 ℃, and the condensation reaction temperature with alkynoic acid is room temperature.
  4. 4. The preparation method of three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 2, wherein in the step S2, the molar ratio of the compound 2 to acetic anhydride to 4-dimethylaminopyridine is 1 (8-12) (5-7), the temperature of the 2, 3-triethyl orthoacetate ring-opening reaction is room temperature, and the temperature of the acetyl protection reaction is room temperature; In the step S3, the molar ratio of the compound 3 to the 1-azidopropanol to the PPh 3 AuOTf to the trifluoromethanesulfonic acid is (1-1.2): (1.5-2): (0.2-0.6): (0.1-0.2), and the temperature of the glycosylation reaction is-40 ℃; In the step S4, the molar ratio of the compound 4 to the triphenylphosphine is (1-1.2): 4-6, and the reaction temperature for reducing the azide group to the amino group is 60 ℃; The reaction temperature in the step S5 is room temperature, and the molar ratio of the compound 6, tetrabutylammonium bromide, o-nitrobenzyl bromide, N-iodosuccinimide, boron trifluoride diethyl ether, iodized acrylic acid, bis-triphenylphosphine palladium dichloride, cuprous iodide, 1-hexyne and triethylamine is (1-1.2): (0.5-1): (4-5): (2-3): (0.05-0.1): (1-2).
  5. 5. The method for preparing three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 2, wherein in the step S6, the molar ratio of the compound 7, the connector compound 8, SPhosAuNTf 2 and the N, N-dimethylformamide is (1-1.2): (1.5-2): (0.2-0.4): (4-6), the temperature of the glycosylation reaction is 0 ℃ to room temperature, the organic solvent is a mixed solution of diethyl ether and dichloromethane, and the volume ratio of diethyl ether and dichloromethane is (3-4): 1; In the step S7, the molar ratio of the compound 9 to the compound 10 to the sodium hydride is (1-1.2): 1.5-2): 4-5, and the reaction temperature is room temperature to 80 ℃; in the step S8, the molar ratio of the compound 11 to triphenylphosphine is (1-1.2): 4-6, the reaction temperature for reducing the azide group to amino is 60 ℃, the solvent is a mixed solution of tetrahydrofuran and water, and the volume ratio of the tetrahydrofuran to the water is (3-5): 1; in the step S9, the molar ratio of the compound 12 to the glutaric acid to the dicyclohexylcarbodiimide to the 4-dimethylaminopyridine is (1-1.2), the molar ratio of the compound to the glutaric acid to the 4-dimethylaminopyridine is (8-10), the molar ratio of the compound to the glutaric acid to the 4-dimethylaminopyridine is (1.5-2), and the temperature of the condensation reaction is room temperature; in the step S10, the molar ratio of the compound 5 to the compound 13 to the 4-dimethylaminopyridine to the carbodiimide is (1-1.2): 0.4-0.5): 1.5-2, and the temperature of the condensation reaction is room temperature; In the step S11, the solvent is a mixed solution of tertiary butanol, water and acetic acid, and the volume ratio of the tertiary butanol to the water to the acetic acid is (3-4) 1 (0.05-0.08).
  6. 6. The preparation method of three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 2, wherein in the step A1, the molar ratio of the compound 9 to the compound 7 to SPhosAuNTf 2 to N, N-dimethylformamide is (1-1.2): (0.2-0.4): (4-6), the temperature of the glycosylation reaction is 0 ℃ to room temperature, the concentration of the solution obtained by dissolving the compound 9 in an organic solvent is 0.05-0.1 mmol/mL, the organic solvent is a mixed solution of diethyl ether and dichloromethane, and the volume ratio of diethyl ether and dichloromethane is (3-4): 1; In the step A2, the molar ratio of the compound 16 to the compound 10 to the sodium hydride is (1-1.2): 1.5-2): 4-5, and the reaction temperature is room temperature to 80 ℃; In the step A3, the molar ratio of the compound 17 to the triphenylphosphine is (1-1.2) (4-6), the temperature of the azide reaction is 60 ℃, the solvent is a mixed solution of tetrahydrofuran and water, and the volume ratio of the tetrahydrofuran to the water is (3-5): 1.
  7. 7. The method for preparing three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 2, wherein in step A4, the molar ratio of the compound 18, glutaric acid, dicyclohexylcarbodiimide and 4-dimethylaminopyridine is (1-1.2): 8-10): 1.5-2): 0.4-0.5, and the condensation reaction temperature is room temperature; In the step A5, the molar ratio of the compound 5 to the compound 19 to the 4-dimethylaminopyridine to the carbodiimide is (1-1.2): 0.4-0.5): 1.5-2, and the condensation reaction temperature is room temperature; in the step A6, the solvent is a mixed solution of tertiary butanol, water and acetic acid, and the volume ratio of the tertiary butanol to the water to the acetic acid is (3-4) 1 (0.05-0.08).
  8. 8. The method for preparing three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 2, wherein in the step B1, the molar ratio of the compound 9, the compound 7, SPhosAuNTf 2 and N, N-dimethylformamide is (1-1.2): 0.2-0.4): 4-6, and the glycosylation reaction temperature is 0 ℃ to room temperature; In the step B2, the molar ratio of the compound 22 to the compound 10 to the sodium hydride is (1-1.2): 1.5-2): 4-5, and the reaction temperature is room temperature to 80 ℃.
  9. 9. The method for preparing three candidate antigen chimeric oligosaccharides of helicobacter pylori according to claim 2, characterized in that in step B3, the molar ratio of the compound 23 to triphenylphosphine is (1-1.2): 4-6, and the temperature of the azide reaction is 60 ℃; In the step B4, the molar ratio of the compound 24 to the glutaric acid to the dicyclohexylcarbodiimide to the 4-dimethylaminopyridine is (1-1.2): 8-10): 1.5-2): 0.4-0.5, the solvent is a mixed solution of tetrahydrofuran and water, the volume ratio of the tetrahydrofuran to the water is (3-5): 1, and the temperature of the condensation reaction is room temperature; In the step B5, the molar ratio of the compound 5 to the compound 25 to the 4-dimethylaminopyridine to the carbodiimide is (1-1.2): 0.4-0.5): 1.5-2, and the condensation reaction temperature is room temperature; in the step B6, the solvent is a mixed solution of tertiary butanol, water and acetic acid, and the volume ratio of the tertiary butanol to the water to the acetic acid is (3-4) 1 (0.05-0.08).
  10. 10. The use of three candidate antigen chimeric oligosaccharides of helicobacter pylori, in particular chimeric oligosaccharide I, chimeric oligosaccharide II, chimeric oligosaccharide III, according to claim 1, for the preparation of helicobacter pylori vaccines or anti-helicobacter pylori drugs.

Description

Three candidate antigen chimeric oligosaccharides of helicobacter pylori and preparation method thereof Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to three candidate antigen chimeric oligosaccharides of helicobacter pylori and a preparation method thereof. Background Helicobacter pylori is a gram-negative pathogen, and unique urease activity and flagellum movement cause the helicobacter pylori to colonize gastric mucosa, induce chronic inflammation and cause peptic ulcer, gastric mucosa-associated lymphoma and gastric cancer. Although eradication therapy with Proton Pump Inhibitors (PPI) in combination with antibiotics is widely used, the drug resistance of first-line drugs such as clarithromycin, levofloxacin, etc. has broken through 30%, and partial areas even exceed 50%, resulting in a year-by-year increase in the failure rate of the treatment. Therefore, the development of novel prevention and control means capable of breaking through the drug resistance limit, especially the vaccine with both prevention and treatment potential, becomes a key break for solving the public health problem. Currently, candidate antigen development of helicobacter pylori vaccines is focused on bacterial virulence factors, urease, cagA proteins, and the like. For example, the Malfertheiner group has conducted an experiment to combine three virulence factor components, namely CagA, vacA and Neutrophil Activating Protein (NAP), as vaccine antigens, and the results prove that the vaccine can induce an immune response of an organism, can generate antigen-specific antibodies, and can also form durable T cell immune memory. Based on the positive findings, researchers select healthy volunteers to carry out subsequent experiments, and the prevention effect and human tolerance of the vaccine to the CagA positive helicobacter pylori are verified. Unfortunately, however, the experimental data show that the vaccine group did not significantly differ from the placebo group in terms of prophylactic effect, failing to achieve the intended objective. Quanming et al successfully developed an oral recombinant helicobacter pylori vaccine by means of DNA recombination technology. The vaccine uses fusion protein of urease B subunit and heat labile enterotoxin B subunit as core antigen, and performs random, double-blind and placebo-controlled phase III clinical test in children. The test result further proves that urease has potential as vaccine candidate antigen, but the oral preparation has the problem of higher antigen dosage, and the protection aging still needs longer tracking verification. In order to further improve the effectiveness and safety of vaccines, synthetic vaccines are becoming a new development direction. In the previous study, we synthesized and screened a series of mannoheptulose antigens in lipopolysaccharide, and found that helicobacter pylori glycoconjugate vaccine made of mannoheptulose can cause high titer antigen specific antibody and strong T cell dependent protective immune reaction in mice, and serum produced after immunization can also be strongly combined with helicobacter pylori NCTC 11637 surface. It was found that helicobacter pylori glycoconjugate vaccines based on alpha-1, 6-glucan in lipopolysaccharide can produce IgG antibodies with cross-reactive capabilities in animals. Thus, mannoheptulose and α -1, 6-glucan may be important epitopes in helicobacter pylori lipopolysaccharide. In view of this, the present invention prepares three chimeric oligosaccharides based on the surface lipopolysaccharide of helicobacter pylori, providing antigen candidates for the development of helicobacter pylori vaccines and drugs. Disclosure of Invention The invention aims at providing three candidate antigen chimeric oligosaccharides of helicobacter pylori and a preparation method thereof. In order to achieve the above purpose, the present invention adopts the following technical scheme: The first aspect of the invention provides three candidate antigen chimeric oligosaccharides of helicobacter pylori, which are chimeric oligosaccharides derived from lipopolysaccharide on the surface of helicobacter pylori, specifically chimeric oligosaccharide I, chimeric oligosaccharide II and chimeric oligosaccharide III, and the chemical structural general formulas are shown in the following formulas (I), (II) and (III) respectively: 。 the second aspect of the present invention provides a method for preparing three candidate antigen chimeric oligosaccharides of helicobacter pylori as described above, comprising (1) preparing chimeric oligosaccharide I, comprising the steps of: S1, under the action of triethyl orthoacetate and paratoluenesulfonic acid monohydrate, performing propylidene protection on 2, 3-hydroxyl in a compound 1 shown in a structural formula (1), reducing an isocephalic position to be hydroxyl under the action of lithium aluminum tri-tert-butoxide hydride, a