Synthetic method of acinetobacter baumannii capsular polysaccharide K128 repeated pentasaccharide fragment

Abstract

The invention belongs to the technical field of natural oligosaccharide chain synthesis, and particularly discloses a synthetic method of an acinetobacter baumannii capsular polysaccharide K128 repetitive pentasaccharide fragment, which develops a synthetic route by taking a capsular polysaccharide repetitive unit from the surface of an acinetobacter baumannii strain KZ-1093 as a target product. And a feasible scheme is provided for chemical synthesis of natural oligosaccharide chains. According to the preparation method provided by the invention, the selectivity and the yield of the reaction can be improved and the generation of byproducts can be reduced by utilizing the adjacent group participation effect, the remote induction effect and the solvent and temperature control strategy. According to the synthesis method provided by the invention, the target product is obtained at the total yield of 8.7% through 11 steps of reactions by taking sugar blocks reported in literatures as raw materials, the product selectivity and yield of glycosylation in each step are relatively high, and a product with an expected configuration can be obtained. Meanwhile, each protecting group of a final reaction product is easy to remove under the conditions of sodium methoxide and catalytic hydrogenation, and side reactions are few.

Description

Technical Field

[0001] The invention belongs to the technical field of natural oligosaccharide chain synthesis, and particularly relates to a method for synthesizing a repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128. Background Art

[0002] Acinetobacter baumannii (AB) is a Gram-negative bacterium widely distributed in nature. It is a conditional pathogen that usually colonizes the human skin, respiratory tract, digestive tract, and urogenital tract, and causes infection when the body's immunity is low, which in turn leads to the occurrence of infectious diseases such as skin and soft tissue infection, acute pneumonia, meningitis, and sepsis, and the mortality rate after infection is high. Thanks to its strong antibiotic tolerance, Acinetobacter baumannii can survive and spread in hospitals as a pathogen, leading to increased morbidity and mortality among critically ill patients in hospitals.

[0003] The pathogenic mechanism of Acinetobacter baumannii is determined by a variety of virulence factors, which allow the bacteria to remain in the host body while causing damage to its cells, thereby causing various diseases. These virulence factors include capsular polysaccharides (CPS), lipopolysaccharides (LPS), outer membrane proteins, and phospholipases. The antigens and pili of Acinetobacter baumannii lipopolysaccharides can help the bacteria adhere to host cells together, which is also the first step for Acinetobacter baumannii to colonize host cells. As a key virulence factor of Acinetobacter baumannii, capsular polysaccharides can resist most interference factors in the external environment and have strong resistance to drying and disinfectants. These resistances give Acinetobacter baumannii widespread and persistent spread in hospital environments. Outer membrane proteins are involved in the invasion and apoptosis of cells. They can bind to the surface of host cells and act on mitochondria and nuclei, thereby inducing apoptosis of host cells. Phospholipases can hydrolyze phospholipids in human cell membranes, and different phospholipases have different pathogenic mechanisms. For example, in the mouse pneumonia model, phospholipase D helps Acinetobacter baumannii survive longer, while phospholipase C is toxic to epithelial cells.

[0004] With the extensive use of antibiotics and the widespread distribution of Acinetobacter baumannii in hospital environments, an increasing number of drug-resistant strains of Acinetobacter baumannii pose a greater threat to critically ill patients in hospitals. The emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR) and even pan-drug-resistant (PDR) strains also makes treatment more difficult. Currently, commonly used clinical treatment methods include selecting appropriate antibiotics for treatment based on drug sensitivity results and combined drug treatment. Common antibiotic treatments include β-lactams, tetracyclines, aminoglycosides and polymyxins. Sulbactam is a β-lactamase inhibitor commonly used in the treatment of Acinetobacter. It has certain intrinsic activity against Acinetobacter baumannii, but the antibacterial activity of sulbactam varies greatly among the currently available clinical isolates of Acinetobacter baumannii. Therefore, it is not recommended for the treatment of patients with severe Acinetobacter baumannii infections. Tetracyclines have in vitro activity against more than 60% of carbapenem-resistant Acinetobacter baumannii strains, but high levels of tetracycline resistance can be detected in some XDR strains, so the therapeutic effect of this type of drug is not ideal. Studies have shown that the sensitivity of aminoglycoside antibiotics to Acinetobacter baumannii isolates is gradually decreasing, which means that larger doses of the antibiotics are needed to have a chance to prevent bacterial regeneration, and large doses of antibiotics are bound to be accompanied by more toxic and side effects. Polymyxin antibiotics are still effective against most isolates of Acinetobacter baumannii, but due to their complex pharmacokinetic and pharmacodynamic properties, the administration of polymyxin is relatively complex, which poses challenges in clinical practice, and the cost of treatment is high. Toxic side effects such as nephrotoxicity and neurotoxicity still have safety issues, making it difficult to use them for a long time. The combination therapy of multiple drugs can overcome the drug resistance of Acinetobacter baumannii to a certain extent. Among them, the three-drug combination therapy of polymyxin, doripenem and sulbactam can effectively enhance and prolong the bactericidal activity, but due to limited clinical trial data support and the complexity of the treatment regimen, these combination therapy regimens are also difficult to be widely used in the actual treatment of Acinetobacter baumannii.

[0005] These treatments have many drawbacks. Not only will they continue to promote the emergence of drug-resistant strains, they may also increase patients' exposure to drug toxicity and side effects, causing damage to other organs in the body. In addition, in 2017, the World Health Organization released a list of bacteria that urgently need new antibiotics, among which carbapenem-resistant Acinetobacter baumannii was listed as the first of the highest urgency level of "extremely important". It can be seen that the development of new antibacterial drugs for Acinetobacter baumannii is urgent.

[0006] Capsular polysaccharides (CPS) are a class of polysaccharide compounds in the outer layer of bacterial cell walls, commonly found in Gram-negative bacteria. They are one of the main virulence factors of bacterial pathogens and play a very important role in the survival and pathogenicity of bacteria. Most capsular polysaccharides are covalently linked to the reducing end of the bacterial outer membrane and are composed of 2-10 oligosaccharide oligomers. These repeating units are then linked by glycosidic bonds to form high molecular weight polymers, which give bacteria the ability to resist various factors affecting the external environment. At the same time, they also have the ability to resist drying and disinfectants, evade the recognition and attachment of host immune system macromolecules and bacteriophages, and promote bacterial adhesion, playing an important role in various physiological activities of bacteria.

[0007] The capsular polysaccharide of Acinetobacter baumannii is a key virulence factor in the pathogenicity of Acinetobacter baumannii. Its structure is determined by a variety of different capsular polysaccharide gene clusters (KL), which are converted into repeating oligosaccharide units, namely K units. Its monosaccharide composition and the connection of glycosidic bonds between monosaccharides are also diverse, including typical glucose, galactose and glucuronic acid, as well as atypical N-acetylmannosamine, N-acetylglucosamine (GlcpNAc), etc. This diversity gives the capsular polysaccharide of Acinetobacter baumannii unique immunogenicity. A study used K1 capsular polysaccharide as a candidate polysaccharide vaccine to immunize a mouse pneumonia model to induce specific antibodies, which was able to play a protective role in the attack of Acinetobacter baumannii isolates. This makes the capsular polysaccharide a target for immunotherapy against Acinetobacter baumannii infection, reflecting the great potential of capsular polysaccharides for the development of polysaccharide vaccines for Acinetobacter baumannii.

[0008] In recent years, researchers' research on polysaccharide vaccines of Acinetobacter baumannii is mostly based on capsular polysaccharides isolated and extracted from natural strains of Acinetobacter baumannii. However, the purification procedure of this natural capsular polysaccharide is very complicated, and there are problems such as uncontrollable and non-repeatable purification. Compared with natural capsular polysaccharides obtained by separation, the synthesis of polysaccharide antigens using chemical synthesis methods is more in line with the high quality requirements of modern polysaccharide vaccine development. Compared with natural capsular polysaccharides directly isolated and purified from strains, chemically synthesized oligosaccharide fragments have the characteristics of high purity, clear chemical structure, and no potential microbial contaminants. In addition, they can controllably synthesize oligosaccharide fragments of different lengths to screen out the minimum effective antigen epitope, thereby reducing the research and development cost of polysaccharide vaccines and accelerating the research progress. Based on the many difficulties in the extraction of capsular polysaccharides of Acinetobacter baumannii, there is an urgent need for a synthesis method that is easy to implement, has a high yield of the target product, and has few side reactions. Summary of the invention

[0009] In order to solve the above technical problems, the present invention proposes a method for synthesizing a repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128, and the steps are as follows:

[0010] S1: Compound a Dissolve in a solvent, add NBS, stir until the reaction is complete, separate and purify to obtain compound b

[0011] S2: Dissolve compound b in a solvent, add 2,2,2-trifluoro-N-phenylacetylimidoyl chloride and cesium carbonate, quench the reaction after the reaction is complete, and separate and purify to obtain the glycosyl donor compound c

[0012] S3: Combine sugar donor compound c and acceptor compound d Mix in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound e

[0013] S4: Dissolve compound e in a solvent, add camphorsulfonic acid CSA, quench the reaction after the reaction is complete, and separate and purify to obtain compound f

[0014] S5: Add the glycosyl donor compound g and receptor compound h Mix in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound i

[0015] S6: Dissolve compound i in a solvent, add NBS and stir until the reaction is complete, separate and purify to obtain compound j

[0016] S7: dissolving compound j in a solvent, adding trichloroacetonitrile and DBU, and continuing to stir until the reaction is complete, separating and purifying to obtain glycosyl donor compound k

[0017] S8: Mix the glycosyl donor compound k and the acceptor compound f in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound l

[0018] S9: Compound l and compound m Mix in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound n

[0019] S10: dissolving compound n in a solvent, adding NaOMe, stirring until the reaction is complete, adjusting the pH of the reaction system to neutral, separating and purifying to obtain an intermediate; dissolving the intermediate in a solvent, catalytically hydrogenating, separating and purifying to obtain the final product o

[0020] Preferably, the molar ratio of compound a to NBS in S1 is (1.0-2.0):(3.0-6.0).

[0021] Preferably, the molar ratio of compound b, 2,2,2-trifluoro-N-phenylacetylimidoyl chloride and cesium carbonate in S2 is (1.0-2.0):(2.0-4.0):(2.0-4.0).

[0022] Preferably, the molar ratio of the donor compound c, the acceptor compound d and the glycosidation reaction catalyst TMSOTf in S3 is (1.0-2.0):(1.0-2.0):(0.1-1.0).

[0023] Preferably, the molar ratio of compound e to CSA in S4 is (1.0-2.0):(0.5-1.0).

[0024] Preferably, the molar ratio of the donor g, the acceptor h and the glycosylation reaction catalyst TMSOTf in S5 is (1.0-2.0):(1.0-2.0):(0.1-1.0).

[0025] Preferably, the molar ratio of compound i to NBS in S6 is (1.0-2.0):(3.0-6.0).

[0026] Preferably, the molar ratio of compound j, trichloroacetonitrile and DBU in S7 is (1.0-2.0):(10.0-20.0):(0.5-1.0).

[0027] Preferably, the molar ratio of the donor compound k, the acceptor compound f and the glycosidation reaction catalyst TfOH in S8 is (1.0-2.0):(1.0-2.0):(0.1-0.5).

[0028] Preferably, the molar ratio of the donor compound l, the acceptor compound m, the solvent, and the glycosylation reaction catalyst TfOH in S9 is (1.0-2.0):(1.0-2.0):(1.0-2.0):(0.1-0.5).

[0029] Preferably, the molar ratio of compound n and NaOMe in S10 is (1.0-2.0):(1.0-2.0); and the catalyst for catalytic hydrogenation is Pd(OH)2 / C.

[0030] Preferably, the solvents used in S5, S7, S8 and S9 are independently dichloromethane; the solvent used in S2 is acetone; and the solvents used in S1 and S6 are independently a mixed solution of acetone and water.

[0031] Preferably, molecular sieves are independently added to the reaction systems of S3, S5, S8 and S9.

[0032] Preferably, the glycosidation reaction temperature in S3, S5, S8 and S9 is below 0°C.

[0033] Preferably, the reaction temperature in S2 and S4 is below 30°C; the reaction temperature in S1 is 20-30°C; and the reaction temperature in S7 is below 25°C.

[0034] The beneficial effects of the present invention are:

[0035] Compared with the prior art, the present invention develops a chemical total synthesis route by targeting the capsular polysaccharide repeating unit derived from the surface of Acinetobacter baumannii strain KZ-1093 as the target product. The oligosaccharide fragments chemically synthesized by this technical route have many advantages such as high purity, clear chemical structure, and no potential microbial contaminants, and can controllably synthesize oligosaccharide fragments of different lengths, thereby screening out the minimum effective antigen epitope, thereby reducing the research and development cost of polysaccharide vaccines and accelerating the research progress. Secondly, the route proposed according to the present invention can simply and efficiently obtain the required oligosaccharide fragments, thereby avoiding separation and extraction from natural Acinetobacter baumannii strains, because the purification procedure of this natural capsular polysaccharide is very complicated, and there are problems such as uncontrollable and non-repeatable purification. Based on the many difficulties in the extraction of current Acinetobacter baumannii capsular polysaccharides, the present invention proposes a synthesis method that is easy to implement and has a high yield of target products and few side reactions, and provides a feasible solution for the chemical synthesis of natural oligosaccharide chains. The synthesis method provided by the present invention uses a sugar building block reported in the literature as a raw material and undergoes 11 steps of reaction to obtain a target product with an overall yield of 8.7%, wherein the selectivity of the product of each glycosylation step is relatively high, and a product of the expected configuration can be obtained. At the same time, each protective group of the final product of the reaction is easily removed under the conditions of sodium methoxide and catalytic hydrogenation, and there are few side reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0037] Figure 1 It is the synthesis route diagram of the present invention. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0039] It should be noted that the synthesis route in the present invention is the key innovation, followed by the ratio of the reactants in each step. Therefore, the ratio of the reactants in each step does not constitute a factor affecting the synthetic product. The capsular polysaccharide repeating unit from the surface of Acinetobacter baumannii strain KZ-1093 was selected as the target product to develop a synthesis route. According to the literature report: its sugar chain sequence is

[0040] The abbreviations used are commonly used in this field. The meanings of some abbreviations are as follows:

[0041] THF: tetrahydrofuran, MeOH: methanol, TMSOTf: trimethylsilyl trifluoromethanesulfonate, TfOH: trifluoromethanesulfonic acid, NBS: N-bromosuccinimide, NIS: N-iodosuccinimide, DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene, CSA: camphorsulfonic acid, TLC: thin layer chromatography.

[0042] Example 1

[0043] See also Figure 1 The synthetic route shown is a method for synthesizing a repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128, comprising the following steps:

[0044] S1: Compound a (1.6 g, 2.6 mmol) was dissolved in a mixed solvent of 50 mL acetone-water (4:1), NBS (1.4 g, 7.9 mmol) was added at 0 ° C, and the mixture was stirred at room temperature for 30 minutes after 5 minutes. When TLC (thin layer chromatography) showed that the starting material disappeared, a saturated aqueous sodium thiosulfate solution was added to quench the reaction, and 100 mL ethyl acetate was added to dilute the reaction system, and then washed with a saturated aqueous sodium thiosulfate solution and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 2:1) to obtain compound b (1.1 g, 2.2 mmol) with a yield of 83%.

[0045] S2: The product compound b (1.1 g, 2.2 mmol) obtained in the previous step was dissolved in 40 mL of acetone, and 2,2,2-trifluoro-N-phenylacetylimidoyl chloride (0.9 mL, 5.5 mmol) and cesium carbonate (1.8 g, 5.5 mmol) were added at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. When TLC showed that the raw material disappeared, the reaction solution was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a residue, which was then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 20:1) to obtain compound c (1.6 g, 2.1 mmol) with a yield of 95%.

[0046] S3: Glycosyl donor compound c (1.6 g, 2.1 mmol) and compound d (700 mg, 1.1 mmol) were mixed in a flask, azeotroped with toluene (3 mL) three times, and dissolved in 20 mL of a mixed solvent of ether-dichloromethane (1:1). Powdered new activated molecular sieves (100 mg / mL solvent) were added, and the mixture was stirred at 0°C for 30 minutes, TMSOTf (16 uL, 0.1 mmol) was slowly added at 0°C, and the reaction was continued to stir at 0°C for 3 hours, triethylamine was added to quench the reaction and diluted with dichloromethane, and then filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain a residue, which was then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 5:1) to obtain the desired compound e (907 mg, 0.72 mmol) with a yield of 65%.

[0047] S4: The product compound e (907 mg, 0.72 mmol) obtained in the previous step was dissolved in 10 mL of a mixed solvent of dichloromethane-methanol (1:1), and CSA (335 mg, 1.44 mmol) was added. The reaction mixture was stirred at room temperature for 6 hours. When TLC showed that the starting material disappeared, triethylamine was added to the reaction to quench the reaction. The reaction solution was concentrated under reduced pressure to obtain a residue, which was then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 1:1) to obtain compound f (627 mg, 0.58 mmol) with a yield of 80%.

[0048] S5: The glycosyl donor compound g (688 mg, 1.1 mmol) and the glycosyl acceptor compound h (539 mg, 0.9 mmol) were mixed in a flask, azeotroped with toluene (3 mL) three times, and dissolved in 10 mL of ultra-dry dichloromethane. Powdered new activated molecular sieves (100 mg / mL solvent) were added, and the mixture was stirred at -20°C for 30 minutes. TMSOTf (16 uL, 0.1 mmol) was slowly added at -20°C, and the reaction was continued at -20°C for 3 hours. When TLC showed that the glycosyl acceptor disappeared, triethylamine was added to quench the reaction and diluted with dichloromethane, and then filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain a residue, which was then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 5:1) to obtain the desired product i (923 mg, 0.86 mmol) with a yield of 95%.

[0049] S6: Compound i (923 mg, 0.86 mmol) was dissolved in a mixed solvent of 20 mL acetone-water (20:1), NBS (455 mg, 2.6 mmol) was added at 0 ° C, and the mixture was stirred at room temperature for 30 minutes after 5 minutes. When TLC showed that the starting material disappeared, a saturated aqueous sodium thiosulfate solution was added to quench the reaction, and 50 mL ethyl acetate was added to dilute the reaction system, and then washed with a saturated aqueous sodium thiosulfate solution and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 2:1) to obtain compound j (614 mg, 0.65 mmol) with a yield of 75%.

[0050] S7: Compound j (614 mg, 0.65 mmol) was dissolved in 10 mL of dichloromethane, trichloroacetonitrile (0.65 mL, 6.5 mmol) and DBU (10 uL, 0.06 mmol) were added at 0°C, and the reaction mixture was stirred at room temperature for 3 hours. When TLC showed that the starting material disappeared, the reaction solution was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a residue, which was then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 3:1) to obtain compound k (712 mg, 0.65 mmol) with a yield of 99%.

[0051] S8: The glycosyl donor compound k (712 mg, 0.65 mmol) and the glycosyl acceptor compound f (627 mg, 0.58 mmol) were mixed in a flask, azeotroped with toluene (3 mL) three times, and dissolved in 10 mL of ultra-dry dichloromethane. Powdered new activated molecular sieves (100 mg / mL solvent) were added, and the mixture was stirred at -20°C for 30 minutes. TMSOTf (16 uL, 0.1 mmol) was slowly added at -20°C, and the reaction was continued at -20°C for 3 hours. When TLC showed that the glycosyl acceptor disappeared, triethylamine was added to quench the reaction and diluted with dichloromethane, and then filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain a residue, which was then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 5:1) to obtain the desired product l (868 mg, 0.43 mmol) with a yield of 74%.

[0052] S9: Mix the donor compound l (868 mg, 0.43 mmol) and the glycosyl acceptor compound m (461 mg, 0.65 mmol) in a flask, co-boil with toluene (3 mL) three times, and dissolve in 10 mL of ultra-dry dichloromethane. Add powdered new activated molecular sieves (100 mg / mL solvent), and stir the mixture at 0°C for 30 minutes. Then add NIS (97 mg, 0.43 mmol) at 0°C, slowly add TMSOTf (12 uL, 0.08 mmol) after 10 minutes, react at 0°C for 12 hours, add triethylamine to quench the reaction and dilute with dichloromethane, and then filter through diatomaceous earth. The filtrate is concentrated under reduced pressure to obtain a residue, which is then purified by silica gel flash chromatography (petroleum ether-ethyl acetate = 2:1) to obtain the desired compound n (616 mg, 0.23 mmol) with a yield of 54%.

[0053] The spectrum information of compound n is as follows:

[0054] 1H NMR(500MHz,CDCl3)δ7.89(d,J=7.7Hz,2H),7.80–7.70(m,6H),7.65(d,J=7.7Hz,1H),7.61(d,J=7.7Hz,1H),7.58–7.53(m,2H),7.46(d,J=8.4Hz,1H),7.37(s,12H),7.33–7.26(m,20H),7.22–7.16(m,12H),7.16–7.04(m,8H),7.04–6.95(m,2H),5.87(dd,J=10.3,7.9Hz,1H),5.47(s,1H),5.21(dd,J=10.4,3.1Hz,1H),5.12(d,J=15.3Hz,2H),5.04(t,J=9.7Hz,2H),4.99–4.94(m,1H),4.88(d,J=8.4Hz,1H),4.85–4.77(m,6H),4.75(d,J=7.7Hz,2H),4.71(dd,J=9.2,4.6Hz,2H),4.69–4.55(m,6H),4.54–4.47(m,2H),4.47–4.43(m,2H),4.41(q,J=5.1Hz,4H),4.35(d,J=11.6Hz,1H),4.31(d,J=7.9Hz,1H),4.28(d,J=3.1Hz,1H),4.11(dd,J=10.7,8.0Hz,1H),4.09–3.95(m,5H),3.92(q,J=5.6Hz,5H),3.84(t,J=6.8Hz,1H),3.77(dt,J=18.6,6.2Hz,5H),3.72–3.66(m,2H),3.66–3.59(m,4H),3.57(dd,J=9.5,6.5Hz,2H),3.51(t,J=6.2Hz,1H),3.47–3.40(m,1H),3.31(s,2H),3.12(d,J=35.0Hz,2H),1.93(s,3H),1.51–1.32(m,6H),0.86(dt,J=18.6,6.4Hz,4H).

[0055] 13C NMR (126MHz, CDCl3) δ169.62,166.31,165.51,161.87,161.74,138.62,138.47,138.28,138.11,137.97,1 37.88,137.40,136.19,135.42,133.44,133.24,133.10,132.98,132.87,129.89,129.75,129.59,101.05 ,100.64,100.48,100.43,99.39,99.04,92.95,92.49,82.94,78.99,78.44,78.03,76.16,75.11,74.72,74.51,73.91,73.73,73.39,73.12,72.92,72.06,71.79,54.88,29.72,29.23,23.31,22.71,21.16,14.13.

[0056] S10: Compound n (100 mg, 0.04 mmol) was dissolved in 4 mL of a mixed solvent of dichloromethane-methanol (1:1), sodium methoxide (22 mg, 0.4 mmol) and DBU (10 uL, 0.06 mmol) were added at room temperature, and the reaction mixture was stirred at room temperature for 2 days. When TLC showed that the starting material disappeared, a hydrogen ion exchange resin was added to adjust the pH to neutral, and then filtered. The residue obtained by concentrating the filtrate was purified by silica gel flash chromatography (dichloromethane-methanol = 20:1) to obtain 69 mg of an intermediate product, which was dissolved in a mixed solvent of 4 mL of THF / MeOH / H2O (2 / 1 / 1), and 20 mg of Pd(OH)2 / C was added for catalytic hydrogenation. After 3 days of reaction, the final compound o (28 mg, 0.0.03 mmol) was obtained, and the overall yield of the two-step reaction was 75%.

[0057] The spectrum information of compound o is as follows:

[0058] 1H NMR(500MHz,D2O)δ4.89(s,1H),4.46(d,J=7.9Hz,1H),4.44(d,J=5.4Hz,1H),4.42(d,J=7.8Hz,1H),4.37(d,J=7 .1Hz,1H),4.10(d,J=3.0Hz,1H),4.03–3.94(m,3H),3.91(t,J=6.7Hz,4H),3.86(d,J=12.1Hz,4H),3.83–3.76(m ,5H),3.75(s,2H),3.71–3.64(m,5H),3.61(dt,J=10.1,5.2Hz,3H),3.42(td,J=14.8,7.2Hz,3H),3.32(t,J=9.3 Hz,1H),3.23(t,J=8.6Hz,1H),2.97–2.89(m,2H),1.97(d,J=12.4Hz,6H),1.68–1.58(m,4H),1.41–1.26(m,3H).

[0059] 13 C NMR(126MHz,D2O)δ174.72,104.79,102.82,102.06,101.39,98.41,80.13,75.97,75.74 ,74.83,74.73,73.86,73.12,72.51,70.91,70.64,70.00,69.69,69.48,69.36,69.26,6 8.52,68.17,68.03,67.89,66.10,61.37,61.11,60.79,58.68,52.32,51.30,39.45,39.40,34.56,33.79,30.69,29.86,26.48,26.39,25.50,24.98,22.46,22.32,22.13,22.06.

[0060] The present invention proposes a synthesis method that is easy to implement, has a high yield of the target product, and has few side reactions, and provides a feasible scheme for the chemical synthesis of natural oligosaccharide chains. According to the preparation method provided by the present invention, the strategies of neighboring group participation, remote induction effect, solvent, and temperature control are utilized to improve the selectivity and yield of the reaction and reduce the generation of by-products. The synthesis method provided by the present invention uses sugar building blocks that have been reported in the literature as raw materials and undergoes 11 steps of reaction to obtain the target product with an overall yield of 8.7%, wherein the selectivity of the product of each glycosylation step is relatively high, and the product of the expected configuration can be obtained. At the same time, the various protective groups of the final product of the reaction are easily removed under the conditions of sodium methoxide and catalytic hydrogenation, and there are few side reactions.

[0061] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all details in detail, nor do they limit the invention to only the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification.

Claims

1. A method for synthesizing a repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128, characterized in that: The steps are as follows: S1: Compound a Dissolve in a solvent, add NBS, stir until the reaction is complete, separate and purify to obtain compound b S2: Dissolve compound b in a solvent, add 2,2,2-trifluoro-N-phenylacetylimidoyl chloride and cesium carbonate, quench the reaction after the reaction is complete, and separate and purify to obtain the glycosyl donor compound c S3: Combine sugar donor compound c and acceptor compound d Mix in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound e S4: Compound e is dissolved in a solvent, CSA (camphorsulfonic acid) is added, and after the reaction is complete, the reaction is quenched, and compound f is obtained by separation and purification. S5: Add the glycosyl donor compound g and receptor compound h Mix in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound i S6: Dissolve compound i in a solvent, add NBS and stir until the reaction is complete, separate and purify to obtain compound j S7: dissolving compound j in a solvent, adding trichloroacetonitrile and DBU, and continuing to stir until the reaction is complete, separating and purifying to obtain glycosyl donor compound k S8: Mix the glycosyl donor compound k and the acceptor compound f in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound l S9: Compound l and compound m Mix in a solvent, add a glycosylation reaction catalyst, stir until the reaction is complete, separate and purify to obtain compound n S10: dissolving compound n in a solvent, adding NaOMe, stirring until the reaction is complete, adjusting the pH of the reaction system to neutral, and isolating and purifying to obtain an intermediate; The intermediate is dissolved in a solvent, catalytically hydrogenated, separated and purified to obtain the final product.

2. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The molar ratio of compound a and NBS in S1 is (1.0-2.0):(3.0-6.0); the molar ratio of compound b, 2,2,2-trifluoro-N-phenylacetimidochloride and cesium carbonate in S2 is (1.0-2.0):(2.0-4.0):(2.0-4.0).

3. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The molar ratio of the donor compound c, the acceptor compound d and the glycosylation reaction catalyst TMSOTf in S3 is (1.0-2.0):(1.0-2.0):(0.1-1.0); the molar ratio of the compound e and CSA in S4 is (1.0-2.0):(0.5-1.0).

4. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The molar ratio of the donor g, the acceptor h and the glycosylation reaction catalyst TMSOTf in S5 is (1.0-2.0):(1.0-2.0):(0.1-1.0).

5. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The molar ratio of compound i and NBS in S6 is (1.0-2.0):(3.0-6.0); the molar ratio of compound j, trichloroacetonitrile and DBU in S7 is (1.0-2.0):(10.0-20.0):(0.5-1.0).

6. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The molar ratio of the donor compound k, the acceptor compound f and the glycosylation reaction catalyst TfOH in S8 is (1.0-2.0):(1.0-2.0):(0.1-0.5); the molar ratio of the donor compound l, the acceptor compound m, the solvent and the glycosylation reaction catalyst TfOH in S9 is (1.0-2.0):(1.0-2.0):(1.0-2.0):(0.1-0.5).

7. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The molar ratio of compound n and NaOMe in S10 is (1.0-2.0):(1.0-2.0); the catalyst for catalytic hydrogenation is Pd(OH)2 / C.

8. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The solvent used in S5, S7, S8 and S9 is independently dichloromethane; the solvent used in S2 is acetone; the solvent used in S1 and S6 is independently a mixed solution of acetone and water.

9. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: Molecular sieves are also independently added to the reaction systems of S3, S5, S8 and S9.

10. The method for synthesizing the repeating pentasaccharide fragment of Acinetobacter baumannii capsular polysaccharide K128 according to claim 1, characterized in that: The glycosidation reaction temperature in S3, S5, S8 and S9 is below 0°C; the reaction temperature in S2 and S4 is below 30°C; the reaction temperature in S1 is 20-30°C; and the reaction temperature in S7 is below 25°C.

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