Synthesis method of Baliella chensinensis lipopolysaccharide O-antigen and application of Baliella chensinensis lipopolysaccharide O-antigen in immunocompetence aspect

Abstract

The invention belongs to the technical field of organic synthesis, and particularly relates to a synthesis method of a Baliella chensinensis lipopolysaccharide O-antigen and application of the Baliella chensinensis lipopolysaccharide O-antigen in the aspect of immunocompetence. The raw materials such as monosaccharide, disaccharide, trisaccharide, tetrasaccharide and the like for preparing the glycoconjugate vaccine can be efficiently synthesized, and the synthesis selectivity and the synthesis efficiency are improved. According to the invention, unprotected ammonia is selected to construct uronic acid amide, and hydrogenation is successfully carried out through the method to obtain the target molecular trisaccharide. Subsequently, monosaccharide, disaccharide and tetrasaccharide target molecules are constructed. Wherein in the construction process of the tetrasaccharide, a method of combining TEMPO and NaOCl is adopted to oxidize the tetrasaccharide into aldehyde, then Pinnick is adopted to oxidize the aldehyde into carboxylic acid, and a two-step one-pot method is adopted to construct the tetrasaccharide. According to the invention, glycoconjugate is prepared by conjugation with HSA, and the glycoconjugate lays a raw material foundation for research and development of the Babyte's fermentum lipopolysaccharide vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and specifically relates to a method for synthesizing Bordetella hinsonii lipopolysaccharide O-antigen and its application in immune activity. Background Art

[0002] Regarding the stereoselectivity of constructing amino sugar glycosidic bonds, the construction of β-configuration glycosidic bonds can usually be achieved through the participation of the amino protecting group at the C-2 position through the neighboring group. The amide bond and the C-1 position will form an oxazoline intermediate, and most of the amino glucosamine existing in nature is mainly in the β-configuration. Therefore, the construction of α-configuration glycosidic bonds is relatively difficult.

[0003] The synthesis of 1,2-cis glycosides focuses on the glycosylation of 2-azido-2-deoxy sugars. For the type of donor anomeric group, almost all leaving groups that can be used in the synthesis of common sugars can be used for the synthesis of 2-amino-2-deoxy sugar glycosides. Among them, common leaving groups such as trichloroacetyl, alkyl, aryl, acetyl or halogen atoms can be applied. When more complex or sterically hindered glycoside acceptors react, thioglycosides and imines are usually better choices.

[0004] Whooping cough is an acute respiratory infectious disease common in children caused by Bordetella pertussis. It is highly contagious. Bordetella hinzii is the pathogen that causes whooping cough and is one of the seven Bordetella species, including B. pertussis, B. parapertussis, B. bronchiseptica, B. avium, B. holmesii and B. trematum. Unlike the familiar Bordetella pertussis, Bordetella hinzii can produce a smooth lipopolysaccharide (LPS) with O-specific polysaccharide chains.

[0005] Evgeny Vinogradov et al. analyzed the structure of the polysaccharide chain of Bordetella hinzii ATCC 51730 lipopolysaccharide (Vinogradov, Evgeny. "Structure of the O-specific polysaccharide chain of the lipopolysaccharide of Bordetella hinzii." Carbohydrate research 337.10 (2002): 961-963.). In the structure of Bordetella hinzii ATCC 51730 lipopolysaccharide O-antigen, the monosaccharide building blocks of the trisaccharide repeating unit skeleton are connected by D-galactose and D-glucose through 1,4-β glycosidic bonds, all C-6 positions are oxidized and exist in the form of amides, and each monosaccharide skeleton is 2,3-dideoxy-2,3-diamino sugar.

[0006] Based on this, the inventors designed a synthetic route for the lipopolysaccharide O-antigen of Bordetella hindschildi, and efficiently synthesized the target compound by constructing a 2,3-dideoxy-2,3-diamino-1,2-cis glycosidic bond. At the same time, the present invention also provides the use of the prepared lipopolysaccharide O-antigen in the synthesis of glycoconjugates. Summary of the invention

[0007] The object of the present invention is to provide a method for synthesizing Bordetella hinsonii lipopolysaccharide O-antigen, which can efficiently synthesize monosaccharides, disaccharides, trisaccharides, tetrasaccharides and other raw materials for preparing glycoconjugate vaccines, and improve the selectivity and efficiency of the synthesis.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] The present invention first provides a trisaccharide compound, including a compound shown in formula I, and the specific structure is as follows:

[0010]

[0011] Further, R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, or TBSO-; R3 is -AcNH, TrocNH- or -NO2; R4 is -O(CH2) n NH2, -O(CH2) n NCbz(Bn) or -O(CH2) n NHCbz; n is an integer between 2 and 6.

[0012] Furthermore, Bn is benzyl, Ac is acetyl, TBS is tert-butyldimethylsilyl, Troc is 2,2,2-trichloroethoxycarbonyl, and Cbz is benzyloxycarbonyl.

[0013] Specifically, the trisaccharide compound shown in Formula I is 2-7, 2-9, 2-2, 2-2a or 2-2b, and the structural formula is:

[0014]

[0015] Further, based on a general inventive concept, the present invention also provides a method for synthesizing a trisaccharide compound, which specifically comprises the following steps:

[0016] 1) reacting a monosaccharide donor and a monosaccharide acceptor in an organic solvent I and a catalyst I at -40°C to -20°C (preferably -40°C) for 1-2h, quenching after the reaction, drying the solvent, and purifying by silica gel column chromatography to obtain a disaccharide intermediate I;

[0017] 2) dissolving the disaccharide intermediate I in THF and Py, and then reacting under HF / Py conditions at 25° C. to 35° C. (preferably 30° C.) for 2-4 days (preferably 3 days), washing, extracting, filtering, spin drying, and purifying by silica gel column chromatography to obtain the disaccharide intermediate II;

[0018] 3) reacting the monosaccharide donor and the disaccharide intermediate II in the presence of an organic solvent I and a catalyst I at -40°C to -20°C (preferably -40°C) for 1-2h, quenching after the reaction, drying the solvent, and purifying by silica gel column chromatography to obtain a trisaccharide product.

[0019] Specifically, the monosaccharide donor in step 1) is

[0020] Specifically, the monosaccharide receptor in step 1) is

[0021] Specifically, the mass ratio of the monosaccharide donor to the monosaccharide acceptor in step 1) is (1.1-1.2):1.

[0022] Specifically, in step 3), the mass ratio of the monosaccharide donor to the disaccharide intermediate II is (1.2-1.4):1.

[0023] Specifically, the organic solvent I is dichloromethane or tetrahydrofuran.

[0024] Specifically, catalyst I is tert-butyldimethylsilyl trifluoromethanesulfonate.

[0025] Further, based on a general inventive concept, the present invention also provides a method for synthesizing compound 2-9, which specifically comprises the following steps:

[0026] (1) Compound 3-3 or 2-7 is mixed with zinc powder, organic solvent I, and organic solvent II, reacted at room temperature (25±5°C) overnight (12h), filtered, washed, and the solvent is dried by spin drying, and reacted at room temperature (25±5°C) for 2-5h (preferably 3h) under the conditions of organic solvent I, organic solvent III, and organic solvent IV. After the reaction is completed, the mixture is quenched, the solvent is removed, the mixture is concentrated by spin drying, and purified by silica gel column chromatography to obtain trisaccharide intermediate I;

[0027] (2) The trisaccharide intermediate I and potassium carbonate are reacted in an organic solvent V at room temperature (25±5° C.) for 1-2 h (preferably 1 h), concentrated by spin drying, and purified by silica gel column chromatography to obtain the trisaccharide intermediate II; the trisaccharide intermediate II is compound 2-9.

[0028] Specifically, in step (1), the molar ratio of compound 3-3 or 2-7 to zinc powder is 1:(80-100).

[0029] Specifically, in step (2), the molar ratio of trisaccharide intermediate I to potassium carbonate is (1-1.5):1.

[0030] Further, based on a general inventive concept, the present invention also provides a method for synthesizing compound 2-2, 2-2a or 2-2b, which specifically comprises the following steps:

[0031] 1) Adding catalyst II and catalyst III to trisaccharide intermediate II in organic solvent I, organic solvent V and water, reacting at room temperature (25±5°C) for 5-8h (preferably 6h), removing the solvent, then adding HATU and organic solvent VI and dissolving them, then adding catalyst IV and catalyst V, reacting at room temperature (25±5°C) for 3-6h, concentrating by spin drying, and purifying by silica gel column chromatography to obtain trisaccharide intermediate III;

[0032] 2) dissolving the trisaccharide intermediate III in THF and Py, and then reacting under HF / Py conditions at room temperature (25±5° C.) for 24-48 hours, neutralizing, filtering, washing, spin-drying and concentrating, and purifying by gel chromatography to obtain the trisaccharide intermediate IV;

[0033] 3) The trisaccharide intermediate IV is reacted in the presence of Pd(OH)2 / C, an organic solvent V and water at room temperature (25±5°C) for 24-48 hours (preferably 24 hours), filtered, concentrated by spin drying, and purified by gel chromatography to obtain a trisaccharide product.

[0034] Specifically, organic solvent I is dichloromethane or tetrahydrofuran; specifically, organic solvent II is anhydrous acetic acid; specifically, organic solvent III is pyridine; specifically, organic solvent IV is acetic anhydride; specifically, organic solvent V is methanol or tert-butanol; specifically, organic solvent VI is DMF.

[0035] Specifically, catalyst II is TEMPO; specifically, catalyst III is BAIB; specifically, catalyst IV is DIPEA; specifically, catalyst V is NH3 dissolved in dioxane.

[0036] Secondly, the present invention provides a tetrasaccharide compound, including a compound represented by formula II and / or a compound represented by formula III, and the specific structure is as follows:

[0037]

[0038] Further, R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, TBSO- or R3 is -AcNH, TrocNH- or -NO2; R4 is -O(CH2) n NH2, -O(CH2) n NCbz(Bn) or -O(CH2) n NHCbz; n is an integer between 2 and 6.

[0039] Furthermore, Bn is benzyl, Ac is acetyl, TBS is tert-butyldimethylsilyl, t Bu is tert-butyl, Troc is 2,2,2-trichloroethoxycarbonyl, and Cbz is benzyloxycarbonyl.

[0040] Specifically, the tetrasaccharide compound includes the following compounds:

[0041]

[0042] Further, based on a general inventive concept, the present invention also provides a method for synthesizing the compound of formula II and / or the tetrasaccharide compound of formula III, comprising the following steps:

[0043] a. dissolving the trisaccharide compound in THF and Py, then reacting under HF / Py conditions at room temperature (25±5°C) for 24-48h, neutralizing, washing, filtering, spin drying and concentrating, and purifying by gel chromatography to obtain the trisaccharide intermediate V;

[0044] b. Dissolve the trisaccharide intermediate V and the monosaccharide acceptor in an organic solvent I, react at -30°C to -20°C (preferably -20°C) for 10-30 min, then add the catalyst VI, react at -30°C to -20°C (preferably -20°C) for 1-3 h (preferably 2 h), quench after the reaction, filter, spin dry and concentrate, and purify by silica gel column chromatography to obtain a tetrasaccharide intermediate I;

[0045] c. Mixing tetrasaccharide intermediate I with zinc powder, organic solvent I and organic solvent II, reacting at room temperature (25±5° C.) for 6-10 h (preferably 8 h), filtering, washing, removing the solvent, and reacting under the conditions of organic solvent I, organic solvent III and organic solvent IV for 3-8 h (preferably 5 h). After the reaction is completed, quenching, concentrating by spin drying, and purifying by silica gel column chromatography to obtain tetrasaccharide intermediate II;

[0046] d. reacting the tetrasaccharide intermediate II and potassium carbonate in an organic solvent V at room temperature (25±5° C.) for 1-2 h (preferably 2 h), concentrating the mixture by spin drying, and purifying the mixture by silica gel column chromatography to obtain the tetrasaccharide intermediate III;

[0047] e. The tetrasaccharide intermediate III is dissolved in THF and Py, and then reacted under HF / Py conditions at room temperature (25±5°C) for 2-3h, neutralized, filtered, washed, concentrated by spin drying, and purified by gel chromatography to obtain a crude product I;

[0048] The crude product I is dissolved in an organic solvent I, saturated NaHCO3, water, and NaClO, and catalysts VII, VIII, and IX are added, and the reaction is carried out at room temperature (25±5°C) for 1-2 hours (preferably 1 hour), and the pH is adjusted to 6-8, and tert-butyl alcohol, NaClO2, and a solution of NaH2PO4 dissolved in distilled water and 2-methyl-2-butene are added in sequence, and the reaction is carried out at room temperature (25±5°C) for 1-2 hours (preferably 2 hours), and the reaction is diluted, and the solvent is removed by spin drying, and the reaction is purified by gel chromatography to obtain a crude product II;

[0049] The crude product II is dissolved in an organic solvent VI and HATU and catalysts IV and V are added, and the mixture is reacted at room temperature (25±5° C.) for 6-10 hours (preferably 8 hours), concentrated by spin drying, and purified by gel chromatography to obtain a tetrasaccharide intermediate IV;

[0050] f. The tetrasaccharide intermediate IV is reacted under the conditions of Pd(OH)2 / C, organic solvent V and water at room temperature (25±5°C) for 24-48h (preferably 24h), the Pd(OH)2 / C is removed, the mixture is concentrated by spin drying, and purified by gel chromatography to obtain the tetrasaccharide product.

[0051] Specifically, in step b, the mass ratio of the trisaccharide intermediate V to the monosaccharide acceptor is 1:(1.4-1.8).

[0052] Specifically, in step c, the mass ratio of the tetrasaccharide intermediate I to the zinc powder is 1:(3-4).

[0053] Specifically, in step d, the mass ratio of tetrasaccharide intermediate II to potassium carbonate is (7-8):1.

[0054] Specifically, catalyst VI is TBSOTf; specifically, catalyst VII is TEMPO; specifically, catalyst VIII is NaBr; specifically, catalyst IX is nBu4NBr.

[0055] The present invention also provides a disaccharide compound, including a compound shown in formula IV, and the specific structure is as follows:

[0056]

[0057] Further, R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, TBSO- or R3 is -AcNH, TrocNH- or -NO2; R4 is -O(CH2) n NH2, -O(CH2) n NCbz(Bn) or -O(CH2) n NHCbz; n is an integer between 2 and 6.

[0058] Furthermore, Bn is benzyl, Ac is acetyl, TBS is tert-butyldimethylsilyl, t Bu is tert-butyl, Troc is 2,2,2-trichloroethoxycarbonyl, and Cbz is benzyloxycarbonyl.

[0059] Specifically, the disaccharide compound includes the following compounds:

[0060]

[0061] The present invention also provides a monosaccharide compound, including a compound represented by Formula V, Formula VI and / or Formula VII, and the specific structure is as follows:

[0062]

[0063] Further, R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, TBSO- or R3 is -AcNH, TrocNH-, AcO-, N3- or -NO2; R4 is -OAll, -O(CH2)nNH2, -O(CH2)nNCbz(Bn) or -O(CH2)nNHCbz; n is an integer between 2 and 6.

[0064] Furthermore, Bn is benzyl, Ac is acetyl, TBS is tert-butyldimethylsilyl, tBu is tert-butyl, Troc is 2,2,2-trichloroethoxycarbonyl, and Cbz is benzyloxycarbonyl.

[0065] Specifically, the monosaccharide compounds include the following compounds:

[0066]

[0067] Furthermore, based on a general inventive concept, the present invention also provides the use of the monosaccharide compound, disaccharide compound, trisaccharide compound or tetrasaccharide compound in improving immune activity.

[0068] Further, based on a general inventive concept, the present invention also provides the use of the monosaccharide compound, disaccharide compound, trisaccharide compound or tetrasaccharide compound in the synthesis of glycoconjugates.

[0069] Further, based on a general inventive concept, the present invention also provides a method for synthesizing a glycoconjugate using the monosaccharide compound, disaccharide compound, trisaccharide compound or tetrasaccharide compound, comprising the following steps:

[0070] Step 1: Dissolve di(N-succinimidyl) glutarate (DSG) in DMSO, add triethylamine, and stir to mix; Dissolve the monosaccharide compound, disaccharide compound, trisaccharide compound or tetrasaccharide compound in DMSO;

[0071] Step 2: adding a monosaccharide compound, a disaccharide compound, a trisaccharide compound or a tetrasaccharide compound dissolved in DMSO to a DMSO solution of DSG, reacting at room temperature (25±5°C) for 1-2 hours (preferably 2 hours), rinsing with PBS and collecting the liquid, and collecting the upper aqueous phase by centrifugation;

[0072] Step 3: Dissolve HSA in PBS, add the upper aqueous phase described in step 2, stir and react at room temperature (25±5°C) for 16-24h (preferably 18h), add water to the obtained reaction product, centrifuge and concentrate to obtain a glycoconjugate.

[0073] Specifically, in step 1, the usage ratio of DSG to triethylamine is (8-30) mol: (3-9) L.

[0074] Specifically, in step 2, the molar ratio of DSG to the monosaccharide compound, disaccharide compound, trisaccharide compound or tetrasaccharide compound is (8-12):1, preferably 10:1.

[0075] Specifically, in step three, the mass ratio of HSA to the monosaccharide compound, disaccharide compound, trisaccharide compound or tetrasaccharide compound is (0.2-0.8):1.

[0076] Specifically, the sugar conjugate is a product of the monosaccharide compound, disaccharide compound, trisaccharide compound or tetrasaccharide compound combined with HSA.

[0077] Compared with the prior art, the advantages of the present invention are:

[0078] According to the synthesis method of 2-nitro sugar ene, the present invention proposes a method of using 2-nitro sugar ene as a raw material, obtaining a pyranose 2-nitro-3-azido-1,2-cis-glycosylated product through a two-step one-pot method, and then reducing it to obtain 2,3-dideoxy-2,3-diamino sugar. The optimal reaction conditions are determined through condition screening. The method has mild conditions, high overall efficiency, simple operation, good selectivity, and a wide range of applications.

[0079] The present invention selects unprotected ammonia to construct uronic acid amide, constructs trisaccharide 2-27 by this method, and successfully hydrogenates to obtain the target molecule trisaccharide. Subsequently, monosaccharide, disaccharide and tetrasaccharide target molecules are constructed. Among them, in the construction process of tetrasaccharide, TEMPO and NaOCl are used to oxidize to aldehyde, and then Pinnick is used to oxidize to carboxylic acid, and the construction is carried out by a two-step one-pot method.

[0080] The present invention also prepares a sugar conjugate by conjugating with HSA, and the sugar conjugate lays a raw material foundation for the research and development of Bordetella hinsonii lipopolysaccharide vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The synthesis and characterization of the glycoconjugate HSA-1 in Example 3;

[0082] Figure 2 The synthesis and characterization of the glycoconjugate HSA-2 in Example 3;

[0083] Figure 3 The synthesis and characterization of the glycoconjugate HSA-3 in Example 3;

[0084] Figure 4 The synthesis and characterization of the glycoconjugate HSA-4 in Example 3;

[0085] Figure 5 The synthesis and characterization of the glycoconjugate HSA-5 in Example 3;

[0086] Figure 6 The synthesis and characterization of the glycoconjugate HSA-6 in Example 3;

[0087] Figure 7 The results of ELISA detection of the recognition of six sugar conjugates by the antiserum (1:4000 dilution) produced by mice immunized with inactivated Bordetella hinschweitii for 35 days and Freund's adjuvant in Example 3. DETAILED DESCRIPTION

[0088] The technical solution of the present invention is further described in detail below through specific embodiments, but the protection scope of the present invention is not limited thereto.

[0089] The abbreviations in the following embodiments are: Ac is acetyl, Ac2O is acetic anhydride, AcOH is glacial acetic acid, All is allyl, ABz is 2-(2-cyclopropylethynyl)benzoyl, AgOTf is silver trifluoromethanesulfonate, BAIB is bis(acetoxy)iodobenzene, Bn is benzyl, Bu2SnO is di-n-butyltin oxide, Bz is benzoyl, t Bu is tert-butyl, Cbz is benzyloxycarbonyl, DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene, DCM is dichloromethane, DDQ is 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, DIPEA is diisopropylethylamine, DMAP is 4,4-dimethylaminopyridine, DME is ethylene glycol dimethyl ether, DMF is dimethylformamide, DMSO is dimethyl sulfoxide, DTBMP is 2,6-di-tert-butyl-4-methylpyridine, Et is ethyl, EA is ethyl acetate, HATU is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, Im(imid) is imidazole, i- Pr is isopropyl, Me is methyl, MPF is N-methylformanilide, MS molecular sieve, Nap is 2-(methyl)naphthalene, NBS is N-bromosuccinimide, NIS is N-iodosuccinimide, PE is petroleum ether, Ph is phenyl, PIFA is [bis(trifluoroacetoxy)iodo]benzene, PivOH is trimethylacetic acid, PMB is methoxybenzyl, PMBCl is 4-methoxybenzyl chloride, PPY is 4-pyrrolidinylpyridine, Py is pyridine, TBAB is tetrabutylammonium bromide, TBAI is tetrabutylammonium iodide, TBAN is tetrabutylammonium nitrate, TBAF is tetrabutylammonium fluoride, TBDPS is tert-butyldiphenylsilyl, TBDPSCl is diphenyl tert-butylchlorosilane, TEMPO is 2,2,6,6-tetramethylpiperidinyloxide, TBS is tert-butyldimethylsilyl, TBSCl is dimethyl tert-butylchlorosilane, TCA is trichloroacetic acid, Tf is trifluoromethanesulfonate, TIPS is triisopropylsilyl, TIPSCl is triisopropylchlorosilane, TEA is triethylamine, TFA is trifluoroacetic acid, THF is tetrahydrofuran, TMS is trimethylsilyl, TMSE is trimethylsilylethyl, TMSOTf is trimethylsilyl trifluoromethanesulfonate, TBSOTf is tert-butyldimethylsilyl trifluoromethanesulfonate, Tol is toluene, Ts is p-toluenesulfonyl, p-TsOH is p-toluenesulfonic acid, TMSCl is trimethylchlorosilane, TMSN3 is trimethylsilylazide, TrocCl is 2,2,2-trichloroethyl chloroformate, Troc is 2,2,2-trichloroethoxycarbonyl, and Lev is levulinyl. In the following examples, 3,4,6-O-acetylglucose ene (Compound 3-13) and Compound 3-23 are conventional commercially available products.

[0090] Example 1 Study on the Methodology of 2,3-dideoxy-2,3-diamino-1,2-cis-glycosylation

[0091] 1.1 Synthesis of monosaccharide donors 3-6

[0092]

[0093] The raw material compound 3,4,6-O-acetylglucose ene 3-13 (20 g, 73.462 mmol) was added to a 500 mL round-bottom flask, potassium carbonate solid powder (3.046 g, 22.039 mmol) was added, and methanol (180 mL) was added to dissolve, and the reaction was stirred at room temperature for 2 h. After the reaction was completed by TLC monitoring (EA), it was filtered, spin-dried, and dried to obtain a waxy solid 3-14.

[0094]

[0095] Compound 3-14 (21 g, 0.144 mol) was added to a 250 mL round-bottom flask, and 100 mL of tert-butanol was added to dissolve it. Then, activated 4AMS (5 g) and Novozymes 435 (4 g) were added at room temperature, and finally vinyl acetate (60 mL) was added. The reaction was continued at room temperature for 18 h. The reaction time did not exceed 20 h. After the reaction was complete as monitored by TLC (PE: EA = 1: 1), it was filtered and the solvent was dried to obtain a crude product 3-17, which was directly carried out to the next step without purification.

[0096] After adding 80 mL of DMF to the reaction bottle containing the crude product 3-17 to dissolve, add imidazole (19 g, 0.288 mol) and tert-butyldimethylsilyl chloride (43 g, 0.288 mol), react at room temperature for 4 h, monitor the reaction by TLC (PE: EA = 5: 1), wash with water, wash with saturated sodium bicarbonate, extract with ethyl acetate, dry, filter, spin dry, and column chromatography (PE: EA = 15: 1) to obtain a white waxy solid 3-18 (43 g, 87%).

[0097]

[0098] Compound 3-18 (10 g, 29.069 mmol) was added to a 500 mL round-bottom flask, tetrabutylammonium nitrate (17.702 g, 58.13 9 mmol), DTBMP (11.938 g, 58.139 mmol), 300 mL of dry dichloromethane was added to dissolve, trifluoroacetic anhydride (8.08 mL, 58.139 mmol) was slowly added under nitrogen protection at -70 ° C, and the reaction was continued at -70 ° C. Solids were generated during the reaction and the system gradually became viscous. After half an hour of reaction, TLC monitored that the reaction was complete (PE: EA = 4: 1), the system was quickly added to an ice-water bath for washing, and then washed with a saturated NaCl solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, the organic layer was spin-dried, dry mixed, and column chromatography (PE: EA = 12: 1 ~ 4: 1) was performed to obtain a white solid powder 3-19 (6.793 g, 60%).

[0099]

[0100] Compound 3-19 (2.1 g, 5.392 mmol) was added to a 50 mL round-bottom flask, and 20 mL of dry dichloromethane was added to dissolve it. Then, 4-pyrrolidinylpyridine (80 mg, 0.539 mmol) and TMSN3 (0.85 mL, 6.47 mmol) were added in sequence at room temperature. During the reaction, the color of the system gradually turned yellow. After reacting for 30 min under nitrogen protection at room temperature, TLC monitored the reaction to be complete (PE: EA = 5: 1). Trimethylacetic acid (165 mg, 1.617 mmol), thiourea catalyst (808 mg, 1.617 mmol), and allyl alcohol (0.74 mL, 10.78 mmol) were directly added to the system in sequence, and reacted at 30 ° C for two days. After TLC monitored the reaction to be complete (PE: EA = 5: 1), the organic layer was spin-dried, the sample was dry mixed, and column chromatography (PE: EA = 12: 1 ~ 4: 1) was performed to obtain a colorless viscous liquid 3-20 (1.508 g, 65%).

[0101]

[0102] Compound 3-20 (430 mg, 1.000 mmol) was added to a 50 mL round-bottom flask, and zinc powder (1.3 g, 20.000 mmol) and copper sulfate (159 mg, 1.000 mmol) were added. A mixed solvent of THF / H2O (15 mL / 1.5 mL) (THF / H2O=10 / 1) was added. After dissolution, concentrated hydrochloric acid (0.83 mL, 10.000 mmol) was slowly added in an ice-water bath. The temperature was slowly raised to room temperature for 2 h, and the reaction was monitored by TLC until completion (DCM:CH3OH =20:1), filter to remove zinc powder, wash the filter residue with THF, then spin off a small amount of THF, add solid sodium bicarbonate powder (504 mg, 6.000 mmol) in the presence of water, slowly add TrocCl (0.412 mL, 3.000 mmol) under ice-water bath, react at room temperature for 2 h, monitor the reaction until complete (PE: EA = 3:1) by TLC, filter, spin dry, dry mix, column chromatography (PE: EA = 12:1 ~ 6:1), to obtain colorless viscous liquid 3-21 (558 mg, 77%).

[0103]

[0104] Compound 3-21 (2.37 g, 3.27 mmol) was added to a 250 mL round-bottom flask, and DCM / CH3OH (60 mL / 60 mL) was added to dissolve it, and palladium chloride (116 mg, 0.654 mmol) was added. The mixture was reacted at room temperature for 2 h under nitrogen protection. After the reaction was complete (PE:EA=3:1) after monitoring by TLC, the mixture was filtered and dried by rotation. The mixture was washed with saturated ammonium chloride solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and the organic layer was dried by rotation. The colorless viscous liquid 3-22 (1.567 g, 70%) was obtained by flash column chromatography.

[0105]

[0106] Compound 3-22 (860 mg, 1.255 mmol) was added to a 25 mL round-bottom flask, and solid potassium carbonate powder (191 mg, 1.38 mmol) was added. After adding 10 mL of acetone to dissolve, CF3CN(Ph)Cl (391 mg, 1.88 mmol) was slowly injected. The reaction was carried out under nitrogen protection at room temperature for 2 to 3 hours. After the reaction was completed (PE:EA=3:1) monitored by TLC, a small amount of triethylamine was added to quench, filtered, spin-dried, and flash column chromatography (PE:EA=15:1~10:1) was performed to obtain a white solid powder 3-6 (861 mg, 77%).

[0107] 1.2 Synthesis of monosaccharide receptor 3-12

[0108]

[0109] Compound 3-23 (800 mg, 2.94 mmol) was added to a 100 mL round-bottom flask, tetrabutylammonium nitrate (1.253 g, 4.116 mmol), DTBMP (845 mg, 4.116 mmol), 15 mL of dry dichloromethane was added to dissolve, trifluoroacetic anhydride (0.692 mL, 4.116 mmol) was slowly added under nitrogen protection at -70 ° C, and the reaction was continued at -70 ° C. Solids were generated during the reaction and the system gradually became viscous. After half an hour of reaction, TLC monitored that the reaction was complete (PE: EA = 2: 1), the system was quickly added to an ice-water bath for washing, and then washed with a saturated NaCl solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, the organic layer was spin-dried, dry mixed, and rapid column chromatography (PE: EA = 10: 1 ~ 3: 1) was performed to obtain a colorless viscous liquid 3-24 (667 mg, 72%).

[0110] White solid powder HO(CH2)5NHCbz was synthesized by the method in reference (Gammon, David W., et al. "A new procedure for highly regio-andstereoselective iodoacetoxylation of protected glycals and α-1,2-cyclopropanated sugars." Journal of Carbohydrate Chemistry 26.3 (2007): 141-157.).

[0111] Compound 3-24 (50 mg, 1.5762 mmol) was added to a 50 mL round-bottom flask, and 10 mL of dry dichloromethane was added to dissolve it. Then, 4-pyrrolidinylpyridine (23 mg, 0.158 mmol) and TMSN3 (0.25 mL, 1.890 mmol) were added in sequence at room temperature. During the reaction, the color of the system gradually turned yellow. After reacting for 30 min under nitrogen protection at room temperature, the reaction was completed (PE:EA=3:1) by TLC monitoring. Trimethylacetic acid (165 mg, 1.617 mmol), thiourea catalyst (808 mg, 1.617 mmol), and HO(CH2)5NHCbz (600 mg, 2.522 mmol) were added in sequence, and the mixture was reacted at 30°C for two days. After the reaction was completed by TLC monitoring (PE:EA=3:1), the organic layer was spin-dried, the sample was dry-mixed, and column chromatography (PE:EA=12:1-4:1) was performed to obtain a colorless viscous liquid 3-25 (498 mg, 59%).

[0112]

[0113] Compound 3-25 (54 mg, 0.100 mmol) was added to a 10 mL round-bottom flask, and DCM / CH3OH (2 mL / 2 mL) was added to dissolve it. Acetyl chloride (0.2 mL) was added under an ice-water bath, and the mixture was reacted at room temperature for 2 h under nitrogen protection. After the reaction was complete by TLC monitoring (PE: EA = 1: 1), the mixture was filtered and dried. The mixture was washed with a saturated sodium bicarbonate solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and the organic layer was dried and subjected to column chromatography to obtain a colorless viscous liquid 3-26 (43 mg, 94%).

[0114]

[0115] Compound 3-26 (453 mg, 1.000 mmol) was added to a 50 mL round-bottom flask, and zinc powder (1.3 g, 20.000 mmol) and copper sulfate (159 mg, 1.000 mmol) were added. After dissolving in a THF / H2O (15 mL / 1.5 mL) mixed solvent (THF / H2O=10 / 1), concentrated hydrochloric acid (0.83 mL, 10.000 mmol) was slowly added under an ice-water bath, and the temperature was slowly raised to room temperature for 2 h. The reaction was monitored by TLC until completion (DCM:CH3OH =20:1), filter to remove zinc powder, wash the filter residue with THF, then spin off a small amount of THF, add solid sodium bicarbonate powder (840 mg, 10.000 mmol) in the presence of water, slowly add TrocCl (0.412 mL, 3.000 mmol) under ice-water bath, react at room temperature for 2 h, monitor the reaction until complete (PE: EA = 2:1) by TLC, filter, spin dry, dry mix, column chromatography (PE: EA = 3:1 ~ 1:1), to obtain colorless viscous liquid 3-27 (701 mg, 83%).

[0116]

[0117] Compound 3-27 (100 mg, 1.134 mmol) was added to a 10 mL round-bottom flask, BAIB (216 mg, 0.670 mmol) was added, and DCM / H2O (1 mL / 0.5 mL) was added to dissolve it. Then, TEMPO (4.2 mg, 0.027 mmol) was added in an ice-water bath, and the temperature was slowly raised to room temperature for 2 h. After TLC monitoring until the reaction was complete (PE:EA:CH3OH=1:2:0.5), it was quenched and washed with saturated sodium thiosulfate, extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate and spin-dried, and the next step was carried out without purification.

[0118] HATU (60 mg, 0.220 mmol) and DIPEA (111 μL, 0.670 mmol) were added to the reaction bottle containing the crude product in the previous step, and finally benzylamine (18 μL, 0.161 mmol) was added. The reaction was allowed to react at room temperature for 24 h. After TLC monitoring until the reaction was complete (PE: EA: CH3OH = 1:2:0.5), the system was washed with distilled water, then washed with saturated NaCl solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, the organic layer was spin-dried, dry-mixed, and column chromatography (DCM: CH3OH = 20:1-10:1) was performed to obtain a white solid powder 3-12 (73 mg, 64%). 1 H NMR (400MHz, CDCl3) δ7.39–7.26(m,10H),7.08–6.97(m,1H),5.87(d,J=7.5Hz,1 H),5.45(d,J=8.3Hz,1H),5.18–5.05(m,2H),4.93(s,1H),4.90–4.82(m,1H),4.7 9–4.59(m,4H),4.54–4.42(m,3H),4.36(s,1H),4.22–4.11(m,2H),3.73–3.60(m, 1H),3.51–3.38(m,1H),3.25–3.11(m,2H),1.67–1.47(m,4H),1.44–1.32(m,2H). 13 C NMR (101MHz, CDCl3) δ168.67,156.48,155.10,154.92,137.44,137.31,136.58,128.82,128.54,128.13,127.7 2,127.66,97.49,95.40,74.59,71.80,68.89,68.03,66.67,51.04,50.50,43.18,40.81,29.78,28.89,23.30.

[0119] 1.3 Synthesis of trisaccharide 3-5

[0120]

[0121] Compound 3-24 (1400 mg, 0.4413 mmol) was added to a 10 mL round-bottom flask, and 2 mL of dry dichloromethane was added to dissolve it. Then, 4-pyrrolidinylpyridine (7 mg, 0.044 mmol) and TMSN3 (0.07 mL, 0.529 mmol) were added in sequence at room temperature. During the reaction, the color of the system gradually turned yellow. After reacting for 30 min under nitrogen protection at room temperature, the reaction was completed (PE:EA=3:1) by TLC monitoring. Trimethylacetic acid (14 mg, 0.133 mmol), thiourea catalyst (66 mg, 0.133 mmol), HO(CH2)5NBn(Cbz) (289 mg, 0.880 mmol) were added in sequence, and the mixture was reacted at 30°C for two days. After the reaction was completed by TLC monitoring (PE:EA=3:1), the organic layer was spin-dried, the sample was dry-mixed, and column chromatography (PE:EA=12:1~4:1) was performed to obtain a white solid powder 3-38 (203 mg, 73%).

[0122]

[0123] Compound 3-38 (160 mg, 0.255 mmol) was added to a 25 mL round-bottom flask, and DCM / CH3OH (2 mL / 2 mL) was added to dissolve it. Acetyl chloride (0.2 mL) was added under an ice-water bath, and the mixture was reacted at room temperature for 2 h under nitrogen protection. After the reaction was complete (PE: EA = 2: 1) after monitoring by TLC, the mixture was dried by rotation. The mixture was washed with a saturated sodium bicarbonate solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and the organic layer was dried by rotation. The white solid powder 3-39 (121 mg, 88%) was obtained by column chromatography.

[0124]

[0125] Compound 3-39 (2.12 g, 3.900 mmol) was added to a 50 mL round-bottom flask, and zinc powder (5.1 g, 78.000 mmol) and copper sulfate (622 mg, 3.900 mmol) were added. After dissolving in THF / H2O (40 mL / 4 mL) mixed solvent (THF / H2O=10 / 1), concentrated hydrochloric acid (3.250 mL, 39.000 mmol) was slowly added under an ice-water bath, and the temperature was slowly raised to room temperature for 2 h. The reaction was monitored by TLC until completion (DCM:CH3OH=1 0:1), filter to remove zinc powder, wash the filter residue with THF, then spin off a small amount of THF, add solid sodium bicarbonate powder (840 mg, 10.000 mmol) in the presence of water, slowly add TrocCl (1.610 mL, 11.700 mmol) under ice-water bath, react at room temperature for 2 h, monitor the reaction until complete (PE: EA = 1: 1) by TLC, filter, spin dry, dry mix, column chromatography (PE: EA = 4: 1 ~ 1: 1), to obtain colorless viscous liquid 3-40 (2.324 g, 71%).

[0126]

[0127] Compound 3-40 (190 mg, 0.227 mmol) was added to a 25 mL round-bottom flask, and the catalyst diphenylaminoboronic acid ethyl ester (15 mg, 0.068 mmol) was added. After acetonitrile (5 mL) was added to dissolve, DIPEA (56 μL, 0.340 mmol) was added at room temperature, and acetyl chloride (24 μL, 0.340 mmol) was added under an ice-water bath. The temperature was slowly raised to room temperature and reacted for 2 hours. After TLC monitoring until the reaction was complete (PE: EA = 3:2), it was washed with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, the organic layer was spin-dried, and column chromatography (PE: EA = 4:1 to 1:1) was performed to obtain a white solid powder 3-8 (170 mg, 86%). 1H NMR (400MHz, CDCl3) δ7.45–7.15(m,10H),5.92(d,J=9.0Hz,1H),5.66(d,J=9.0Hz,0.2H),5. 43(d,J=9.3Hz,0.4H),5.30(d,J=9.6Hz,0.4H),5.18(d,J=16.4Hz,2H),4.83(d,J=13.5Hz,1 H),4.79–4.60(m,4H),4.51(d,J=6.4Hz,2H),4.39(dd,J=11.4,7.0Hz,1H),4.18–3.87(m,4H ),3.73–3.54(m,1H),3.46–3.17(m,3H),2.08(s,3H),1.67–1.46(m,4H),1.38–1.20(m,2H). 13 C NMR (101MHz, CDCl3) δ171.36,155.06,155.00,137.85,136.71,128.59,128.52,128.47,128.02,127.99,127.88,127.82,127.41,127.34 ,127.23,97.13,95.40,74.58,68.33,67.96,67.59,67.23,62.59,51 .95,50.59,50.28,47.05,46.14,29.01,27.96,27.44,23.44,20.87.

[0128]

[0129] The donor compound 3-6 (321 mg, 0.382 mmol) and the acceptor compound 3-8 (280 mg, 0.318 mmol) were added to a 25 mL round-bottom flask, and dry dichloromethane (5 mL) was added to dissolve. After adding activated 4AMS (500 mg), the system was dried for 30 minutes, and tert-butyldimethylsilyl trifluoromethanesulfonate (15.0 μL, 0.637 mmol) was added at -40 ° C, and stirred at -40 ° C for 1-2 hours under nitrogen protection. Be careful not to react for too long. After the TLC plate detected that the donor reaction was complete, the reaction bottle was taken out of the low temperature environment and 4AMS was filtered out. An appropriate amount of triethylamine was added to quench the reaction system, the solvent was spin-dried, and column chromatography was separated (PE: EA = 5: 1 to 1: 1) to obtain a white powder solid compound 3-41 (305 mg, 49.2%).

[0130]

[0131] The raw material compound 3-41 (118 mg, 0.076 mmol) was added to a 10 mL plastic reaction bottle, and solvents THF (2 mL) and Py (2 mL) were added to dissolve, and then HF / Py (0.2 mL) was added at 0°C and reacted at 30°C for 3 d. After the reaction was completed as monitored by TLC plate (PE: EA = 1: 1), it was washed with saturated sodium bicarbonate solution and saturated NaCl solution in turn, extracted with ethyl acetate, and the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. Column chromatography (PE: EA = 5: 1 to 1: 1) gave a white solid powder 3-7 (100 mg, 92%).

[0132]

[0133] The donor compound 3-6 (444 mg, 0.520 mmol) and the acceptor compound 3-7 (344 mg, 0.240 mmol) were added to a 25 mL round-bottom flask, and dry dichloromethane (5 mL) was added to dissolve. After adding activated 4AMS (500 mg), the system was dried for 30 minutes, and tert-butyldimethylsilyl trifluoromethanesulfonate (12.0 μL, 0.052 mmol) was added at -20°C, and stirred under nitrogen protection at -20°C for 1-2 hours. Note that the reaction time should not be too long. After the TLC plate detected that the donor reaction was complete, the reaction bottle was taken out of the low temperature environment and 4AMS was filtered off. An appropriate amount of triethylamine was added to quench the reaction system, the solvent was spin-dried, and column chromatography was separated (PE: EA = 5: 1 to 1: 1) to obtain a white powder solid compound 3-5 (353 mg, 71%). 1H NMR (400MHz, CDCl3) δ7.45–7.12(m,10H),6.44(s,1H),5.92(s,1H),5.69(d,J=8.5Hz,2H),5.17(d,J=17.2Hz,2H),5.00(dd,J=35.7,10.6Hz,2H), 4.90–4.42(m,17H),4.32–3.93(m,9H),3.84–3.15(m,12H),2.17–1.99(m ,9H),1.68–1.48(m,4H),1.38–1.25(m,2H),0.87(s,9H),0.08(s,6H).13C NMR (101MHz, CDCl3) δ171.21,170.52,156.69,156.17,155.98,155.24,154.95,154.67,154.65,154.42,137.78,128.55,128.42,1 27.93,127.82,127.73,127.31,127.17,103.06,101.24,96.64,95.62,95.58,95.49,95.41,95.24,95.03,76.03,75.14,75.01,74 .79,74.61,74.44,74.24,73.93,70.22,68.00,67.17,64.29,63.73,62.02,60.45,57.68,55.90,55.23,53.31,50.56,50.24,49.54,46.96,46.08,29.67,28.93,27.86,27.38,26.88,25.64,23.45,23.34,21.08,20.91,20.88,20.85,17.89,14.18,-3.94,-4.56.

[0134] 1.4 Synthesis of Monosaccharide Building Block 3-49

[0135]

[0136] Compound 3-24 (0.500 g, 1.576 mmol) was added to a 25 mL round-bottom flask, and 5 mL of dry dichloromethane was added to dissolve it. Then, 4-pyrrolidinylpyridine (23 mg, 0.158 mmol) and TMSN3 (0.250 mL, 1.891 mmol) were added in sequence at room temperature. During the reaction, the color of the system gradually turned yellow. After reacting for 30 min under nitrogen protection at room temperature, the reaction was monitored by TLC until it was complete (PE:EA=3:1). Trimethylacetic acid (64 mg, 0.473 mmol), thiourea catalyst (230 mg, 0.473 mmol) and allyl alcohol (0.215 mL, 3.152 mmol) were directly added to the system in sequence, and the mixture was reacted at 30°C for two days. After the reaction was completed by TLC monitoring (PE: EA = 3:1), the organic layer was spin-dried, the sample was dry-mixed, and column chromatography (PE: EA = 10:1-6:1) was performed to obtain a white solid powder 3-50 (0.401 g, 71%).

[0137]

[0138] Compound 3-50 (32 mg, 0.100 mmol) was added to a 25 mL round-bottom flask, and DCM / CH3OH (2 mL / 2 mL) was added to dissolve it. Acetyl chloride (0.2 mL) was added under an ice-water bath, and the mixture was reacted at room temperature for 2 h under nitrogen protection. After the reaction was complete under TLC monitoring (PE: EA = 2: 1), the mixture was spin-dried. The mixture was washed with a saturated sodium bicarbonate solution, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. The organic layer was spin-dried, and column chromatography (PE: EA = 5: 1 to 1: 1) was performed to obtain a white solid powder 3-51 (25 mg, 99%).

[0139]

[0140] Compound 3-51 (0.858 g, 3.130 mmol) was added to a 100 mL round-bottom flask, 30 mL of dry dichloromethane was added to dissolve, pyridine (1.5 mL, 18.770 mmol) was added at room temperature, di-tert-butylsilylbis(trifluoromethanesulfonic acid) was added at 0°C, the temperature was gradually raised to room temperature and the reaction was carried out under nitrogen protection for 1 hour. After TLC monitoring until the reaction was complete (PE:EA=3:1), the mixture was washed with water and saturated sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, the organic layer was spin-dried, and column chromatography (PE:EA=9:1~2:1) was performed to obtain colorless viscous liquid 3-52 (0.791 g, 61%).

[0141]

[0142] Compound 3-52 (500 mg, 1.576 mmol) was added to a 25 mL round-bottom flask, zinc powder (1.3 g, 20.000 mmol), copper sulfate (159 mg, 1.000 mmol), THF / H2O (15 mL / 1.5 mL) mixed solvent (THF / H2O = 10 / 1) was added, and after dissolution, concentrated hydrochloric acid (0.83 mL, 10.000 mmol) was slowly added in an ice-water bath, and the temperature was slowly raised to room temperature for 2 h, and then monitored by TLC (DCM: CH3OH = 20:1). After the reaction is complete, the zinc powder is filtered off, and the filter residue is washed with THF. A small amount of THF is then removed by spinning. Solid sodium bicarbonate powder (504 mg, 6.000 mmol) is added in the presence of water. TrocCl (0.412 mL, 3.000 mmol) is slowly added under an ice-water bath. After reacting for 2 h at room temperature, TLC is monitored to determine the completion of the reaction (PE: EA = 3: 1). The mixture is filtered, spin-dried, dry-mixed, and subjected to column chromatography (PE: EA = 12: 1 to 6: 1) to obtain a colorless viscous liquid 3-50 (558 mg, 77%).

[0143]

[0144] The preparation method of compound 3-54 is the same as that of compound 3-22. Compound 3-53 (1.563 g, 2.155 mmol) is added to a 250 mL round-bottom flask, CH3OH (60 mL) is added to dissolve, palladium chloride (76 mg, 0.431 mmol) is added, and the mixture is reacted at room temperature for 2 h under nitrogen protection. After the reaction is complete (PE: EA = 3: 1) after monitoring by TLC, the mixture is filtered and dried by rotation. The mixture is washed with saturated ammonium chloride solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, the organic layer is dried by rotation, and white solid powder 3-54 (1.507 g, 68%) is obtained by rapid column chromatography.

[0145]

[0146] The preparation method of compound 3-49 is the same as that of compound 3-6. Compound 3-54 (137 mg, 0.205 mmol) is added to a 25 mL round-bottom flask, solid cesium carbonate powder (98 mg, 0.310 mmol) is added, and 3 mL of E dichloromethane is added to dissolve it. Then CF3CN(Ph)Cl (62 mg, 0.310 mmol) is slowly injected. The mixture is reacted under nitrogen protection at room temperature for 2 to 3 hours. After the reaction is complete as monitored by TLC (PE:EA=3:1), a small amount of triethylamine is added to quench the mixture, the mixture is filtered, and the mixture is spin-dried. 3-49 (131 mg, 76%) is obtained by rapid column chromatography (PE:EA=15:1 to 10:1).

[0147] In summary, according to the synthesis method of 2-nitro sugar ene, Example 1 proposes to use 2-nitro sugar ene as a raw material, and obtain a pyranose type 2-nitro-3-azido-1,2-cis glycosylation product through a two-step one-pot method, and then reduce it to obtain 2,3-dideoxy-2,3-diamino sugar. Through condition screening, the optimal reaction conditions are determined. The method has mild conditions, high overall efficiency, simple operation, good selectivity, and a wide range of applications. At the same time, this method is also applied to the synthesis of Bordetella hinziiATCC 51730 lipopolysaccharide O-antigen repeating units, and 2,3-diaminopyranoside can be obtained efficiently, greatly reducing the difficulty of its synthetic route.

[0148] Example 2 Synthesis of Bordetella hinzii ATCC 51730 LPS O-antigen fragment

[0149] 1.1 Synthesis of trisaccharides

[0150] In the method for synthesizing the lipopolysaccharide O-antigen of Bordetella hinzii ATCC 51730 in Example 1, the synthesis of compound 3-49 (i.e., monosaccharide 2-8 in Example 2), compound 3-41 (i.e., disaccharide 2-33 in Example 2), compound 3-5 (i.e., trisaccharide 2-7 in Example 2), and compound 3-23 (i.e., disaccharide 2-38 in Example 2) has been completed. In order to facilitate the description of the compound synthesis route, some of the same compounds as those in Example 1 have been renamed and numbered in Example 2, which are described here.

[0151]

[0152] Compound 2-7 (210 mg, 0.10 mmol) and zinc powder (650 mg, 10.0 mmol, 100 eq) were weighed into a 25 mL reaction flask, tetrahydrofuran (3 mL) was added and stirred in a stirrer, and then anhydrous acetic acid (1 mL) was slowly added and allowed to react at room temperature overnight. TLC monitored the complete reaction of the raw material, filtered to remove the zinc powder, and the filter cake was washed with tetrahydrofuran and methanol 2-3 times respectively to obtain a filtrate, and the solvent was removed by rotary evaporator to obtain a crude product. Tetrahydrofuran (1 mL), pyridine (2 mL) and acetic anhydride (1 mL) were added to the reaction flask containing the crude product in sequence, and the reaction was carried out at room temperature for 3 hours. TLC monitored the complete reaction of the crude product, and methanol (2 mL) was added under an ice bath to quench the reaction. The rotary evaporator removed most of the solvent and concentrated, the sample was dry mixed, and column chromatography was separated (DCM / MeOH=10 / 1) to obtain a white solid powder 2-17 (84 mg, 0.10 mmol, 65%).

[0153]

[0154] Compound 2-17 (52 mg, 0.04 mmol) and potassium carbonate (10 mg, 0.06 mmol, 1.5 eq) were weighed into a reaction flask in sequence, methanol (2 mL) was added and stirred in a stirrer, and the reaction was carried out at room temperature for 1 hour. The reaction of the raw materials was completed by TLC monitoring, and most of the solvent was removed by rotary evaporator and concentrated. The sample was mixed by dry method and separated by column chromatography (DCM / MeOH=8 / 1) to obtain white solid powder 2-9 (44 mg, 0.038 mmol, 94%).

[0155]

[0156] Compound 2-9 (220 mg, 0.19 mmol) was added to a reaction flask, and dichloromethane (2 mL), distilled water (1 mL) and tert-butyl alcohol (0.5 mL) were added. The mixture was stirred on a stirrer, and TEMPO (30 mg, 0.19 mmol, 1.0 eq) and BAIB (612 mg, 1.9 mmol, 10 eq) were added in sequence. The mixture was reacted at room temperature for 6 hours. TLC detected that the reaction raw materials were complete, and the solvent was removed by a rotary evaporator to obtain a crude product. HATU (288 mg, 4 eq) was added to the reaction flask containing the crude product, and DMF (2 mL) was dissolved. DIPEA (0.16 mL) was added and stirred on a stirrer. Then NH3 (in 1,4-dioxane, 0.4 M, 4.2 mL) was slowly added and the mixture was reacted at room temperature for 3-6 hours. The crude product was completely reacted as monitored by TLC, concentrated by rotary evaporator, mixed by dry method, and separated by column chromatography (DCM / MeOH=6 / 1) to obtain white solid powder 2-26 (157 mg, 0.13 mmol, 69%).

[0157]

[0158] Compound 2-26 (55 mg, 0.045 mmol) was added to a plastic reaction bottle, tetrahydrofuran (1 mL) and pyridine (1 mL) were added, and the mixture was stirred in an ice bath for 5 min. HF / Py (0.14 mL) was slowly added dropwise, and the mixture was reacted at room temperature for 24-48 hours. TLC monitored the complete reaction of the raw materials, and solid sodium bicarbonate was added to neutralize the mixture, filtered, and the filter cake was washed with methanol. An appropriate amount of toluene was added to the system, and the mixture was concentrated on a rotary evaporator. The salt was removed and purified by Sephadex LH-20 gel column (DCM / MeOH=1 / 1) to obtain a white solid powder 2-27 (39 mg, 0.035 mmol, 80%).

[0159]

[0160] Compound 2-27 (45 mg, 0.04 mmol) was added to a reaction flask, followed by Pd(OH)2 / C (100 mg), tert-butyl alcohol (7 mL) and distilled water (3 mL). The system was placed in an ice bath, and the air in the reaction system was replaced with hydrogen from a hydrogen balloon, and the reaction was continued at room temperature for 24 hours. TLC monitored the complete reaction of the raw materials, filtered to remove Pd(OH)2 / C, obtained the filtrate, and concentrated on a rotary evaporator. Because the product was too polar, it was purified using a Sephadex LH-20 gel column (H2O / MeOH=1 / 1) to obtain a white solid 2-2 (32 mg, 0.036 mmol, 89%). 1 H NMR(400MHz,D2O)δ4.93(d,J=3.2Hz,1H),4.67(s,1H),4.55(d,J=8.3Hz,1H),4.38(s,2H),4.28–4.13(m,2H),4.01–3.80(m,5H),3.75–3 .57(m,4H),3.55–3.48(m,1H),2.91(t,J=7.4Hz,2H),1.94(s,6H),1.93–1.88(m,9H),1.87(s,3H),1.66–1.52(m,4H),1.43–1.30(m,2H). 13 C NMR(100MHz,D2O)δ174.7,174.6,174.5,174.3,174.3,174.1,172.7,172.3,171.3,101.6,101.1,96.6,76.9,76.2,75.2,74.6,69.6 ,69.2,68.3,54.6,54.3,53.6,53.3,49.7,47.4,39.3,28.0,26.5,22.3,22.2,22.18,22.08,22.06,21.9,21.7.HRMS(ESI-TOF)calcd for C 35 H 58 N 10 O 16 [M+Na] + 897.3924, found897.3923.

[0161] 1.2 Synthesis of Tetrasaccharide

[0162] Drawing on the synthetic strategy of the target molecule trisaccharide 2-2, the target molecule tetrasaccharide 2-1 was synthesized using a one-pot two-step method, first oxidizing it to an aldehyde using a combination of TEMPO and NaOCl, and then oxidizing it to a carboxylic acid through Pinnick, thereby preparing the target compound.

[0163]

[0164] Compound 2-7 (344 mg, 0.164 mmol) was added to a plastic reaction bottle, tetrahydrofuran (5 mL) and pyridine (5 mL) were added, and the mixture was stirred in an ice bath for 5 min. HF / Py (0.5 mL) was slowly added dropwise, and the mixture was reacted at room temperature for 24 hours. TLC monitored the complete reaction of the raw materials, saturated sodium bicarbonate solution was added to the system for neutralization, ethyl acetate was diluted, water and saturated sodium chloride were washed, anhydrous sodium sulfate was dried, filtered, concentrated by rotary evaporator, dry mixed, and separated by column chromatography (PE / EA=2 / 1) to obtain a white solid 2-28 (318 mg, 0.160 mmol, 98%).

[0165]

[0166] Compound 2-8 (150 mg, 0.179 mmol, 1.5 eq) and 2-28 (237 mg, 0.119 mmol, 1.0 eq) were added to the reaction flask, and freshly dried MS, dry dichloromethane was added under nitrogen protection, and the mixture was placed in a low-temperature reactor at -20°C and stirred for 10 min. TBSOTf (7 μL, 0.036 mmol, 0.3 eq) was added and the reaction was allowed to proceed for 2 hours. TLC monitored that the reaction of the raw material was complete, and triethylamine was added at -20°C to quench the reaction. The mixture was transferred to room temperature and filtered. The filtrate was concentrated on a rotary evaporator, and the sample was dry mixed and separated by column chromatography (PE / EA=1 / 2) to obtain a white solid 2-6 (244 mg, 0.14 mmol, 78%).

[0167]

[0168] Compound 2-6 (150 mg, 0.057 mmol) and zinc powder (582 mg, 8.96 mmol, 160 eq) were weighed into a 25 mL reaction flask, tetrahydrofuran (3 mL) was added and stirred in a stirrer, and then anhydrous acetic acid (1 mL) was slowly added and allowed to react at room temperature for 8 hours. TLC monitored the complete reaction of the raw material, filtered to remove the zinc powder, and the filter cake was washed with tetrahydrofuran and methanol 2-3 times respectively to obtain a filtrate, and the solvent was removed by rotary evaporator to obtain a crude product. Tetrahydrofuran (1 mL), pyridine (2 mL) and acetic anhydride (1 mL) were added to the reaction flask containing the crude product in sequence, and the reaction was reacted at room temperature for 5 hours. TLC monitored the complete reaction of the crude product, and methanol (2 mL) was added under an ice bath to quench the reaction. The rotary evaporator was concentrated, the sample was dry mixed, and column chromatography separation (DCM / MeOH=10 / 1) obtained a white solid 2-29 (51 mg, 0.032 mmol, 57%).

[0169]

[0170] Compound 2-29 (72 mg, 0.046 mmol) and potassium carbonate (10 mg, 0.069 mmol, 1.5 eq) were weighed into a reaction flask in sequence, methanol (2 mL) was added and stirred in a stirrer, and the reaction was carried out at room temperature for 2 hours. The reaction of the raw materials was completed by TLC monitoring, and most of the solvent was removed by rotary evaporator and concentrated. The sample was mixed by dry method and separated by column chromatography (DCM / MeOH=7 / 1) to obtain white solid powder 2-30 (52 mg, 0.038 mmol, 78%).

[0171]

[0172] Compound 2-30 (110 mg, 0.076 mmol) was added to a plastic reaction bottle, tetrahydrofuran (1 mL) and pyridine (1 mL) were added, and the mixture was stirred for 5 min in an ice bath, HF / Py (0.1 mL) was slowly added dropwise, and the mixture was reacted at room temperature for 2-3 hours. The reaction of the raw materials was completed after TLC monitoring. Solid sodium bicarbonate was added to neutralize the mixture. The mixture was filtered and the filter cake was washed with methanol. An appropriate amount of toluene was added to the system. The mixture was concentrated on a rotary evaporator and preliminarily purified by Sephadex LH-20 gel column (DCM / MeOH=1 / 1) to remove inorganic salts to obtain a crude product (the first step). The crude product (the first step) was transferred to a 25 mL round-bottom flask. TEMPO (14 mg, 0.091 mmol, 1.2 eq), NaBr (100 μL, 1 M), nBu4NBr (200 μL, 1 M) and saturated NaHCO3 (500 μL) were added in sequence. After adding dichloromethane (4 mL) and distilled water (0.64 mL), the mixture was moved to an ice bath and stirred for 5 min. NaClO (0.6 mL) was slowly added dropwise. After the addition was complete, the mixture was moved to room temperature and reacted for 1 hour. 1 M HCl solution was used to adjust the pH of the system to 6-8, and tert-butyl alcohol (2.8 mL), NaClO2 (200 mg) and NaH2PO4 (160 mg) dissolved in 0.8 mL of distilled water, and 2-methyl-2-butene (0.74 mL) were added in sequence, and the reaction was carried out at room temperature for 2 hours. TLC monitored the complete reaction of the raw materials, methanol was added for dilution, and the solvent was removed by rotary evaporator. Because the product has moderate polarity and the main purpose is to remove salts, it is not suitable to use methanol / water as the mobile phase, so Sephadex LH-20 gel column (DCM: MeOH = 1: 1) was used for preliminary purification to remove inorganic salts to obtain a crude product (the second step); HATU (144 mg, 5 eq) was added to the reaction flask containing the crude product (the second step), DMF (2 mL) was dissolved, DIPEA (80 μL, 6 eq) was added, and the mixture was stirred on a stirrer, and then NH3 (in 1,4-dioxane, 0.4 M, 2.2 mL, 12 eq) was slowly added, and the mixture was reacted at room temperature for 8 hours. The crude product was completely reacted by TLC monitoring, concentrated by rotary evaporator, mixed by dry method, and separated by column chromatography (DCM / MeOH=1 / 1) to obtain a crude product containing a small amount of silica gel (the third step). The crude product (the third step) was removed by Sephadex LH-20 gel column (DCM / MeOH=1 / 1) to obtain a white solid 2-5 (45 mg, 0.033 mmol, 45%).

[0173]

[0174] Compound 2-5 (20 mg, 0.022 mmol) was added to a reaction flask, followed by Pd(OH)2 / C (120 mg), tert-butyl alcohol (7 mL) and distilled water (3 mL). The system was placed in an ice bath, and the air in the reaction system was replaced with hydrogen from a hydrogen balloon. The reaction was allowed to proceed at room temperature for 1 day. TLC monitored the complete reaction of the raw material, and Pd(OH)2 / C was removed by filtration to obtain the filtrate, which was concentrated by a rotary evaporator. Considering that the product had good solubility in a methanol / water mobile phase, it was purified using a Sephadex LH-20 gel column (H2O / MeOH=1 / 1) to obtain a white solid 2-1 (20 mg, 0.018 mmol, 80%). 1 H NMR(400MHz,D2O)δ5.21(d,J=3.5Hz,1H),4.95(d,J=3.4Hz,1H),4.68(s,1H),4.6 0(d,J=8.3Hz,1H),4.39(s,2H),4.27–4.10(m,8H),4.02–3.90(m,4H),3.86–3.80 (m,2H),3.77–3.48(m,6H),2.95–2.90(m,2H),1.95(s,6H),1.92(s,6H),1.91(s, 3H),1.89(s,3H),1.86(s,3H),1.84(s,3H),1.64–1.55(m,4H),1.43–1.34(m,2H). 13 C NMR(100MHz,D2O)δ174.7,174.5,174.3,174.29,174.2,174.1,173.8,173.2 ,172.8,171.8,171.4,101.4,101.1,96.8,96.6,77.3,75.5,75.1,74.6,73.0 ,71.4,69.7,68.4,66.9,54.5,54.3,53.3,49.7,48.5,47.4,46.7,39.3,28.0 ,26.5,22.3,22.2,22.1,22.07,22.0,21.8,21.7,21.7.HRMS(ESI-TOF)calcd for C 45 H 73 N 13 O 21 [M+Na] + 1331.4936, found 1331.4980.

[0175] 1.3 Synthesis of monosaccharides and disaccharides

[0176] Monosaccharide 2-4 and disaccharide 2-3 are both building blocks of the target molecules trisaccharide and tetrasaccharide, so the same synthesis strategy is adopted to complete the synthesis of the target molecules monosaccharide 2-4 and disaccharide 2-3.

[0177]

[0178] Compound 2-23 (1g, 1.59mmol) and zinc powder (2g, 32mmol, 20eq) were weighed into a 25mL reaction flask in sequence, tetrahydrofuran (10mL) was added and stirred in a stirrer, and then anhydrous acetic acid (3mL) was slowly added and allowed to react at room temperature for 4 hours. TLC monitored the complete reaction of the raw material, filtered to remove the zinc powder, and the filter cake was washed with tetrahydrofuran and methanol 2-3 times respectively to obtain a filtrate, and the solvent was removed by rotary evaporator to obtain a crude product. Tetrahydrofuran (4mL), pyridine (8mL) and acetic anhydride (4mL) were added to the reaction flask containing the crude product in sequence, and the reaction was reacted at room temperature for 2 hours. TLC monitored the complete reaction of the crude product, and methanol (4mL) was added under ice bath to quench the reaction. The rotary evaporator was concentrated, the sample was dry mixed, and column chromatography separation (DCM / MeOH=30 / 1) obtained a white solid 2-31 (846mg, 1.29mmol, 81%).

[0179]

[0180] Compound 2-31 (846 mg, 1.29 mmol) and potassium carbonate (663 mg, 4.8 mmol, 3 eq) were weighed into a reaction flask in sequence, methanol (16 mL) was added and stirred in a stirrer, and the reaction was carried out at room temperature for 1 hour. The reaction of the raw materials was completed by TLC monitoring, and most of the solvent was removed by rotary evaporator and concentrated. The sample was mixed by dry method and separated by column chromatography (DCM / MeOH=12 / 1) to obtain a white solid 2-32 (617 mg, 1.07 mmol, 90%).

[0181]

[0182] Compound 2-32 (250 mg, 0.42 mmol) was added to a reaction flask, and dichloromethane (3 mL), distilled water (1.5 mL) and tert-butyl alcohol (0.7 mL) were added. The mixture was stirred on a stirrer, and TEMPO (13 mg, 0.084 mmol, 0.2 eq) and BAIB (541 mg, 1.68 mmol, 4 eq) were added in sequence. The mixture was reacted at room temperature for 4 hours. TLC detected that the reaction raw materials were complete, and the solvent was removed by a rotary evaporator to obtain a crude product. HATU (319 mg, 2 eq) was added to the reaction flask containing the crude product, and DMF (4.2 mL) was dissolved. DIPEA (0.14 mL, 3 eq) was added and stirred on a stirrer. Then NH3 (in 1,4-dioxane, 0.4 M, 4 mL, 3 eq) was slowly added and reacted at room temperature for 3-6 hours. The crude product was completely reacted as monitored by TLC, concentrated by rotary evaporator, mixed by dry method, and separated by column chromatography (DCM / MeOH=8 / 1) to obtain white solid powder 2-25 (150 mg, 0.25 mmol, 59%).

[0183]

[0184] Compound 2-25 (30 mg, 0.051 mmol) was added to a reaction flask, followed by Pd(OH)2 / C (100 mg), tert-butyl alcohol (7 mL) and distilled water (3 mL). The system was placed in an ice bath, and the air in the reaction system was replaced with hydrogen from a hydrogen balloon. The reaction was allowed to proceed at room temperature for 12 hours. TLC monitored the complete reaction of the raw material, and Pd(OH)2 / C was removed by filtration to obtain the filtrate, which was concentrated by a rotary evaporator. Considering that the product had good solubility in a methanol / water mobile phase, it was purified using a Sephadex LH-20 gel column (H2O / MeOH=1 / 1) to obtain a white solid 2-4 (15 mg, 0.042 mmol, 83%). 1 H NMR(400MHz,D2O)δ4.94(s,1H),4.38(s,1H),4.23(d,J=12.3Hz,1H),4.20–4.15(m,2H),3.72–3.62(m, 1H),3.54–3.45(m,1H),2.89(t,J=7.6Hz,2H),1.95–1.91(m,6H),1.69–1.54(m,4H),1.46–1.32(m,2H). 13 C NMR(100MHz,D2O)δ174.4,174.3,173.8,96.6,70.7,68.2,67.7,48.9,47.3,39.4,28.0,26.7,22.3,21.8,21.7.HRMS(ESI-TOF)calcd for C 30 H40 N4O8[M+Na] + 360.2082, found 361.2080.

[0185]

[0186] Compound 2-33 (100 mg, 0.064 mmol) and zinc powder (209 mg, 3.2 mmol, 50 eq) were weighed into a 25 mL reaction flask, tetrahydrofuran (3 mL) was added and stirred in a stirrer, and then anhydrous acetic acid (1 mL) was slowly added and allowed to react at room temperature for 4 hours. TLC monitored the complete reaction of the raw material, filtered to remove the zinc powder, and the filter cake was washed with tetrahydrofuran and methanol 2-3 times respectively to obtain a filtrate, and the solvent was removed by rotary evaporator to obtain a crude product. Tetrahydrofuran (1 mL), pyridine (2 mL) and acetic anhydride (1 mL) were added to the reaction flask containing the crude product in sequence, and the reaction was reacted at room temperature for 3 hours. TLC monitored the complete reaction of the crude product, and methanol (1 mL) was added under ice bath to quench the reaction. The rotary evaporator was concentrated, the sample was dry mixed, and column chromatography separation (DCM / MeOH=12 / 1) obtained a white solid 2-35 (47 mg, 0.046 mmol, 72%).

[0187]

[0188] Compound 2-34 (230 mg, 0.226 mmol) and potassium carbonate (31 mg, 0.22 mmol, 1 eq) were weighed into a reaction flask in sequence, methanol (4 mL) was added and stirred in a stirrer, and the reaction was carried out at room temperature for 2 hours. TLC monitored the complete reaction of the raw materials, and most of the solvent was removed by rotary evaporator and concentrated. The sample was mixed by dry method and separated by column chromatography (DCM / MeOH=11 / 1) to obtain a white solid 2-35 (202 mg, 0.21 mmol, 96%).

[0189]

[0190] Compound 2-35 (202 mg, 0.21 mmol) was added to a reaction flask, and dichloromethane (3 mL), distilled water (1.5 mL) and tert-butanol (0.7 mL) were added. The mixture was stirred on a stirrer, and TEMPO (13 mg, 0.084 mmol, 0.4 eq) and BAIB (541 mg, 1.68 mmol, 8 eq) were added in sequence. The mixture was reacted at room temperature for 5 hours. TLC detected that the reaction raw materials were complete, and the solvent was removed by a rotary evaporator to obtain a crude product. HATU (239 mg, 3 eq) was added to the reaction flask containing the crude product, and DMF (2.5 mL) was dissolved. DIPEA (0.15 mL) was added and stirred on a stirrer. Then NH3 (in 1,4-dioxane, 0.4 M, 3.1 mL) was slowly added and the mixture was reacted at room temperature for 6 hours. The crude product was completely reacted as monitored by TLC, concentrated by rotary evaporator, mixed by dry method, and separated by column chromatography (DCM / MeOH=10 / 1) to obtain white solid powder 2-36 (144 mg, 0.15 mmol, 72%).

[0191]

[0192] Compound 2-36 (55 mg, 0.057 mmol) was added to a plastic reaction bottle, tetrahydrofuran (1 mL) and pyridine (1 mL) were added, and the mixture was stirred in an ice bath for 5 min. HF / Py (0.1 mL) was slowly added dropwise, and the mixture was reacted at room temperature for 2 days. TLC monitored the complete reaction of the raw materials, and solid sodium bicarbonate was added to neutralize the mixture, filtered, and the filter cake was washed with methanol. An appropriate amount of toluene was added to the system, and the mixture was concentrated on a rotary evaporator, and the sample was dry mixed. Column chromatography (DCM / MeOH=6 / 1) was used to separate the mixture to obtain a white solid 2-37 (39.5 mg, 0.046 mmol, 82%).

[0193]

[0194] Compound 2-37 (35 mg, 0.041 mmol) was added to a reaction flask, followed by Pd(OH)2 / C (100 mg), tert-butyl alcohol (7 mL) and distilled water (3 mL). The system was placed in an ice bath, and the air in the reaction system was replaced with hydrogen from a hydrogen balloon. The reaction was allowed to proceed overnight at room temperature. TLC monitored the complete reaction of the raw material, and Pd(OH)2 / C was removed by filtration to obtain the filtrate, which was concentrated by a rotary evaporator and purified by a Sephadex LH-20 gel column (H2O / MeOH=1 / 1) to obtain a white solid 2-3 (22 mg, 0.035, 86%). 1H NMR(400MHz,D2O)δ4.94(d,J=3.3Hz,1H),4.40(d,J=7.5Hz,2H),4.29–4.17(m,2H),3.96(t,J=10.5Hz,1H),3.79(d,J=9.7Hz, 1H),3.68–3.49(m,4H),2.95–2.85(m,2H),1.96(d,J=2.5Hz,6H),1.92(d,J=3.5Hz,6H),1.64–1.54(m,4H),1.41–1.31(m,2H). 13 C NMR(100MHz,D2O)δ174.73,174.70,174.6,174.2,173.3,173.1,100.7,96.6,74.8,74.5,70.0,69.8 ,68.4,54.5,54.3,49.7,47.3,39.3,28.0,26.5,22.3,22.2,22.1,21.9,21.7.HRMS(ESI-TOF)calcd for C 46 H 69 N7O 13 Si[M+H] + 656.2652, found 656.2644.

[0195] 1.4 Synthesis of trisaccharide derivatives

[0196]

[0197] Compound 2-9 (55 mg, 0.047 mmol) was added to a reaction flask, and dichloromethane (2 mL) and distilled water (1 mL) were added. The mixture was stirred on a stirrer, and TEMPO (8 mg, 0.047 mmol, 1.0 eq) and BAIB (150 mg, 0.47 mmol, 10 eq) were added in sequence. The mixture was placed on a magnetic stirrer and reacted at room temperature for 6 hours. TLC detected that the reaction raw materials were complete, and the solvent was removed by a rotary evaporator to obtain a crude product. KHCO3 (56 mg, 0.56 mmol, 12.0 eq) and DMF (1.0 mL) were added to the reaction flask containing the crude product, and BnBr (28 μL, 0.234 mmol, 5.0 eq) was added under an ice bath and stirred on a stirrer. The mixture was reacted at room temperature for 4 hours. The crude product was completely reacted as monitored by TLC, diluted with dichloromethane, washed with water, saturated NaHCO3 solution and saturated NaCl solution, dried over anhydrous Na2SO4, concentrated, dry mixed, and separated by column chromatography (DCM / MeOH=20 / 1) to give a white solid 2-38 (51 mg, 0.034 mmol, 73%).

[0198]

[0199] Compound 2-38 (24 mg, 0.016 mmol) was added to a plastic reaction bottle, tetrahydrofuran (1 mL) and pyridine (1 mL) were added, and the mixture was stirred in an ice bath for 5 min. HF / Py (0.3 mL) was slowly added dropwise, and the mixture was reacted at room temperature for 1-2 days. TLC monitored the complete reaction of the raw materials, and solid sodium bicarbonate was added to neutralize the mixture, filtered, and the filter cake was washed with methanol. An appropriate amount of toluene was added to the system, and the mixture was concentrated on a rotary evaporator, and the sample was dry mixed. Column chromatography (DCM / MeOH=12 / 1) was used to obtain a white solid 2-39 (20 mg, 0.014 mmol, 91%).

[0200]

[0201] Compound 2-39 (18 mg, 0.013 mmol) was added to a reaction flask, followed by Pd(OH)2 / C (100 mg), tert-butyl alcohol (7 mL) and distilled water (3 mL). The system was placed in an ice bath, and the air in the reaction system was replaced with hydrogen from a hydrogen balloon. The reaction was allowed to proceed at room temperature for 3 days. TLC monitored the complete reaction of the raw material, and Pd(OH)2 / C was removed by filtration to obtain the filtrate, which was concentrated by a rotary evaporator. Considering that the product had good solubility in a methanol / water mobile phase, it was purified using a Sephadex LH-20 gel column (H2O / MeOH=1 / 1) to obtain a white solid 2-2a (9 mg, 0.010 mmol, 82%). 1 H NMR(600MHz,D2O)δ4.78(dd,J=13.2,3.1Hz,1H),4.47(d,J=8.4Hz,1H),4.40(d,J=8.3Hz,1H),4.30(s,1H),4.17(m,2H),4.09–4.05(m,1H),3 .97(d,J=9.8Hz,1H),3.92–3.39(m,13H),2.83(t,J=7.6Hz,2H),1.88– 1.85(m,6H),1.83–1.79(m,11H),1.55–1.46(m,4H),1.33–1.27(m,2H). 13C NMR (100MHz, D2O) δ174.70,174.67,174.63,174.57,174.5,174.4,174.1,173.4,101.7,101.6,96.6,77.6,77.4,75.74,75.66,69. 7,68.2,54.6,54.5,53.6,52.9,49.8,47.4,46.7,39.3,28.0,26.4,22.3,22.2,22.11,22.07,21.9,21.7,8.2.HRMS(ESI-TOF)calcd for C 35 H 55 N7O 19 [M+H] + 878.3625, found 878.3626.

[0202]

[0203] Compound 2-9 (83 mg, 0.071 mmol) was added to a plastic reaction bottle, tetrahydrofuran (2 mL) and pyridine (2 mL) were added, and the mixture was stirred in an ice bath for 5 min. HF / Py (0.2 mL) was slowly added dropwise, and the mixture was reacted at room temperature for 1-2 days. TLC monitored the complete reaction of the raw materials, and solid sodium bicarbonate was added to neutralize the mixture, filtered, and the filter cake was washed with methanol. An appropriate amount of toluene was added to the system, and the mixture was concentrated on a rotary evaporator, and the sample was dry mixed. Column chromatography (DCM / MeOH=12 / 1) was used to separate the mixture to obtain a white solid 2-40 (70 mg, 0.066 mmol, 93%).

[0204]

[0205] Compound 2-40 (70 mg, 0.066 mmol) was added to a reaction flask, followed by Pd(OH)2 / C (200 mg), tert-butyl alcohol (7 mL) and distilled water (3 mL). The system was placed in an ice bath, and the air in the reaction system was replaced with hydrogen from a hydrogen balloon. The reaction was allowed to proceed at room temperature for 2 days. TLC monitored the complete reaction of the raw material, and Pd(OH)2 / C was removed by filtration to obtain the filtrate, which was concentrated by a rotary evaporator. Considering that the product had good solubility in a methanol / water mobile phase, it was purified using a Sephadex LH-20 gel column (H2O / MeOH=1 / 1) to obtain a white solid 2-2b (52 mg, 0.062, 94%). 1H NMR(600MHz,D2O)δ4.80(d,J=3.6Hz,1H),4.62(d,J=8.3Hz,1H),4.48(d,J=8.2Hz,1H),4.16(dd,J=11.9,2.4 Hz,1H),4.13–4.07(m,2H),3.99(t,J=10.5Hz,1H),3.92(t,J=6.4Hz,1H),3.89–3.82(m,2H),3.79(d,J=11.0H z,1H),3.74–3.68(m,1H),3.69–3.57(m,7H),3.58–3.51(m,1H),3.48–3.39(m,3H),3.35(t,J=9.7Hz,1H),2.9 1(t,J=7.6Hz,2H),1.97(s,4H),1.95(s,5H),1.93(s,5H),1.90(s,9H),1.64–1.55(m,4H),1.42–1.34(m,2H). 13 C NMR(150MHz,D2O)δ174.8,174.7,174.6,174.5,174.3,174.2,101.2,101.0,96.3,77.1,76.0,75.2,73.5,70.5,67.9,67.8,61.1 ,60.9,60.1,54.8,54.5,54.2,53.0,50.2,47.7,39.3,28.0,26.5,22.4,22.14,22.12,21.92,21.90,21.7.HRMS(ESI-TOF)calcd for C 35 H 61 N7O 16 [M+H] + 836.4248, found836.4253.

[0206] In summary, this example successfully completed the synthesis of LPS O-antigen of Bordetella hinsonii ATCC 51730 by adjusting the synthesis strategy, including monosaccharide, disaccharide, trisaccharide, tetrasaccharide and two trisaccharide derivative molecules.

[0207] The present invention selects unprotected ammonia to construct uronic acid amide, and constructs trisaccharide 2-27 by this method, and successfully hydrogenates to obtain the target molecule trisaccharide. Subsequently, monosaccharide, disaccharide and tetrasaccharide target molecules are constructed. Among them, in the construction process of tetrasaccharide, TEMPO and NaOCl are used to oxidize to aldehyde, and then Pinnick is used to oxidize to carboxylic acid, and the construction is carried out by a two-step one-pot method. Finally, two trisaccharide derivative molecules that have not been oxidized and have only been oxidized without condensation can be synthesized through multiple steps through trisaccharide 2-9.

[0208] After completing the synthesis of the O-antigen fragment of the lipopolysaccharide of Bordetella hinskeli ATCC 51730, these molecules will be screened for antigenic activity to identify key sugar epitopes. Understanding the effects of carboxylic acid and formamide functional groups on sugars on antigenicity will lay the foundation for the development of sugar vaccines against Bordetella hinskeli.

[0209] Example 3 Synthesis of glycoconjugates

[0210] 1.1 Synthesis and characterization of glycoconjugates

[0211] In order to facilitate the description of the synthetic route of the glycoconjugate, some of the compounds identical to those in Example 2 were renamed and numbered in Example 3, and are explained here. Compound 1 in Example 3 is 2-4 in Example 2, compound 2 in Example 3 is 2-3 in Example 2, compound 3 in Example 3 is 2-2b in Example 2, compound 4 in Example 3 is 2-2a in Example 2, compound 5 in Example 3 is 2-2 in Example 2, and compound 6 in Example 3 is 2-1 in Example 2.

[0212] 1.1.1 Synthesis of glycoconjugates

[0213] In a 5 mL round-bottom flask, dissolve di(N-succinimidyl)glutarate (DSG; 9.06 mg, 27.8 μmol; 5.28 mg, 16.2 μmol; 3.72 mg, 11.4 μmol; 3.72 mg, 11.4 μmol; 3.91 mg, 12 μmol; 2.87 mg, 8.8 μmol) in DMSO (160 μL), add triethylamine (9 μL; 6 μL; 4 μL; 4 μL; 4 μL; 3 μL) and stir at 450 rpm for 5 min. Dissolve sugar (compound 1: 1 mg, 2.78 μmol; compound 2: 1 mg, 1.62 μmol; compound 3: 1 mg, 1.2 μmol; compound 4: 1 mg, 1.14 μmol; compound 5: 1 mg, 1.14 μmol; compound 6: 1 mg, 0.88 μmol) in DMSO (160 μL) using a 1.5 mL EP tube. Slowly add sugar dropwise to DSG (the entire dropwise addition process is greater than 10 min), and react at 450 rpm for 2 h at room temperature. Transfer the reaction system to a 15 mL centrifuge tube, rinse the original bottom flask with 800 μL 10× PBS and collect the liquid in a 15 mL centrifuge tube, then add 5 mL chloroform and centrifuge (1800 g, 2 min), take the upper aqueous phase, and repeat 3 times. Take the aqueous phase in a 1.5 mL EP tube for the last time, centrifuge (14500 g, 1 min), take the upper aqueous phase, and repeat 2 times. After centrifugation, observe that there is no stratification of the solution. Take the supernatant from the capillary tube and apply it to the TLC plate to show the sugar color. Finally, collect the upper aqueous phase. Take HSA (human serum albumin, 0.73mg, 11nmol; 0.4mg, 6nmol; 0.33mg, 5nmol; 0.33mg, 5nmol; 0.33mg, 5nmol; 0.27mg, 4nmol, MKBio) and add 1mL 10×PBS to dissolve, add the above upper aqueous phase, and stir at 380rpm for 18h at room temperature. Fill the inner and outer tubes of the ultrafiltration tube (30kDa MWCO, Millipore) with ultrapure water and precool on ice for 3min. Discard the ultrapure water in the inner and outer tubes, and transfer the solution after the above reaction for 18h into the ultrafiltration tube. Add 2mL of ultrapure water and centrifuge (4000rpm, 6min, 4℃), repeat the above operation 3 times, and finally concentrate the solution volume to about 200μL to obtain the sugar conjugate.

[0214] 1.1.2 Characterization of glycoconjugates

[0215] According to the concentration of sugar conjugates determined by the BCA method, the sugar conjugate solution and the original HSA protein were diluted to 1 mg / mL with ultrapure water, and 20 μL of each was used for the detection of the molecular weight of the conjugate by time-of-flight mass spectrometry (MALDI-TOF-MS). The conjugation reaction was qualitatively determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A 1.5 mL EP tube was taken, and 20 μL of sample solution and 5 μL of 5×SDS-PAGE loading buffer were added to each tube. After heating at 100°C for 3 min, the tube was centrifuged, and each sample solution and 4 μL of Marker were added to the lanes. Cover the electrophoresis tank cover and pay attention to the electrode correspondence. Turn on the electrophoresis instrument, wipe off the moisture of the electrode, and perform electrophoresis in two sections. The first section: constant voltage 70V, time 30min; the second section: constant voltage 120V, time 60min. After the electrophoresis, the gel was taken out and placed in Coomassie Brilliant Blue R250 protein rapid staining solution for 10 minutes at 45°C water bath. After the staining, it was rinsed repeatedly with ultrapure water for 2-3 times, and then added to 25mL of decolorizing solution for 15 minutes at 45°C water bath, and then the remaining 25mL of decolorizing solution was added and placed on a shaker for shaking. After 4 hours, the decolorizing solution was replaced with ultrapure water and continued to be placed on a shaker overnight for shaking until the background was transparent and without blue. The decolorized gel was placed in an automatic gel imager for scanning and imaging, and the picture was saved. By MALDI-TOF-MS and SDS-PAGE determination, the sugar conjugates HSA-1, HSA-2, HSA-3, HSA-4, HSA-5 and HSA-6 contained an average of 3.3, 3.6, 2.5, 1.1, 2.5 and 1.6 sugars on each HSA molecule, respectively.

[0216] 1.2 Inactivated Bordetella hindsii immunized mice to obtain serum antibodies

[0217] Female BALB / c mice aged 6 to 8 weeks (4 mice per group, two groups in total: immunization group and control group) were subcutaneously immunized with inactivated B. hindsii. According to the immunization process, each mouse was immunized with inactivated bacteria (80 μg / mouse) containing complete Freund's adjuvant (CFA) on day 0, and boosted with inactivated bacteria (80 μg / mouse) containing incomplete Freund's adjuvant (IFA) on days 14 and 28. The volume ratio of bacterial liquid to Freund's adjuvant was 1:1. The control group mice were injected with PBS only. Blood was collected from mice through the orbital vein on day 35, and serum was prepared.

[0218] 1.3 ELISA screening of the best glycoconjugate against Bordetella hindschildi

[0219] HSA, HSA-1, HSA-2, HSA-3, HSA-4, HSA-5, and HSA-6 (10 μg / mL) were dissolved in carbonate buffer (0.05 M, pH = 9.6), and then the antigen was added to a high-binding 96-well ELISA plate (Corning) at 100 μL / well. Three replicate wells were set for each sample and left to stand overnight at 4°C. The next day, the liquid in the wells was discarded, 300 μL PBST was added to each well, and the wells were shaken for 5 minutes, and the wells were washed three times. The PBST was discarded, and the antiserum (primary antibody) was diluted with 1% BSA-PBS at a ratio of 1:4000, and incubated at 37°C for 2 hours. The primary antibody in the wells was discarded, and the wells were washed three times with PBST, and HRP-labeled goat anti-mouse IgG (secondary antibody) diluted with 1% BSA-PBS at a ratio of 1:10000 was added, and the wells were incubated at 37°C in the dark for 1 hour. Discard the secondary antibody in the wells, wash 3 times with PBST (same as above), add 100 μL TMB colorimetric solution (solution A: solution B = 1:1) to each well, and incubate at 37°C in the dark for 20 min until color is developed. Keep the colorimetric solution, add 50 μL 2% sulfuric acid to each well to terminate the reaction, and immediately use an enzyme reader to detect the absorbance value of each well at 450 nm.

[0220] 2.1 Results and Discussion

[0221] 2.1.1 Preparation of sugar-HSA protein glycoconjugates

[0222] In order to screen out the best antigens among compounds 1, 2, 3, 4, 5 and 6, it is necessary to first combine the above six sugars with HSA carrier protein to form a series of sugar conjugates, which are convenient for fixation on the bottom of the wells of the ELISA plate and used as coatings for ELISA screening of the best antigens to detect the binding ability with anti-Bordetella hindschildi serum antibodies. After preparing a series of sugar conjugates, SDS-PAGE and MALD-TOF-MS were used for qualitative and quantitative characterization to calculate the sugar content of each conjugate.

[0223] 2.1.2 Synthesis and characterization of sugar-HSA conjugates

[0224] (1) Synthesis and characterization of HSA-1

[0225] HSA was conjugated with compound 1 by the activated ester method to obtain HSA-1 conjugate, which was analyzed by SDS-PAGE and MALD-TOF-MS results (see Figure 1 ), the conjugation effect of HSA and compound 1 can be analyzed and evaluated.

[0226] like Figure 1The SDS-PAGE results show that compared with the simple carrier protein HSA, the migration path of the band of the sugar conjugate HSA-1 on the gel is shorter. Due to the molecular sieving effect of polyacrylamide, proteins with small molecular weight can pass through the gel pores more easily, with small resistance and fast migration speed, while proteins with large molecular weight are subject to greater resistance and are lagged behind. Therefore, it can be seen from the figure that the molecular weight of the HSA-1 conjugate is larger than that of HSA, and the preliminary characterization shows that compound 1 is successfully conjugated with HSA.

[0227] observe Figure 1 The MALDI-TOF-MS results showed that the molecular weight of HSA was 66406.715Da and that of HSA-1 was 67893.954Da, which further characterized the successful conjugation of compound 1 with HSA. Calculations showed that approximately 3.3 sugar molecules were conjugated to each HSA protein molecule.

[0228] (2) Synthesis and characterization of HSA-2

[0229] DSG was connected to the amino group on compound 2 by the activated ester method to form an amide bond, and then it was conjugated to HSA protein to form a glycoprotein conjugate. Finally, ultrafiltration and concentration were performed, and the conjugation results were verified by SDS-PAGE and MALDI-TOF-MS (see Figure 2 ), it was calculated that approximately 3.6 sugar molecules were conjugated to each HSA protein molecule.

[0230] (3) Synthesis and characterization of HSA-3

[0231] DSG was connected to the amino group on compound 3 by the activated ester method to form an amide bond, and then it was conjugated to HSA protein to form a glycoprotein conjugate. Finally, ultrafiltration and concentration were performed, and the conjugation results were verified by SDS-PAGE and MALDI-TOF-MS (see Figure 3 ), it was calculated that approximately 2.5 sugar molecules were conjugated to each HSA protein molecule.

[0232] (4) Synthesis and characterization of HSA-4

[0233] DSG was connected to the amino group on compound 4 by the activated ester method to form an amide bond, and then it was conjugated to HSA protein to form a glycoprotein conjugate. Finally, ultrafiltration and concentration were performed, and the conjugation results were verified by SDS-PAGE and MALDI-TOF-MS (see Figure 4 ), it was calculated that approximately 1.1 sugar molecules were conjugated to each HSA protein molecule.

[0234] (5) Synthesis and characterization of HSA-5

[0235] DSG was connected to the amino group on compound 5 by the activated ester method to form an amide bond, and then it was conjugated to HSA protein to form a glycoprotein conjugate. Finally, ultrafiltration and concentration were performed, and the conjugation results were verified by SDS-PAGE and MALDI-TOF-MS (see Figure 5 ), it was calculated that approximately 2.5 sugar molecules were conjugated to each HSA protein molecule.

[0236] (6) Synthesis and characterization of HSA-6

[0237] DSG was connected to the amino group on compound 6 by the activated ester method to form an amide bond, and then it was conjugated to HSA protein to form a glycoprotein conjugate. Finally, ultrafiltration and concentration were performed, and the conjugation results were verified by SDS-PAGE and MALDI-TOF-MS (see Figure 6 ), it was calculated that approximately 1.6 sugar molecules were conjugated to each HSA protein molecule.

[0238] 2.1.3 Sugar conjugation results and analysis

[0239] By MALDI-TOF-MS and SDS-PAGE, the glycoconjugates HSA-1, HSA-2, HSA-3, HSA-4, HSA-5 and HSA-6 contained an average of 3.3, 3.6, 2.5, 1.1, 2.5 and 1.6 sugars per HSA molecule, respectively. The results showed that the average number of sugars bound to each HSA molecule decreased with increasing sugar chain length. It is noteworthy that the sugar content of these glycoconjugates is comparable, all within a narrow range of 1.4-3.2% (Table 1).

[0240] Table 1 Sugar content of sugar conjugates

[0241]

[0242] 2.1.4 Serum antibodies obtained from mice immunized with inactivated Bordetella hindsoe

[0243] Female BALB / c mice aged 6 to 8 weeks (4 mice per group, two groups in total: immunization group and control group) were subcutaneously immunized with inactivated B. hindsii. On day 0, each mouse was immunized with inactivated bacteria (80 μg / mouse) containing complete Freund's adjuvant (CFA), and boosted with inactivated bacteria (80 μg / mouse) containing incomplete Freund's adjuvant (IFA) on days 14 and 28, with a volume ratio of 1:1 between bacterial solution and Freund's adjuvant. The control group mice were injected with PBS only. Blood was collected from mice through the orbital vein on day 35, and serum was prepared.

[0244] 2.1.5 ELISA screening of the best glycoconjugate against Bordetella hindschildi

[0245] In order to evaluate the antigenicity of the six sugars, ELISA assays were performed using HSA as a negative control and synthetic sugar conjugates as coating antigens. First, inactivated Bordetella hinsonii was mixed with Freund's adjuvant (FA) as the positive immune group and BALB / c mice were subcutaneously immunized. The mice in the negative non-immune group received only PBS. The positive immune group received a primary vaccine on day 0, a booster on day 14, and another booster on day 28. The mouse sera from the positive immune group and the negative non-immune group were extracted on day 35 for ELISA antibody titer analysis. The ability of the antibodies produced by the mice to recognize the antigen (sugar conjugate) was determined by the ratio (P / N) of the absorbance of the serum of the positive immune mouse (P) to the absorbance of the serum of the negative non-immune mouse (N). As Figure 7 As shown, the serum of mice immunized with inactivated B. hinschweig on the 35th day had almost no binding to HSA protein. Among the six synthetic sugar conjugates, HSA-6 was significantly recognized by the serum of the immunized group on the 35th day, followed by HSA-3 and HSA-1, which were able to better recognize antibodies. These results also show that the length of the sugar chain has a significant effect on the recognition of positive immune mouse serum, and after the introduction of hydroxyl groups on the sugar chain, the ability of antigen recognition antibodies can be increased to a certain extent, while the introduction of carboxyl groups has the opposite effect. Therefore, compound 6 can be used as a promising antigen for the development of candidate vaccines against B. hinschweig.

Claims

1. A trisaccharide compound, characterized in that Including the compound shown in formula I, the specific structure is as follows: R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, or TBSO-; R3 is -AcNH, TrocNH- or -NO2; R4 is -O(CH2) n NH2, -O(CH2) n NCbz(Bn) or -O(CH2) n NHCbz; n is an integer between 2 and 6.

2. The compound according to claim 1, characterized in that The trisaccharide compound shown in formula I is 2-7, 2-9, 2-2, 2-2a or 2-2b, and has the structural formula:

3. A method for synthesizing the trisaccharide compound according to claim 1 or 2, characterized in that: The specific steps include: 1) reacting a monosaccharide donor and a monosaccharide acceptor in an organic solvent I and a catalyst I at -40°C to -20°C for 1-2h, quenching after the reaction, drying the solvent, and purifying by silica gel column chromatography to obtain a disaccharide intermediate I; 2) dissolving the disaccharide intermediate I in THF and Py, and then reacting at 25°C to 35°C for 2-4 days under HF / Py conditions, washing, extracting, filtering, spin drying, and purifying by silica gel column chromatography to obtain the disaccharide intermediate II; 3) reacting the monosaccharide donor and the disaccharide intermediate II in the presence of an organic solvent I and a catalyst I at -40°C to -20°C for 1-2h, quenching after the reaction, drying the solvent, and purifying by silica gel column chromatography to obtain a trisaccharide product; The monosaccharide donor in step 1) is The monosaccharide acceptor in step 1) is The mass ratio of the monosaccharide donor to the monosaccharide acceptor in step 1) is (1.1-1.2):1; In step 3), the mass ratio of the monosaccharide donor to the disaccharide intermediate II is (1.2-1.4):1; The organic solvent I is dichloromethane or tetrahydrofuran; Catalyst I is tert-butyldimethylsilyl trifluoromethanesulfonate.

4. A tetrasaccharide compound, characterized in that Including the compound represented by formula II and / or the compound represented by formula III, the specific structure is as follows: R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, TBSO- or R3 is -AcNH, TrocNH- or -NO2; R4 is -O(CH2) n NH2, -O(CH2) n NCbz(Bn) or -O(CH2) n NHCbz; n is an integer between 2 and 6.

5. The compound according to claim 4, characterized in that The tetrasaccharide compounds include the following compounds:

6. A disaccharide compound, characterized in that Including the compound shown in formula IV, the specific structure is as follows: R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, TBSO- or R3 is -AcNH, TrocNH- or -NO2; R4 is -O(CH2) n NH2, -O(CH2) n NCbz(Bn) or -O(CH2) n NHCbz; n is an integer between 2 and 6.

7. The compound according to claim 6, characterized in that The disaccharide compounds include the following compounds:

8. A monosaccharide compound, characterized in that Including compounds represented by formula V, formula VI and / or formula VII, the specific structure is as follows: R1 is -CONH2, -CONHBn, -COOH, -OH or R2 is -OH, -AcNH, TBSO- or R3 is -AcNH, TrocNH-, AcO-, N3- or -NO2; R4 is -OAll, -O(CH2) n NH2, -O(CH2) n NCbz(Bn) or -O(CH2) n NHCbz; n is an integer between 2 and 6.

9. The compound according to claim 8, characterized in that The monosaccharide compounds include the following compounds:

10. Use of the monosaccharide compound according to claim 8, the disaccharide compound according to claim 6, the trisaccharide compound according to claim 1 or the tetrasaccharide compound according to claim 4 in synthesizing a glycoconjugate.

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