A method for the preparation of a mycobacterium bovis polysaccharide antigen
By preparing bovine mycobacterium polysaccharide antigen through specific chemical synthesis steps, the problem of difficult preparation in existing technologies has been solved, enabling efficient vaccine preparation and quality control, and promoting the research and development process of bovine tuberculosis vaccines.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YANCHENG TEACHERS UNIV
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies make it difficult to effectively prepare bovine mycobacterium polysaccharide antigens, leading to difficulties in vaccine preparation and quality control, which affects the prevention and control of bovine tuberculosis.
Compounds 2 and 3 were reacted in dichloromethane under argon protection using NIS-TMSOTf catalysis. Subsequently, compound 4 was deprotected in sodium methoxide solution, and compound 6 was reacted with iodomethane. Then, the mixture was hydrogenated under hydrogen by carbon-supported 20% palladium hydroxide to obtain bovine mycobacterium polysaccharide antigen 1 with a well-defined chemical structure.
This study achieved efficient synthesis of bovine mycobacterium polysaccharide antigen, solved the quality control problem in vaccine preparation, and provided a reliable approach to vaccine research and development.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing bovine mycobacterium polysaccharide antigen, belonging to the field of vaccine research and development technology. Background Technology
[0002] Chronic infectious diseases caused by mycobacterial infections, such as tuberculosis, remain a significant public health problem worldwide. Bovine tuberculosis, caused by *Mycobacterium bovis*, is a zoonotic infectious disease and a notifiable animal disease mandated by the World Organisation for Animal Health (OIE). In my country, it is classified as a Class II animal infectious disease, posing a significant threat to the cattle industry, food safety, and human health. Statistics show that approximately 5% of human tuberculosis cases are caused by *Mycobacterium bovis*. Therefore, controlling the spread of bovine tuberculosis is of great public health importance.
[0003] Specialized polysaccharide complexes in the cell walls of mycobacteria play a crucial role in maintaining cell wall structure and pathogenicity. Phenolic glycolipids (PGLs) are important components of the mycobacterial cell wall and are also important virulence factors for mycobacterial infection of hosts (Journal of Shanghai Jiaotong University, 2013, 33, 7). Bovine mycobacterial phenolic glycolipid PGL-bovis is composed of a polysaccharide moiety (oligosaccharide), a long-chain β-diol lipid core, and methyl branched-chain fatty acids (mycocerosic acids), etc. Acta Leprol , 1989, 7, 81). The sugar structure of Mycobacterium bovis PGL-bovis plays an important role in the pathogenic mechanism of Mycobacterium bovis and can serve as a good candidate target antigen (Adv Exp Med Biol, 2021, 1313, 155). Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention develops a method for preparing a bovine mycobacterium polysaccharide antigen, wherein the polysaccharide is the glycosyl portion of a phenolic glycolipid in the cell wall of Mycobacterium tuberculosis, and the structure of this polysaccharide antigen 1 is shown in formula (I): Formula (I).
[0005] In addition, the present invention relates to a method for preparing the aforementioned bovine mycobacterium polysaccharide antigen 1, comprising the following steps: (1) Under argon protection and in the presence of molecular sieves, NIS-TMSOTf catalyzed the reaction of compound 2 and compound 3 in dichloromethane. After terminating the reaction, compound 4 was obtained by separation and purification. .
[0006] (2) Compound 4 was deprotected in a methanol solution of sodium methoxide to give compound 5: .
[0007] (3) Compound 5 reacts with iodomethane under sodium hydrogen catalysis to give compound 6:
[0008] (4) Under oxygen-free conditions, compound 6 was added to carbon-supported palladium hydroxide, hydrogenated with hydrogen gas, filtered under nitrogen protection, and purified to obtain Mycobacterium bovis polysaccharide antigen 1: .
[0009] The present invention has the following advantages: The chemical structure of the bovine mycobacterium surface polysaccharide antigen 1 described in this invention is well-defined and singular. It can be synthesized in large quantities by chemical methods and can be used to prepare vaccines by conjugation with carrier proteins. This overcomes the difficulties in vaccine preparation and quality control caused by natural polysaccharides and is expected to accelerate vaccine research and development. Attached Figure Description
[0010] Figure 1 This is the 1H NMR spectrum of Mycobacterium bovis polysaccharide antigen 1; Figure 2 This is the carbon NMR spectrum of Mycobacterium bovis polysaccharide antigen 1. Detailed Implementation Plan
[0011] The present invention will now be analyzed in more detail with reference to the accompanying drawings and embodiments.
[0012] Example 1 Preparation of Mycobacterium bovis surface polysaccharide antigen 1 .
[0013] (1) Compound 2 (0.176 g, 0.367 mmol) and compound 3 (0.099 g, 0.367 mmol) were dried and dehydrated and dissolved in anhydrous dichloromethane (2 mL). A 4 Å molecular sieve was added to the system. The mixture was stirred under argon atmosphere at room temperature for 10 minutes, then cooled to -20 °C. NIS (0.099 g, 0.440 mmol) was added, followed by the dropwise addition of TMSOTf (0.05 mL, 0.05 mmol). The reaction was stirred at -20 °C for 2 hours, quenched with phosphate buffer (pH=7), and filtered through a diatomaceous earth column. The diatomaceous earth was washed with dichloromethane, and a saturated Na2S2O3 solution was added to the filtrate to make it colorless. The organic phase was separated, and the aqueous phase was extracted three times with dichloromethane. The combined organic phase was washed with water, dried with anhydrous MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1) to give compound 4 (0.25 g, 96% yield).
[0014] 1 H NMR (400 MHz, CDCl3): δ 1.34 (d, 3H, 3 J = 6.0 Hz), 2.22 (s, 3H,OAc), 2.79 (t, 2H, 3 J = 7.0 Hz), 3.39-3.51 (m, 2H), 3.55 (dd,1H, 3 J = 9.0 Hz, 3 J = 9.0 Hz), 3.86-3.96 (m, 1H), 4.17 (dd, 1H, 3 J = 9.5 Hz, 3 J = 3.5 Hz), 5.45(d, 1H, 3 J = 2.0 Hz), 5.56 (dd, 1H, 3 J = 3.5 Hz, 3 J = 2.0 Hz), 6.98 (d, 2H, 3 J = 8.4 Hz), 7.11 (d, 2H, 3 J = 8.0 Hz).
[0015] (2) Freshly prepared sodium methoxide solution (1 mL, 0.5 M methanol solution) was added to a methanol (20 mL) solution of compound 4 (0.227 g, 0.33 mmol). The reaction mixture was stirred at room temperature for 1 hour and monitored by TLC. Methanol was removed under vacuum, and the residue was extracted three times with dichloromethane. The combined organic phases were washed with water, dried over anhydrous MgSO4, filtered, and concentrated under vacuum. The product was separated by silica gel (petroleum ether / ethyl acetate = 1:1) to give compound 5 (0.213 g, 99% yield).
[0016] 1 H NMR (400 MHz, MeOD): δ 1.21 (d, 3H, 3 J = 6.2 Hz), 2.22 (s, 3H), 2.57 (t, 2H, 3 J = 7.0 Hz), 3.18-3.28 (m, 2H), 3.61 (dd,1H, 3 J = 9.5 Hz, 3 J =9.5 Hz,), 3.69-3.78 (m, 1H), 3.97 (dd, 1H, 3 J = 9.5 Hz, 3 J = 3.2 Hz), 4.24 (dd, 1H, 3 J = 3.0 Hz, 3 J = 2.0 Hz), 5.42 (d, 1H, 3 J = 2.0 Hz), 6.98 (d, 2H, 3 J = 8.8 Hz), 7.12 (d, 2H, 3 J = 8.0 Hz).
[0017] (3) Sodium hydride (60% dispersed in mineral oil, washed twice with petroleum ether) (0.247 g, 0.618 mmol) was added in portions to a THF (5 mL) solution of compound 5 (0.20 g, 0.309 mmol) under argon atmosphere at 0 °C. After stirring for half an hour, iodomethane (0.38 mL, 0.618 mmol) was added to the solution. The mixture was stirred overnight at room temperature, quenched with methanol, and extracted three times with ethyl acetate. The organic phase was washed with water and brine, dried over anhydrous MgSO4, and concentrated under vacuum. The product was separated by chromatography on silica gel (petroleum ether / ethyl acetate = 4:1) to give compound 6 (0.2 g, 80% yield).
[0018] 1 H NMR (400 MHz, CDCl3): δ 1.29 (d, 3H, 3 J = 6.4 Hz), 2.74 -2.86 (m,2H), 2.87-2.93 (m, 3H), 3.44 -3.52 (m, 2H), 3.59 (s, 3H), 3.61 (dd,1H, 3 J =9.5 Hz, 3 J = 9.5 Hz), 3.71 (dd, 1H, 3 J = 3.0 Hz, 3 J = 2.0 Hz), 3.74-3.84 (m,1H), 4.08 (dd, 1H, 3 J = 9.5 Hz, 3 J = 3.2 Hz), 5.51 (s, 1H), 6.90-7.00 (m, 2H), 7.00-7.20 (m, 2H).
[0019] (4) Under hydrogen atmosphere at 1 atm, 20% Pd(OH)₂ / C (1.0 mg) was added to a solution of compound 6 (67 mg, 0.10 mmol) in methanol and ethyl acetate (1:1, 2 mL). The reaction mixture was stirred at 25 °C for 72 hours and filtered through a diatomaceous earth column. The diatomaceous earth was washed with methanol, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (dichloromethane / MeOH = 5:1) to give compound 1 (25 mg, 80% yield).
[0020] 1H NMR (400 MHz, MeOD): δ 1.21 (d, 3H, 3 J = 6.0 Hz), 2.91 (s, 3H), 2.96-3.06 (m, 2H), 3.21-3.33 (m, 2H), 3.41 (dd,1H, 3 J = 9.5 Hz, 3 J = 9.5 Hz),3.54 (s, 3H), 3.55-3.63 (m, 1H), 3.64 (dd, 1H, 3 J = 3.5 Hz, 3 J = 1.5 Hz), 3.90(dd, 1H, 3 J = 9.5 Hz, 3 J = 3.5 Hz), 5.58 (s, 1H), 7.07-7.10 (m, 2H), 7.25-7.29(m, 2H). 13 C NMR (100 MHz, MeOD): δ 16.7, 29.8, 42.3, 50.2, 58.1, 69.2, 70.7,72.7, 80.5, 95.2, 116.7, 129.6, 129.9, 155.6.
[0021] HRMS C 16 H 26 NO5: Calcd. [M+ H] + 312.1811, Found 312.1817.
[0022] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for preparing Mycobacterium bovis polysaccharide antigen, characterized in that... The structural formula of the polysaccharide antigen is shown in formula (I): Equation (I) 2. A method for preparing the bovine mycobacterium polysaccharide antigen as described in claim 1, characterized in that, Includes the following steps: (1) Under argon protection and in the presence of molecular sieves, NIS-TMSOTf catalyzed the reaction of compound 2 and compound 3 in dichloromethane. After terminating the reaction, compound 4 was obtained by separation and purification. ； (2) Compound 4 was deprotected in a methanol solution of sodium methoxide to give compound 5: ； (3) Compound 5 reacts with iodomethane under sodium hydride catalysis to give compound 6: ； (4) Under oxygen-free conditions, compound 6 was added to carbon-supported palladium hydroxide, hydrogenated with hydrogen gas, filtered under nitrogen protection, and purified to obtain Mycobacterium bovis polysaccharide antigen 1: 。