Method for chemical synthesis of vibrio vulnificus biotype 2 serotype a o-antigen disaccharide fragment

Abstract

The present invention belongs to the field of chemistry. Disclosed in the present invention is a method for the chemical synthesis of a Vibrio vulnificus biotype 2 serotype A O-antigen disaccharide fragment. In the present invention, D-glucose, L-galactose, etc. are used as raw materials to prepare two glycosylated building blocks, and a set of synthesis routes for efficiently constructing a disaccharide fragment is designed. By means of the optimization of a protecting group and the timing for introducing a modification group, the target disaccharide is successfully prepared. The disaccharide prepared in the present invention has easily obtainable raw materials, and a simple and easily repeatable preparation method, and has good application prospects in the aspects such as the development of new drugs and vaccines for Vibrio vulnificus.

Description

Method for chemical synthesis of Vibrio vulnificus biotype 2, serogroup A O-antigen disaccharide fragment TECHNICAL FIELD

[0001] The present application relates to a method for chemical synthesis of Vibrio vulnificus biotype 2, serogroup A O-antigen disaccharide fragment, in particular a hetero-modified polyamino oligosaccharide with a reducing end assembled amino linker, belongs to the field of chemistry. BACKGROUND

[0002] Vibrio vulnificus is a gram-negative bacterium widely distributed in seawater and seafood (C. Baker-Austin et al., Trends Microbiol. 2020, 28, 81-82), and is one of the three major Vibrio species that threaten human health (C. Baker-Austin et al., Nat. Rev. Dis. Primers. 2018, 4, 1-19). Vibrio vulnificus infection can cause severe sepsis and gangrene and other diseases, with a mortality rate of up to 50% (WHO, Food & Agriculture Org., 2021). The harm of Vibrio vulnificus is attributed to multiple pathogenic factors, including capsular polysaccharide (CPS), lipopolysaccharide (LPS), pili, flagella, cytotoxins, enzymes, hemolysins, and cytolysins. In clinical practice, Vibrio vulnificus can be divided into three biotypes (1-3) (O. James D et al., In The biology of vibrios. 2006, 349-366). Currently, about 40 highly virulent pathogenic strains have been found in clinical practice. Most human infections with Vibrio vulnificus are associated with biotype 1 and 2 strains, and the mortality rate of biotype 2 infection is higher. Notably, biotype 2 strains are the only strain of zoonosis (C. Amaro et al., In Vibrio spp. Infections. 2023, 175-194; F. Roig et al., Appl. Environ. Microbiol. 2023, 18, 373-383). In addition, biotype 2 strains are further divided into serogroups A and E (C. Amaro et al., Appl. Environ. Microbiol. 1996, 62, 1454-1457; E. Sanjuán et al., Appl. Environ. Microbiol. 2004, 70, 7024-7032).

[0003] In general, infection of V. vulnificus can be treated with antibiotics combined with surgery, and amputation is necessary in some cases (K. Coerdt et al., A. Cutis. 2021, 107, E12-E17). Recent reports show that the use of antibiotics to control V. vulnificus infection is very limited, with repeated outbreaks of the disease and the isolation of multiple antibiotic-resistant strains. Therefore, there is an urgent need to develop vaccines against V. vulnificus (F. Roig et al., Appl. Environ. Microbiol. 2023, 18, 373-383). Currently, research on V. vulnificus vaccines is mainly limited to fish (Q. Ji, et al., Immunol. Lett. 2020, 226, 46-54). The existing veterinary vaccine for V. vulnificus is based on whole organisms, consisting of live attenuated or inactivated organisms (S. Lee, et al., Infect. Immun. 2006, 74, 694-702). In recent years, some new V. vulnificus vaccines have appeared, including DNA vaccines and vector vaccines, but have not yet been introduced into the market. Researchers have used a variety of methods to develop human V. vulnificus vaccines, including whole cell and attenuated live vaccines (Q. Ji et al., Aquaculture. 2022, 560, 738560). However, there is still controversy about the long-term preventive efficacy of V. vulnificus, and no effective vaccine against human infection with V. vulnificus has been marketed so far (C. Baker-Austin et al., Nat. Rev. Dis. Primers. 2018, 4, 1-19). On the other hand, since many antigenic determinants in inactivated vaccines can induce non-specific immune responses, subunit vaccines based on polysaccharides and membrane proteins on the surface of V. vulnificus are considered to provide potential and more effective immune protection (G. Pettis et al., Int. J. Mol. Sci. 2020, 21, 3259). The lipopolysaccharide O-antigen of V. vulnificus biotype 1 and 2 shows strong structural specificity, making it a potential immune target for the development of V. vulnificus vaccines (C. Baker-Austin et al., Trends Microbiol. 2020, 28, 81-82). H. Zhang et al. reported that the conjugate of V. vulnificus biotype 1 (MO6-24 and BO62316) O-antigen with carrier protein CRM197 can effectively induce T cell-dependent immune responses in mice (H. Zhang et al., JACS Au. 2021, 2, 97-108.).Collado et al. measured antibody titers and bactericidal / bacteriostatic activity in eel mucus and serum in a study and demonstrated that the O antigen of Vibrio vulnificus biovar 2 is a potential immunization target (R. Collado et al., Dis. Aquat. Org. 2000, 43, 91-101). However, O-antigens obtained via the biological route often exhibit structural heterogeneity, often with residual lipid A and other cellular impurities, which can reduce the safety and efficiency of the candidate vaccine (P. H. Seeberger, Chem. Rev. 2021, 121, 3598-3626; C. Qin et al., Curr. Opin. Chem. Biol. 2024, 78, 102424). Therefore, the preparation of well-defined oligosaccharides corresponding to O-antigens by chemical methods has become a valuable tool for the continuous development of glyco vaccines (C. Whitfield et al., J. Biol. Chem. 2020, 295, 10593-10609; R. van der Put et al., ACS Cent. Sci. 2022, 8, 449-460).

[0004] The O-antigen of Vibrio vulnificus biovar 2 serogroup A is composed of a trisaccharide repeating unit with four aminos and three carboxyls: [→4)-β-D-GlcpNAc3NAcylAN-(1→4)-α-L-GalpNAmA-(1→3)-α-D-QuipNAc-(1→]. The four aminos are functionalized by three different groups, including 2-O-acetyl-4-D-malonyl, ethylamidine and two acetylaminos, and one of the three carboxyls is functionalized to form a carboxamide group (A. Shashkov et al., Carbohydr. Res. 2009, 344, 2005-2006.). The disaccharide fragment [→4)-α-L-GalpNAmA-(1→3)-α-D-QuipNAc-(1→] with the rare ethylamidine modification is considered as a potential immunological target (C. Qin et al., Chin. J. Nat. Med. 2022, 20, 401-420; G. Tian et al., Molecules. 2023, 28, 7112.). The chemical synthesis of oligosaccharides with a single structure and high purity can facilitate the study of structure-activity relationship. So far, people have been committed to the synthesis of rare deoxyamino sugars, the assembly of complex glycans and the installation of rare modification groups. Even so, the chemical synthesis of this disaccharide is still a challenge due to the rare L-galactosamine acid, two 1,2-cis glycosidic bonds and ethylamidine (J. Harjani et al., 2011, 76, 1683-1691). In addition, the selection of reducing end assembly of orthogonal linking arms facilitates conjugation or immobilization in biological research. Considering that the active part of the linking arm may affect the synthesis efficiency of the disaccharide, it is necessary to evaluate the practicability of different linking arms. Therefore, the chemical synthesis of this structure needs overall route design, including the selection of protecting groups, the selection of modification group introduction timing, the efficiency and selectivity of glycosylation reaction and other key links. The synthesis will play a key role in the activity evaluation of Vibrio vulnificus biovar 2 serogroup A O-antigen disaccharide fragment and the development of carbohydrate vaccines.

[0005] SUMMARY

[0006] The present application relates to a chemical synthesis method of a disaccharide fragment of Vibrio vulnificus biovar 2 serogroup A O-antigen assembled with a linking arm, and the chemical structure of the disaccharide fragment is shown as general formula I:

[0007] In the present application, the linking arm L can be a chain structure with 2-40 carbon atoms (including the number of carbon atoms in the side chain) containing 0-6 heteroatoms.

[0008] The main chain of the linking arm in the present application is 4-8 atoms in length, and can contain 1, 2 or 3 heteroatoms (O, N and S) in the chain. When the main chain of the linking arm is 9-14 atoms in length, the chain can contain 1, 2, 3, 4, 5 or 6 heteroatoms (O, N and S).

[0009] The linking arm -L- in the present application can be a cyclic structure which is fully or partially fluorinated. The linking arm -L- can contain a three-, four-, five- or six-membered saturated carbocyclic ring; can contain a five-membered unsaturated carbocyclic ring (non-aromatic ring); can contain a four-, five- or six-membered saturated oxacyclic ring; can contain a four-, five- or six-membered saturated azacyclic ring; or can contain a six-membered aromatic carbocyclic ring.

[0010] The linking arm -L- in the present application can also contain an amide bond and / or a urea group.

[0011] The linking arm -L- in the present application can also contain one or more substituent groups, which can include -F, -Cl, -CH3, -C2H5, -C3H7, -C5H9, -C6H 13 , -OCH3, -OC2H5, -CH2F, -CHF2, -CF3, -C(O)-NH2, -SCH3, -SC2H5, -NHC(O)CH3, -N(CH3)2 and -N(C2H5)2.

[0012] The basic (carboxamidine) and acidic (carboxyl) groups in the synthetic sugar chain structure in the present application can form corresponding salts with organic or inorganic acids or bases. The acids which can be used for salt formation include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, lactic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, methanesulfonic acid, ethanesulfonic acid, mandelic acid, picric acid, adipic acid, gluconic acid, tartaric acid, nitrous acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, hydroxyethanesulfonic acid, vinylsulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, p-aminobenzenesulfonic acid, camphorsulfonic acid, o-methylmandelic acid, hydroxybenzenesulfonic acid, o-toluene-tartaric acid, malonic acid, aminonaphthalenesulfonic acid, and other mineral acids or carboxylic acids. The inorganic or organic bases which can be used for salt formation include sodium hydroxide, potassium hydroxide, aqueous ammonia, tetraalkylammonium hydroxide, lysine, arginine and the like.

[0013] The basic (carboxamidine) and acidic (carboxyl) groups in the synthetic sugar chain structure in the present application can also form a zwitterion by intramolecular proton transfer, i.e. the proton of the acidic group is transferred to the basic group, and the general formula I can be a zwitterion containing -O - and -NH3 + .

[0014] In one embodiment of the present invention, the chemical synthesis method of the disaccharide fragment assembled with the linker arm of the Vibrio vulnificus biotype 2A serotype O-antigen represented by the above general formula (I) uses the following two monosaccharide building blocks 1 (D-quinolamine), 2 (L-aminogalacturonic acid) and linker arm 3 as raw materials:

[0015] in:

[0016] PG 1 The hydroxyl protecting group can be selected from the following groups: acetyl (Ac), benzoyl (Bz), acetylpropionyl (Lev), neopentyl (Piv), allyloxycarbonyl (Alloc), chloroacetyl (ClAc), dichloroacetyl (DCA), trichloroacetyl (TCA), 2-naphthylmethyl (Nap), p-methoxybenzyl (PMB), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triethylsilyl (TES);

[0017] PG 2 The hydroxyl protecting group can be 2-naphthylmethyl (Nap) or benzyl (Bn);

[0018] PG 3 The hydroxyl protecting group can be 2-naphthylmethyl, benzyl, acetyl, acetylpropionyl, benzoyl, chloroacetyl, dichloroacetyl, trichloroacetyl, neopentanoyl, allyloxycarbonyl, benzyl, 2-naphthylmethyl, p-methoxybenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triethylsilyl;

[0019] PG 4 and PG 5 The hydroxyl protecting group can be benzenemethyl acetal (PhCH) or isopropylidene ketal ((CH3)2CH);

[0020] PG 6 and PG 7 The amino protecting group can be benzyl (Bn) or benzyloxycarbonyl (Cbz);

[0021] LG is a leaving group used in glycosylation reactions, which can be bromine, fluorine, ethyl thio, p-toluenethio, phenylthio, trichloroacetylimine, N-phenyltrifluoroacetylimine, or dibutyl phosphate.

[0022] The reactions are as follows:

[0023] Reaction A (glycosylation reaction): Monosaccharide building block 1 undergoes a glycosylation reaction with linker arm 3.

[0024] Reaction B (reductive acetylation of azido group): After the above glycosylation reaction is completed, the azido group at position 2 in the structure is subjected to reductive acetylation,

[0025] Reaction C (deprotection of quinovosamine at position 3): After the reductive acetylation of the azido group is completed, the deprotection of the quinovosamine at position 3 is performed, and the protecting group PG is removed 1 , to obtain a D-quinovosamine acceptor for assembling a disaccharide, and then a glycosylation reaction is performed with the monosaccharide building block 2 to obtain an initial disaccharide;

[0026] Reaction D (deprotection of galactosamine at positions 4 and 6): The deprotection of the galactosamine at positions 4 and 6 of the monosaccharide building block 2 in the above obtained initial disaccharide is performed, and the protecting group PG 4 and PG 5 ,

[0027] Reaction E (oxidation of primary hydroxyl group to carboxyl group): After the deprotection is completed, the primary hydroxyl group at position 6 of the monosaccharide building block 2 is oxidized to a carboxyl group,

[0028] Reaction F (protection of carboxyl group): Subsequently, the oxidized carboxylic acid is protected,

[0029] Reaction G (reduction of azido group to amino group): After the protection of the carboxyl group, the azido group at position 2 of the monosaccharide building block 2 is reduced to an amino group,

[0030] Reaction H (modification of amino group to form acetamidate group): After the reduction to the amino group, the acetamidate group is constructed by a modification reagent,

[0031] Finally, Reaction I (total deprotection): Total deprotection is performed to remove the protecting group PG 2 of the monosaccharide building block 1, the protecting group of the carboxylic acid of the monosaccharide building block 2, the protecting group PG 3 , and the protecting group PG 6 and PG 7 of the linking arm 3, to obtain a disaccharide fragment of the Vibrio vulnificus serogroup O-antigen of serotype 2, type B, which is assembled with a linking arm, represented by the general formula (I).

[0032] In one embodiment of the present application, the above synthesis process is as follows:

[0033] In one embodiment of the present application, in the process of 1) Reaction A (glycosylation reaction):

[0034] When the leaving group used in the glycosylation reaction is ethylthio group, p-toluenethio group, or phenylthio group, the activating agent used in the glycosylation reaction can be any one or more of methyl triflate (TfOMe), dimethylsulfide triflate (DMTST), trifluoromethanesulfonic acid (TfOH), and trimethylsilyl trifluoromethanesulfonate (TMSOTf); the reaction temperature can be between -40°C and room temperature.

[0035] When the leaving group is fluorine, the activating agent in the glycosylation reaction can be silver perchlorate (AgClO4), titanium tetrafluoride (TiF4), trifluoromethanesulfonic anhydride (Tf2O), etc., and the reaction temperature can be between -40°C and room temperature;

[0036] When the leaving group is bromine, the activating agent in the glycosylation reaction can be silver perchlorate (AgClO4), silver trifluoromethanesulfonate (AgOTf), and the reaction temperature can be between -40°C and room temperature;

[0037] When the leaving group is trichloroacetimidate, N-phenyltrifluoroacetimidate, or dibutyl phosphate, the activating agent in the glycosylation reaction can be boron trifluoride etherate complex (BF3·OEt2), trimethylsilyl trifluoromethanesulfonate (TMSOTf), silver trifluoromethanesulfonate (AgOTf), and the reaction temperature can be between -40°C and room temperature.

[0038] The molecular sieve used for water removal in the glycosylation reaction can be molecular sieve or molecular sieve.

[0039] In one embodiment of the present application, during the process of 2) reaction B (reduction of azido group and acetylation):

[0040] The reduction of azido group and acetylation can be directly completed by the thioacetic acid (AcSH) / pyridine method, or by reduction of azido group to amino group and then acetylation. The method for reducing azido group to amino group can be trimethyl phosphine (PMe3) / water, triphenyl phosphine (PPh3) / water, 1,3-propanedithiol / triethylamine, sodium borohydride (NaBH4) / nickel dichloride (NiCl2), tin dichloride (SnCl2) / benzenethiol (PhSH) / triethylamine, zinc / copper / acetic acid, Lindlar catalyst / hydrogen, etc.

[0041] The acetylation method for amino group can be acetic anhydride (Ac2O) / methanol, acetic anhydride / pyridine, acetyl chloride (AcCl) / triethylamine, etc.

[0042] In one embodiment of the present application, during the process of 3) reaction C (deprotection of quinovose amine 3-position):

[0043] When the hydroxyl protecting group is acetyl (Ac), benzoyl (Bz), chloroacetyl (ClAc), dichloroacetyl (DCA), trichloroacetyl (TCA), or pivaloyl (Piv), the deprotection conditions used can be sodium methoxide / methanol, potassium hydroxide / methanol, sodium hydroxide / methanol, etc.

[0044] When the hydroxyl protecting group is levulinoyl (Lev), the deprotection conditions used can be hydrazine acetate / pyridine, etc.

[0045] When the hydroxyl protecting group is allyloxycarbonyl (Alloc), the deprotection condition used can be palladium diacetate (Pd(OAc)2) / diethylamine, etc.

[0046] When the hydroxyl protecting group is 2-naphthylmethyl (Nap), p-methoxybenzyl (PMB), the deprotection condition used can be 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) / water, cerium ammonium nitrate (CAN) / water, etc.

[0047] When the hydroxyl protecting group is tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triethylsilyl (TES), the deprotection condition used can be tetrabutylammonium fluoride (TBAF), hydrofluoric acid, etc.

[0048] In one embodiment of the present application, in the process of 4) reaction D (deprotection of the 4, 6-position of galactosamine), the deprotection condition used can be:

[0049] When the hydroxyl protecting group at the 4, 6-position of galactosamine is benzal acetal (PhCH), the deprotection condition used can be aqueous acetic acid, aqueous sulfuric acid, aqueous trifluoroacetic acid, Pd(OH)2, Pd / C catalytic hydrogenolysis, etc.

[0050] When the hydroxyl protecting group at the 4, 6-position of galactosamine is benzal acetal isopropylidene ketal ((CH3)2CH), the deprotection condition used can be acetic acid in methanol, sulfuric acid in methanol, trifluoroacetic acid in methanol, and all protonic acids and Lewis acids in methanol, silica gel or acidic ion exchange resin, etc.

[0051] In one embodiment of the present application, in the process of 5) reaction E (oxidation of primary hydroxyl to carboxyl), the method of oxidation of primary hydroxyl to carboxyl can be any one or more of the following:

[0052] Jones reagent (CrO3 / H2SO4), Collins reagent (CrO3 / py), pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), etc.

[0053] It can also be based on dimethyl sulfoxide (DMSO), in which the activator can be any one or more of dicyclohexyl carbodiimide (DCC), acetic anhydride, trifluoroacetic anhydride, phosphorus pentoxide, pyridine / sulfur trioxide, and oxalyl chloride, etc.

[0054] It can also be a transition metal under oxygen treatment;

[0055] ​​It can also be treated with (2,2,6,6-tetramethylpiperidinooxy) TEMPO and similar nitrogen-oxygen radical active substances;

[0056] In an embodiment of the present application, 6) during the reaction F (carboxyl protection) process:

[0057] The protecting group of the galacturonic acid carboxyl group can be methyl, t-butyl, allyl, trityl, benzhydryl, t-butyldimethyl (TBS), propargyl, benzyl (Bn), p-methoxybenzyl (PMB), etc.

[0058] In an embodiment of the present application, 7) during the reaction G (azido group reduction to amino group) process:

[0059] The method of reducing azido group to amino group can be trimethyl phosphine (PMe3) / water, triphenyl phosphine (PPh3) / water, 1,3-propanedithiol / triethylamine, sodium borohydride (NaBH4) / nickel dichloride (NiCl2), tin dichloride (SnCl2) / benzenethiol (PhSH) / triethylamine, zinc / copper / acetic acid, Lindlar catalyst / hydrogen, etc.

[0060] In an embodiment of the present application, 8) during the reaction H: modification of amino group to form formamidine group process:

[0061] The method of modifying amino group to form formamidine group can be carried out by treating aryl thioacetimidate halide or alkyl thioacetimidate halide with a base, the aryl group can be benzyl or naphthylmethyl, etc., the alkyl group can be methyl or ethyl, etc., the halide acid can be hydrochloric acid or hydrobromic acid, etc., and the base used can be pyridine, triethylamine, diisopropylethylamine, 1,8-diazabicycloundec-7-ene (DBU), potassium carbonate, sodium carbonate, etc.

[0062] The method of modifying amino group to form formamidine group can be carried out by treating alkyl acetimidate halide with a base, the alkyl group can be ethyl, trifluoroethyl, trichloroethyl, etc., the halide acid can be hydrochloric acid or hydrobromic acid, etc., and the base used can be pyridine, triethylamine, diisopropylethylamine, 1,8-diazabicycloundec-7-ene (DBU), potassium carbonate, sodium carbonate, etc.

[0063] In an embodiment of the present application, 9) during the reaction I: full deprotection process:

[0064] The catalytic hydrogenation full deprotection can be carried out by passing hydrogen gas at room temperature under catalytic conditions. The catalyst used for catalytic hydrogenation can be 10% palladium on carbon catalyst or palladium hydroxide, etc., and the solvent used for the reaction can be water / methanol / dichloromethane / acetic acid mixture, water / t-butyl alcohol / dichloromethane mixture, etc.

[0065] When the protecting group of the carboxyl group is methyl, the carboxyl protecting group needs to be removed by sodium hydroxide aqueous solution first, and then the protecting group is removed by catalytic hydrogenation;

[0066] When the protecting group of the carboxyl group is tert-butyl, tert-butyl dimethyl (TBS), and p-methoxybenzyl (PMB), the carboxyl protecting group needs to be removed by formic acid, acetic acid or trifluoroacetic acid first, and then the protecting group is removed by catalytic hydrogenation;

[0067] When the protecting group of the carboxyl group is allyl, the carboxyl protecting group needs to be removed by palladium acetate, sodium 2-methylhexanoate and triphenylphosphine in acetone first, and then the protecting group is removed by catalytic hydrogenation;

[0068] When the protecting group of the carboxyl group is triphenylmethyl, the carboxyl protecting group needs to be removed by methanol or water / dioxane first, and then the protecting group is removed by catalytic hydrogenation;

[0069] When the protecting group of the carboxyl group is benzyl (Bn), the protecting group is directly removed by catalytic hydrogenation.

[0070] The chemical synthesis method of the Vibrio vulnificus serogroup O-antigen of biological type 2 A with a modified disaccharide fragment of a connecting arm provided by the present application, when the sugar building block 1 (quinovose) is used as a glycosyl donor, the amino group at position 2 can be protected by an azido group, so as to facilitate the generation of a 1,2-cis-alpha-glycosidic bond.

[0071] The chemical synthesis method of the Vibrio vulnificus serogroup O-antigen of biological type 2 A with a modified disaccharide fragment of a connecting arm provided by the present application, when the sugar building block 2 (galactose) is used as a glycosyl donor, the amino group at position 2 can be protected by an azido group, so as to facilitate the generation of a 1,2-cis-alpha-glycosidic bond.

[0072] The chemical synthesis method of the Vibrio vulnificus serogroup O-antigen of biological type 2 A with a modified disaccharide fragment of a connecting arm provided by the present application, the acetylamino group at position 2 in the structure of the sugar building block 1 (quinovose) can be introduced immediately after the glycosylation reaction of the sugar building block 1 (quinovose) is completed, so as to avoid hindering the introduction of the sugar building block thereafter.

[0073] The chemical synthesis method of the Vibrio vulnificus serogroup O-antigen of biological type 2 A with a modified disaccharide fragment of a connecting arm provided by the present application, the carboxyl group at position 6 in the structure of the sugar building block 2 (galactose) can be introduced immediately after the construction of the disaccharide skeleton is completed, so as to avoid the influence of the carboxyl group on the glycosylation efficiency.

[0074] The chemical synthesis method of the Vibrio vulnificus serogroup O-antigen of biological type 2 A with a modified disaccharide fragment of a connecting arm provided by the present application, compared with the disaccharide 17* assembled with a thiol connecting arm and oxidized at the monosaccharide stage, the glycosylation efficiency is obviously improved (Figure 11). The total yield from the synthesis of L-galactose to the similar disaccharide skeleton is increased from 4.5% to 15.8%. Advantages:

[0075] The present invention is the first to accomplish the chemical synthesis of the O-antigen of the Vibrio vulnificus biovar 2, serogroup A with a linker arm modified disaccharide fragment. Furthermore, the introduction of the corresponding linker arm at the reducing end of the saccharide structure provides a basis for the synthesis of oligosaccharide structure coupling carrier molecules or immobilization on the corresponding matrix, which will contribute to the study of the biological effects of the O-antigen of the Vibrio vulnificus biovar 2, serogroup A monosaccharide structure, the development of saccharide vaccines, etc. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1: O-antigen disaccharide fragment of Vibrio vulnificus biovar 2, serogroup A.

[0077] Figure 2: Compounds shown in general formula I.

[0078] Figure 3: Compounds shown in monosaccharide building blocks 1, 2 and linker arm 3.

[0079] Figure 4: Reaction scheme for the synthesis of a-D-pyranquinofuranose 6.

[0080] Figure 5: Reaction scheme for the synthesis of the O-antigen disaccharide fragment I of Vibrio vulnificus biovar 2, serogroup A.

[0081] Figure 6: Chemical synthesis reaction scheme of compound 3*.

[0082] Figure 7: Chemical synthesis reaction scheme of compound 6*.

[0083] Figure 8: Chemical synthesis reaction scheme of compound 12*.

[0084] Figure 9: Chemical synthesis reaction scheme of compound 15*.

[0085] Figure 10: Chemical synthesis reaction scheme of compound 16*.

[0086] Figure 11: Chemical synthesis reaction scheme of compound 17*. DETAILED DESCRIPTION

[0087] All the commercial reagents used in the experiments were used directly without further treatment. The anhydrous solvents used in the reactions were prepared by a MBraun MB-SPS 800 solvent drying system. The solvents used in the column chromatography on silica gel were all of analytical purity and were used after being distilled under reduced pressure. The silica gel plates used in the thin layer chromatography (TLC) were glass-based or aluminum box-based silica gel plates prepared from 60-F254 silica gel. The color reagents used in the thin layer chromatography were sugar color reagent (90.1% (v / v) 3-methoxyphenol, 2.5% (v / v) sulfuric acid ethanol solution), or CAM color reagent (5% (w / v) ammonium molybdate, 1% (w / v) cerium (II) sulfate and 10% (v / v) sulfuric acid aqueous solution), or ninhydrin color reagent (1.5% (w / v) indantrione and 3% (v / v) n-butanol acetic acid solution). The silica gel used in the normal phase silica gel column chromatography was 200-300 mesh silica gel. The filler used in the molecular exclusion chromatography was Sephadex G-10 (GE Healthcare). (GE Healthcare).

[0088] The yield of each reaction step was calculated respectively, and the yield calculation method was: (the amount of substance of the target product / the amount of substance of the raw material) x 100%. The structure of the product was identified by nuclear magnetic spectrum, infrared spectrum, optical rotation, and high resolution mass spectrum, and the purity of the product was analyzed by nuclear magnetic spectrum and high performance liquid chromatography. The hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum were measured by Bruker Ultrashield Plus 400 meganuclear magnetic resonance instrument or Bruker AVIII 600 meganuclear magnetic resonance instrument at 25°C. The high resolution mass spectrum was measured by Agilent 6220 electrospray ion source-time of flight mass spectrometer. The optical rotation was measured by Schmidt & Haensch UniPol L 1000 full-automatic polarimeter at 589 nm, and the concentration (c) unit was g / 100 mL. The infrared spectrum was measured by Thermo Fisher Scientific Nicolet iS5 infrared instrument. The analytical high performance liquid chromatography was performed by Agilent 1200 series liquid phase connected quadrupole electrospray mass spectrometer 6130, and the analytical column used was Thermo Scientific Hypercarb column (150 x 4.6 mm). The preparative high performance liquid chromatography was performed by Agilent 1200 series liquid phase connected quadrupole electrospray mass spectrometer 6130, and the semi-preparative column used was Thermo Scientific Hypercarb column (150 x 10 mm).

[0089] Example 1:

[0090] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-azido-3-O-acetyl-4-O-benzyl-2-deoxy-a-D-quinovopyranoside (1*)

[0091] The reaction equation is shown in Figure 6;

[0092] Under nitrogen protection, 2-azido-3-O-acetyl-4-O-benzyl-2-deoxy-a-D- quinovopyranosyl trichloroacetimidate (C. Qin, J. Am. Chem. Soc. 2018, 140, 3120-3127) (1.13 g, 2.29 mmol) and N-benzyl-N-benzyloxy carbonyl-5-aminopentan-1-ol (B. Schumann, 2014, 5, 1992-2002) (1.13 g, 3.44 mmol) were dissolved in a mixture of anhydrous diethyl ether / anhydrous dichloromethane (57 mL, 3:1, v / v), and activated molecular sieves (Aw-300 type) were added and stirred for 30 min. After the reaction solution was cooled to -40 °C, trimethylsilyl trifluoromethanesulfonate (0.63 mL, 3.44 mmol) was slowly added dropwise, and the reaction solution was stirred at this temperature until the reaction was completed. After the reaction was completed, triethylamine was added dropwise to quench the reaction, and the molecular sieves were removed by filtration, and the solvent was removed under reduced pressure. The obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 15:1, v / v) to obtain the product 1* (1.31 g, 2.07 mmol, 90%, a / β = 2.2:1). IR v max (film) 3030, 2934, 2105, 1749, 1694, 1506, 1496, 1453, 1421, 1362, 1222, 1067, 1044, 898, 767, 733, 697 cm -1 ; 1 H NMR (400 MHz, CDC13) δ = 7.41-7.14 (m, 13H, a-Ph-H, β-Ph-H), 5.51 (t, J = 10.7 Hz, 0.5H, a 3-H), 5.18 (d, J = 12.3 Hz, 1.6H, a-PhCH2, β-PhCH2), 5.00 (t, J = 10.4 Hz, 0.3H, β 3-H), 4.84 (s, 1H, a 1-H), 4.64 (s, 1.1H, a-PhCH2), 4.60 (d, J = 5.50 Hz, 0.4H, β-PhCH2), 4.50 (s, 1.6H, a-PhCH2, β-PhCH2), 4.31 (s, 0.3H, β 1-H), 3.93-3.55 (m, 1.3H, Linker-OCH a , a 5-H), 3.50-3.30 (m, 1.1H, Linker-NCH2, linker-OCH b, β5-H, β2-H), 3.21 (m, 2.4H, α4-H, β4-H), 3.06 (dd, J = 10.6, 3.5 Hz, 0.5H, α2-H), 2.05 (d, 2.4H, β-CH3CO, α-CH3CO), 1.58 (m, 3.2H, Linker-CH2), 1.33 (d, J = 6.2 Hz, 1H, β6-CH3), 1.29 (d, J = 6.3 Hz, 2.2H, α6-CH3); 13 C NMR (100 MHz, CDC13) δ = 170.1, 163.7, 156.8, 156.3, 137.8, 136.9, 128.6, 128.1, 127.9, 127.8, 127.1, 127.4, 101.9 (β anomeric), 97.9 (α anomeric), 92.0, 82.4, 81.6, 77.2, 75.0, 73.9, 72.0, 71.4, 70.1, 67.3, 66.8, 64.6, 61.6, 50.6, 50.3, 47.1, 46.3, 29.3, 27.9, 21.0, 17.9; HR-ESI-MS (m / z): calcd for C 35 H 42 N4O7Na + (M+Na + ): 653.1752, found: 653.1738.

[0093] Example 2:

[0094] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-acetamido-3-0-acetyl-4-0-benzyl-2-deoxy-a-D-quinovopyranoside (2a)

[0095] The reaction equation is shown in Figure 6;

[0096] To a solution of compound 1* (0.81 g, 1.31 mmol) in anhydrous pyridine (22 mL) was added thioacetic acid (22 mL) at 0 °C, and the reaction was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by azeotropic distillation with toluene, and the obtained crude product was purified by silica gel column chromatography (petroleum ether: acetone, 6: 1, v / v) to give the product 2a* (0.75 g, 1.49 mmol, 88%) as colorless syrup. D 20 = +54.2° (c = 1.00, CHCl3); IR v max(film) 3324, 3030, 2933, 1738, 1693, 1518, 1496, 1453, 1422, 1363, 1304, 1233, 1134, 1073, 1046, 905, 735, 698 cm -1 ; 1 H NMR (400 MHz, CDC13) δ = 7.43-7.14 (m, 15H, Ph-H), 5.89-5.62 (d, J = 8.9 Hz, 1H, NH), 5.29-5.11 (m, 3H, 3-H, PhCH2), 4.65 (m, 3H, 1-H, PhCH2), 4.50 (s, 2H, PhCH2), 4.22 (t, J = 10.2, 3.5 Hz, 1H, 2-H), 3.76 (m, 1H, 5-H), 3.58 (m, 1H, Linker-CH a ), 3.27 (m, 4H, 4-H, Linker-CH2, linker-CH b ), 1.98 (s, 3H, CH3CO), 1.92 (s, 3H, CH3CO), 1.52 (m, 4H, Linker-CH2), 1.28 (m, 5H, 6-CH3, Linker-CH2). 13 CNMR (100 MHz, CDC13) δ = 171.3, 170.0, 156.7, 156.2, 137.8, 136.8, 128.5, 128.4, 127.9, 127.3, 97.0 (anomeric), 81.8, 77.4, 77.2, 77.0, 76.7, 75.1, 73.8, 67.8, 67.2, 66.9, 53.4, 52.6, 50.5, 50.2, 47.0, 46.1, 29.0, 27.9, 27.4, 23.5, 23.2, 21.0, 17.8; HR-ESI-MS (m / z): calcd for C 37 H 46 N2O8Na + (M + Na + ): 669.3152, found: 669.3144.

[0097] Example 3:

[0098] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-acetamido-4-0-benzyl-2-deoxy-a-D-quinovopyranoside (3*)

[0099] The reaction equation is shown in Figure 6;

[0100] To a solution of compound 2a* (0.64 g, 0.99 mmol) in methanol (33 mL) was added sodium methoxide (0.03 g, 0.49 mmol) and the resulting reaction was stirred at room temperature. Upon completion of the reaction, the reaction was neutralized with Amberlite IR 120 cation exchange resin and the organic phase was filtered and distilled off under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether: acetone, 4: 1, v / v) to give the product 3* (0.69 g, 0.97 mmol, 98%) as a colorless syrup.[α] D 20 = +30.1 ° (c = 1.00, CHCI3); IR v max (film) 3324, 3030, 2930, 1692, 1543, 1496, 1453, 1422, 1367, 1307, 1227, 1115, 1069, 1043, 847, 732, 697 cm -1 ; 1 H NMR (400 MHz, CDCI3) δ = 7.41 - 7.20 (m, 15H, Ph-H), 6.20 (d, J = 8.5 Hz, 0.5H, NH), 5.84 (d, J = 8.4 Hz, 0.5H, NH), 5.17 (d, J = 12.5 Hz, 2H, PhCH2), 4.93 (d, J = 11.1 Hz, 1H, PhCH2), 4.71 (d, J = 11.1 Hz, 1H, PhCH2), 4.68 (s, 1H, 1-H), 4.49 (d, J = 11.0 Hz, 2H, PhCH2), 4.09 (t, J = 9.5 Hz, 1H, 2-H), 3.81 (t, J = 9.7 Hz, 1H, 3-H), 3.72 - 3.48 (m, 2H, 5-H, Linker-CH a ), 3.37 - 3.17 (m, 3H, Linker-CH b , Linker-CH2), 3.12 (t, J = 9.1 Hz, 1H, 4-H), 3.03 (s, 1H, 3-OH), 2.03 (s, 3H, CH3CO), 1.66 - 1.42 (m, 4H, Linker-CH2), 1.28 (m, 5H, 6-CH3, Linker-CH2); 13C NMR (100 MHz, CDC13) δ = 172.2, 156.4, 138.5, 137.9, 136.8, 128.7, 128.6, 128.4, 128.3, 128.1, 127.9, 127.5, 127.3, 97.0, 84.5, 77.5, 77.4, 77.2, 76.8, 75.3, 74.8, 67.6, 66.8, 54.6, 50.6, 47.0, 46.2, 29.2, 28.0, 27.4, 23.7, 18.0; HR-ESI-MS (m / z): calcd for C 35 H 44 N2O7Na + (M+Na + ): 627.3046, found: 627.3052.

[0101] Example 4:

[0102] Synthesis of phenyl 2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-l-seleno-a-L- galactopyranoside (4*)

[0103] The reaction equation is shown in Figure 7.

[0104] Phenyl 2-azido-4,6-O-benzylidene-2-deoxy-l-seleno-a-L-galactopyranoside (Chemical synthesis of a synthetically useful L-galactosaminuronic acid building block. C. Qin, J. Chin. J. Nat. Med. 2022, 20, 387-392) (0.51 g, 1.18 mmol) was dissolved in anhydrous DMF (4 mL), sodium hydride (0.09 g, 2.36 mmol) was added, and stirred at room temperature overnight. After the reaction was completed, the reaction solution was diluted with ethyl acetate, extracted with water and saturated brine respectively, and the obtained organic phase was concentrated after anhydrous sodium sulfate dehydration. The obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 10:1, v / v) to obtain the product 4* (0.54 g, 1.03 mmol, 87%) as a white solid. D 20 = -132.1 ° (c = 1.00, CHCl3); IR v max (film) 3062, 3038, 2864, 2111, 1454, 1362, 1312, 1248, 1213, 1161, 1092, 1083, 1022, 913, 845, 740, 696 cm -1 ; 1H NMR (400 MHz, CDC13) δ = 7.68-7.21 (m, 15H, Ph-H), 6.06 (s, 1H, 1-H), 5.51 (s, 1H, PhCH), 4.78 (d, J = 5.0 Hz, 2H, PhCH2), 4.43 (dd, J = 10.3, 5.2 Hz, 1H, 2-H), 4.25 (d, J = 3.2 Hz, 1H, 4-H), 4.13 (d, J = 11.8 Hz, 1H, 6-CH a ), 4.04 (s, 1H, 5-H), 4.01 (d, J = 2.2 Hz, 1H, 6-CH b ), 3.79 (dd, J = 10.3, 3.4 Hz, 1H, 3-H); 13 C NMR (100 MHz, CDC13) δ = 137.7, 134.6, 134.0, 129.3, 129.2, 129.0, 128.7, 127.9, 126.3, 101.1, 100.9, 91.9, 85.7 (anomeric), 77.6, 77.5, 77.4, 76.9, 76.8, 75.6, 73.4, 72.7, 71.6, 69.6, 65.3, 60.0, 44.3; HR-ESI-MS (m / z): calcd for C 26 H 25 N3O4SeNa + (M + Na + ): 546.0908, found: 546.0920.

[0105] Example 5:

[0106] Synthesis of 2-azido-3-0-benzyl-4,6-0-benzylidene-2-deoxy-a-L-galactopyranoside (5*)

[0107] The reaction equation is shown in Figure 7.

[0108] Compound 4* (0.52 g, 0.99 mmol) was dissolved in THF / water mixture (2.5 mL, 1 : 1, v / v), after the addition of bromo succinimide (0.29 g, 2.49 mmol), the reaction solution was stirred at room temperature for 6 hours. After the reaction was completed, the reaction solution was diluted with dichloromethane, and the organic phase was extracted with 10% Na2S2O3 / 1M NaHCO3 mixture (1 : 1, v / v). After removing the solvent under reduced pressure, the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 5: 1, v / v) to obtain the product 5* (0.38 g, 0.94 mmol, 95%) in colorless syrup form. D 20= -112.7 ° (c = 1.00, CHCI3); IR v max (film), 2921, 2852, 2108, 1681, 1597, 1338, 1299, 1229, 1132, 1110, 1065, 738, 697 cm -1 ; 1 H NMR (400 MHz, CDCI3) δ = 7.58-7.22 (m, 10H, Ph-H), 5.46 (s, 1 H, PhCH), 5.35 (t, J = 3.0 Hz, 1 H, 1-H), 4.79-4.69 (s, 2H, PhCH2), 4.19 (s, 1 H, 6-CH a ), 4.16 (s, 1 H, 3-H), 4.02-3.93 (m, 3H, 6-CH b , 2-H, 5-H), 3.76 (s, 1 H, 4-H), 3.33 (s, 1 H, 1-OH); 13 C NMR (100 MHz, CDCI3) δ = 137.8, 129.1, 128.6, 126.3, 101.0, 96.3, 92.6, 78.0, 77.4, 76.8, 74.5, 73.0, 72.3, 71.7, 69.4, 66.7, 63.6, 62.6, 59.4, 29.7; HR-ESI-MS (m / z): calcd for C 20 H 21 N3O5Na + (M+Na + ): 406.1379, found: 406.1388.

[0109] Example 6:

[0110] Synthesis of 2-azido-3-0-benzyl-4,6-0-benzylidene-2-deoxy-a-L-galactopyranosyl trifluoroacetimidate (6*)

[0111] The reaction equation is shown in Figure 7.

[0112] To compound 5* (0.09 g, 0.22 mmol) dissolved in anhydrous dichloromethane (2.5 mL), potassium carbonate (0.25 g, 1.77 mmol) and N-phenyltrifluoroacetimidoyl chloride (0.27 mL, 1.78 mmol) were added under nitrogen protection, and stirred at room temperature overnight. After the reaction was completed, the solid was removed by filtration, and washed with dichloromethane, and the obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 5: 1, v / v) to obtain product 6* (0.15 g, 0.28 mmol, 79%). 1HNMR (400 MHz, CDC13) δ = 7.68-7.01 (m, 14H, Ph-H), 6.84 (d, J = 7.7 Hz, 2H, Ph-H, 1-H), 5.49 (s, 1H, Ph-CH), 4.76 (s, 2H, Ph-CH2), 4.31 (d, J = 12.6 Hz, 1H, 3-H), 4.11 (s, 2H, 5-H, 6-CH a ), 3.99 (d, J = 12.7 Hz, 1H, 3-H), 3.58-3.16 (m, 2H, 4-H, 6-CH b ); 13 C NMR (100 MHz, CDC13) δ = 143.4, 137.4, 129.2, 128.7, 128.6, 128.3, 128.1, 127.9, 126.3, 124.4, 119.3, 101.2, 101.1, 77.9, 77.3, 77.2, 77.0, 76.7, 72.0, 71.9, 68.8, 67.4, 61.0, 29.7; HR-ESI-MS (m / z): calcd for C 28 H 25 F3N4O5Na + (M+Na + ): 577.1675, found: 577.1688.

[0113] Example 7:

[0114] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-acetamido-4-0-benzyl-3-0-(2-azido-3-0-benzyl-4,6-0-benzylidene-2-deoxy-a-L-galacto-pyranosyl)-2-deoxy-a-D- quinofo-ranose (7*)

[0115] The reaction equation is shown in Figure 8.

[0116] Under nitrogen atmosphere, the triflimide sugar donor 6* (0.09 g, 0.16 mmol) and acceptor 3* (0.14 g, 0.23 mmol) were dissolved in a mixture of dry dichloromethane / dry diethyl ether (5.2 mL, 1 :3, v / v), after addition of thiophene (0.15 mL, 0.19 mmol) and activated molecular sieves (AW-300 type), the resulting reaction was stirred at room temperature for 30 min. After cooling to -40 °C, trimethylsilyl triflate (3 μL, 0.016 mmol) was added, followed by gradual increase of the reaction temperature to room temperature. After TLC confirmed the end of the reaction, triethylamine was added dropwise at 0 °C to quench the reaction, and the molecular sieves were removed by filtration on celite. After extraction of the organic phase with saturated sodium bicarbonate solution, the resulting crude was concentrated and purified by column chromatography on silica gel (petroleum ether: acetone, 6:1-3:1, v / v) to give the target disaccharide 7* (0.11 g, 0.11 mmol, 68%, α only).[α] D 20 = -152.8 ° (c = 1.00, CHCl3); IR v max (film) 2917, 2849, 2110, 1680, 1540, 1496, 1454, 1423, 1362, 1229, 1130, 1072, 735, 698 cm -1 ; 1 H NMR (400 MHz, CDCl3) δ = 7.46-7.06 (m, 25H, Ph-H), 6.19-5.75 (d, 1H, NH), 5.35 (d, J = 3.5 Hz, 1H, 1'-H), 5.24 (s, 1H, PhCH), 5.18-5.07 (m, 2H, PhCH2), 4.66 (d, J = 8.8 Hz, 2H, PhCH2), 4.61 (d, J = 3.4 Hz, 1H, 1-H), 4.53 (t, J = 8.8 Hz, 2H, PhCH2), 4.45 (t, J = 5.5 Hz, 2H, PhCH2), 4.29 (d, J = 10.6 Hz, 1H, 2-H), 3.96-3.78 (m, 5H, 3-H; 2'-H; 3'-H; 4'-H; 6'-CH a ), 3.70 (m, 2H, 5-H, 5'-H), 3.53 (s, 1H, Linker-CH a ), 3.41-3.14 (m, 4H, Linker-CH b , Linker-CH2, 6'-CH b ), 3.11 (t, J = 9.4 Hz, 1H, 4-H), 1.95 (d, J = 9.6 Hz, 3H, CH3CO), 1.63-1.36 (m, 5H, Linker-CH2, 6-CH3).13 C NMR (100 MHz, CDC13) δ = 138.0, 129.1, 128.7, 128.6, 128.3, 128.1, 128.0, 127.9, 127.5, 126.3, 100.9, 83.8, 77.4, 77.2, 76.9, 73.1, 71.4, 69.2, 67.4, 53.6, 32.0, 29.8, 29.5, 29.2, 24.9, 22.8, 18.2, 14.3; HR-ESI-MS (m / z): calcd for C 55 H 63 N5O 11 Na + (M+Na + ): 992.4422, found: 992.4439.

[0117] Example 8:

[0118] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-acetamido-4-0-benzyl-3-0-(2-azido-3-0-benzyl-2-deoxy-a-L-galacto-pyranosyl)-2-deoxy-a-D- quinofo-ranose (8*)

[0119] The reaction equation is shown in Figure 8.

[0120] Compound 7* (0.10 g, 0.1 mmol) was added to 80% aqueous acetic acid (2.5 mL) and heated to 55 °C with stirring until the reaction was complete. After removing the solvent by distillation under reduced pressure, purification by silica gel column chromatography (dichloromethane:methanol, 60:1-40:1-30:1, v / v) gave 4,6-dihydroxyl sugar 8* (0.08 g, 0.09 mmol, 91%) as a white solid. [a] D 20 = -57.0° (c = 1.00, CHCI3); IR v max (film) 3350, 2917, 2849, 2110, 1680, 1540, 1517, 1496, 1454, 1423, 1362, 1229, 1130, 1072, 735, 698 cm -1 ; 1H NMR (600 MHz, CDC13) δ = 7.55-7.11 (m, 20H, Ph-H), 6.20 (d, J = 9.3 Hz, 0.5H, NH), 5.92 (d, J = 9.1 Hz, 0.5H, NH), 5.32 (s, 1H, 1'-H), 5.17 (s, 2H, PhCH2), 4.73-4.58 (m, 5H, 1-H, 2PhCH2), 4.50 (t, J = 10.1 Hz, 2H, PhCH2), 4.32 (d, J = 10.5 Hz, 1H, 2-H), 3.92 (m, 2H, 3-H, 5'-H), 3.86-3.55 (m, 5H, 5-H, 2'-H, 3'-H, 4'-H Linker-CH a ), 3.54 (d, J = 4.9 Hz, 2H, 6'-CH2), 3.29 (m, 3H, Linker-CH b 2), 3.17 (t, J = 9.4 Hz, 1H, 4-H), 2.77 (m, 1H, 4'-OH), 1.97 (d, J = 11.9 Hz, 3H, CH3CO), 1.70 (s, 1H, 6'-OH), 1.55 (m, 4H, Linker-CH2), 1.31 (m, 5H, Linker-CH2, 6-CH3); 13 C NMR (100 MHz, CDC13) δ = 170.0, 156.5, 137.9, 137.3, 136.8, 128.9, 128.7, 128.6, 128.5, 128.1, 127.9, 127.4, 98.2, 97.9, 97.4, 83.9, 77.4, 77.2, 76.9, 76.2, 75.9, 75.1, 72.1, 69.5, 67.9, 63.1, 60.0, 53.6, 50.4, 47.3, 46.1, 29.8, 29.2, 28.9, 28.0, 27.5, 23.9, 23.5, 18.2; HR-ESI-MS (m / z): calcd for C 48 H 59 N5O 11 Na + (M+Na + ): 904.4109, found: 904.4116.

[0121] Example 9:

[0122] Synthesis of N-benzyl-N-carbobenzyloxy-5-aminopentyl 2-acetamido-4-0-benzyl-3-0-(2-azido-3-0-benzyl-2-deoxy-a-L-galactopyranosyl benzyl)-2-deoxy-a-D- quino pyranoside (9*)

[0123] The reaction equation is shown in Figure 8;

[0124] To a solution of compound 8* (38 mg, 43 μmol) in dichloromethane (1 mL) was added water (1 mL), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) (1.5 mg, 8.6 μmol) and diacetoxyliodobenzene (BAIB) (42 mg, 1.3 mmol) at 0 °C. After stirring at room temperature for 4 h, the reaction mixture was purified by silica gel column chromatography, concentrated and the obtained crude was directly used in the next step. The crude carboxylic acid compound was dissolved in dry DMF (5 mL), and sodium bicarbonate (14 mg, 0.1 mmol) and benzyl bromide (46 μL, 0.38 mmol) were added successively. The reaction mixture was stirred at room temperature. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the obtained crude was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 5:1, v / v) to give benzyl glycuronide 9* (28 mg, 28 μmol, 65% yield for two steps) as a yellow syrup.[α] D 20 = -42.5° (c = 1.00, CHCl3); IR v max (film) 2957, 2920, 2849, 2113, 1721, 1500, 1457, 1360, 1213, 1161, 1094, 1073, 1028, 907, 740, 700 cm -1 ; 1 H NMR (400 MHz, CDCl3) δ = 7.41-7.15 (m, 25H, Ph-H), 5.98 (d, J = 9.4 Hz, 1H, NH), 5.43 (s, 1H, 1'-H), 5.20 (d, J = 12.3 Hz, 2H, PhCH2), 5.16 (s, 1H, PhCH a ), 4.98 (d, J = 12.4 Hz, 1H, PhCH a ), 4.72-4.69 (m, 2H, PhCH a , 5'-H), 4.68 (d, J = 5.1 Hz, 1H, PhCH a ), 4.63 (d, J = 5.4 Hz, 2H, PhCH b , 1-H), 4.55 (d, J = 10.7 Hz, 1H, PhCH b), 4.50 (d, J = 5.4 Hz, 2H, NPhCH2), 4.30 (t, J = 11.7 Hz, 1H, 2-H), 4.16 (s, 1H, 4'-H), 3.93 (t, J = 8.6 Hz, 1H, 3-H), 3.85 (dd, J = 10.3, 3.1 Hz, 1H, 3'-H), 3.72 (m, 2H, 2'-H, 5-H), 3.55 (d, J = 20.5 Hz, 1H, Linker-CH a ), 3.37-3.19 (m, 3H, Linker-CH2, Linker-CH b ), 3.16 (t, J = 9.3 Hz, 1H, 4-H), 2.36 (s, 1H, 4'-OH), 1.97 (d, J = 7.6 Hz, 3H, CH3CO), 1.53 (m, 4H, Linker-CH2), 1.32-1.24 (m, 5H, 6-CH3, Linker-CH2); 13 C NMR (100 MHz, CDC13) δ = 168.1, 137.9, 137.5, 137.0, 135.3, 128.7, 128.6, 128.5, 128.2, 128.0, 127.8, 127.3, 97.7, 97.2, 83.8, 77.2, 76.8, 76.3, 75.5, 75.1, 72.3, 70.3, 67.8, 67.2, 59.5, 53.3, 50.3, 47.1, 46.1, 29.0, 27.8, 27.4, 23.5, 18.0; HR-ESI-MS (m / z): calcd for C 55 H 63 N5O 12 Na + (M + Na + ): 1008.4371, found: 1008.4366.

[0125] Example 10:

[0126] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-acetamido-4-0-benzyl-3-0-(2-amino-3-0-benzyl-2-deoxy-a-L-galactopyranosyl benzyl ester)-2-deoxy-a-D- quinovopyranoside (10*)

[0127] The reaction equation is shown in Figure 8;

[0128] Compound 9* (25 mg, 25.4 μmol) was dissolved in pyridine (2.6 mL) under nitrogen, followed by the addition of water (0.24 mL, 13.2 mmol), triethylamine (0.05 mL, 0.4 mmol) and 1,3-propanedithiol (0.05 mL, 0.5 mmol). The reaction was stirred at room temperature for 6 h. After TLC confirmed the complete consumption of starting material, the solvent was removed by distillation under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether: acetone, 5: 1 to 3: 1, v / v) to give amino compound 10* (18.5 mg, 19.0 μmol, 75%) as a colorless syrup.[α] D 20 = -57.1 ° (c = 1.00, CHCI3); IR v max (film) 3405, 3033, 2922, 1700, 1598, 1500, 1469, 1360, 1280, 1096, 1025, 741, 698 cm -1 ; 1 H NMR (600 MHz, CDCI3) δ = 7.39-7.13 (m, 25H, Ph-H), 6.70 (s, 0.5H, NH), 6.58 (s, 0.5H, NH), 5.30 (s, 1H, 1'-H), 5.22-5.12 (m, 3H, Ph-CH2, PhCH a ), 4.87 (d, J = 12.5 Hz, 1H, PhCH b ), 4.69 (d, J = 1.8 Hz, 1H, 5'-H), 4.68-4.62 (m, 2H, PhCH a , PhCH a ), 4.60-4.53 (m, 2H, PhCH b , PhCH b ), 4.50 (d, J = 10.3 Hz, 2H, NPhCH2), 4.22 (s, 1H, 2-H), 4.14 (t, J = 2.4 Hz, 1H, 4'-H), 4.00 (d, J = 10.5 Hz, 1H, 3-H), 3.73 (d, J = 15.2 Hz, 1H, 5-H), 3.57 (s, 1H, 3'-H), 3.52 (s, 1H, Linker-CH a ), 3.26 (s, 1H, Linker-CH b), 3.23-3.18 (m, 3H, 2'-H, Linker-CH2), 3.15 (t, J = 9.4 Hz, 1H, 4-H), 1.94 (d, J = 15.3 Hz, 3H, CH3CO), 1.58-1.45 (m, 4H, Linker-CH2), 1.26 (m, 5H, Linker-CH2, 6-CH3); 13 C NMR (150 MHz, CDC13) δ = 168.7, 137.8, 137.5, 135.4, 128.7, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.5, 127.3, 127.2, 99.5, 97.2, 83.6, 77.9, 77.2, 77.0, 76.8, 75.3, 74.9, 74.6, 71.8, 70.6, 67.8, 67.2, 67.1, 66.8, 66.6, 53.9, 50.2, 50.1, 47.1, 46.0, 31.9, 29.6, 29.3, 29.3, 29.2, 27.4, 23.5, 22.7, 18.0, 14.1; HR-ESI-MS (m / z): calcd for C 55 H 65 N3O 12 Na + (M+Na + ): 982.4466, found: 982.4450.

[0129] Example 11:

[0130] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-acetamido-4-O-benzyl-3- O-(2-acetamido-3-O-benzyl-2-deoxy-a-L-galactopyranosyl benzyl ester)-2-deoxy-a-D- quinovopyranoside (12*)

[0131] The reaction equation is shown in Figure 8.

[0132] The amino compound 10* (18 mg, 18.74 μmol) was dissolved in 1 mL of anhydrous pyridine under argon protection, and then cooled to 0 °C. Benzyl thioacetimidate hydrochloride (5.7 mg, 28.12 μmol) was added. The reaction solution was stirred at 0 °C for 5 h, and then concentrated. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol, 20:1, v / v) to give compound 11* (13.4 mg, 13.3 μmol, 71%) as colorless syrup. D 20 = -80.2° (c = 0.50, CHCI3); IR v max(film) 3368, 3062, 2918, 1748, 1690, 1649, 1542, 1496, 1454, 1422, 1385, 1309, 1218, 1136, 1063, 911, 814, 735, 697, cm -1 ; 1 HNMR (600 MHz, CDC13) δ = 7.51-6.95 (m, 25H, Ph-H), 6.77 (s, 1H, 2-NH), 5.46 (s, 1H, 1'-H), 5.16 (m, 2H, PhCH2), 5.08 (d, J = 12.3 Hz, 1H, PhCH a ), 4.79 (d, J = 12.4 Hz, 1H, PhCH a ), 4.69 (d, J = 11.6 Hz, 1H, PhCH b ), 4.64 (s, 1H, 5'-H), 4.58 (d, J = 11.3 Hz, 1H, PhCH a ), 4.54 (s, 1H, 1-H), 4.49 (m, 3H, Ph-CH2, PhCH b ), 4.44 (d, J = 11.3 Hz, 1H, PhCH b ), 4.26 (td, J = 9.8, 3.6 Hz, 1H, 2-H), 4.17 (s, 1H, 4'-H), 4.05 (t, J = 10.3 Hz, 1H, 3-H), 3.83 (s, 1H, 2'-H), 3.76 (m, 2H, 5-H, 3'-H), 3.52 (m, 1H, Linker-CH a ), 3.29 (m, 3H, Linker-CH2, Linker-CH b ), 3.16 (d, J = 9.3 Hz, 1H, 4-H), 2.33 (s, 3H, Am-CH3), 1.97 (s, 3H, CH3CO), 1.55 (q, J = 13.3 Hz, 4H, Linker-CH2), 1.25 (d, J = 6.6 Hz, 5H, Linker-CH2, 6-CH3); 13C NMR (150 MHz, CDC13) δ = 166.5, 137.9, 137.3, 137.1, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.2, 97.6, 82.8, 77.2, 77.0, 76.8, 74.3, 71.0, 70.9, 70.1, 68.0, 67.2, 67.0, 66.6, 53.8, 51.6, 29.7, 29.0, 19.7, 17.9; HR-ESI-MS (m / z): calcd for C 57 H 68 N4O 12 Na + (M+Na + ): 1023.4731, found: 1023.4724.

[0133] Example 12:

[0134] Synthesis of 5-Aminopentyl 2-acetamido-3-0-(2-acetamidino-2-deoxy-a-L- galactopyranosyluronic acid)-2-deoxy-a-D-gluco-pyranose (12*)

[0135] The reaction equation is shown in Figure 8.

[0136] The disaccharide 11* (13 mg, 12.9 μmol) was dissolved in a mixture of tert-butanol / water / dichloromethane (4 mL, 5:2:1, v / v / v), and the reaction system was replaced with nitrogen, then 10% palladium-carbon hydrogenation catalyst was added, and the nitrogen replacement was continued for 5 minutes. After further replacing the reaction system with hydrogen for 5 minutes, the reaction solution was stirred under hydrogen for 48 hours, and then filtered with diatomite. The obtained crude product was preliminarily purified with a C18 column (Macherey-Nagel, Diiren, Germany) (eluent: water and methanol), and the product was further purified with reverse-phase high-performance liquid chromatography (semi-preparative Thermo Scientific Hypercarb column) to obtain the target product 12* (5.8 mg, 11.4 μmol, 88%) in the form of a white solid. D 20 = -78.16° (c = 0.10, H20); 1H NMR (400 MHz, D20) δ = 5.34 (d, J = 3.8 Hz, 1H, 1'-H), 5.09 (d, J = 1.4 Hz, 1H, 5'-H), 4.71 (d, J = 3.7 Hz, 1H, 1-H), 4.40 (d, J = 3.2 Hz, 1H, 4'-H), 4.16 (dd, J = 10.5, 3.2 Hz, 1H, 3'-H), 4.11 (dd, J = 10.3, 3.6 Hz, 1H, 2-H), 3.95 (dd, J = 10.4, 3.8 Hz, 1H, 2'-H), 3.87 (m, 1H, 3-H), 3.79 (dd, J = 9.8, 6.2 Hz, 1H, 5-H), 3.71 - 3.64 (m, 1H, Linker-CH a ), 3.50 - 3.43 (m, 1H, Linker-CH b ), 3.31 (t, J = 9.3 Hz, 1H, 4-H), 3.01 (t, J = 7.7 Hz, 2H, Linker-CH2), 2.29 (s, 3H, Am-CH3), 2.01 (s, 3H, CH3CO), 1.68 (dt, J = 13.5, 7.0 Hz, 4H, Linker-CH2), 1.47 (p, J = 7.0 Hz, 2H, Linker-CH2), 1.28 (d, J = 6.2 Hz, 3H, 6-CH3); 13 C NMR (100 MHz, D20) δ = 173.7, 172.6, 166.3, 96.8, 95.5, 75.6, 73.8, 70.7, 69.0, 67.8, 67.7, 66.9, 53.8, 52.6, 39.5, 28.2, 26.6, 22.5, 22.0, 19.0, 16.7; HR-ESI-MS (m / z): calcd for C 21 H 39 N4O 10 + (M+H + ): 507.2661, found: 507.2645.

[0137] Comparative Example 1

[0138] Synthesis of 6-(benzylthio)hexanol 2-azido-3-0-acetyl-4-0-benzyl-2-deoxy-a-D- quinovopyranose (13*)

[0139] The reaction equation is shown in Figure 9;

[0140] Under nitrogen atmosphere, 2-azido-3-O-acetyl-4-O-benzyl-2-deoxy-a-D- quinovopyranosyl trichloroacetimidate (C. Qin, J. Am. Chem. Soc. 2018, 140, 3120-3127) (1.25 g, 2.54 mmol) and 6-(benzylthio)hexanol (B. Schumann, 2014, 5, 1992-2002) (0.85 g, 3.81 mmol) were dissolved in a mixture of dry diethyl ether / dry dichloromethane (65 mL, 3:1, v / v) with activated molecular sieves (Aw-300 type) and stirred for 30 min. The reaction was cooled to -40 °C and trimethylsilyl trifluoromethanesulfonate (0.69 mL, 3.81 mmol) was added dropwise slowly. The reaction was stirred at this temperature until the reaction was completed. The reaction was quenched by the addition of triethylamine and the molecular sieves were removed by filtration. The solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 10:1, v / v) to give product 13* (1.17 g, 2.20 mmol, 87%, a / β = 2.4:1). IR v max (film) 3029, 2936, 2009, 1750, 1691, 1501, 1489, 1451, 1420, 1361, 1225, 1065, 1041, 892, 767, 732, 696 cm -1 ; 1 H NMR (400 MHz, CDC13) δ = 7.45-7.10 (m, 12H, a-Ph-H, β-Ph-H), 5.52 (t, J = 10.6 Hz, 0.5H, a3-H), 5.00 (t, J = 10.5 Hz 0.3H, β3-H), 4.86 (d, J = 3.5 Hz, 0.6H, a1-H), 4.63 (s, 1H, a-PhCH2), 4.59 (d, J = 3.4 Hz, 0.9H, β-PhCH2), 4.33 (d, J = 7.9 Hz, 0.3H, β1-H), 3.92-3.80 (m, 1H, a5-H), 3.69 (s, 2H, Linker-OCH a 2-H), 3.56-3.30 (m, 2H, Linker-SCH2, Linker-OCH b, β5-H, β2-H), 3.21 (m, 1H, α4-H), 3.05 (dd, J = 10.6, 3.7 Hz, 0.8H, α2-H), 2.41 (t, J = 7.3 Hz, 1H, β4-H), 2.04 (s, 2.9H, β-CH3CO, α-CH3CO), 1.55 (m, 7H, Linker-CH2), 1.36 (m, 3H, Linker-CH2), 1.33 (d, 6.1 Hz, 1H, β6-CH3), 1.29 (d, J = 6.2 Hz, 2.4H, α6-CH3); HR-ESI-MS (m / z): calcd for C 28 H 37 N3O5SNa + (M+Na + ): 550.2352, found: 550.2339.

[0141] Comparative Example 2:

[0142] The reaction equation for the synthesis of 6-(benzylthio)hexanol 2-acetylamino-3-0-acetyl-4-0-benzyl-2-deoxy-a-D-quinovose (14a) is shown in Figure 9.

[0143] To a solution of compound 13* (1.16 g, 2.20 mmol) in anhydrous pyridine (37 mL) was added thioacetic acid (37 mL) at 0 °C, and the reaction was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by azeotropic distillation with toluene, and the crude product was purified by column chromatography on silica gel (petroleum ether: acetone, 5: 1, v / v) to give the product 14a (1.09 g, 2.00 mmol, 91%) as a colorless syrup. D 20 = +62.3° (c = 1.00, CHCl3); IR v max (film) 3325, 3031, 2937, 1740, 1699, 1520, 1495, 1450, 1423, 1361, 1301, 1236, 1130, 1075, 1046, 907, 733, 699 cm -1 ; 1H NMR (400 MHz, CDC13) δ = 7.42-7.13 (m, 10H, Ph-H), 5.71 (d, J = 9.3 Hz, 1H, NH), 5.21 (dd, J = 10.8, 9.2 Hz, 1H, 3-H), 4.69 (d, J = 3.7 Hz, 1H, 1-H), 4.64 (s, 2H, PhCH2), 4.31 (t, J = 6.7 Hz, 0.5H, PhCH2), 4.21 (m, 1H, 2-H), 3.78 (m, 1H, 5-H), 3.70 (s, 1.5H, PhCH2), 3.62 (m, 1H, 4-H), 3.39-3.23 (m, 2H, Linker-CH2), 2.41 (t, J = 7.3 Hz, 2H, Linker-CH2), 1.98 (s, 3H, CH3CO), 1.92 (s, 3H, CH3CO), 1.62-1.50 (m, 6H, Linker-CH2), 1.28 (m, 5H, 6-CH3, Linker-CH2); 13 C NMR (100 MHz, CDC13) δ = 206.0 171.7, 166.3, 135.1, 134.5, 133.4, 133.3, 129.2, 128.8, 128.4, 128.1, 127.9, 127.4, 126.3, 126.2, 118.3, 100.8, 77.4, 77.1, 72.6, 72.1, 70.6, 67.9, 66., 62.4, 38.1, 29.8, 28.1; HR-ESI-MS (m / z): calcd for C 30 H 41 NO6SNa + (M+Na + ): 566.7088, found: 566.7079.

[0144] Comparative Example 3:

[0145] Synthesis of 6-(benzylthio)hexanol 2-acetylamino-4-O-benzyl-2-deoxy-a-D- quinovose (15*)

[0146] The reaction equation is shown in Figure 9;

[0147] To a solution of compound 14a* (0.81 g, 1.49 mmol) in methanol (50 mL) was added sodium methoxide (0.04 g, 0.75 mmol) and the resulting reaction was stirred at room temperature. Upon completion of the reaction, the reaction was neutralized with Amberlite IR 120 cation exchange resin and the organic phase was filtered and distilled under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether: acetone, 4: 1, v / v) to give the product 15* (0.71 g, 1.42 mmol, 95%) as a colorless syrup.[α] D 20 = +29.3° (c = 1.00, CHCl3); IR v max (film) 3328, 3025, 2931, 1688, 1540, 1492, 1457, 1421, 1360, 1305, 1223, 1117, 1066, 1043, 849, 730, 699 cm -1 ; 1 H NMR (400 MHz, CDC13) δ = 7.41 - 7.06 (m, 10H, Ph-H), 5.81 (d, J = 8.6 Hz, 1H, NH), 4.89 (d, J = 11.1 Hz, 1H, PhCH2), 4.68 (d, J = 11.2 Hz, 1H, PhCH2), 4.65 (d, J = 3.9 Hz, 1H, 1-H), 4.05 (m, 1H, 2-H), 3.76 (t, J = 9.2 Hz, 1H, 3-H), 3.69 - 3.58 (m, 3H, PhCH2, Linker-CH a ), 3.32 (m, 1H, Linker-CH b ), 3.08 (t, J = 9.1 Hz, 1H, 4-H), 2.54 (s, 1H, 3-OH), 2.38 (m, 2H, Linker-CH2), 2.00 (s, 3H, CH3CO), 1.52 (m, 4H, Linker-CH2), 1.31 - 1.15 (m, 7H, 6-CH3, Linker-CH2); HR-ESI-MS (m / z): calcd for C 28 H 39 NOSNa + (M+Na + ): 524.6718, found: 524.6724.

[0148] Comparative Example 4:

[0149] Synthesis of 2-azido-3-0-2-naphthylmethyl-4-0-acetylpropionyl-2-deoxy-a-L- galactopyranosyluronic acid benzyl ester-trifluoroacetimidate (16*)

[0150] The reaction equation is shown in Figure 10;

[0151] Benzyl 2-azido-3-O-2-naphthalenylmethyl-4-O-acetylpropionyl-2-deoxy-a-L- galactopyranoside (Chemical synthesis of a synthetically useful L-galactosaminuronic acid building block. C. Qin, Chin. J. Nat. Med. 2022, 20, 387-392) (10.7 mg, 0.02 mmol) was dissolved in anhydrous dichloromethane (0.5 mL) under nitrogen protection, after the addition of potassium carbonate (21.6 mg, 1.56 mmol) and N-phenyltrifluoroacetimidoyl chloride (23.3 μL, 1.56 mmol), it was stirred at room temperature overnight. After the reaction was completed, the solid was removed by filtration, and washed with dichloromethane, and the obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 5:1, v / v) to obtain the product 16* (10.1 mg, 0.014 mmol, 88%). 1 H NMR (400 MHz, CDC13) δ = 7.90-6.79 (m, 17H, Ph-H), 5.84 (s, 1H, 4-H), 5.34-5.14 (m, 2H, PhCH2), 4.91 (d, J = 11.5 Hz, 1H, PhCH2), 4.67 (d, J = 11.5 Hz, 1H, PhCH2), 4.12 (d, J = 7.2 Hz, 1H, 5-H), 3.88 (t, J = 9.2 Hz, 1H, 2-H), 3.53 (d, J = 9.4 Hz, 1H, 3-H), 2.75-2.48 (m, 4H, Lev-CH2), 2.12 (s, 3H, CH3CO); 13 C NMR (100 MHz, CDC13) δ = 206.0, 171.5, 165.4, 134.9, 134.1, 133.3, 129.3, 128.9, 128.8, 128.5, 128.1, 127.9, 127.6, 126.4, 126.3, 126.2, 124.6, 119.4, 77.4, 77.1, 73.3, 72.2, 68.1, 66.3, 61.3, 38.0, 29.8, 28.0; HR-ESI-MS (m / z): calcd for C 37 H 33 F3N4O8Na + (M+Na + ): 741.2148, found: 741.2155.

[0152] Comparative Example 5:

[0153] 6-(Benzylthio)hexanol 2-acetamido-4-O-benzyl-3-O-(2-azido-3-O-2- naphthalenylmethyl-4-O-acryloyl-2-deoxy-α-L-galactopyranosyl benzyl ester)-2- deoxy-α-D-gluco-pyranose (17*) Synthesis

[0154] The reaction equation is shown in Figure 11.

[0155] Under nitrogen atmosphere, the glycosyl donor 16* (9 mg, 0.013 mmol) and acceptor 15* (14 mg, 0.028 mmol) were dissolved in a mixture of dry dichloromethane / dry diethyl ether (0.5 mL, 1:3, v / v), after the addition of thiophene (15 μL, 0.15 mmol) and activated molecular sieves (AW-300 type), the resulting reaction solution was stirred at room temperature for 30 minutes. After cooling to -40 °C, trimethylsilyl triflate (0.25 μL, 12.5 μmol) was added, and then the reaction temperature was gradually increased to room temperature. After TLC confirmed the end of the reaction, triethylamine was added dropwise at 0 °C to quench the reaction, and the molecular sieves were removed by filtration with diatomite. After the organic phase was extracted with saturated sodium bicarbonate solution, the resulting crude product was concentrated and purified by silica gel column chromatography (petroleum ether: acetone, 6:1-3:1, v / v) to obtain the target disaccharide 17* (3.1 mg, 0.003 mmol, 23%, α / β = 10:1).[α] D 20 = -170.3° (c = 0.50, CHCl3); IR v max (film) 3329, 2930, 2110, 1740, 1677, 1375, 1230, 1040, 970, 750, 698 cm -1 ; 1 H NMR (400 MHz, CDCl3) δ = 7.85-7.10 (m, 22H, Ph-H), 5.86 (d, J = 9.4 Hz, 1H, NH), 5.67 (dd, J = 3.3, 1.8 Hz, 1H, 4'-H), 5.45 (d, J = 3.4 Hz, 1H, 1'-H), 5.16 (d, J = 11.9 Hz, 1H, PhCH a ), 4.94 (d, J = 12.0 Hz, 1H, PhCH b ), 4.86-4.79 (m, 2H, PhCH a , 5'-H), 4.66-4.50 (m, 4H, 1-H, PhCH b, 4.31 (td, J = 9.9, 3.8 Hz, 1H, 2-H), 3.98-3.87 (m, 2H, 3'-H, 3-H), 3.79-3.68 (m, 3H, 5-H, SCH2Ph), 3.68-3.54 (m, 2H, 2'-H, Linker-CH a ), 3.33 (dt, J = 9.8, 6.7 Hz, 1H, Linker-CH b ), 3.16 (t, J = 9.3 Hz, 1H, 4-H), 2.63-2.30 (m, 6H, Lev-CH2, Linker-CH2), 2.02 (s, 3H, Lev-CH3), 1.94 (s, 3H, CH3CO), 1.57 (m, 4H, Linker-CH2), 1.46-1.16 (m, 5H, 6-CH3, Linker-CH2). 13 C NMR (100 MHz, CDC13) δ = 171.4, 167.3, 137.6, 134.6, 133.4, 133.2, 129.2, 129.0, 128.7, 128.6, 128.4, 128.1, 127.8, 127.3, 127.0, 126.2, 97.8 (C1'), 97.3 (C1), 83.9, 77.5, 77.4, 77.2, 77.0, 76.8, 75.3, 73.9, 72.2, 69.6, 68.0, 67.7, 67.4, 59.8, 53.5, 38.1, 36.5, 31.4, 29.8, 29.4, 29.2, 28.7, 28.0, 26.0, 23.6, 18.2.; HR-ESI-MS (m / z): calcd for C 57 H 66 N4O 12 SNa + (M+Na + ): 1053.4296, found: 1053.4282.

[0156] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application and any person skilled in the art, without departing from the spirit and scope of the present application, can make various modifications and modifications, therefore, the scope of protection of the present application should be defined by the claims.

Claims

1. A method for the chemical synthesis of a di-saccharide fragment of the O-antigen of Vibrio vulnificus bio 2, serotype O4, assembled with a linker, characterized in that, The chemical structure of this disaccharide fragment is shown in general formula I: The connecting arm L is a chain structure with 2-40 carbon atoms containing 0-6 heteroatoms, a substituted or unsubstituted three-six membered ring structure, an amide bond, or a urea group; The chemical synthesis method is as follows: two monosaccharide building blocks 1, 2 and a connecting arm 3 are used as raw materials: wherein: PG 1 PG is a hydroxyl protecting group selected from the group consisting of acetyl, benzoyl, pivaloyl, allyloxycarbonyl, chloroacetyl, dichloroacetyl, trichloroacetyl, 2-naphthylmethyl, p-methoxybenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triethylsilyl; PG 2 is a hydroxyl protecting group selected from: 2-naphthylmethyl, benzyl; PG 3 PG is a hydroxyl protecting group selected from the group consisting of: 2-naphthylmethyl, benzyl, acetyl, pivaloyl, benzoyl, chloroacetyl, dichloroacetyl, trichloroacetyl, pivaloyl, allyloxycarbonyl, benzyl, 2-naphthylmethyl, p-methoxybenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triethylsilyl; PG 4 and PG 5 is a hydroxyl protecting group selected from the group consisting of: benzal acetal, isopropylidene ketal; PG 6 and PG 7 an amino protecting group selected from the group consisting of benzyl, benzyloxycarbonyl; LG is a leaving group for the glycosylation reaction, selected from the group consisting of bromine, fluorine, ethylthio, p-toluenesulfenyl, phenylthio, trichloroacetyl imidate, N-phenyl trifluoroacetyl imidate, dibutyl phosphate; The method comprises the following steps in sequence: Reaction A: glycosylation reaction of monosaccharide building block 1 with connecting arm 3, Reaction B: after the glycosylation reaction, the azido group at position 2 in the structure is reduced and acetylated, Reaction C: After the reduction of the azido group and acetylation is complete, the 3 position is deprotected and the protecting group PG is removed 1 to give the D-quinoxamine acceptor which is used to assemble the disaccharide, which is then glycosylated with monosaccharide building block 2 to give the initial disaccharide; Reaction D: Deprotection of the 4,6 positions of the monosaccharide building block 2 and removal of the protecting group PG from the initial disaccharide obtained above 4 and PG 5 , Reaction E: after the deprotection, the primary hydroxyl group at position 6 in the monosaccharide building block 2 is oxidized to a carboxyl group, Reaction F: then the oxidized carboxylic acid is protected, Reaction G: after the carboxyl group is protected, the azido group at position 2 in the monosaccharide building block 2 is reduced to an amino group, Reaction H: after the amino group is reduced, the imidamide group is constructed by a modification reagent, Finally, reaction I: global deprotection, removal of the protecting groups PG in the monosaccharide building block 1 2 , the protecting group of the carboxylic acid in the monosaccharide building block 2, the protecting group PG 3 , and the protecting group PG in the linker 3 6 and PG 7 , to obtain the assembled disaccharide fragment of the Vibrio vulnificus serogroup 2 O-antigen of serotype O of general formula (I) with the linker.

2. The method of chemical synthesis of claim 1, wherein, The synthetic route is shown below:

3. The method of chemical synthesis of claim 1, wherein, In the process of reaction A, an activating agent is used to promote the glycosylation reaction; the activating agent is selected from any one or more of methyl triflate, dimethyl methylthio sulfonium triflate, trifluoromethanesulfonic acid, and trimethylsilyl triflate.

4. The method of chemical synthesis of claim 1, wherein, The temperature of the glycosylation reaction in the process of reaction A is -40°C to room temperature.

5. The method of chemical synthesis of claim 1, wherein, In the process of reaction B, the azido group is directly reduced and acetylated by using thioacetic acid and pyridine; or the azido group is first reduced to an amino group, and then acetylated.

6. The method of chemical synthesis of claim 1, wherein, In the reaction E, the primary hydroxyl group is oxidized into carboxyl group by using an oxidizing agent; the oxidizing agent is Jones reagent, Collins reagent, pyridinium chlorochromate any one or more of the salts, or pyridinium dichromate any one or more of the salts Alternatively, the primary hydroxyl group is oxidized to a carboxyl group under the action of dimethyl sulfoxide and an activating agent; the activating agent is any one or more of cyclohexyl carbodiimide, acetic anhydride, trifluoroacetic anhydride, phosphorus pentoxide, pyridine / sulfur trioxide, and oxalyl chloride.

7. The method of chemical synthesis of claim 1, wherein, In the process of reaction G, the azido group is reduced to an amino group by using any one of the following systems: trimethyl phosphine / water, triphenyl phosphine / water, 1,3-propanedithiol / triethylamine, sodium borohydride / nickel dichloride, tin dichloride / benzene thiol / triethylamine, zinc / copper / acetic acid, and Lindlar catalyst / hydrogen.

8. The method of chemical synthesis of claim 1, wherein, In the process of reaction H, the amino group is modified to an imidamide group by treating the aryl ester halide of thioacetyl imidate or the alkyl ester halide of thioacetyl imidate with a base; Alternatively, the amino group is modified to an imidamide group by treating the alkyl ester halide of acetyl imidate with a base.

9. A method for the chemical synthesis of a disaccharide fragment with a linker arm assembled with Vibrio vulnificus serotype 2A O-antigen obtained by the method according to any one of claims 1-8, wherein the chemical structural formula of the disaccharide fragment is shown in general formula I: The connecting arm L is a chain structure with 2-40 carbon atoms containing 0-6 heteroatoms, a substituted or unsubstituted three-six membered ring structure, an amide bond, or a urea group.

10. Use of the Vibrio vulnificus biovar 2 type A serogroup O antigen assembled with a disaccharide fragment of a connecting arm in the development or preparation of a Vibrio vulnificus vaccine or a drug for diseases caused by Vibrio vulnificus infection according to claim 9.

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