WO2020155519A1 - 一种幽门螺旋杆菌o2血清型o抗原寡糖类化合物的合成 - Google Patents

一种幽门螺旋杆菌o2血清型o抗原寡糖类化合物的合成 Download PDF

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WO2020155519A1
WO2020155519A1 PCT/CN2019/091200 CN2019091200W WO2020155519A1 WO 2020155519 A1 WO2020155519 A1 WO 2020155519A1 CN 2019091200 W CN2019091200 W CN 2019091200W WO 2020155519 A1 WO2020155519 A1 WO 2020155519A1
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compound
helicobacter pylori
hydrogen
reaction
serotype
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尹健
胡静
刘中华
秦春君
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the synthesis of a Helicobacter pylori O2 serotype O antigen oligosaccharide compound, which belongs to the field of organic synthesis.
  • Helicobacter pylori is a micro-aerobic gram-negative bacterium. About half of the world’s population is infected with this pathogen because of its low socioeconomic status and poor medical and health conditions. A proportion is as high as 80%. It is reported that 47% and 66% of the population in China's urban and rural areas are infected with Helicobacter pylori. Oral-oral transmission and fecal-oral transmission are the most important modes of transmission of this pathogen, and mother-to-child transmission is also one of the modes of transmission of this pathogen.
  • Helicobacter pylori infections occur during childhood. Once infected, the bacteria that cure the disease may live on the gastric mucosa for a lifetime. Although 70% of infected persons are clinically asymptomatic, this pathogen has been confirmed to be related to a series of gastrointestinal diseases. 15%-20% of long-term infections will cause gastritis, stomach and tense A series of gastrointestinal diseases such as didenal ulcer, and 1%-2% of long-term infections will develop into gastric cancer.
  • the main treatment for gastrointestinal diseases associated with Helicobacter pylori infection is a combination of two antibiotics (metronidazole and clarithromycin) and a proton pump inhibitor, called triple therapy.
  • This method has achieved certain results in the treatment of Helicobacter pylori, but there are still many shortcomings.
  • the main limitations are: 1) The metabolic time of antibiotics in the gastrointestinal tract is relatively short, and 2) The special antibiotics in the stomach Relatively unstable in a low pH environment, 3) Helicobacter pylori generally resides in the gastric mucosa, while the concentration of antibiotics in the mucosa is very low, 4) More importantly, the therapeutic effect of antibiotics gradually weakens with the emergence of resistant bacteria the trend of. Based on the above shortcomings, it has been reported that the one-year recurrence rate of Helicobacter pylori after treatment with triple therapy has reached 15%-30%.
  • the cell surface of Helicobacter pylori is mainly composed of lipopolysaccharide, which plays a very important role in the integrity of the bacterial biofilm structure and function, and it also exists in bacterial secretions. , Is one of the pathogenic and virulence factors of the bacteria.
  • the current research on Helicobacter pylori lipopolysaccharide is mainly extracted from inactivated bacteria.
  • the disadvantage of this method is that there are very few products obtained at one time.
  • the extracted lipopolysaccharide also exists The structure is not specific enough, and it is easy to have the characteristics of similar impurities in the structure, and the experiment repeatability is poor, which interferes with the research to a certain extent.
  • glycosyl building blocks Through the design and construction of glycosyl building blocks and the selection and optimization of glycosylation reactions, we obtain O antigen disaccharides, tetrasaccharides and hexasaccharides.
  • the reducing ends of the synthesized disaccharides, tetrasaccharides and hexasaccharides are all assembled with amino linking arms, which can be linked to carrier proteins to form glycoconjugates for further immunological research.
  • the technical problem to be solved by the present invention is to synthesize Helicobacter pylori O2 serotype O antigen oligosaccharide fragment by chemical method.
  • the construction of ⁇ -1,2 cis-glycosidic bond is a key step in the synthesis of the target structure.
  • the present invention uses diacetone glucose as the only starting material, and finally obtains the protected fully ⁇ -linked protected di-, tetra-, and hexasaccharides through the construction of glycosyl building blocks and the optimization of glycosylation reaction conditions, and the final goal is obtained by deprotecting.
  • the compounds assemble disaccharides, tetrasaccharides and hexasaccharides of Helicobacter pylori O2 serotype O antigen with amino link arms.
  • the disaccharides, tetrasaccharides and hexasaccharides synthesized by this method are all assembled with amino linking arms at their reducing ends, which can be combined with carrier proteins for immunological research.
  • n is the number of repeating units of the disaccharide fragment, which can be 2-12;
  • the first object of the present invention is to provide a method for preparing Helicobacter pylori O2 serotype O antigen oligosaccharide compound, which uses sugar building blocks A, B and C to construct Helicobacter pylori O2 serotype O antigen oligosaccharide Compounds, including 1,2- ⁇ -cis-glycosidic bonds and 1,3- ⁇ -cis-glycosidic bonds between building sugar blocks;
  • the sugar building blocks A, B, and C are compounds represented by structural formula II and III respectively:
  • LG is a glycosylation leaving group, including trichloroacetimide group and p-methylphenylthio; linker includes -(CH2) n -NY 1 Y 2 or -(CH2) n -NY 1 Y 2 (linker), Y 1 is hydrogen (H) or an alkoxy group such as benzyl (Bn), Y 2 is hydrogen (H) or an alkoxycarbonyl group such as benzyloxycarbonyl (Cbz);
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are hydrogen or ether group; R 7 is hydrogen or acyl group.
  • the construction of 1,3- ⁇ -cis-glycosidic bonds in the method is carried out in a mixed solvent of dichloromethane and ether.
  • the volume ratio of dichloromethane to ether in the mixed solvent is 1: (2.5-5).
  • the construction of the 1,3- ⁇ -cis-glycosidic bond in the method also requires the introduction of additives.
  • the additive includes thiophene.
  • the molar ratio of the additive to the sugar cuts is (80-120):1.
  • the construction of the 1,2- ⁇ -cis-glycosidic bond in the method is carried out in a dichloromethane solvent.
  • the method for constructing 1,3-glucose ⁇ -glycosidic bond is preferably: the glycosyl donor and the glycosyl acceptor are azeotropically boiled in toluene, dichloromethane and ether are added, and the concentration of the reaction substrate is 0.01-0.1M, using molecular sieve as desiccant, adding thiophene equivalent to 100 times the equivalent of glycosyl donor, stirring at room temperature for 30 minutes and then cooling to 0°C, reaction time 12h, and terminating the reaction with pyridine.
  • the method includes the following steps:
  • the method further includes: referring to step (3), constructing a 1,3- ⁇ -cis-glycosidic bond between the polysaccharide compound and the polysaccharide compound to prepare Helicobacter pylori O2 serum Type O antigen oligosaccharide compound; the polysaccharide compound is a compound represented by formula IV;
  • m is the number of repeating units of the disaccharide fragment, ranging from 2 to 12;
  • Linker includes -(CH2) n -NY 1 Y 2 or -(CH2) n -NY 1 Y 2 (linker), Y 1 is hydrogen (H) or alkoxy such as benzyl (Bn), Y 2 is hydrogen ( H) or an alkoxycarbonyl group such as benzyloxycarbonyl (Cbz);
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are hydrogen or ether group; R 7 is hydrogen or acyl group.
  • the method for synthesizing monosaccharide building block A in the step (1) includes: using diacetone glucose as the starting material, in sodium hydride (NaH) and benzyl bromide (BnBr) Under the conditions, 3-benzyl-diacetone glucose is generated, and then the two propylidene rings are hydrolyzed by acid; the resulting 3-benzyl compound is protected with acetyl groups for the exposed 4 hydroxyl groups, and then is used in p-methylthiophenol and Lewis Under the action of acid (BF 3 ⁇ E 2 tO), the terminal group is protected with a thiophenyl group; in the next step, the remaining acetyl groups at positions 2, 4, and 6 are hydrolyzed in an alkaline environment, followed by a benzylidene group At the same time, the 4 and 6 hydroxyl groups are protected, and 2-OH is freed to obtain sugar building block A.
  • NaH sodium hydride
  • BnBr benzyl bromide
  • the method for synthesizing monosaccharide building block A in step (1) specifically includes: using commercially available diacetone glucose as a starting material, adding sodium hydride (NaH) and allyl Under the action of bromine (AllBr), it is protected with an allyl group at position 3, and then the two propylidene groups of the 3-allyl compound are removed with allyl alcohol (AllOH) and hydrochloric acid. Protect with allyl groups. Allyl glucose is then used under the action of sodium hydride (NaH) and benzyl bromide (BnBr) to protect the remaining three hydroxyl groups of glucose with benzyl groups.
  • the two allyl groups at the 1, 3 positions of glucose are removed by the action of palladium chloride (PdCl 2 ) and methanol, and then the 1.3 position is protected with acetyl under the conditions of pyridine and acetic anhydride.
  • hydrazine acetate is used to selectively remove the acetyl group at position 1 of glucose, and the obtained 1-OH compound is made into a sugar group donor with trichloroacetonitrile and DBU, which is sugar building block A.
  • the synthesis of monosaccharide building block A is carried out in an organic solvent;
  • the organic solvent includes dichloromethane, ethyl acetate, methanol, N,N-dimethylformamide, One or more of tetrahydrofuran, pyridine and chloroform.
  • the substrate concentration is 0.02-0.5M.
  • the acid is hydrochloric acid, acetic acid or p-toluenesulfonic acid.
  • the reaction temperature is -78° C. to the reflux temperature of the solvent, and the reaction time is 1-48 h.
  • sugar building block A The structural formula of sugar building block A is shown in formula II, and its terminal position is a thiophenyl group, which is a leaving group.
  • the method for synthesizing monosaccharide building block B in the step (1) includes: protecting the 2-OH of the above-mentioned monosaccharide building block A with an acetyl group to obtain the 2-OAc compound React with the linking arm to obtain a glucose compound with a linking arm at the end group, and finally remove 2-OAc in methanol and sodium methoxide to obtain glucose building block B.
  • the disaccharide compound in step (2) includes the disaccharide donor D and the disaccharide acceptor E as shown in formula V:
  • linker includes -(CH2) n -NY 1 Y 2 or -(CH2) n -NY 1 Y 2 (linker), Y 1 is hydrogen (H) or an alkoxy group such as benzyl (Bn), and Y 2 is Hydrogen (H) or an alkoxycarbonyl group such as benzyloxycarbonyl (Cbz);
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are hydrogen or ether group; R 7 is hydrogen or acyl group.
  • the step (2) includes: taking sugar building blocks A and C as raw materials, using dichloromethane as a solvent, adding a desiccant, and performing it at -78°C to prepare the disaccharide Donor D;
  • the desiccant is Molecular sieve, Molecular sieve, One or more of molecular sieve, anhydrous sodium sulfate, anhydrous magnesium sulfate, and anhydrous calcium sulfate.
  • the mass ratio of the desiccant to the reactant is 1.0-4.0.
  • a TLC plate is used to monitor the reaction progress of the reaction. After the reaction, the reaction is terminated with pyridine, and the product is purified by a silica gel column.
  • the step (3) includes: synthesizing a tetrasaccharide compound with a disaccharide donor D and a disaccharide acceptor E. ,
  • the tetrasaccharide compound includes a tetrasaccharide donor F and a tetrasaccharide acceptor G as shown in formula VI,
  • the step (3) includes: adding thiophene as an additive in a mixed solvent of DCM and Et 2 O, and using Molecular sieve is used as a desiccant and reacted at 0°C to room temperature to prepare tetrasaccharide F;
  • Tetrasaccharide E is removed from 3"’-OAc in sodium methoxide solution to obtain tetrasaccharide G.
  • an accelerator may be added when preparing the tetrasaccharide F.
  • the accelerator includes N-iodosuccinimide NIS, trimethylsilyl trifluoromethanesulfonate TMSOTf, TfOH trifluoromethanesulfonate, silver trifluoromethanesulfonate One or more of AgOTf.
  • the volume ratio of DCM and Et 2 O in the mixed solvent is preferably 1:3.
  • the oligosaccharide chain can extend from the reducing end to the non-reducing end in a disaccharide-binding mode, including two steps: 1) The oligosaccharide with a linker at the reducing end removes the non-reducing end. Lipid group; 2) Glycosylation reaction with disaccharide donor 16.
  • a 1,3- ⁇ -cis-glycosidic bond is used to synthesize a hexasaccharide compound using disaccharide and tetrasaccharide;
  • the hexasaccharide compound includes a compound represented by formula VII;
  • the synthesis conditions of the disaccharide compound are: monosaccharide building blocks A and B (LG/linker) are used as substrates, the reaction solvent is methylene chloride, a desiccant is used, and the reaction temperature is -78 °C, the glycosylation reaction is carried out under the catalysis of Lewis acid. The reaction process is protected by inert gas. After the reaction raw materials disappear, the reaction is quenched with pyridine or triethylamine, filtered, washed, extracted, dried, and the product is purified with a silica gel column to obtain the disaccharide I/II.
  • the method for assembling the tetrasaccharide using the disaccharide compound I as the donor and the disaccharide compound II as the acceptor to assemble the tetrasaccharide, and the reaction conditions are using the disaccharide compounds I and II as the substrates,
  • the reaction solvent is a mixed solvent of dichloromethane and ether, and thiophene is added at the same time.
  • the reaction temperature is room temperature.
  • the reaction is carried out under the catalysis of Lewis acid.
  • the reaction process is protected by inert gas. After the reaction raw materials disappear, filter, wash, extract and dry, and the products are purified by silica gel column to obtain tetrasaccharides.
  • the hexasaccharide assembly conditions are as follows: the tetrasaccharide compound removes the non-reducing ester group under alkaline conditions, and then assembles the hexasaccharide compound with the disaccharide compound I as a substrate.
  • the reaction conditions are the same as when assembling the tetrasaccharide compound.
  • the method further includes deprotection.
  • the deprotection is performed by using Pd/C, reacting under hydrogen for 24 hours to remove all aromatic groups, and purifying the Helicobacter pylori O2 serotype O antigen oligo by C18 reversed phase column. Carbohydrates.
  • the deprotection specifically includes: 1)
  • the solvent used is methanol, tetrahydrofuran, ethyl acetate or a mixed solvent consisting of one or more; the reaction is completed with H+ resin for neutralization, the reaction temperature is room temperature ⁇ 40 °C, The reaction time is 2h ⁇ 2d to obtain semi-deprotected carbohydrate compounds;
  • the reaction solvent is one or more of tetrahydrofuran, dichloromethane, ethyl acetate, tert-butanol, water, methanol, and acetic acid.
  • Palladium is used The carbon is 10% palladium on carbon, the mass ratio of palladium on carbon to the reactant is 0.1:1 to 0.5:1, the hydrogen pressure used for debenzylation and benzylidene is 1 to 100 atm, and the reaction temperature is normal temperature. The time is 2 ⁇ 48h.
  • the second object of the present invention is to provide a Helicobacter pylori O2 serotype O antigen oligosaccharide compound assembled with amino linking arms, the structural formula of which is shown in formula VII, and the compound is prepared by the above method;
  • the structure of an O-antigen oligosaccharide of Helicobacter pylori O2 serotype assembled with amino linking arms is:
  • the third object of the present invention is to provide a method for preparing a sugar-protein conjugate, which uses the above-mentioned Helicobacter pylori O2 serotype O antigen oligosaccharide compound assembled with amino linking arms.
  • the fourth object of the present invention is to apply the above-mentioned Helicobacter pylori O2 serotype O antigen oligosaccharide compound assembled with amino linking arms in the development or preparation of Helicobacter pylori vaccine or the treatment of diseases caused by Helicobacter pylori infection Application in medicine.
  • the present invention is to obtain Helicobacter pylori O2 serotype O-antigen disaccharide, tetrasaccharide and hexasaccharide through chemical synthesis.
  • the present invention finds a method which is very conducive to the formation of glucose ⁇ -glycosidic bonds through protective agent strategy, temperature effect, solvent effect and additive effect, and applies this method to Helicobacter pylori O2 serotype O-antigen disaccharide, four During the synthesis of sugar and hexasaccharides.
  • the synthetically obtained Helicobacter pylori O2 serotype O-antigen disaccharide, the reducing ends of the tetrasaccharide and the hexasaccharide are assembled with amino linking arms, which can be made into glycoconjugates with carrier proteins for immunological research and prevent development And the treatment of Helicobacter pylori has an important role.
  • Figure 7 Deprotection of disaccharides, tetrasaccharides and hexasaccharides
  • the commercially available diacetone glucose 1 is used as the starting material, and it reacts with benzyl bromide under the action of sodium cyanide to generate 3-OBn glucose compound 2.
  • the propylidene group is removed in 80% acetic acid solution, 4 newly generated hydroxyl groups are protected with acetyl groups under the action of pyridine and acetic anhydride to obtain compound 3.
  • Subsequent compound 3 reacts with thiophenol under the action of boron trifluoride ⁇ ether (BF 3 ⁇ Et 2 O) to produce 1-glycoside compound 4.
  • the new compound obtained in the previous step was dissolved in acetic anhydride (360ml), and anhydrous sodium acetate (18.9g) was added at the same time.
  • the reaction solution was stirred at 80°C for 4h.
  • the reaction solution was poured into ice water Then it was extracted with ethyl acetate and saturated sodium bicarbonate, the organic phase was separated, dried with anhydrous sodium sulfate and concentrated.
  • the concentrated solution was purified with a silica gel column (petroleum ether/ethyl acetate, 4:1) to obtain compound 3 (69 g, 157 mmol, 86%).
  • Compound 4 Dissolve compound 3 (42g, 96mmol) in anhydrous DCM (240ml), add p-methylthiophenol (17.9g, 144mmol) and an appropriate amount of freshly activated molecular sieves, stir at room temperature for 30 minutes, and then cool to 0 °C. After stirring for 20 minutes, boron trifluoride ether (60ml) was slowly added. The reaction solution was stirred at room temperature for 24 hours, and the reaction progress was monitored by TLC. After the raw materials were completely reacted, the reaction solution was extracted with dichloromethane and saturated sodium bicarbonate solution.
  • Compound 5 Compound 4 (27g, 54mmol) was dissolved in methanol (150ml), 5M sodium methoxide (22ml) was added at room temperature, and the reaction solution was stirred at room temperature for 2h. The reaction was monitored by TLC, and the reaction solution was neutralized to neutrality with Amberlite IR120 H + resin after the raw materials were completely reacted. The resin was filtered off, the filtrate was spin-dried to obtain a new compound ( ⁇ 20g), and the oil pump was vacuumed overnight.
  • the new compound ( ⁇ 20g, 53mmol) was dissolved in anhydrous DMF, and benzaldehyde dimethyl acetal (8.3ml) and p-toluenesulfonic acid (1.0g) were added sequentially.
  • the reaction solution was stirred at 70° C. for 6 h, and the reaction was monitored by TLC. After the reaction of the raw materials was completed, the reaction solution was neutralized with triethylamine.
  • the reaction solution was concentrated and extracted with dichloromethane and saturated brine, the organic phase was separated, dried with anhydrous sodium sulfate and concentrated, and the concentrated solution was purified by silica gel column to obtain compound 5 (19.1 g, 41 mmol).
  • compound 8 is an analog of compound 5 with an amino linker assembled at the reducing end.
  • compound 5 first protect 2-OH with an acetyl group to obtain compound 6, then use p-methylphenylthio as the leaving group, and react with the five-carbon amino linker under the accelerator NIS and TMSOTf to obtain compound 7.
  • the 2-OAc of compound 7 was removed with methanol and sodium methoxide to obtain sugar building block 8.
  • the diacetone glucose 1 is the starting material and reacts with allyl bromide under the action of NaH to obtain the 3-allyl compound 9.
  • Compound 9 undergoes ring-opening reaction in allyl alcohol added with hydrochloric acid to obtain 1,3-diallyl compound 10.
  • use benzyl to protect the remaining three naked hydroxyl groups of glucose to obtain compound 11.
  • Compound 11 uses palladium chloride in dry methanol to specifically remove the 1,3 allyl groups to obtain 1-OH compound 12.
  • the two hydroxyl groups generated in the previous reaction were protected with acetyl groups to obtain compound 13.
  • the 1-OAc group was selectively removed under the action of hydrazine acetate to obtain 1-OH compound 14.
  • trichloroacetonitrile and DBU are used to react with compound 14 to obtain trichloroacetimide ester glycosyl building block 15.
  • the disaccharide compound 16 was synthesized from monosaccharide donor 15 and monosaccharide acceptor 5 under the condition of TMSOTf as a promoter.
  • the monosaccharide donor 15 and the monosaccharide acceptor 8 were used to synthesize the disaccharide compound 17 in a similar manner, and then the 3'-OAc of 17 was removed under the condition of sodium methoxide to obtain the disaccharide compound 18.
  • Compound 16 The glycosyl donor 15 (3.1 g, 4.8 mmol) and the glycosyl acceptor (2.8 g, 6.1 mmol) were mixed, azeotroped with toluene 3 times under the protection of nitrogen, and vacuumed overnight under the oil pump. Add dichloromethane (100ml) and appropriate molecular sieves, stir at room temperature for 30 minutes and then cool to -78°C, continue stirring for 30 minutes, add accelerator TMSOTf (100 ⁇ L), and stir at low temperature for 5 hours. The progress of the reaction was monitored by TLC, and after all the glycosyl donor was consumed, the reaction was quenched with triethylamine.
  • reaction solution was extracted with dichloromethane and saturated sodium bicarbonate solution, the organic phase was separated, dried with anhydrous sodium sulfate and concentrated.
  • concentrated solution was purified with a silica gel column (petroleum ether/ethyl acetate, 10:1) to obtain compound 16 (2.9 g, 3.2 mmol).
  • Compound 17 Mix glycosyl donor 15 (1.42g, 2.3mmol) and glycosyl acceptor 8 (1.11g, 1.6mmol) with an amino linking arm, azeotrope with toluene 3 times under the protection of nitrogen, and in the oil pump Vacuum overnight. Then add anhydrous DCM (100ml) and appropriate molecular sieves, stir at room temperature for 30 minutes and then cool to -78°C, continue stirring for 30 minutes, add accelerator TMSOTf (37 ⁇ L), and continue to stir the reaction solution at low temperature for 6 hours. The reaction was monitored by TLC, and after the glycosyl donor was completely consumed, the reaction was quenched with triethylamine.
  • reaction solution was extracted with dichloromethane and saturated sodium bicarbonate solution, the organic phase was separated, dried with anhydrous sodium sulfate and concentrated.
  • concentrated solution was purified with a silica gel column (petroleum ether/ethyl acetate, 4:1) to obtain compound 17 (1.35 g, 1.2 mmol).
  • Compound 18 Compound 17 (1.1 g, 0.96 mmol) was dissolved in anhydrous methanol (20 ml), and sodium methoxide (100 mg) was added. The reaction solution was stirred at 40° C. for 24 hours, and the reaction was monitored by TLC. After all the raw materials were reacted, the reaction solution was neutralized with Amberlite IR120 H + resin. The resin was filtered off, concentrated and purified by silica gel column (petroleum ether/ethyl acetate, 3:1) to obtain compound 18 (898 mg, 0.82 mmol).
  • Compound 19 Mix disaccharide glycosyl donor 15 (287 mg, 0.3 mmol) and disaccharide glycosyl acceptor 18 (230 mg, 0.12 mmol), azeotrope with toluene 3 times under the protection of nitrogen, and vacuum overnight under the oil pump . Then add anhydrous DCM (2ml) ether (4ml), thiophene (12.6mmol) and appropriate molecular sieves, stir at room temperature for 30 minutes and then cool to 0°C, continue stirring for 30 minutes, add accelerators NIS (135mg) and AgOTf (26mg), The reaction solution was continuously stirred for 12h under ice bath. The reaction was monitored by TLC.
  • reaction solution was extracted with 10% sodium thiosulfate solution and dichloromethane, the organic phase was separated, dried with anhydrous sodium sulfate and concentrated.
  • concentrated solution was purified with a silica gel column (petroleum ether/ethyl acetate, 4:1) to obtain compound 19 (121 mg, 0.064 mmol).
  • Example 5 the solvents were replaced with the solvents shown in Table 1, and the content of 1,3- ⁇ -cis glycosidic bonds in the resulting product was determined. The results are shown in Table 1.
  • Example 5 the accelerators were replaced with the additives shown in Table 2, and the content of 1,3- ⁇ -cis glycosidic bonds in the resulting product was determined. The results are shown in Table 2.
  • Tetrasaccharide compound 19 is reacted for two days under the conditions of methanol and sodium methoxide to remove 3""-OAc to obtain tetrasaccharide compound 20. Subsequently, compound 20 is used as the glycosyl acceptor to react with the disaccharide donor 16 under the catalysis of a Lewis acid promoter to obtain the hexasaccharide compound 21.
  • Compound 20 Compound 19 (90 mg, 0.047 mmol) was dissolved in a mixed solvent of methanol (9 ml) and ethyl acetate (1 ml), and sodium methoxide (30 mg) was added. The reaction was stirred at 40°C for 2 days, and the reaction was monitored by TLC. After all the raw materials were reacted, the reaction solution was neutralized with Amberlite IR120 H + resin to neutrality. The resin was filtered off, concentrated and purified by silica gel column (petroleum ether/ethyl acetate, 3:1) to obtain compound 21 (68 mg, 0.037 mmol).
  • Disaccharide donor 15 60 mg, 0.064 mmol
  • tetrasaccharide acceptor 20 50 mg, 0.027 mmol
  • anhydrous DCM 0.5ml
  • thiophene 0.6ml
  • appropriate molecular sieves stir at room temperature for 30min, then cool to 0°C, continue stirring for 30min
  • accelerator NIS 29mg
  • AgOTf 3mg
  • reaction solution was extracted with 10% sodium thiosulfate solution and dichloromethane, the organic phase was separated, dried with anhydrous sodium sulfate and concentrated.
  • concentrated solution was purified with a silica gel column (petroleum ether/ethyl acetate, 4:1) to obtain compound 21 (33.4 mg, 0.012 mmol).
  • a series of Helicobacter pylori O2-serotype oligosaccharide derivatives can be synthesized, such as octasaccharide, decasaccharide and dodecose.
  • the oligosaccharide chain can extend from the reducing end to the non-reducing end in a +2 sugar mode, including two steps. 1) The oligosaccharide with a linking arm at the reducing end removes the lipid group at the non-reducing end; 2) the glycosylation reaction with the disaccharide donor 16.
  • the method of removing the non-reducing lipid groups of oligosaccharides dissolve the compound (-50mg) in methanol (10-15ml), and add a small amount of ethyl acetate or tetrahydrofuran to increase the solubility. Sodium methoxide (20-30mg) was added, and the reaction solution was stirred at 40°C for 12h-2d. Use TLC to monitor the progress of the reaction. After all the raw materials have been reacted, use Amberlite IR120 H + resin to neutralize the reaction solution to neutrality. The resin was filtered off, concentrated and purified by silica gel column.
  • the method of glycosylation reaction using disaccharide 16 as the glycosyl donor the glycosyl donor and the glycosyl acceptor were azeotroped with toluene three times under the protection of nitrogen, and the oil pump was vacuumed overnight.
  • the reactant was dissolved in a mixed solvent of dichloromethane and ether (1:1), and 100 equivalents of thiophene and an appropriate amount of molecular sieve were added to the glycosyl donor.
  • the reaction concentration is 0.02M (relative to the glycosyl donor), and the reaction time is 12-24h.
  • the progress of the reaction was monitored by TLC.
  • the reaction solution was extracted with 10% sodium thiosulfate, dichloromethane and saturated sodium bicarbonate solution, the organic phase was separated, dried with anhydrous sodium sulfate, and concentrated on silica gel. Column purification obtains new oligosaccharide compounds.
  • Example 7 Deprotection of disaccharides, tetrasaccharides and hexasaccharides
  • the deprotection of disaccharide, tetrasaccharide and hexasaccharide compounds is divided into two steps. First, remove non-reducing acetyl groups under alkaline conditions; second, hydrogenate all aromatic groups under the catalysis of palladium-carbon to obtain disaccharides, tetrasaccharides and hexasaccharides with five-carbon amino linking arms assembled at the reducing end. sugar.
  • the method for removing the non-reducing disaccharide acetyl group The compound is dissolved in a mixed solvent of methanol and ethyl acetate (9:1) at a concentration of 0.02M, and 0.5 equivalent of sodium methoxide relative to the raw material is added. The reaction solution was stirred at 40°C for 12h-2d. The progress of the reaction was monitored by TLC. After all the raw materials have been reacted, the reaction solution is neutralized with Amberlite IR120 H + resin to neutrality. The resin was filtered off, concentrated and purified by silica gel column.
  • the method of hydrogenation reaction The compound (6-10mg) was dissolved in a mixed solution of isobutanol (3ml), methanol (1ml) and water (1ml), and glacial acetic acid (0.1ml) and palladium carbon (50mg) were added.
  • the reaction liquid is carried out in a medium-pressure hydrogen reactor, the reaction pressure is 0.4MPa, and the reaction time is 12h.
  • the reaction solution is filtered with celite, concentrated and purified by a C18 column to obtain the target compound.
  • sugar building blocks preferably the commercially available diacetone glucose as the starting material, and re-synthesized two glucose building blocks for the assembly of Helicobacter pylori O2 serotype O-antigen oligosaccharides (corresponding to Example 1 and Example 2 respectively) ).
  • glycosylation promoters including iodine band succinimide, Lewis acid trimethylsilyl triflate, trifluoromethanesulfonic acid Or silver trifluoromethanesulfonate, etc., and the superimposed use of various accelerators, and finally found that the application of sulfoglycoside 124 as a sugar donor, supplemented by iodine with succinimide, silver triflate and trimethyl Silicon trifluoromethanesulfonate was used together as a promoter, and reacted in a mixed solvent of DCM and ether (1/3) and added thiophene, and ⁇ / ⁇ , 10:1 could be obtained with a yield of 59%. ⁇ tetrasaccharide compound 133.
  • the yield of tetrasaccharide obtained with 127 as the donor is only 62%, and the ⁇ -stereoselectivity can also reach ⁇ / ⁇ >10:1.
  • the yield of disaccharide TFAI donor 137 is a little higher than that of disaccharide glucosinolate donor 124, the ⁇ -stereoselectivity is not significantly improved, only 2% higher.
  • a two-step reaction is required, and the yield is only 49%.
  • the increased yield of tetrasaccharide compound 133 is insignificant, and it is not worth it from the perspective of overall synthesis.
  • the easiest way to think of is to first open the benzylidene ring on the glucose residue at the reducing end of the disaccharide 124, thereby vacating the C-4 and C-6 hydroxyl groups for Protect the ester group. Since there are many hydrophobic groups on 124 and insoluble in the water phase, the benzylidene group is hydrolyzed in dichloromethane with trifluoroacetic acid to obtain the 2-hydroxy compound 139, and then the vacant hydroxyl group is protected with benzoyl. Compound 140. The total yield of the two-step reaction was 61%.
  • the newly synthesized disaccharide donor 140 was used for glycosylation reaction with the disaccharide acceptor 138, and it was found that the donor 140 could not be activated by the promoter. No matter whether it was to increase the reaction temperature to 40°C or increase the amount of the promoter, Can activate 140. During the reaction, 140 was neither hydrolyzed nor reacted with the receptor. After the reaction, sufficient amount of 140 could be recovered from the reaction solution.
  • the donor 142 and the glycosyl acceptor 138 are used for glycosylation to generate tetrasaccharides, and trimethylsilyl trifluoromethanesulfonate is used as an accelerator to obtain a pure ⁇ -stereoselective tetrasaccharide compound 143, which is also beneficial For the long-range effect of benzoyl.
  • the efficiency of the glycosylation reaction depends on the donor of the disaccharide, and we consider the disaccharide 124 Beginning to the utilization of tetrasaccharide 133 or tetrasaccharide 143, the total yield of 133 obtained by the direct glycosylation reaction of glucosinolate 124 is 53% (59% ⁇ 90%), via glycosyl TFAI donor 127 Glycosylation with acceptor 138 gives a total yield of 133 of 28.5% (71% ⁇ 72% ⁇ 62% ⁇ 90%), and the third route is obtained by glycosylation of benzyloxyglycosyl donor 142 The total yield of 143 is only 15.5%, (84% ⁇ 73% ⁇ 63% ⁇ 73% ⁇ 55% ⁇ 100%).

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Abstract

本发明公开了一种幽门螺旋杆菌O2血清型O抗原寡糖类化合物的合成，属于有机合成领域。本发明通过化学合成得到幽门螺旋杆菌O2血清型O-抗原二糖至二十四糖。本发明利用保护剂策略、温度效应、溶剂效应和添加剂效应，开发了一种非常有利于葡萄糖α糖苷键生成的化学合成方法。将此方法用于合成组装有氨基连接臂的幽门螺旋杆菌O2血清型O-抗原寡糖类化合物；合成得到的组装有氨基连接臂的幽门螺旋杆菌O2血清型O-抗原寡糖类化合物可以与载体蛋白制成糖缀合物，用于免疫学研究，对发展预防和治疗幽门螺旋杆菌具有重要作用。

Description

一种幽门螺旋杆菌O2血清型O抗原寡糖类化合物的合成 技术领域
本发明涉及一种幽门螺旋杆菌O2血清型O抗原寡糖类化合物的合成，属于有机合成领域。
背景技术
幽门螺旋杆菌是一种微需氧型的革兰氏阴性菌，全球约有一半人口感染了这种致病菌，因为较低的社会经济地位和较差的医疗卫生条件，在发展中国家中这一比例更是高达80％，据报道在我国城市和农村分别有47％和66％的人口感染了幽门螺旋杆菌。口-口传播和粪-口传播是该致病菌最主要的传播方式，母婴传播也是该致病菌的传播方式之一。
大部分的幽门螺旋菌的感染发生儿童时期，一旦感染，治病菌可能在胃粘膜上存在一生。尽管70％的被感染者在临床上是无症状的，但是这种致病菌已经被证实与一系列胃肠道疾病有关，15％-20％的长期感染者将会引发胃炎，胃和十二指肠溃疡等一系列胃肠道疾病，另外还有1％-2％的长期感染者将发展成胃癌。胃癌已成为全球发病率第五高(～952000例，占癌症病人的6.2％，2012年WHO数据)和死亡率第三高的癌症(～723000例，占癌症病人的8.8％，2012年WHO数据)1994年WHO下属的国际癌症研究机构(IACR)将幽门螺旋杆菌分类为I类致癌物。
目前针对幽门螺旋杆菌感染相关的胃肠道疾病的主要治疗方法是一种利用两种抗生素(甲硝咪唑和克拉霉素)和一种质子泵抑制剂的联合治疗方法，称之为三联疗法。这种方法在治疗幽门螺旋杆菌上取得了一定的效果，但是仍有不少不足之处，主要限制表现在：1)抗生素在胃肠道中的代谢时间比较短，2)抗生素在胃这种特殊pH低环境下相对不稳定，3)幽门螺杆菌一般寄居在胃黏膜处，而抗生素浓度在黏膜浓度很低，4)更重要的是随着耐药性细菌的出现抗生素的治疗效果有逐渐减弱的趋势。基于以上缺陷，有报道使用三联疗法治疗后的幽门螺旋杆菌的一年复发率已达15％-30％。
因此，为了克服这一缺陷，研究针对这一致病菌的疫苗相关研究越来越受到重视。近年来，针对幽门螺旋杆菌疫苗的研究表现在的毒力因素抗原上，如尿酶B，细胞毒素相关毒素(CagA)，空泡毒素(VacA)，鞭毛，热休克蛋白，黏附素，中性粒细胞活性蛋白等。另外，针对灭活全菌细胞和菌溶产物的研究也是疫苗的热点之一。
与其他革兰氏阴性菌一样，幽门螺旋杆菌的细胞表面主要是有脂多糖组成，脂多糖对该细菌的生物膜结构和功能的完整性具有相当重要的作用，也存在于细菌的分泌物中，是该细菌的致病因素和毒力因素之一。
Monteiro系统地研究了各类幽门螺旋杆菌的脂多糖结构发现其结构与典型的革兰氏阴性菌一致，包含三个不同的结构单元，分别是多聚糖O糖链，核心寡糖链和富含脂肪酸的脂质A。在幽门螺旋杆菌中，核心寡糖链结构相对保守而多聚糖O糖链表现出特异性的表型。根据多聚糖O糖链中具体单糖种类和连接方式的不同可以将幽门螺旋杆菌分类6种不同的血清型(O1-O6)。2015年Monteiro报道了幽门螺旋杆菌的O2血清型O聚糖链的具体结构，该血清型的O-抗原是由葡萄糖残基依次通过α-(1→2)-和α-(1→3)-糖苷键依次连接而成的重复单元结构，最长可达12个重复单元的24糖链。
目前针对幽门螺旋杆菌脂多糖的研究主要是通过从灭活细菌中提取，这一方法的不足在于一次提取得到的产物极少，另外受细菌基因表达和修饰的特性，提取得到的脂多糖也存在结构不够专一，并且容易附带结构类似杂质的特点，实验重复性差，对研究存在一定的干扰。我们利用化学合成方法，以幽门螺旋杆菌O2血清型O抗原寡糖为目标化合物，通过糖基砌块的设计和构建以及糖基化反应的选择和优化得到O抗原二糖，四糖和六糖，并且所合成的二糖，四糖和六糖还原端均组装有氨基连接臂，可以与载体蛋白连接成糖缀合物，开展进一步的免疫学研究。
发明内容
本发明所要解决的技术问题是通过化学方法合成幽门螺旋杆菌O2血清型O抗原寡糖片段。α-1,2顺式糖苷键的构建是合成目标结构的关键步骤。本发明利用二丙酮葡萄糖为唯一起始原料通过糖基砌块的构建和糖基化反应条件的优化最终得到保护的全α连接的保护二，四，六糖，最终脱去保护得到最后的目标化合物组装有氨基连接臂的幽门螺旋杆菌O2血清型O抗原二糖，四糖和六糖。同时，用该方法合成得到的二糖，四糖和六糖还原端均组装有氨基连接臂，可以和载体蛋白结合用于免疫学研究。
Figure PCTCN2019091200-appb-000001
其中m为二糖片段的重复单元数，可以为2～12；
R为-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，n＝1～10，N为氮Y 1为氢(H)或者烷氧基如苄基(Bn)，Y 2为氢(H)或者烷氧羰基如苄甲氧羰基(Cbz)，R1，R2，R3，R4，R5，R6为氢或者醚类基团如苄基(Bn)。
本发明的第一个目的是提供一种幽门螺旋杆菌O2血清型O抗原寡糖类化合物的制备方法，所述方法利用糖砌块A、B、C构建幽门螺旋杆菌O2血清型O抗原寡糖类化合物，包括构建糖砌块之间的1,2-α-顺式-糖苷键和1,3-α-顺式-糖苷键；
其中糖砌块A、B、C分别为结构式II、III所示的化合物：
Figure PCTCN2019091200-appb-000002
其中，LG为糖基化反应离去基团，包括三氯乙酰亚胺基、对甲基苯硫基；linker包括-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，Y 1为氢(H)或者烷氧基如苄基(Bn)，Y 2为氢(H)或者烷氧羰基如苄甲氧羰基(Cbz)；
R 1，R 2，R 3，R 4，R 5，R 6为氢或者醚类基团；R 7为氢或者酰基类基团。
在本发明的一种实施方式中，所述方法中1,3-α-顺式-糖苷键的构建是在二氯甲烷和乙醚的混合溶剂中进行的。
在本发明的一种实施方式中，所述混合溶剂中二氯甲烷与乙醚的体积比为1：(2.5-5)。
在本发明的一种实施方式中，所述方法中1,3-α-顺式-糖苷键的构建还需要引入添加剂。
在本发明的一种实施方式中，所述添加剂包括噻吩。
在本发明的一种实施方式中，所述添加剂与糖切块的摩尔比为(80-120)：1。
在本发明的一种实施方式中，所述方法中1,2-α-顺式-糖苷键的构建是在二氯甲烷溶剂中进行的。
在本发明的一种实施方式中，1,3-葡萄糖α糖苷键的构建方法优选：糖基供体和糖基受体在甲苯中共沸，加入二氯甲烷和乙醚，反应底物的浓度为0.01～0.1M，以分子筛作为干燥剂，加入相当于糖基供体当量100倍的噻吩，在室温下搅拌30min后冷却至0℃，反应时间为12h，用吡啶终止反应。
在本发明的一种实施方式中，所述方法包括以下步骤：
(1)合成葡萄糖单糖砌块A、B、C；
(2)合成二糖化合物：构建糖砌之间的1,2-α-顺式-糖苷键；
(3)合成四糖化合物：构建步骤(2)所得二糖化合物之间的1,3-α-顺式-糖苷键。
在本发明的一种实施方式中，所述方法还包括：参照步骤(3)，构建多糖化合物与多糖化合物之间的1,3-α-顺式-糖苷键，制备得到幽门螺旋杆菌O2血清型O抗原寡糖类化合物；所述多糖化合物为式IV所示的化合物；
Figure PCTCN2019091200-appb-000003
其中，m为二糖片段的重复单元数，为2～12；
linker包括-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，Y 1为氢(H)或者烷氧基如苄基(Bn)，Y 2为氢(H)或者烷氧羰基如苄甲氧羰基(Cbz)；
R 1，R 2，R 3，R 4，R 5，R 6为氢或者醚类基团；R 7为氢或者酰基类基团。
在本发明的一种实施方式中，所述步骤(1)中单糖砌块A的合成方法包括：以二丙酮葡萄糖为起始原料，在氢化钠(NaH)和溴化苄(BnBr)的条件下生成3-苄基-二丙酮葡萄糖，随后用酸水解掉两个丙叉环；得到的3-苄基化合物用乙酰基保护裸露的4个羟基，接着在对甲基苯硫酚和路易斯酸(BF 3·E 2tO)的作用下给端基位用苯硫基保护；下一步在碱性环境下水解掉剩余的2,4,6号位上的乙酰基，随后用苄叉基同时保护住4,6号位羟基，空出2-OH得到糖砌块A。
在本发明的一种实施方式中，所述步骤(1)中单糖砌块A的合成方法具体包括：以市售的二丙酮葡萄糖为起始原料，在氢化钠(NaH)和烯丙基溴(AllBr)的作用下在3号位用烯丙基保护住，随后用烯丙醇(AllOH)和盐酸将3-烯丙基化合物的两个丙叉基脱去，同时在一号位也用烯丙基保护。烯丙基葡萄糖随用在氢化钠(NaH)和溴化苄(BnBr)作用下将葡萄糖剩余的三个羟基用苄基保护。下一步用氯化钯(PdCl 2)和甲醇作用下将葡萄糖1,3号位的两个烯丙基脱除，接着在吡啶和醋酸酐的条件下用乙酰基保护住1.3号位。最后用醋酸肼选择性的脱除葡萄糖1号位的乙酰基，得到的1-OH化合物用三氯乙腈和DBU做成糖基供体，即为糖砌块A。
在本发明的一种实施方式中，单糖砌块A的合成是在有机溶剂中进行的；所述有机溶剂包括二氯甲烷、乙酸乙酯、甲醇，N,N-二甲基甲酰胺、四氢呋喃，吡啶、氯仿中的一种或者几种。
在本发明的一种实施方式中，单糖砌块A的合成反应中，底物浓度为0.02～0.5M。
在本发明的一种实施方式中，酸为盐酸，乙酸或者对甲苯磺酸。
在本发明的一种实施方式中，反应温度为-78℃～溶剂回流温度，反应时间为1-48h。
糖砌块A的结构式如式II所示，其端基位为苯硫基，是一种离去基团。
在本发明的一种实施方式中，所述步骤(1)中单糖砌块B的合成方法包括：将上述的单糖砌块A的2-OH用乙酰基保护，得到的2-OAc化合物与连接臂反应，得到端基位有连接臂的葡萄糖化合物，最后在甲醇和甲醇钠中脱去2-OAc得到葡萄糖砌块B。
在本发明的一种实施方式中，所述步骤(2)中的二糖化合物包括如式V所示的二糖供体D和二糖受体E：
Figure PCTCN2019091200-appb-000004
其中，linker包括-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，Y 1为氢(H)或者烷氧基如苄基(Bn)，Y 2为氢(H)或者烷氧羰基如苄甲氧羰基(Cbz)；
R 1，R 2，R 3，R 4，R 5，R 6为氢或者醚类基团；R 7为氢或者酰基类基团。
在本发明的一种实施方式中，所述步骤(2)包括：以糖砌块A和C作为原料，以二氯甲烷为溶剂，加入干燥剂，在-78℃下进行，制备得到二糖供体D；
利用糖砌块B和C合成二糖受体E，糖苷化反应方法和合成二糖供体D一致，糖苷化反应完成，产物纯化后用甲醇钠溶液脱去3’-OAc得到二糖受体E。
所述的干燥剂为
Figure PCTCN2019091200-appb-000005
分子筛，
Figure PCTCN2019091200-appb-000006
分子筛，
Figure PCTCN2019091200-appb-000007
分子筛，无水硫酸钠，无水硫酸镁，无水硫酸钙中的一种或者几种。
所述的干燥剂与反应物的质量比为1.0～4.0。
在本发明的一种实施方式中，所述步骤(2)中反应用TLC板监测反应进程反应结束后用吡啶终止反应，产物用硅胶柱纯化。
在本发明的一种实施方式中，所述步骤(3)包括：以二糖供体D和二糖受体E合成四糖化合物。，
在本发明的一种实施方式中，所述四糖化合物包括如式VI所示的四糖供体F和四糖受体G，
Figure PCTCN2019091200-appb-000008
在本发明的一种实施方式中，所述步骤(3)包括：在DCM、Et 2O混合溶剂中，加入添加剂噻吩，使用
Figure PCTCN2019091200-appb-000009
分子筛作为干燥剂，0℃～室温进行反应，制备得到四糖F；
四糖E在甲醇钠溶液中脱去3”’-OAc得到四糖G。
在本发明的一种实施方式中，制备得四糖F时还可以加入促进剂。
在本发明的一种实施方式中，促进剂包括N-碘代丁二酰亚胺NIS、三氟甲基磺酸三甲基硅酯TMSOTf、三氟甲磺酸TfOH、三氟甲烷磺酸银AgOTf中的一种或者几种。
在本发明的一种实施方式中，混合溶剂中的DCM、Et 2O的体积比优选1：3。
在本发明的一种实施方式中，参照四塘的构建方法，还可以进一步合成八糖，十糖，十二糖等一系列幽门螺旋杆菌O2-血清型寡糖衍生物。
在本发明的一种实施方式中，寡糖链可以以结合二糖的模式由还原端向非还原端延伸，包括两个步骤：1)还原端带有连接臂的寡糖脱去非还原端的脂类基团；2)与二糖供体16的糖基化反应。
在本发明的一种实施方式中，利用二糖和四糖构建1,3-α-顺式-糖苷键合成六糖化合物；所述六糖化合物包括式VII所示的化合物；
Figure PCTCN2019091200-appb-000010
在本发明的一种实施方式中，二糖化合物的合成条件：以单糖砌块A和B(LG/linker)为底物，反应溶剂为二氯甲烷，使用干燥剂，反应温度为-78℃，在路易斯酸的催化下进行糖基化反应。反应过程都为惰性气体保护，反应原料消失后，用吡啶或者三乙胺淬灭反应，过滤，洗涤，萃取，干燥，产物用硅胶柱纯化分别得到二糖I/II。
在本发明的一种实施方式中，四糖的组装方法：以二糖化合物I为供体，二糖化合物II为受体组装四糖，反应条件为以二糖化合物I和II为底物，反应溶剂为二氯甲烷和乙醚的混 合溶剂，同时添加噻吩。使用干燥剂，反应温度为室温。反应在路易斯酸的催化下进行。反应过程都为惰性气体保护，反应原料消失后，过滤，洗涤，萃取，干燥，产物用硅胶柱纯化分别得到四糖。
在本发明的一种实施方式中，六糖组装的条件：四糖化合物在碱性条件下脱去非还原酯基，然后和二糖化合物I作为底物组装六糖化合物。反应条件与组装四糖化合物时相同。
在本发明的一种实施方式中，所述方法还包括脱保护。
在本发明的一种实施方式中，所述脱保护是利用Pd/C，在氢气下反应24h，脱除所有的芳香基团，经C18反相柱纯化得到幽门螺旋杆菌O2血清型O抗原寡糖类化合物。
在本发明的一种实施方式中，所述脱保护具体包括：1)
1)在碱性条件下将酰基脱除，所用溶剂为甲醇，四氢呋喃，乙酸乙酯中的一种或者几种组成的混合溶剂；反应结束用H+树脂中和，反应温度为室温～40℃，反应时间为2h～2d得到半脱保护的糖类化合物；
2)利用钯/碳、氢气对半脱保护产物进行全脱保护，反应溶剂为四氢呋喃，二氯甲烷，乙酸乙酯，叔丁醇，水，甲醇，醋酸中的一种或者多种，所用钯碳为10％的钯碳，钯碳与反应物的质量比为0.1:1～0.5:1，脱苄基和苄叉基所用的氢气压力为1～100atm，所述的反应温度为常温，反应时间为2～48h。
本发明的第二个目的是提供一种组装有氨基连接臂的幽门螺旋杆菌O2血清型O抗原寡糖类化合物，其结构式如式VII所示，所述化合物是利用上述方法制备得到的；
Figure PCTCN2019091200-appb-000011
在本发明的一种实施方式中，一种组装有氨基连接臂的幽门螺旋杆菌O2血清型的O-抗原寡糖的结构为：
α-D-Glc-(1-2)-α-D-Glc-(1-3)-α-D-Glc-(1-2)-α-D-Glc-(1-3)-α-D-Glc-(1-2)-α-D-Glc-linker。
本发明的第三个目的是提供一种糖-蛋白缀合物的制备方法，所述方法是利用上述的组装有氨基连接臂的幽门螺旋杆菌O2血清型O抗原寡糖类化合物。
本发明的第四个目的是将上述的组装有氨基连接臂的幽门螺旋杆菌O2血清型O抗原寡糖类化合物应用于在开发或制备疗幽门螺旋杆菌疫苗或者疗幽门螺旋杆菌感染导致的疾病的药物中的应用。
本发明有益效果：
本发明是通过化学合成得到幽门螺旋杆菌O2血清型O-抗原二糖，四糖和六糖。本发明通过保护剂策略，温度效应，溶剂效应和添加剂效应，发现了一种非常有利于葡萄糖α糖苷键生成的方法，并且将此方法应用在幽门螺旋杆菌O2血清型O-抗原二糖，四糖和六糖的合成之中。合成得到的幽门螺旋杆菌O2血清型O-抗原二糖，四糖和六糖的还原端均组装有氨基连接臂，可以与载体蛋白制成糖缀合物，用于免疫学研究，对发展预防和治疗幽门螺旋杆菌具有重要作用。
附图说明
图1：单糖砌块5的合成；
图2：单糖砌块8的合成；
图3：单糖砌块15的合成；
图4：二糖化合物16和18的合成；
图5：四糖化合物19的合成；
图6；六糖化合物21的合成；
图7：二糖、四糖和六糖的脱保护；
图8：化合物24的氢谱；
图9：化合物24的碳谱；
图10：化合物24的氢-氢相关谱；
图11：化合物24的碳-氢相关谱。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
糖砌块5的合成如图1：
如图1所示，以市售双丙酮葡萄糖1为起始原料，在氰化钠和作用下与溴化苄反应生成3-OBn葡萄糖化合物2。丙叉基在80％醋酸溶液中脱除后，在吡啶和醋酸酐的的作用下用乙酰基保护4个新生成的羟基得到化合物3。随后化合物3在三氟化硼·乙醚(BF 3·Et 2O)的的作用下与苯硫酚反应，生成1-硫苷化合物4。用甲醇甲醇钠溶液脱去化合物4的三个乙酰基，接着在苯甲醛二甲缩醛和对甲基苯环酸的作用下用苄叉基保护4,6号位羟基，留出2号位裸露羟基得到糖砌块5。
具体试验操作和步骤。
化合物2：将二丙酮葡萄糖(50g，192mmol)溶于DMF(480ml)中，在0℃下搅拌20min，缓缓加入氰化钠(15.4g，284mmol)，继续在0℃下搅拌30min，接着向反应液中加入溴化苄(34.2ml，288mmol)，反应恢复到室温，继续搅拌3h。用TLC检测反应液待原料反应完全后，用冰水淬灭反应，用二氯甲烷和饱和食盐水萃取，分离有机相，经无水硫酸钠干燥后浓缩，用硅胶柱纯化(石油醚/乙酸乙酯，15:1)得到化合物2(67g，183mmol，95％)。 1H NMR(400MHz,Chloroform-d)δ7.55–7.04(m,5H,Ar-H),5.90(d,J＝3.7Hz,1H,H-1),4.68(d,J＝11.8Hz,1H,Bn-H),4.63(d,J＝11.8Hz,1H,Bn-H),4.58(d,J＝3.7Hz,1H,H-2),4.37(dt,J＝7.7,6.1Hz,1H,H-5),4.15(dd,J＝7.8,3.2Hz,1H,H-6a),4.11(dd,J＝9.1,6.7Hz,1H,H-6b),4.01-3.99(m,2H,H-3,H-4),1.49(s,3H,Me-H),1.43(s,3H,Me-H),1.37(s,3H,Me-H),1.31(s,3H,Me-H)。 13C NMR(101MHz,Chloroform-d)δ137.65,128.40(2C),127.84,127.65(2C),111.79,108.98,105.30,82.67,81.72,81.33,72.54,72.39,67.40,26.85,26.79,26.26,25.45。
化合物3：将化合物2(67g，183mmol)溶解在1M盐酸溶液(480ml)中，80℃下搅拌2h，用TLC检测反应，待原料反应完全后，用饱和碳酸氢钠溶液调节反应液至中性后，用旋蒸旋干溶剂，得到的产物用甲苯共沸三次，在油泵上抽真空过夜。
将上一步得到的新化合物溶于醋酸酐(360ml)，同时加入无水乙酸钠(18.9g)，反应液在80℃下搅拌4h，用TLC检测原料反应完全之后，将反应液倒入冰水中，接着用乙酸乙酯和饱和碳酸氢钠萃取，分离有机相，用无水硫酸钠干燥之后浓缩。浓缩液用硅胶柱纯化(石油醚/乙酸乙酯，4:1)得到化合物3(69g，157mmol，86％)。-构型： 1H NMR(400MHz,Chloroform-d)δ7.36–7.19(m,5H,Ar-H),6.31(d,J＝3.6Hz,1H,1-H),5.16(t,J＝9.8Hz,1H,4-H),5.05(dd,J＝10.0,3.7Hz,1H,2-H),4.71(d,J＝11.8Hz,1H,Bn-H),4.63(d,J＝11.8Hz,1H,Bn-H),4.24–4.16(m,1H,6a-H),4.09–3.92(m,3H,3-H,5-H,6b-H),2.16(s,3H,Me-H), 2.07(s,3H,Me-H),1.99(s,3H,Me-H),1.97(s,3H,Me-H)。 13C NMR(101MHz,Chloroform-d)δ170.58(d,J＝2.1Hz),169.56,169.25,168.73,137.94,128.39(2C),127.78,127.48(2C),89.41,76.97,74.81,71.54,70.24,69.14,61.82,20.79(2C),20.63,20.50。-构型 1H NMR(400MHz,Chloroform-d)δ7.33–7.13(m,5H,Ar-H),5.63(d,J＝8.2Hz,1H,1-H),5.12(td,J＝9.2,4.2Hz,2H,2-H,4-H),4.59(s,2H,2Bn-H),4.19(dd,J＝12.5,5.0Hz,1H,6a-H),4.05(dd,J＝12.4,2.3Hz,1H,6b-H),3.76–3.66(m,2H,3-H,5-H),2.05(s,3H,Me),2.02(s,3H,Me),1.94(s,6H,2Me)。 13C NMR(101MHz,Chloroform-d)δ170.55,169.26,169.08,169.02,137.59,128.43,127.89,127.76,91.92,79.90,74.19,72.89,71.54,69.09,61.78,20.75,20.62.
化合物4：将化合物3(42g，96mmol)溶于无水DCM(240ml)中，加入对甲基苯硫酚(17.9g，144mmol)和适量新鲜活化的分子筛，室温下搅拌30min，随后降温至0℃。继续搅拌20min后缓缓加入三氟化硼乙醚(60ml)。反应液在室温下搅拌24h，用TLC监测反应进程，待原料完全反应之后，用二氯甲烷和饱和碳酸氢钠溶液萃取反应液。分离有机相，经无水硫酸钠干燥后浓缩，浓缩液使用硅胶柱纯化(石油醚/乙酸乙酯，4:1)得到化合物4。(40.5g，81mmol)。 1H NMR(400MHz,Chloroform-d)δ7.45–6.91(m,9H,Ar-H),5.05(t,J＝9.7Hz,1H,4-H),5.02(t,J＝9.5Hz,1H,2-H),4.64–4.51(m,3H,1-H,2Bn-H),4.16(t,J＝3.4Hz,2H,6ab-H),3.71(t,J＝9.2Hz,1H,3-H),3.60(ddd,J＝9.9,5.0,3.1Hz,1H,5-H),2.33(s,1H,Me-H),2.07(s,3H,Me-H),2.04(s,3H,Me-H),1.95(s,3H,Me-H)。 13C NMR(101MHz,Chloroform-d)δ170.59,169.32,169.17,138.38,137.73,133.26,129.60,128.44,127.85,127.76,86.41,81.59,77.46,77.14,76.82,76.06,74.26,71.40(d,J＝3.1Hz),69.71,62.56,21.14,20.96,20.75,20.72。
化合物5：化合物4(27g，54mmol)溶于甲醇(150ml)，室温下加入5M甲醇钠(22ml)，反应液在室温下继续搅拌2h。用TLC监测反应，待原料彻底反应完全之后用Amberlite IR120 H +树脂中和反应液至中性。滤去树脂，旋干滤液得到新化合物(～20g)，油泵上抽真空过夜。
将新化合物(～20g，53mmol)溶于无水DMF，依次加入苯甲醛二甲缩醛(8.3ml)和对甲基苯磺酸(1.0g)。反应液在70℃下搅拌6h，用TLC监测反应，待原料反应完全之后用三乙胺中和反应液。反应液浓缩后用二氯甲烷和饱和食盐水萃取，分离有机相，用无水硫酸钠干燥后浓缩，浓缩液经硅胶柱纯化得到化合物5(19.1g，41mmol)。 1H NMR(400MHz,Chloroform-d)δ7.68–7.21(m,13H,Ar-H),7.13(d,J＝7.9Hz,2H,Ar-H),5.56(s,1H,PhCH-H),4.94(d,J＝11.5Hz,1H,Bn-H),4.78(d,J＝11.5Hz,1H,Bn-H),4.56(d,J＝9.7Hz,1H,1-H),4.38(dd,J＝10.5,4.9Hz,1H,6a-H),3.78(t,J＝10.3Hz,1H,6b-H),3.72–3.65(m,1H,3-H),3.63(t,J＝9.0Hz,1H,4-H),3.47(td,J＝9.3,9.0,5.8Hz,2H,2-H,5-H),2.56–2.49(m,1H,2OH-H),2.34(s,3H,Me)。 13C NMR(101MHz,Chloroform-d)δ138.75,138.20,137.20,133.83,129.81,129.00,128.45,128.25,128.11,127.86,127.20,125.99,101.24,88.58,81.61,81.13,74.81,72.13,70.73,68.64,21.17。
实施例2
糖砌块8的合成
糖砌块8的合成如图2
如图2所示，化合物8是化合物5还原端组装有氨基连接臂的类似物。从化合物5出发，先用乙酰基保护2-OH得到化合物6，接着以对甲基苯硫基作为离去基团，在促进剂NIS和TMSOTf下与五碳氨基连接臂反应得到化合物7。最后，用甲醇和甲醇钠脱去化合物7的2-OAc得到糖砌块8。
具体试验操作和步骤：
化合物6：将化合物5(9.7g，21mmol)溶于干燥吡啶(60ml)中，冰浴下缓缓加入乙酸酐(40ml)。反应液在室温下搅拌6h，用TLC检测原料反应完全之后，用二氯甲烷稀释反应液，用1M盐酸溶液洗涤，饱和碳酸氢钠溶液和二氯甲烷萃取，分离有机相，用无水硫酸 钠干燥后浓缩。浓缩液经硅胶柱(石油醚/乙酸乙酯，7:1)纯化得到化合物6(7.3g，14.4mmol)。 1H NMR(400MHz,Chloroform-d)δ7.54–7.19(m,12H,Ar-H),7.11(d,J＝7.9Hz,2H,Ar-H)5.56(s,1H,PhCH-H),4.99(dd,J＝10.0,8.6Hz,1H,2-H),4.85(d,J＝12.0Hz,1H,Bn-H),4.68–4.60(m,2H,1-H,Bn-H),4.37(dd,J＝10.5,5.0Hz,1H,6a-H),3.79(t,J＝10.3Hz,1H,6b-H),3.75–3.69(m,2H,3-H,4-H),3.47(dq,J＝7.9,5.0Hz,1H,5-H),2.33(s,3H,Me),2.03(s,3H,Me)。 13C NMR(101MHz,Chloroform-d)δ169.22,138.26,137.12,133.39(2C),129.65(2C),128.99,128.29(2C),128.24(2C),128.21,127.87(2C),127.66,125.96(2C),101.19，86.96,81.30,79.77,74.31,71.35,70.46,68.53,21.13,20.95。
化合物7：将化合物6(7.3g，14.4mmol)和氮-苄基-氮-甲酸苄酯-戊醇(8.62g，28.8mmol)溶于无水DCM(100ml)中，加入适量新鲜活化的分子筛，室温下搅拌30min，随后转移至冰浴。30min之后往反应液中一次加入NIS(5.6g，17.3mmol)和TMSOTf(0.3ml，1.7mmol)。继续冰浴下反应10h，用TLC监测反应进程，待原料反应完全之后，依次用10％的硫代硫酸钠，饱和碳酸氢钠溶液和二氯甲烷萃取反应液，分离有机相，用无水硫酸钠干燥之后浓缩。浓缩液经硅胶柱纯化(石油醚/乙酸乙酯，3:1)得到化合物7(7.5g，11.1mmol)。
化合物8：将化合物7(7.0g，10.3mmol)溶于甲醇(50ml)中，室温下加入甲醇钠(110mg)，反应液在室温下搅拌8h，用TLC监测反应，待原料全部反应完毕，用Amberlite IR120H +树脂中和反应液至中性。滤去树脂，浓缩后经硅胶柱纯化(石油醚/乙酸乙酯，3:1)得到化合物8(4.6g，8.45mmol)。
实施例3
糖砌块15的合成
如图3所示，其二丙酮葡萄糖1为起始原料，在NaH的作用下与烯丙基溴反应得到3-烯丙基化合物9。化合物9在加有盐酸的烯丙醇中进行开环反应，得到1,3-二烯丙基化合物10。接下来用苄基保护葡萄糖剩下的三个裸露羟基得到化合物11。化合物11在干燥甲醇中利用氯化钯专属性地脱去1,3号位的烯丙基得到1-OH化合物12。用乙酰基保护前一步反应生成的两个羟基得到化合物13。在醋酸肼的作用下选择性脱除1-OAc基，得到1-OH化合物14。最后用三氯乙腈和DBU与化合物14反应得到三氯乙酰亚胺酯糖基砌块15。
具体试验操作和步骤：
化合物9：将市售的二丙酮葡萄糖1(50g，191mmol)溶于无水DMF(380ml)，冰浴下搅拌30min，随后分批加入氢化钠(23g)和烯丙基溴(34ml)。反应在冰浴下搅拌2h，用TLC监测反应，待原料反应完毕之后用冰水淬灭反应，用二氯甲烷和饱和食盐水萃取反应液，用无水硫酸钠干燥后浓缩，浓缩液经硅胶柱纯化(石油醚/乙酸乙酯，20:1)得到化合物2(57.1g，190mmol)。1H NMR(400MHz，CDCl3):δ＝5.95–5.77(m,2H,1-H,Ally-H),5.31(dd,J＝17.2,1.7Hz,1H,Ally-H),5.20(dt,J＝10.4,1.5Hz,1H,Ally-H),4.54(d,J＝3.7Hz,1H,2-H),4.31(dt,J＝7.7,6.0Hz,1H,5-H),4.34-4.07(m,4H,4-H,6a-H,2Ally-H),4.00(dd,J＝8.6,5.8Hz,1H,6b-H),3.94(d,J＝3.0Hz,1H,3-H),1.50(s,3H,Me),1.43(s,3H,Me),1.35(s,3H,Me),1.31(s,3H,Me)。 13C NMR(101MHz,Chloroform-d)δ134.28,117.41,111.88,109.07,105.36,82.91,81.53,81.30,72.62,71.47,67.41,26.96,26.93,26.37,25.54.
化合物10：将化合物9(52.8g，176mmol)溶于烯丙醇(350ml)中，加入浓盐酸(10.5ml)。反应液在80℃下搅拌2h，用TLC监测反应，待原料反应完全之后，用饱和碳酸氢钠溶液中和反应液至中性。浓缩反应液，经硅胶柱纯化(二氯甲烷/甲醇，25：1)得到化合物10(40.3g，155mmol)。 1H NMR(400MHz,Chloroform-d)δ6.01–5.74(m,2H，2Ally-H),5.24(dt,J＝17.3,1.7Hz,2H,2Ally-H),5.15(ddt,J＝13.5,10.4,1.4Hz,2H,2Ally-H),4.83(d,J＝3.7Hz,1H,H-1),4.38(ddt,J＝12.7,5.7,1.5Hz,1H,Ally-H),4.22(ddt,J＝12.7,6.0,1.4Hz,1H,Ally-H),4.15(ddt,J＝12.8,5.3,1.5Hz,1H,Ally-H),3.97(ddt,J＝12.8,6.3,1.4Hz,1H,Ally-H).3.75(d,J＝3.6Hz,2H,H-6ab),3.59(dt,J＝9.2,3.6Hz,1H,H-5),3.53(dd,J＝9.1,3.8 Hz,1H,H-2),3.51–3.42(m,2H,H-3,H-4)。 13C NMR(101MHz,Chloroform-d)δ135.13,133.49,118.12,117.33,97.73,82.36,73.93,72.52,71.38,69.84,68.60,62.04。
化合物11：将化合物10(38g，146mmol)溶于无水DMF(365ml)中，冰浴下搅拌30min，分批缓缓加入氢化钠(35g)，继续冰浴下搅拌30min，加入溴化苄(61ml)。反应液在冰浴下继续搅拌5h，用TLC监测反应。待原料全部反应后，用冰水淬灭反应，用饱和食盐水和二氯甲烷萃取，分离有机相，用无水硫酸钠干燥后浓缩，浓缩液经硅胶柱纯化得到化合物11(67.8g，127.7mmol)。 1H NMR(400MHz,Chloroform-d)δ7.37(ddt,J＝21.5,14.3,6.6Hz,13H,Ar-H),7.24(d,J＝7.1Hz,2H，Ar-H),6.01(dddt,J＝34.9,17.4,11.7,5.6Hz,1H,Ally-H),5.35(dt,J＝16.1,7.7Hz,1H,Ally-H),5.23(t,J＝10.2Hz,1H,Ally-H),4.87(td,J＝14.4,12.4,6.9Hz,2H,1-H,Bn-H),4.79(d,J＝6.0Hz,1H,Bn-H),4.73–4.56(m,2H,2Bn-H),4.50(t,J＝11.7Hz,3H,Ally-H,2Bn-H),4.35(td,J＝15.1,13.9,5.8Hz,0H)4.19(dd,J＝13.2,5.6Hz,Ally-H),4.05(dd,J＝12.9,6.8Hz,Ally-H),3.91(q,J＝7.8,6.1Hz,1H,3-H),3.85–3.71(m,2H,5-H,6a-H),3.65(dt,J＝14.7,7.7Hz,2H,4-H,6b-H),3.60–3.51(m,1H,2-H)。 13C NMR(101MHz,CDCl 3)δ138.54,138.35,138.24,137.99,135.40,135.21,134.12,133.81,128.43,128.39,128.37,128.25,128.09,128.05,128.03,127.95,127.93,127.83,127.80,127.72,127.62,118.15,117.20,116.74,116.54,102.69,95.82,84.40,82.19,81.81,79.65,77.83,77.68,77.42,77.33,77.09,77.01,76.78,76.70,75.12,75.05,74.93,74.85,74.52,74.42,73.50,73.22,70.32,70.20,69.01,68.48,68.19.
化合物12：将化合物11(10.2g，19.3mmol)溶于无水甲醇(644ml)中，加入氯化钯(684.5mg)，反应液在40℃下搅拌20min，用TLC监测反应，待原料完全反应之后，用饱和碳酸氢钠和二氯甲烷萃取反应液，分离有机相，用无水硫酸钠干燥之后浓缩，浓缩液经硅胶柱纯化(石油醚/乙酸乙酯，3:1)得到化合物12(6.5g，14.4mmol)。 1H NMR(400MHz,Chloroform-d)δ7.31–7.15(m,15H,Ar-H),5.18(d,J＝3.5Hz,1H,1-H),4.92(d,J＝11.5Hz,1H,Bn-H),4.77(d,J＝11.3Hz,1H,Bn-H),4.65(d,J＝11.8Hz,1H,Bn-H),4.59(d,J＝11.8Hz,1H,Bn-H),4.54(d,J＝12.2Hz,1H,Bn-H),4.43(d,J＝12.2Hz,1H,Bn-H),4.04(t,J＝9.2Hz,1H,3-H),3.96(ddd,J＝10.1,3.9,2.5Hz,1H,5-H),3.66–3.58(m,2H,6ab-H),3.46(dd,J＝10.0,8.9Hz,1H,4-H),3.36(dd,J＝9.5,3.5Hz,1H,2-H)2.91(s,2H,OH-H)。 13C NMR(101MHz,CDCl 3)δ138.36,137.80,137.74,128.62,128.58,128.44,128.42,128.20,128.13,128.10,128.05,128.00,127.95,127.91,127.81,127.78,127.76,97.11,90.71,82.19,79.54,77.65,77.45,77.41,77.29,77.09,76.77,76.56,74.60,74.52,74.29,73.50,73.27,72.83,69.78,68.72.
化合物13：将化合物12(6.1g，14mmol)溶于吡啶(40ml)，冰浴下搅拌30min，缓缓加入乙酸酐(30ml)。反应液在冰浴下搅拌过夜。用TLC监测反应进程，待原料全部反应完全后，用1M盐酸溶液淬灭反应。用饱和碳酸氢钠溶液和乙酸乙酯萃取反应液，分离有机相，用无水硫酸钠干燥后浓缩，浓缩液经硅胶柱纯化(石油醚/乙酸乙酯，6:1)得到化合物13。(6.77g，12.66mmol)。 1H NMR(400MHz,Chloroform-d)δ7.52–7.15(m,13H,Ar-H),7.12(dd,J＝7.6,1.9Hz,2H,Ar-H),5.63(d,J＝8.1Hz,1H,H-1),5.26(t,J＝9.4Hz,1H,H-3),4.69(d,J＝12.0Hz,1H,Bn-H),4.65(d,J＝12.0Hz,1H,Bn-H),4.58(d,J＝12.0Hz,1H,Bn-H),4.47(t,J＝6.0Hz,3H,Bn-H),3.80–3.67(m,3H,H-4,H-6a,H-6b),3.60(dt,J＝9.9,2.5Hz,1H,H-5),3.51(dd,J＝9.5,8.1Hz,1H,H-2),2.07(s,3H,Me),1.84(s,3H,Me)。 13C NMR(101MHz,Chloroform-d)δ169.80,168.98,137.88,137.79,128.43,128.11,127.88,127.85,127.83,94.07,78.45,75.61,75.53,75.34,74.38,74.31,73.64,67.85,21.07,20.96。
化合物14：将化合物13(1.6g，3.0mmol)溶于无水DMF(30ml)，加入乙酸肼(305mg)。反应液在40℃下搅拌2h，用TLC监测反应，待原料反应完毕以后将反应液浓缩。用饱和食盐水和DCM萃取，分离有机相，经无水硫酸钠干燥后浓缩，浓缩液用硅胶柱纯化(石油醚/乙酸乙酯，2:1)得到化合物14(1.36g，2.76mmol)。 1H NMR(400MHz,Chloroform-d)δ7.37–7.21(m,13H,Ar-H),7.13(d,J＝7.2Hz,2H,Ar-H),5.50(t,J＝9.6Hz,1H,3-H),5.27(d,J＝3.6 Hz,1H,1-H),4.67–4.37(m,6H,Bn-H),4.07(d,J＝10.0Hz,1H,6a-H),3.78(s,1H,1-OH),3.73–3.58(m,3H,4-H,5-H,6b-H),3.48(dd,J＝9.8,3.4Hz,1H,2-H),1.91(s,3H,Me)。 13C NMR(101MHz,CDCl 3)δ170.14,137.96,137.77,137.64,128.53,128.46,128.43,128.34,128.13,128.12,128.03,128.00,127.95,127.90,127.86,127.78,90.86,77.58,77.50,77.18,76.86,76.10,74.36,74.28,73.90,73.58,73.55,73.45,72.56,69.91,68.37,21.12.
化合物15：将化合物14(3 00mg，0.61mmol)溶于无水DCM(6ml)中，冰浴下加入三氯乙腈(0.6ml)和DBU(91μL)。反应液在冰浴下搅拌2h，用TLC监测反应，待原料全部反应完毕，用饱和食盐水和二氯甲烷萃取反应液，分离有机相，经无水硫酸钠干燥后浓缩，浓缩液用硅胶柱纯化得到化合物15(357mg，0.56mmol)。 1H NMR(400MHz,Chloroform-d)δ8.59(s,1H,NH-H),7.28(dt,J＝19.8,5.5Hz,13H,Ar-H),7.19–7.14(m,2H,Ar-H),6.54(d,J＝3.5Hz,1H,H-1),5.59(t,J＝9.7Hz,1H,H-3),4.65(d,J＝12.4Hz,1H,Bn-H),4.60(d,J＝11.9Hz,1H,Bn-H),4.55–4.48(m,3H,Bn-H),4.46(d,J＝11.8Hz,1H,Bn-H),4.04(dt,J＝10.1,2.5Hz,1H,H-6a),3.84–3.73(m,2H,H-6b,H-4),3.67(dd,J＝10.0,3.6Hz,2H,H-2,H-5),1.91(s,3H,Me)。 13C NMR(101MHz,Chloroform-d)δ169.87,161.34,137.82,137.77,137.74,93.96,91.20,76.51,75.48,74.66,73.67,73.19,72.99,72.53,67.90,21.12。
实施例4
二糖化合物的16和18合成
如图4所示，二糖化合物16由单糖供体15和单糖受体5在TMSOTf作为促进剂的条件下合成。以单糖供体15和单糖受体8用类似的方法合成二糖化合物17，随后17在甲醇甲醇钠的条件下脱去3’-OAc得到二糖化合物18。
化合物16：将糖基供体15(3.1g，4.8mmol)和糖基受体(2.8g，6.1mmol)混合，在氮气保护下用甲苯共沸3次，在油泵下抽真空过夜。加入二氯甲烷(100ml)和适量分子筛，室温下搅拌30min后冷却至-78℃，继续搅拌下30min，加入促进剂TMSOTf(100μL)，反应在低温下搅拌5h。用TLC监测反应进程，待糖基供体全部反应消耗之后，用三乙胺淬灭反应。用二氯甲烷和饱和碳酸氢钠溶液萃取反应液，分离有机相，用无水硫酸钠干燥后浓缩。浓缩液用硅胶柱纯化(石油醚/乙酸乙酯，10:1)得到化合物16(2.9g，3.2mmol)。 1H NMR(400MHz,Chloroform-d)δ7.52–7.45(m,2H,Ar-H),7.41–7.23(m,21H,Ar-H),7.14(td,J＝7.5,1.6Hz,3H,Ar-H),7.07(d,J＝7.9Hz,2H,Ar-H),7.02(t,J＝7.6Hz,2H,Ar-H),5.99(d,J＝3.7Hz,1H,1’-H),5.60(s,1H,PhCH),5.55(t,J＝9.7Hz,1H,3’-H),4.99(d,J＝10.1Hz,1H,Bn-H),4.90(d,J＝8.9Hz,1H,1-H),4.85(d,J＝12.2Hz,1H,Bn-H),4.64(d,J＝10.2Hz,1H,Bn-H),4.61–4.51(m,2H,2Bn-H),4.47(d,J＝11.2Hz,1H,Bn-H),4.41–4.35(m,1H,6A-H),4.34(d,J＝11.0Hz,1H,Bn-H),4.26(dd,J＝11.1,5.3Hz,2H,5’-H,Bn-H),3.93–3.83(m,2H,2-H,3-H),3.80(d,J＝10.3Hz,1H,6A-H),3.74(dd,J＝12.3,3.3Hz,1H,4-H),3.71–3.66(m,1H,4’-H),3.56(dd,J＝10.3,3.6Hz,1H,2’-H),3.51(dd,J＝9.6,5.0Hz,1H,5-H),3.17–3.06(m,2H,6’-H),2.35(s,3H,Me),1.96(s,3H,Me)。 13C NMR(101MHz,CDCl 3)δ192.42,170.09,163.32,138.36,138.26,138.17,138.15,137.86,137.83,137.77,137.47,137.19,136.42,134.48,132.49,132.33,129.85,129.81,129.76,129.02,128.56,128.44,128.37,128.34,128.32,128.29,128.25,128.19,127.85,127.82,127.76,127.75,127.68,127.55,125.99,101.28,95.59,88.02,85.23,81.98,81.09,78.88,76.04,75.95,75.58,74.84,74.42,74.39,74.32,73.59,73.47,72.48,72.36,71.33,70.10,69.92,69.78,68.67,67.73,67.41,62.62,21.14,21.12。
化合物17：将糖基供体15(1.42g，2.3mmol)和带有氨基连接臂的糖基受体8(1.11g，1.6mmol)混合，在氮气保护下用甲苯共沸3次，在油泵下抽真空过夜。之后加入无水DCM(100ml)和适量分子筛，室温下搅拌30min后降温至-78℃，继续搅拌30min后，加入促进剂TMSOTf(37μL)，反应液在低温下继续搅拌6h。用TLC监测反应，待糖基供体完全消耗之后，用三乙胺淬灭反应。用二氯甲烷和饱和碳酸氢钠溶液萃取反应液，分离有机相，用无水硫酸钠干燥后浓缩。浓缩液用硅胶柱纯化(石油醚/乙酸乙酯，4:1)得到化合物17(1.35g， 1.2mmol)。 1H NMR(400MHz,Chloroform-d)δ7.48–6.97(m,35H,Ar-H),5.64(s,1H,1’-H),5.55(d,J＝7.7Hz,2H,PhCH,3’-H),5.16(d,J＝10.3Hz,2H,2Bn-H),4.92(d,J＝10.2Hz,1H,Bn-H),4.65(t,J＝12.3Hz,2H,2Bn-H),4.55(t,J＝10.5Hz,2H,1-H,Bn-H),4.50–4.40(m,4H,2Cbz-H,2Bn-H),4.32(d,J＝10.8Hz,1H,6a-H,Bn-H),4.23(t,J＝9.4Hz,2H,5’-H,Bn-H),3.73(ddt,J＝37.9,19.0,9.3Hz,6H,2-H,4-H,5-H,6b-H,4’-H,linker-OCH),3.50(d,J＝9.7Hz,1H,2’-H),3.41(q,J＝8.7Hz,2H,3-H,linker-OCH),3.18(q,J＝11.6,10.7Hz,4H,6’ab-H,linker-NCH 2),1.91(s,3H,Me),1.70–1.34(m,4H,linker-CH2),1.22(d,J＝27.7Hz,2H,linker-CH 2)。
化合物18：将化合物17(1.1g，0.96mmol)溶于无水甲醇(20ml)，加入甲醇钠(100mg)。反应液在40℃下搅拌24h，用TLC监测反应，待原料全部反应完全之后用Amberlite IR120 H +树脂中和反应液至中性。滤去树脂，浓缩后经硅胶柱纯化(石油醚/乙酸乙酯，3:1)得到化合物18(898mg，0.82mmol)。 1H NMR(400MHz,Chloroform-d)δ7.67–6.95(m,35H,Ar-H),5.68(s,1H,1’-H),5.62(s,1H,PhCH-H),5.21(d,J＝11.2Hz,2H,Bn-H),4.94(d,J＝10.1Hz,1H,Bn-H),4.83(d,J＝11.3Hz,2H,Bn-H),4.60(t,J＝11.1Hz,4H,1-H,1Bn-H,2Cbz-H),4.50(t,J＝10.5Hz,3H,3Bn-H),4.39(d,J＝9.8Hz,1H,6a-H),4.29(d,J＝12.1Hz,1H,Bn-H),4.25–4.08(m,2H,3’-H,5’-H),3.79(dq,J＝18.3,9.5Hz,5H,2-H,3-H,4-H,6b-H,linker-OCH),3.65(t,J＝9.5Hz,1H,4’-H),3.57–3.37(m,3H,5-H,2’-H,linker-OCH),3.34–3.14(m,4H,6’ab-H,linker-NCH 2),2.50(s,1H,3’-OH),1.52(ddt,J＝22.4,15.3,7.9Hz,4H,linker-CH 2),1.41–1.14(m,2H,linker-CH2)。 13C NMR(101MHz,CDCl 3)δ156.72,156.22,138.90,138.05,137.93,137.66,137.30,129.03,128.59,128.49,128.32,128.28,128.26,128.14,127.98,127.89,127.73,127.68,127.62,127.54,127.34,125.99,104.00,101.23,95.10,82.28,79.60,79.03,77.63,77.42,77.31,77.10,76.79,75.65,75.57,74.63,73.51,73.40,71.97,69.62,69.49,68.81,67.86,67.21,65.95,50.53,50.24,47.05,46.15,29.74,29.63,27.96,27.54,23.33。
实施例5
四糖19的合成如图5所示。
化合物19：将二糖糖基供体15(287mg，0.3mmol)和二糖糖基受体18(230mg，0.12mmol)混合，在氮气保护下用甲苯共沸3次，在油泵下抽真空过夜。之后加入无水DCM(2ml)乙醚(4ml)和噻吩(12.6mmol)以及适量分子筛，室温下搅拌30min后降温至0℃，继续搅拌30min后，加入促进剂NIS(135mg)和AgOTf(26mg)，反应液在冰浴下继续搅拌12h。用TLC监测反应，待糖基供体完全消耗之后，用10％硫代硫酸钠溶液和二氯甲烷萃取反应液，分离有机相，用无水硫酸钠干燥后浓缩。浓缩液用硅胶柱纯化(石油醚/乙酸乙酯，4:1)得到化合物19(121mg，0.064mmol)。 1H NMR(400MHz,Chloroform-d)δ5.81(s,1H，a1-H),5.73(t,J＝9.7Hz,1H,c3-H),5.63(s,1H,b1-H),5.55(d,J＝5.6Hz,2H,2PhCH),5.19(d,J＝8.9Hz,3H,1c-H,2Bn-H),4.90(d,J＝10.9Hz,1H,Bn-H),4.79(d,J＝15.8Hz,3H,3Bn-H),4.54(ddt,J＝47.2,27.9,9.9Hz,13H,d1-H,a3-H,2Cbz-H,9Bn-H),4.38–4.14(m,8H,d3-H,b5-H,c5-H,c6-H2,3Bn-H),4.03(t,J＝9.3Hz,1H,b3-H),3.74(td,J＝30.8,27.3,8.7Hz,9H,a6-H2,a2-H,b4-H,d2-H,d5-H,c4-H,a5-H,linker-OCH),3.49(ddd,J＝42.6,21.7,9.6Hz,6H,d6-H2,d4-H,b2-H,a2-H,a4-H),3.20(d,J＝34.7Hz,5H,b6-H2,linker-NH 2,linker-OCH),1.89(s,3H,Me)。 13C NMR(101MHz,CDCl 3)δ169.61,138.61,138.32,138.16,138.02,137.96,137.68,137.61,137.34,136.87,130.92,128.96,128.80,128.75,128.69,128.56,128.48,128.38,128.32,128.26,128.21,128.09,128.03,127.95,127.86,127.73,127.66,127.62,127.59,127.56,127.51,127.47,127.25,126.18,125.99,103.93,101.22,95.04,94.72,94.50,82.61,82.21,79.44,78.54,78.31,77.36,77.04,76.72,76.25,75.72,75.49,75.37,75.18,74.15,73.44,73.38,73.16,72.13,71.40,69.92,69.43,69.17,68.91,68.81,68.16,67.93,67.18,65.80,65.58,62.36,50.48,47.05,46.15,29.72,29.55,27.89,27.49,23.20,21.09,19.20,13.74。
实施例6 1,3-α-顺式糖苷键构建体系的优化：
1)溶剂对1，3-α-顺式糖苷键构建的影响：
参照实施例5，经溶剂分别替换为表1中所示的溶剂，测定所得产物中1，3-α-顺式糖苷键的含量。结果见表1。
表1 不同溶剂和添加剂体系构建1,3-α-顺式糖苷键
Figure PCTCN2019091200-appb-000012
2)促进剂对1,3-α-顺式糖苷键构建的影响：
参照实施例5，将促进剂分别替换为表2中所示的添加剂，测定所得产物中1，3-α-顺式糖苷键的含量。结果见表2。
表2 不同促进剂条件构建1,3-α-顺式糖苷键
Figure PCTCN2019091200-appb-000013
实施例7
六糖21的合成如图6所示。
四糖化合物19在甲醇和甲醇钠的条件下反应两天脱去3””-OAc得到四糖化合物20。随后以化合物20为糖基受体与二糖供体16在路易斯酸促进剂的催化下反应得到六糖化合物21。
化合物20：将化合物19(90mg，0.047mmol)溶于甲醇(9ml)和乙酸乙酯(1ml)的混合溶剂中，加入甲醇钠(30mg)。反应在40℃下搅拌2d，用TLC监测反应，用待原料全部反应完全之后用Amberlite IR120 H +树脂中和反应液至中性。滤去树脂，浓缩后经硅胶柱纯化(石油醚/乙酸乙酯，3:1)得到化合物21(68mg，0.037mmol)。 1H NMR(400MHz,Chloroform-d)δ5.79(d,J＝3.4Hz,1H,a1-H),5.68(d,J＝5.3Hz,1H,b1-H),5.55(d,J＝2.7Hz,2H,2PhCH),5.19(d,J＝9.2Hz,2H,2Bn-H),5.06(d,J＝3.4Hz,1H,c1-H),4.97(d,J＝11.1Hz,1H,Bn-H),4.79(ddd,J＝21.1,11.9,7.4Hz,4H,4Bn-H),4.68–4.44(m,11H,d1-H,b3-H,a4-H,6Bn-H,2Cbz-H),4.46–4.37(m,2H,2Bn-H),4.36–4.23(m,6H,d3-H,a5-H,2Bn-H,6-H2),4.17(t,J＝9.3Hz,1H,c3-H),4.12–4.01(m,2H,c4-H,a3-H),3.87–3.68(m,6H,d2-H,b4-H,c5-H,6-H2,linker-OCH),3.68–3.62(m,2H,a2-H,d5-H),3.60(d,J＝9.4Hz,2H,b2-H,b5-H),3.45(dd,J＝11.1,3.1Hz,1H,6-H),3.40–3.33(m,2H,c2-H,d4-H),3.30–3.11(m,6H,linker-NCH 2,linker-OCH,6-H,6-H2),1.56–1.34(m,4H,2linker-CH 2),1.23–1.06(m,4H,linker-CH 2)。 13C NMR(101MHz,CDCl 3)δ156.70,156.18,138.86,138.70,138.24,138.20,138.14,138.07,138.01,137.93,137.78,137.62,137.44,137.31,136.91,129.79,129.23,129.01,128.81,128.71,128.69,128.65,128.59,128.50,128.42,128.32,128.29,128.25,128.22,128.20,128.16,128.15,128.02,127.97,127.88,127.84,127.75,127.66,127.62,127.60,127.56,127.48,127.42,127.35,126.16,126.00,103.92,102.01,101.26,101.21,95.76,95.36,94.59,82.79,82.11,81.12,79.58,79.43,78.86,78.25,78.07,77.40,77.28,77.08,76.76,75.35,75.31,74.79,74.67,74.47,73.85,73.57,73.45,73.39,73.29,71.93,71.75,69.92,69.23,68.77,68.21,67.90,67.20,65.81,62.70,50.50,50.20,47.05,46.13,29.74,29.58,27.91,27.50,23.23.
化合物21：将二糖供体15(60mg，0.064mmol)和四糖受体20(50mg，0.027mmol)在氮气保护下用甲苯共沸3次，在油泵下抽真空过夜。之后加入无水DCM(0.5ml)乙醚(1ml)和噻吩(0.6ml)以及适量分子筛，室温下搅拌30min后降温至0℃，继续搅拌30min后，加入促进剂NIS(29mg)和AgOTf(3mg)，反应液在冰浴下继续搅拌12h。用TLC监测反应，待糖基供体完全消耗之后，用10％硫代硫酸钠溶液和二氯甲烷萃取反应液，分离有机相，用无水硫酸钠干燥后浓缩。浓缩液用硅胶柱纯化(石油醚/乙酸乙酯，4:1)得到化合物 21(33.4mg，0.012mmol)。 1H NMR(400MHz,Chloroform-d)δ5.91(s,1H,a1-H),5.75(d,J＝12.4Hz,2H,b1-H,b3-H),5.58(s,2H,c1-H,PhCH-H),5.52(s,1H,PhCH),5.50(s,1H,PhCH),5.36(s,1H,d1-H),5.22(s,3H,e1-H,2Bn-H),4.94–4.72(m,7H,7Bn-H),4.53(ddt,J＝44.2,17.1,10.5Hz,18H,f1-H,c3-H,d3-H,2Cbz-H,13Bn-H),4.34(dt,J＝23.0,10.9Hz,7H,f4-H,4Bn-H,6-H2),4.22(d,J＝11.1Hz,2H,f5-H,b5-H),4.08(q,J＝11.8,9.3Hz,3H,a3-H,e3-H,6-H),3.90(d,J＝9.6Hz,1H,a2-H,6-H),3.88–3.64(m,10H,f2-H,f3-H,b4-H,d4-H,e4-H,a5-H,c3-H,6-H2,linker-OCH),3.53(dp,J＝32.8,11.4,10.7Hz,10H,a4-H,c2-H,b2-H,e2-H,c5-H,d2-H,d5-H,e5-H,6-H2),3.36–3.10(m,5H,b6-H2,linker-NCH 2,linker-OCH),1.93(s,3H,Me),1.57–1.40(m,4H,2linker-CH 2),1.23–1.11(m,2H,linker-CH 2)。 13C NMR(176MHz,CDCl 3)δ206.98,192.41,169.83,169.74,156.70,138.41,137.94,137.90,137.71,137.29,136.90,136.44,134.48,130.93,129.77,129.02,128.86,128.84,128.62,128.57,128.50,128.43,128.38,128.34,128.28,128.24,128.05,128.01,127.91,127.87,127.75,127.70,127.65,127.59,127.56,127.53,127.50,127.47,127.45,127.40,127.37,127.33,127.29,126.24,126.21,126.16,126.01,103.95,101.28,101.18,94.82,94.36,94.12,93.96,82.53,82.21,79.40,78.67,77.22,77.04,76.86,76.16,75.78,75.59,75.38,75.20,74.89,74.27,74.18,73.45,73.42,73.39,73.27,72.97,72.13,71.17,70.51,70.07,69.81,69.24,68.98,68.80,68.60,68.15,67.83,67.20,65.83,62.13,50.47,50.16,47.03,46.10,30.96,29.73,29.54,27.88,27.47,23.20,22.72,21.13,21.11,14.15。
依照此方法可以合成八糖，十糖，十二糖等一系列幽门螺旋杆菌O2-血清型寡糖衍生物。
寡糖链可以以+2糖的模式由还原端向非还原端延伸，包括两个步骤。1)还原端带有连接臂的寡糖脱去非还原端的脂类基团；2)与二糖供体16的糖基化反应。
脱去寡糖非还原端脂类基团的方法：将化合物(～50mg)溶于甲醇(10～15ml)中，可以适量添加少量乙酸乙酯或者四氢呋喃增加溶解性。加入甲醇钠(20～30mg)，反应液在40℃下搅拌12h～2d。用TLC监测反应进程，待原料全部反应完全之后用Amberlite IR120 H +树脂中和反应液至中性。滤去树脂，浓缩后经硅胶柱纯化。
以二糖16为糖基供体进行糖苷化反应的方法：将糖基供体和糖基受体在氮气保护下用甲苯共沸三次，在油泵上抽真空过夜。将反应物溶于二氯甲烷和乙醚(1:1)的混合溶剂中，同时加入相对于糖基供体100当量的噻吩和适量分子筛。加入NIS和AgOTf，TMSOTf和TfOH中的一种或者几种作为促进剂。反应浓度为0.02M(相对于糖基供体)，反应时间为12～24h。用TLC监测反应进程，待糖基供体反应完全之后用10％硫代硫酸钠、二氯甲烷和饱和碳酸氢钠溶液萃取反应液，分离有机相，用无水硫酸钠干燥，浓缩后经硅胶柱纯化得到新的寡糖化合物。
实施例7：二糖，四糖和六糖的脱保护
如图7所示，二糖，四糖和六糖化合物的脱保护分为两个步骤。第一，在碱性条件下脱除非还原端的乙酰基；第二，在钯碳的催化下氢化掉所有的芳香基团，得到还原端组装有五碳氨基连接臂的二糖、四糖和六糖。
脱除非还原端二糖乙酰基的方法：将化合物溶于甲醇和乙酸乙酯(9:1)的混合溶剂中，浓度为0.02M，加入相对于原料0.5当量的甲醇钠。反应液在40℃下搅拌12h～2d。用TLC监测反应进程。待原料全部反应完全之后用Amberlite IR120 H +树脂中和反应液至中性。滤去树脂，浓缩后经硅胶柱纯化。
氢化反应的方法：将化合物(6～10mg)溶于异丁醇(3ml)、甲醇(1ml)和水(1ml)的混合溶液中，加入冰乙酸(0.1ml)和钯碳(50mg)。反应液在中压氢气反应釜中进行，反应压力为0.4MPa，反应时间为12h。待原料反应完全之后用硅藻土过滤反应液，浓缩后经C18小柱纯化得到目标化合物。
具体试验和操作步骤：
化合物22：以二糖化合物17为原料，经过脱乙酰基和氢化两个步骤得到脱保护的二糖化合物22。 1H NMR(400MHz,Deuterium Oxide)δ5.37(d,J＝3.8Hz,1H,1’-H),4.61(d,J＝7.9 Hz,1H,1-H),4.05(dt,J＝10.2,3.3Hz,1H,5’-H),4.00–3.89(m,2H,6-H,linker-OCH),3.81(d,J＝3.4Hz,2H,6’-H2),3.77–3.67(m,3H),3.58(t,J＝9.0Hz,1H,4-H),3.52(dd,J＝9.9,3.9Hz,1H,2’-H),3.49–3.36(m,4H,4’-H,2-H,3-H,5-H),3.02(t,J＝7.6Hz,2H,linker-NCH 2),1.70(q,J＝7.2Hz,4H,2linker-CH 2),1.57–1.39(m,2H,linker-CH 2)。 13C NMR(101MHz,D 2O)δ102.77,97.61,77.02,75.71,74.50,72.91,71.63,71.43,70.18,69.84,69.30,60.81,60.23,39.37,28.28,26.43,22.28。
化合物23：以四糖化合物19为起始原料，经过脱乙酰基和氢化两个步骤得到脱保护的四糖化合物23。 1H NMR(400MHz,Deuterium Oxide)δ5.44(d,J＝3.6Hz,1H,a1-H),5.28(d,J＝3.9Hz,1H,b1-H),5.08(d,J＝3.7Hz,1H,c1-H),4.53(d,J＝8.1Hz,1H,d1-H),3.96(d,J＝9.6Hz,2H,a3-H,c4-H),3.92–3.65(m,12H,linker-OCH,b3-H,c3-H,d3-H,b4-H,d5-H,6*6-H),3.65–3.44(m,7H,linker-OCH,a2-H,a5-H,b2-H,c2-H,2*6-H),3.43–3.22(m,5H,a4-H,b5-H,d2-H,d4-H,c5-H),2.90(t,J＝7.6Hz,2H,linker-NCH 2),1.58(q,J＝7.4Hz,4H,linker-CH 2),1.36(q,J＝8.0Hz,2H,linker-CH 2)。 13C NMR(101MHz,D 2O)δ102.75,97.71,96.49,95.98,79.83,76.87,75.74,75.37,74.50,72.84,71.82,71.54,71.28,70.21,69.88,69.51,69.29,60.84,60.32,39.38,28.30,26.45,22.29。
化合物24：以六糖化合物21为起始原料，经过脱乙酰基和氢化两个步骤得到脱保护的六糖化合物24。 1H NMR(400MHz,Deuterium Oxide)δ5.51(d,J＝3.7Hz,1H,a1-H),5.48(d,J＝3.7Hz,1H,b1-H),5.31(d,J＝3.9Hz,1H,c1-H),5.13(d,J＝3.8Hz,1H,d1-H),5.11(d,J＝3.7Hz,1H,e1-H),4.55(d,J＝8.0Hz,1H,f1-H),4.09–3.25(m,38H,6*2-H,6*3-H,6*4-H,6*5-H,6*6-H2,linker-OCH 2),2.93(t,J＝7.6Hz,2H,linker-NCH 2),1.61(h,J＝6.9,6.2Hz,4H,linker-CH 2),1.43–1.30(m,2H,linker-CH 2)。 13C NMR(101MHz,D 2O)δ102.77,96.58,95.99,79.76,79.48,76.78,75.76,75.34,75.03,74.51,72.85,71.82,71.63,71.29,71.14,70.22,70.04,69.87,69.72,69.54,69.31,60.84,60.58,60.33,60.05,39.38,28.32,26.46,22.31。相应核磁图谱如图8-11所示。
实施例8：单糖砌块制备的优化
为了得到C-2和C-3分别为羟基的葡萄糖砌块，我们从市售D-葡萄糖出发，先用乙酸酐和乙酸钠在高温下给葡萄糖的羟基上保护住乙酰基，随后通过结晶得到的全乙酰基葡萄糖108大都为β-异头物。随后在路易斯酸的辅助下全乙酰葡萄糖与乙硫醇反应得到硫糖苷葡萄糖109。继续用甲醇钠水解掉剩余四个乙酰基之后得到110，将硫糖苷葡萄糖110的4,6号位羟基用苄叉基保护，得到空出2,3号位羟基的硫糖苷葡萄糖107。
Figure PCTCN2019091200-appb-000014
由于葡萄糖C2和C3号位性质接近，无法用化合物107通过随机上苄基得到，这样也让我们需要重新设计糖砌块路线，而且需要从一开始就将C-2和C-3的结构区别开。为此，我们选择市售的二丙酮葡萄糖为起始原料重新设计反应路线，与前期设计路线是所选取的D-葡萄糖相比，二丙酮葡萄糖最大的利处在于C-3自留一个羟基，这就从一开始区别了C-2和C-3羟基的性质。因此我们重新设计糖砌块，优选市售二丙酮葡萄糖为起始原料，重新合成两个 葡萄糖砌块用于组装幽门螺旋杆菌O2血清型O-抗原寡糖(分别对应实施例1和实施例2)。
实施例9：糖基化反应的优化
在有二糖124和138的基础上，进一步探寻了连接四糖的条件。我们首先在纯二氯甲烷中尝试了糖苷化反应，以最常用的碘带丁二酰亚胺和一种路易斯酸三甲基硅三氟甲磺酸酯反应得到四糖133。但遗憾的是这个反应的选择性不够，α/β的选择性仅仅有1∶1.8。为了尽可能多的得到更多的目标化合物所需要的α构型的四糖133，我们尝试了各种不同的溶剂，发现向反应溶剂中添加适量乙醚可以适量地提高α构型，例如将反应溶剂换做二氯甲烷与乙醚(1∶3)的31％混合溶剂之后，四糖α构型的选择性提高了8倍，达到了α/β，4∶1的结果，但是产率下降了。通过向二氯甲烷与乙醚混合溶剂中添加相对于糖基供体100倍当量噻吩发现得到的四糖中α构型的比例进一步提高，达到的α/β＞10∶1，在选择性上满足实验需求，不过反应产率较低，仅有44％。为了尝试提高这个糖基化反应的产率，我们尝试不同糖基化反应的促进剂，包括碘带丁二酰亚胺与路易斯酸三甲基硅三氟甲磺酸酯，三氟甲磺酸或者三氟甲磺酸银等，以及多种促进剂的叠加使用，最终发现在应用硫糖苷124为糖基供体，辅以碘带丁二酰亚胺、三氟甲磺酸银和三甲基硅三氟甲磺酸酯共同使用作为促进剂，并且在DCM与乙醚的混合溶剂(1/3)且添加噻吩的溶剂中反应，能够以59％的产率得到α/β，10∶1的四糖化合物133。
表3 合成四糖化合物133的条件优化
Figure PCTCN2019091200-appb-000015
Figure PCTCN2019091200-appb-000016
为了进一步提高反应的产率和α-立体选择性，我们对四糖的反应做了一些优化，两条主要的优化路线如下：1)尽管二糖化合物124上的硫苷能够用于被碘带丁二酰亚胺和路易斯酸活化用于糖基化反应，但是活化效率较低。尽管逐步加大了促进剂的当量，但是依然从糖基化反应后能回收得到未活化的受体。为此，将硫苷换作一种反应活性更好的离去基团不失为一种提高四糖合成产率的方法；2)此外，发明人经过实验发现在供体上引入强吸电子的酯类基团对糖基化反应的α-立体选择性，因此可以考虑在二糖供体上引入酯类基团来提高四糖反应的α-立体选择性。
不过，按照表3(编号6)的方法，以127为供体得到的四糖产率也仅为62％，α-立体选择性也可以达到α/β>10:1。尽管在产率上，二糖TFAI供体137比起二糖硫糖苷供体124在产率上高出一点点，但是在α-立体选择性并没有明显的提升，仅仅高出2％。而且，从124到127需要经过两步反应，产率仅为49％。比起所损失的糖的比例，四糖化合物133所提升的产率显得微不足道，从总体合成的角度来考虑，显得并不值得这么做。
为了在二糖供体上引入脂类基团，最容易想到的方式就是先将二糖124还原端葡萄糖残基上的苄叉环打开，从而空出C-4和C-6位羟基用来保护酯基。由于124上的疏水基团较多，不溶于水相，因而采用在二氯甲烷中以三氟乙酸方式水解掉苄叉基得到2羟基化合物139，随后将空出的羟基用苯甲酰基保护得到化合物140。两步反应总产率为61％。
Figure PCTCN2019091200-appb-000017
接下来利用新合成的二糖供体140与二糖受体138糖基化反应，结果发现供体140并不能被促进剂活化，不管是提高反应温度至40℃还是提高促进剂的用量都不能够活化140。反应的过程中140既没有被水解，也没有与受体反应，反应后也能从反应液中回收到足量的140。140之所以难活化的原因发明人推测有以下两点：1)本身硫苷就较为稳定，这个在124用作供体的时候就活化不完全可以得出；2)苯甲酰基具有极强的吸电子能力，从而降低了端基位的电子云密度，使糖基供体钝化。因此，为了提高140的反应活性，必须将硫苷换成反应性更好的离去基团。类似从124到127的过程，140也经历相同的两步反应得到糖基TFAI供体142。
Figure PCTCN2019091200-appb-000018
随后应用供体142与糖基受体138糖基化反应生成四糖，应用三甲基硅三氟甲磺酸酯为促进剂，得到纯α-立体选择的四糖化合物143，这也是得益于苯甲酰基的远程作用。
Figure PCTCN2019091200-appb-000019
不过，尽管在立体选择性上看，苯甲酰基二糖供体在142糖苷化反应中表现出优异的选择性，但是从合成路线的效率上来看，这一改进的意义并不大。我们默认二糖受体在糖基化反应中是不损失的，除了参与反应的部分都可以回收回来，那么讨论糖基化反应的效率就取决于二糖的供体，我们考虑从二糖124开始到四糖133或者四糖143的利用率，以硫苷二糖124直接糖基化反应的得到133的总产率为53％，(59％×90％),经由糖基TFAI供体127与受体138糖基化反应得到133的总产率为28.5％，(71％×72％×62％×90％)，而第三种途径经由苄氧糖基供体142糖基化反应得到143的总产率仅为15.5％，(84％×73％×63％×73％×55％×100％)。因此，基于糖基化反应的效率来说，我们依然选用尽管在糖基化反应中产率不是最高，但α-立体选择性较好，且总路线最短，总产率遥遥领先另外两个路径的方法。因此我们采用这一方法完成四糖133的合成。
虽然本发明以较佳实例公开如上，但其并非用以限定本发明，任何熟悉此技术的人在不脱离本发明的精神和范围内都可作各种改动与修饰，因此本发明的保护内容应该以权力要求书所限定的为准。

Claims (17)

  1. 一种幽门螺旋杆菌O2血清型O抗原寡糖类化合物的合成方法，其特征在于，所述方法是利用糖砌块A、B、C构建幽门螺旋杆菌O2血清型O抗原寡糖类化合物，包括构建糖砌块之间的1,2-α-顺式-糖苷键和1,3-α-顺式-糖苷键；
    其中糖砌块A、B、C分别为结构式II、III所示的化合物：
    Figure PCTCN2019091200-appb-100001
    其中，LG为糖基化反应离去基团，包括三氯乙酰亚胺基、对甲基苯硫基；linker包括-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，Y 1为氢或者烷氧基，Y 2为氢或者烷氧羰基；
    R 1，R 2，R 3，R 4，R 5，R 6为氢或者醚类基团；R 7为氢或者酰基类基团；
    所述方法中1,3-α-顺式-糖苷键的构建是在二氯甲烷和乙醚的混合溶剂中进行的；所述方法中1,3-α-顺式-糖苷键的构建还需要引入添加剂噻吩，添加剂与糖砌块的摩尔比为(80-120)：1。
  2. 根据权利要求1所述的方法，其特征在于，所述混合溶剂中二氯甲烷与乙醚的体积比为1：(2.5-5)。
  3. 一种幽门螺旋杆菌O2血清型O抗原寡糖类化合物的合成方法，其特征在于，所述方法是利用糖砌块A、B、C构建幽门螺旋杆菌O2血清型O抗原寡糖类化合物，包括构建糖砌块之间的1,2-α-顺式-糖苷键和1,3-α-顺式-糖苷键；
    其中糖砌块A、B、C分别为结构式II、III所示的化合物：
    Figure PCTCN2019091200-appb-100002
    其中，LG为糖基化反应离去基团，包括三氯乙酰亚胺基、对甲基苯硫基；linker包括-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，Y 1为氢或者烷氧基，Y 2为氢或者烷氧羰基；
    R 1，R 2，R 3，R 4，R 5，R 6为氢或者醚类基团；R 7为氢或者酰基类基团。
  4. 根据权利要求3所述的方法，其特征在于，所述方法中1,3-α-顺式-糖苷键的构建是在二氯甲烷和乙醚的混合溶剂中进行的。
  5. 根据权利要求4所述的方法，其特征在于，所述混合溶剂中二氯甲烷与乙醚的体积比为1：(2.5-5)。
  6. 根据权利要求3所述的方法，其特征在于，所述方法中1,3-α-顺式-糖苷键的构建还需要引入添加剂。
  7. 根据权利要求4所述的方法，其特征在于，所述方法中1,3-α-顺式-糖苷键的构建还需要引入添加剂。
  8. 根据权利要求5所述的方法，其特征在于，所述方法中1,3-α-顺式-糖苷键的构建还需要引入添加剂。
  9. 根据权利要求6所述的方法，其特征在于，所述添加剂与糖砌块的摩尔比为(80-120)：1。
  10. 根据权利要求6所述的方法，其特征在于，所述添加剂包括噻吩。
  11. 根据权利要求7所述的方法，其特征在于，所述添加剂包括噻吩。
  12. 根据权利要求8所述的方法，其特征在于，所述添加剂包括噻吩。
  13. 根据权利要求9所述的方法，其特征在于，所述添加剂包括噻吩。
  14. 根据权利要求1-13任一所述的方法，其特征在于，所述方法包括以下步骤：
    (1)合成葡萄糖单糖砌块A、B、C；
    (2)合成二糖化合物：构建糖砌之间的1,2-α-顺式-糖苷键；
    (3)构建多糖化合物与多糖化合物之间的1,3-α-顺式-糖苷键，制备得到幽门螺旋杆菌O2血清型O抗原寡糖类化合物；所述多糖化合物为式IV所示的化合物；
    Figure PCTCN2019091200-appb-100003
    其中，m为二糖片段的重复单元数，为2～12；
    linker包括-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，Y 1为氢(H)或者烷氧基，Y 2为氢或者烷氧羰基；
    R 1，R 2，R 3，R 4，R 5，R 6为氢或者醚类基团；R 7为氢或者酰基类基团。
  15. 一种组装有氨基连接臂的幽门螺旋杆菌O2血清型O抗原寡糖类化合物，其特征在于，其结构式如式VII所示，所述化合物是利用权利要求1-13任一所述的方法制备得到的；
    Figure PCTCN2019091200-appb-100004
    其中，linker包括-(CH2) n-N-Y 1Y 2或者-(CH2) n-N-Y 1Y 2(linker)，Y 1为氢或者烷氧基，Y 2为氢或者烷氧羰基。
  16. 一种糖-蛋白缀合物的制备方法，其特征在于，所述方法是利用权利要求15所述的组装有氨基连接臂的幽门螺旋杆菌O2血清型O抗原寡糖类化合物。
  17. 权利要求15所述的组装有氨基连接臂的幽门螺旋杆菌O2血清型O抗原寡糖类化合物在开发或制备疗幽门螺旋杆菌疫苗或者疗幽门螺旋杆菌感染导致的疾病的药物中的应用。
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