Chemical synthesis method for vibrio cholerae serotype o100 o-antigen oligosaccharide, and use

Abstract

Disclosed are a chemical synthesis method for Vibrio cholerae serotype O100 O-antigen oligosaccharide, and a use, belonging to the technical field of chemistry. The present invention uses three monosaccharide building blocks and five kinds of carboxylic acid derivative, and under the effects of solvent, temperature and neighboring group participation, through orthogonal protection, selective assembly and amide coupling, five Vibrio cholerae serotype O100 O-antigen oligosaccharide fragments are synthesized. Using the synthesized oligosaccharide fragments, combined with NMR analysis and carbohydrate chip technology, the absolute configuration and immunological function of the 3,5-dihydroxyhexanoyl group in the O-antigen trisaccharide are clarified, providing a theoretical basis for further structure-activity research and minimal antigen epitope screening. The present invention has promising applications in the synthesis of Vibrio cholerae glycoconjugate vaccines and the development of new drugs.

Description

A chemical synthesis method and application of oligosaccharides containing O antigen from Vibrio cholerae O100 serotype. Technical Field

[0001] This invention relates to a chemical synthesis method and application of oligosaccharides containing O antigen from Vibrio cholerae O100 serotype, belonging to the field of chemical technology. Background Technology

[0002] Vibrio cholerae is the pathogen of cholera, which can cause cholera pandemics. Cholera is an acute diarrheal disease characterized by watery diarrhea and potentially fatal dehydration (Qadri et al. Clin. Microbiol. Rev. 2022, 35(3), e00211-00221). According to WHO's outbreak briefing, 30 countries reported cholera cases in 2023, with 40,900 cases and 775 deaths reported in January 2024 alone.

[0003] Antibiotics and oral cholera vaccines (OCVs) have been widely used to treat Vibrio cholerae infection. Studies have shown that Vibrio cholerae has developed resistance to all of these antibiotics. While OCVs have proven effective, they also have many limitations, including poor immunogenicity in young children, short protection period, and delayed immune induction (Waldor et al. Annu. Rev. Microbiol. 2022, 76(1), 681-702). A cholera vaccine that can play a crucial role in global cholera control is still needed.

[0004] Glycoprotein conjugate vaccines have shown promising potential, with several marketed glycoprotein conjugate vaccines already available for disease prevention and treatment, such as the pneumococcal 13-valent glycoconjugate vaccine, the meningococcal glycoconjugate vaccine, and the Salmonella conjugate vaccine. Using structurally well-defined synthetic oligosaccharides conjugated to protein carriers can enhance the immunogenicity of oligosaccharides and induce T-cell-dependent immune responses, and has been proven safe and reliable (Seeberger, Chem. Rev. 2021, 121(7), 3598-3626).

[0005] Based on the different lipopolysaccharide O antigens on the bacterial surface, Vibrio cholerae is classified into more than 200 serotypes. In 2019, Perepelov et al. isolated and identified the structure of the trisaccharide repeat unit of the O antigen in Vibrio cholerae serotype O100, which is [→3)-β-d-QuipNAc4N(dHh)-(1→3)-α-d-Fucp4N(RHb)-(1→3)-α-l-FucpNAc-(1→], where RHb and dHh represent (R)-3-hydroxybutyryl and 3,5-dihydroxyhexanoyl, respectively (Perepelov et al. Carbohydr. Res. 2019, 472, 98-102).

[0006] This O-antigen trisaccharide contains two difficult-to-construct 1,2-cis-α-fucoside bonds and one easily broken 1,2-trans-β-D-quinoside bond, with four nitrogen atoms coupled to two rare modifying groups. These factors make the synthesis of the repeating unit of this O-antigen trisaccharide extremely challenging, and it has not yet been totally synthesized. Notably, the bacterial surface glycan modifying group is considered a potential immune target. The absolute configuration of the two chiral centers in the unique dHh modifying group remains unclear, and its immunological function is also poorly understood. The chemical synthesis of a homogeneous O-antigen oligosaccharide, the assignment of its absolute configuration, and preliminary immunological studies are of great significance for the development of Vibrio cholerae O100 serotype glycoconjugate vaccines and related drugs.

[0007] Summary of the Invention Technical issues:

[0008] To address the aforementioned problems, this invention relates to a chemical synthesis method for an oligosaccharide of the O antigen of Vibrio cholerae O100 serotype; and to the application of the synthesized oligosaccharide to elucidate the absolute configuration and immunological function of the dHh modified group.

[0009] Technical solution:

[0010] Since the absolute configuration of the two chiral centers in dHh is not yet determined, theoretically, there are four possible isomers of this O antigen trisaccharide. Furthermore, to facilitate the study of the immunological effects of dHh, it is necessary to synthesize a trisaccharide derivative without dHh modification. Introducing corresponding linkers at the reducing ends of the synthesized oligosaccharides can provide a basis for further immunological investigations. Therefore, this invention utilizes three monosaccharide building blocks and five carboxylic acid derivatives, through a series of orthogonal protection, selective assembly, and amide coupling processes, to synthesize four possible trisaccharide isomers and one derivative. Using NMR technology, error analysis was performed on the four trisaccharide isomers and the extracted lipopolysaccharide O antigen to clarify the absolute configuration of dHh. The five oligosaccharide fragments were combined with a microarray to create a sugar chip, and the immunological effects of dHh were evaluated through screening using the sugar chip.

[0011] One object of the present invention is to provide a chemical synthesis method for oligosaccharides of cholerae O100 serotype O antigen, the method using three monosaccharide building blocks and five carboxylic acid derivatives as raw materials;

[0012] The structure of the cholerae arc O100 serum type O antigen oligosaccharide is shown in formulas (1) to (5) below:

[0013] The structures of the three monosaccharide building blocks are shown in formulas (6) to (8), and the structures of the five carboxylic acid derivatives are shown in formulas (9) to (13).

[0014] PG2, PG3, PG4, PG6, and PG7 are temporary hydroxyl protecting groups, which can be one of benzyl (Bn), 2-naphthylmethyl (Nap), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), or triethylsilyl (TES).

[0015] PG8, PG9, PG 11 PG 12 PG 14 PG 15 PG 17 PG 18 PG 20 It is a temporary hydroxyl protecting group, which can be one of benzyl (Bn), 2-naphthylmethyl (Nap), acetyl (Ac), benzoyl (Bz), neopentanoyl, 9-pentanemethoxycarbonyl (Fmoc), or 2-p-methoxybenzyl (PMB);

[0016] PG 10 PG 13 PG 16 PG 19 PG 21 Each of the following is independently selected from hydroxyl (OH), chlorine (Cl), bromine (Br), fluorine (F), and C1-4 alkoxy groups;

[0017] PG1 is a temporary amino protecting group, which can be one of trichloroacetyl (TCA), dichloroacetyl (DCA), or chloroacetyl (CIAc);

[0018] PG5 is a temporary amino protecting group, which can be one of acetyl (Ac), trichloroacetyl (TCA), dichloroacetyl (DCA), chloroacetyl (CIAc), trichloroethoxycarbonyl (Troc), phthaloyl (Phth), 9-fluorenylmethoxycarbonyl (Fmoc), or tert-butyloxycarbonyl (Boc);

[0019] The linker is -(CH2). nN-Y1Y2 or -(CH) n S-Y1, where n = 1–25, Y1 and Y2 are one of hydrogen, acyl, benzyl (Bn), 2-naphthylmethyl (Nap), or benzylmethoxycarbonyl (Cbz); Linker* is -(CH2). n NH2 or -(CH) n SH, where n = 1 to 25;

[0020] The leaving group LG1 is N-phenyltrifluoroacetylimide ester (CF3C(=NPh)O-);

[0021] The leaving group LG2 is selected from one of the following: trichloroacetylimide ester (CCl3C(=NH)O-), N-phenyltrifluoroacetylimide ester (CF3C(=NPh)O-), methylthio (SMe), selenylphenyl (SePh), ethylthio (SEt), phenylthio (SPh), p-toluenethio (STol), and dibutylphosphonic acid (-P(=O)-(OBu)2);

[0022] The synthesis method includes the following steps:

[0023] (1) Construction of disaccharide receptor: The monosaccharide building block 8 is de-protected by the hydroxyl protecting group PG6 at position 3 to obtain receptor 14; receptor 14 is glycosylated with monosaccharide building block 7 to obtain disaccharide 15; the azide group in disaccharide 15 is reduced to an amino group using a reducing agent, and compound 13 is added for amidation to obtain compound 16; the hydroxyl protecting group PG4 at position 3 on monosaccharide building block 7 is removed from compound 16 to obtain disaccharide receptor 17;

[0024] (2) Constructing the target trisaccharide:

[0025] Disaccharide acceptor 17 and monosaccharide building block 6 undergo glycosylation reaction under the action of an activator to construct trisaccharide 18; then the azide group of trisaccharide 18 is reduced by a reducing agent, and any one of the carboxylic acid derivatives in formulas (9) to (12) is added for amidation to obtain compounds 19 to 22; compounds 19 to 22 are subjected to catalytic hydrogenation and deprotection to obtain target compounds 1 to 4.

[0026] Alternatively, trisaccharide 18 is reduced and acylated to convert the azide group to an acetamino group, followed by catalytic hydrogenation and deprotection to give target compound 5;

[0027] PG a PG b The hydroxyl temporary protecting group is independently selected from benzyl, 2-naphthylmethyl, acetyl, benzoyl, neopentanoyl, 9-pentomethoxycarbonyl, and 2-p-methoxybenzyl.

[0028] In one embodiment of the present invention, the C1-4 alkoxy group includes methyl (Me), ethyl (Et), and tert-butyl (t-Bu); when the PG 10 PG 13 PG 16 PG 19 PG 21 When the protecting group is C1-4 alkoxy, it must be removed before coupling the amide.

[0029] In one embodiment of the present invention, the disaccharide acceptor 17 is synthesized by the following method: under the catalysis of an activator and the effect of a solvent, acceptor 14 and donor 7 undergo a glycosylation reaction to obtain disaccharide 15; then, under the action of a reducing agent, the azide group in disaccharide 15 is reduced to an amino group to obtain an aminobiose intermediate; (R)-3-hydroxybutyric acid derivative 13 is activated by a condensing agent or prepared into an acyl halide, and condensed with the aminobiose intermediate amide to obtain compound 16; the PG4 protecting group in compound 16 is removed to obtain acceptor 17; the corresponding synthetic route is shown below:

[0030] In one embodiment of the present invention, the disaccharide 15 is synthesized by utilizing solvent effects, catalysis of activating reagents, and orthogonal protection of non-participating groups.

[0031] In one embodiment of the present invention, the concentration of the glycosylation reaction is 0.01 to 0.1 M.

[0032] In one embodiment of the present invention, the activating agent is one of TMSOTf, NIS / TMSOTf, and NIS / TfOH.

[0033] In one embodiment of the present invention, the solvent is one or more of anhydrous dichloromethane, diethyl ether, toluene, methanol, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, or water. A mixture of anhydrous dichloromethane, diethyl ether, and toluene is preferred.

[0034] In one embodiment of the present invention, the molar ratio of the donor to the acceptor is (1-3):1 or 1:(1-3).

[0035] In one embodiment of the present invention, the specific reaction conditions for synthesizing the disaccharide are as follows: the glycosyl donor and the glycosyl acceptor are dissolved in a mixed solvent of toluene, dichloromethane and diethyl ether, stirred under argon protection, molecular sieves are added, the reaction temperature is -20℃ to 0℃, 0.1 to 0.3 equivalents (compared to the molar equivalents of the donor) of activating reagent are added, and the reaction time is 2 to 8 hours.

[0036] In one embodiment of the present invention, the reducing agent used to reduce the azide group in the disaccharide 15 is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.

[0037] In one embodiment of the present invention, the condensing agent is one of DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), DPPA (diphenyl azidophosphate), DPC1 (diphenyl phosphoric acid chloride), DECP (diphenyl cyanophosphate), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate).

[0038] In one embodiment of the present invention, the acyl halide includes one of acyl chloride, acyl bromide, and acyl fluoride.

[0039] In one embodiment of the present invention, the method for preparing the acyl chloride includes one of the following: synthesizing acyl chloride using sulfoxide, synthesizing acyl chloride using oxalyl chloride, or preparing acyl chloride using trichlorotriazine.

[0040] In one embodiment of the present invention, trisaccharide 18 is synthesized by: utilizing temperature effects and the neighboring group participation effect at the C2 position, acceptor 17 and donor 6 are glycosylated under the catalysis of an activator to construct a single configuration of trisaccharide 18; the corresponding synthetic route is shown below:

[0041] In one embodiment of the present invention, the construction of the 1,2-trans-β-glycosidic bond in the trisaccharide 18 is achieved by utilizing the temperature effect, the catalysis of the activating reagent, and the participation of the neighboring group of PG1.

[0042] In one embodiment of the present invention, the concentration of the glycosylation reaction is 0.01 to 0.1 M.

[0043] In one embodiment of the present invention, the activator is one of TMSOTf, NIS / TMSOTf, and NIS / TfOH.

[0044] In one embodiment of the present invention, the solvent is one or more of anhydrous dichloromethane, diethyl ether, toluene, methanol, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, or water.

[0045] In one embodiment of the present invention, the molar ratio of the donor to the acceptor is (1-3):1 or 1:(1-3).

[0046] In one embodiment of the present invention, the glycosylation reaction conditions of the trisaccharide 18 include: dissolving the disaccharide acceptor 17 and the donor 6 in dichloromethane solvent, adding molecular sieves, adding an activating reagent in an amount of 0.2 to 1 equivalent (compared to the molar equivalent of the acceptor), and controlling the reaction temperature to gradually increase to room temperature from 0°C for a reaction time of 2 to 8 hours.

[0047] In one embodiment of the present invention, the synthesis method of compound 19 is as follows: reducing the azide group at the non-reducing end of trisaccharide 18 with a reducing agent to obtain an aminotrisaccharide intermediate; activating compound 9 with a condensing agent or preparing it into an acyl halide, and then coupling it with the aminotrisaccharide intermediate amide to obtain compound 19; the corresponding synthetic route is as follows:

[0048] In one embodiment of the present invention, the reducing agent is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.

[0049] In one embodiment of the present invention, the condensing agent is one of DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide)), DPPA (diphenyl azidophosphate), DPPCl (diphenylphosphochloride), DECP (diphenyl cyanophosphate), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate).

[0050] In one embodiment of the present invention, the acyl halide includes one of acyl chloride, acyl bromide, and acyl fluoride.

[0051] In one embodiment of the present invention, the method for preparing the acyl chloride includes one of the following: synthesizing acyl chloride using sulfoxide, synthesizing acyl chloride using oxalyl chloride, or preparing acyl chloride using trichlorotriazine.

[0052] In one embodiment of the present invention, the synthesis methods of compounds 20, 21, and 22 are the same as or similar to those of compound 19. That is, the synthesis of compounds 20, 21, and 22 involves reducing the azide group at the non-reducing end 4 position of trisaccharide 18 with a reducing agent to obtain an aminotrisaccharide intermediate, and then coupling compounds 10, 11, or 12 with an aminotrisaccharide intermediate amide, respectively.

[0053] In one embodiment of the present invention, compounds 1-5 are prepared by the following synthesis: compounds 19, 20, 21, and 22 (PGa and PGb represent the corresponding hydroxyl protecting groups) are catalytically hydrogenated under palladium on carbon conditions, and deprotected to obtain target compounds 1-4; trisaccharide 18 is reduced and acetylated with a reducing agent, and the azide group at the non-reducing end at position 4 is converted to an acetamido group, followed by catalytic hydrogenation and deprotection to obtain target compound 5; the corresponding synthetic route is shown below:

[0054] In one embodiment of the present invention, the method for removing the ester protecting group in the deprotection method can be potassium hydroxide / methanol / water, sodium hydroxide / ethanol / water, lithium hydroxide / methanol / water, sodium hydroxide / methanol / water, potassium hydroxide / ethanol / water, lithium hydroxide / ethanol / water, etc.

[0055] In one embodiment of the present invention, the method for removing the silyl ether protecting group in the deprotection method can be tetrabutylammonium fluoride, hydrofluoric acid, etc.

[0056] In one embodiment of the present invention, the removal of the carbon ether protecting group in the deprotection method can be catalytic hydrogenation, that is, in the presence of a catalyst, hydrogen gas is introduced to carry out the reaction.

[0057] In one embodiment of the present invention, the catalyst used for catalytic hydrogenation may be a 10% palladium on carbon catalyst or palladium hydroxide, etc.

[0058] In one embodiment of the present invention, the solvent used in the deprotection reaction can be a mixture of water / methanol / dichloromethane / acetic acid, a mixture of water / tert-butanol / dichloromethane, a mixture of water / tert-butanol / tetrahydrofuran, etc., and the reaction temperature can be between 0 and 40°C.

[0059] In one embodiment of the present invention, in the synthesis of compound 5, the reducing agent is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.

[0060] In one embodiment of the present invention, in the synthesis of compound 5, acetylation can be performed using pyridine, acetic anhydride; or methanol, acetic anhydride; or acetic acid, acetic anhydride, etc.

[0061] In one embodiment of the present invention, the reductive acylation of compound 5 can also be obtained by directly adding compound 18 to a mixed solution of thioacetic acid and pyridine.

[0062] One application of this invention is to use the synthesized oligosaccharide to elucidate the absolute configuration of dHh.

[0063] In one embodiment of the present invention, the absolute configuration of dHh is determined by NMR error analysis using four synthesized oligosaccharide isomers (1-4) and extracted natural O-polysaccharide (OPS).

[0064] In one embodiment of the present invention, the OPS is extracted from inactivated Vibrio cholerae O100 serotype bacteria.

[0065] In one embodiment of the present invention, in order to minimize errors, the NMR instruments used for the deprotected products (1-5) are all 600M, and the tests are conducted at the same temperature (25°C).

[0066] Another application of this invention is to elucidate the immunological role of dHh using the synthesized oligosaccharides.

[0067] In one embodiment of the present invention, the immunological function of dHh is demonstrated to be achieved through glycan chip technology.

[0068] In one embodiment of the present invention, the preparation process of the sugar chip includes: attaching the linker arms of the reducing ends of five oligosaccharide fragments to the chip, incubating with antiserum, labeling with secondary antibody, and fluorescence scanning.

[0069] In one embodiment of the present invention, the antiserum is derived from animal serum immunized with Vibrio cholerae O100 serotype lipopolysaccharide (LPS) or human serum infected with Vibrio cholerae O100 serotype.

[0070] In one embodiment of the present invention, the results of the sugar chip show that the deletion and conformational changes of dHh do not affect its binding ability with the antibody.

[0071] This invention also provides a Vibrio cholerae glycoprotein conjugate for vaccine development, which is composed of five oligosaccharide fragments (1-5) of the above-mentioned compound conjugated with a protein. Its general formula can be represented as sugar-linker-carrier protein.

[0072] In one embodiment of the present invention, the carrier protein comprises one of the following: diphtheria toxin nontoxic mutant protein (CRM197), hemocyanin (KLID), bovine serum albumin (BSA), meningitis perimeningeal protein (OMPC), tetanus toxoid (TT), or diphtheria toxoid (DT).

[0073] The present invention also provides the use of the glycoconjugate in the preparation of a vaccine for the prevention or treatment of diseases caused by Vibrio cholerae infection.

[0074] Furthermore, the main symptoms of the disease include one or more of the following: diarrhea, vomiting, sunken eyes, dry skin, altered consciousness, shock, and death.

[0075] This invention also provides the application of the above-mentioned chemical synthesis method in the preparation of sugar chips or Vibrio cholerae glycoprotein conjugates, the application comprising the following process:

[0076] S1: The above chemical synthesis method was used to prepare an oligosaccharide fragment of cholerae O100 serum type O antigen with a connecting arm;

[0077] S2: Subsequently, the linker arm of the obtained oligosaccharide fragment is used to bind to the chip or carrier protein to obtain the corresponding sugar chip or Vibrio cholerae glycoprotein conjugate. Beneficial effects

[0078] This invention utilizes three monosaccharide building blocks and five carboxylic acid derivatives, and by leveraging neighboring group participation effect, long-range participation effect, solvent effect, etc., through a series of orthogonal protection, stereoselective assembly and efficient amide coupling, to develop an efficient and concise method for synthesizing O antigen oligosaccharides. Five oligosaccharide fragments were successfully synthesized using this method.

[0079] Using four synthesized oligosaccharide isomers (1-4) and combined with NMR technology, the absolute configuration (3S, 5S) of dHh was elucidated. Determining the absolute configuration laid the foundation for subsequent investigation of structure-activity relationships. Sugar chips were fabricated from the five oligosaccharide fragments (1-5). Sugar chip screening showed that dHh is not a key antigenic epitope component. Next, referring to the method described in this invention, other oligosaccharide fragments can be synthesized to screen for the smallest antigenic epitope.

[0080] In conclusion, this invention will provide a reliable theoretical basis for the design of Vibrio cholerae vaccines, infection diagnosis, and drug development. Attached Figure Description

[0081] Figure 1 shows five oligosaccharide fragments, the three required monosaccharide building blocks, and five carboxylic acid derivatives;

[0082] Figure 2 shows the synthetic routes of four hexanoic acid derivatives 18* to 22*;

[0083] Figure 3 shows the synthetic route of the disaccharide receptor 28*;

[0084] Figure 4 shows the synthetic route of trisaccharide 31*;

[0085] Figure 5 shows the synthetic route of the fully protected trisaccharides 32* to 35*;

[0086] Figure 6 shows the synthetic route of the target compounds (1*~5*);

[0087] Figure 7 shows the target compounds (1*~5*). 13 Full spectrum error analysis diagram of C-NMR;

[0088] Figure 8 shows the target compounds (1*~5*). 13 C-NMR processing spectrum error analysis diagram; 2' to 6' in the figure represent different dHh values. 13 The NMR signal of C, similarly 2" to 4" indicates different NMR signals in RHb. 13 C NMR signal;

[0089] Figure 9 shows a comparison of the results of the sugar chip screening; where A is a schematic diagram of the oligosaccharide structure, B is a spotting pattern diagram, C is the chip scanning result, and D is the quantitative result of the average fluorescence. The error bar comes from the standard deviation between two points of two uniform concentrations.

[0090] Figure 10 shows the NMR-HSQC structural identification of compound 1*;

[0091] Figure 11 shows the NMR-HSQC structural identification of compound 2*;

[0092] Figure 12 shows the NMR-HSQC structural identification of compound 3*;

[0093] Figure 13 shows the NMR-HSQC structural identification of compound 4*;

[0094] Figure 14 shows the NMR-HSQC structural identification of compound 5*. Detailed Implementation

[0095] All commercially available reagents used in the experiments were used directly without any pretreatment. Anhydrous solvents used in the reactions were prepared using an MBraun MB-SPS 800 solvent drying system. All solvents used in silica gel column chromatography were analytical grade and distilled under reduced pressure. Thin-layer chromatography (TLC) used glass-based or aluminum foil-based silica gel plates made from 60-F254 silica gel, and normal-phase silica gel column chromatography used 200-300 mesh silica gel.

[0096] The yields of each reaction step were calculated as follows: (amount of target product / amount of starting material) × 100%. The product structure was identified using NMR spectroscopy, infrared spectroscopy, optical rotation, and high-resolution mass spectrometry. Purity analysis was performed using NMR spectroscopy. Proton, carbon, and two-dimensional NMR spectra were measured at 25°C using Bruker Ascend 600M and 400M NMR spectra. High-resolution mass spectrometry was performed using an Agilent 6220 electrospray ionization source-time-of-flight mass spectrometer. Infrared spectroscopy was performed using a Thermo Fisher Scientific Nicolet iS5 infrared spectrometer, and optical rotation was measured at 589 nm using a Schmidt & Haensch UniPol L10000 fully automated polarimeter. Concentration (c) was measured in g / 100 mL.

[0097] Example 1

[0098] Synthesis of 3,5-dibenzylhexanoic acid

[0099] As shown in Figure 2: Starting from methyl (R)-3-hydroxybutyrate 6* and methyl (S)-3-hydroxybutyrate 7*, respectively, tert-butyl (R)-5-hydroxyhexanoate 8* and tert-butyl (S)-5-hydroxyhexanoate 9* were obtained via Claisen ester condensation. Further, using the method of Narasaka and Evans, four different configurations of tert-butyl 3,5-dihydroxyhexanoate 10*–13* were selectively constructed. Through silver oxide-mediated selective benzylation, compounds 14*–17* were obtained. The tert-butyl group was removed using trifluoroacetic acid to yield 3,5-dibenzylhexanoate 18*–21*.

[0100] Specific experimental procedures and steps:

[0101] Compound 8*: Lithium diisopropylamine (1.25 mL, 2.5 mmol) and tert-butyl acetate (0.34 mL, 2.51 mmol) were added to a tetrahydrofuran (1 mL) solution at -78 °C and stirred for 20 min. Compound 6* (0.1 g, 0.85 mmol) was dissolved in THF (15 mL) solution and then added dropwise to the reaction system. The mixture was stirred at -50 °C for 2 h. After the reaction was complete as detected by TLC, deionized water was added, and the mixture was extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, concentrated by vacuum distillation using a rotary evaporator, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1, v / v) to obtain compound 9* (134 mg, 0.66 mmol, 78%). [α] 25 D =-13.7°(c=0.2,CHCl3); IR νmax(film)2973,2931,1731,1715,1456,1368,1252,1145,959,839,754cm -1 ; 1 H NMR(600MHz,Chloroform-d)δ4.26(ddq,J=9.6,6.4,3.1Hz,1H,5-H),3.37(d,J=2.2Hz,2H,2-CH2),2.8 5(d,J=3.4Hz,1H,5-OH),2.76–2.60(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.21(d,J=6.3Hz,3H,6-CH3). 13C NMR(151MHz,Chloroform-d)δ204.2,166.1,63.8,51.1,28.0,22.4; HRMS(ESI)calculated for C 10 H 18 O4Na + [M+Na] + :225.1097,found:225.1083

[0102] Compound 9*: Lithium diisopropylamine (1.25 mL, 2.5 mmol) and tert-butyl acetate (0.34 mL, 2.51 mmol) were added to a tetrahydrofuran (1 mL) solution at -78 °C and stirred for 20 min. Compound 7* (0.1 g, 0.85 mmol) was dissolved in THF (15 mL) solution and then added dropwise to the reaction system. The mixture was stirred at -50 °C for 2 h. After the reaction was complete as determined by TLC, deionized water was added, and the mixture was extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, concentrated by vacuum distillation using a rotary evaporator, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1, v / v) to obtain compound 9* (137 mg, 0.68 mmol, 80%). [α] 25 D =+9.9°(c=0.3,CHCl3); IR νmax(film)2971,2925,2853,1731,1714,1456,1368,1257,1145,958,939,754cm -1 ; 1 H NMR(400MHz,Chloroform-d)δ4.27(dtd,J=12.6,6.3,3.0Hz,1H,5-H),3.39(d,J=1.1Hz,2 H,2-CH2),2.79–2.60(m,2H,4-CH2),1.49(s,9H,tBu-CH3),1.23(d,J=6.4Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ204.3,166.1,63.8,51.1,50.9,28.0,22.3; HRMS(ESI)calculated for C 10 H 18 O4Na + [M+Na] + :225.1097,found:225.1083

[0103] Compound 10*: Compound 8* (150 mg, 0.74 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (4:1, v / v, 7.5 mL) at -70 °C, followed by the addition of diethylmethoxyborane (0.8 mL, 0.8 mmol). After one hour, sodium borohydride (31 mg, 0.82 mmol) was added to the solution. The reaction was then continued at -70 °C for 22 hours. After the reaction was confirmed to be complete by TLC, a saturated ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with a saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel column chromatography (petroleum ether: ethyl acetate, 1:1, v / v) to give 10* (105.7 mg, 0.52 mmol, 70%). [α] 25 D =-165.5°(c=0.1,CHCl3);IR νmax(film)3028,2976,2933,1715,1368,1272,1154,1069,843,760,605cm -1 ; 1 H NMR (600MHz, Chloroform-d) δ4.23 (dq, J=9.0, 5.6Hz, 1H, 3-H), 4.07 (dqd, J=8.9, 6.2, 2.5Hz, 1H, 5-H), 3.80 (s,1H,3-OH),3.43(s,1H,5-OH),2.40(d,J=6.1Hz,2H,2-CH2),1.61-1.49(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.20(d,J=6.2Hz,3H,6-CH3). 13 C NMR(151MHz,Chloroform-d)δ172.2,81.6,69.2,68.2,44.0,42.6,28.1,23.7; HRMS(ESI)calculated for C 10 H 20 O4Na + [M+Na] + :227.1254,found:227.1283

[0104] Compound 11*: Triacetoxytetramethylborane (3.0 g, 11.5 mmol) was added to a mixed solution of dry acetonitrile (9.1 mL) and acetic acid (9.1 mL) at room temperature and stirred for 30 min. Then, compound 9* (370 mg, 1.83 mmol) was dissolved in acetonitrile (2.7 mL) and added dropwise to the reaction system at -40 °C, and stirred for 94 h. After the reaction was complete as determined by TLC, the mixture was quenched with saturated potassium sodium tartrate solution, diluted and extracted with dichloromethane; the organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and the product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 1:1, v / v) to give dihydroxy ester 11* (317 mg, 1.56 mmol, 85%). [α] 25 D =+63.1°(c=0.9,CHCl3);IR νmax(film)2974,2931,1727,1393,1367,1257,1152,1084,954,842,760,664cm -1 ; 1 H NMR(400MHz,Chloroform-d)δ4.33(dq,J=8.4,4.0Hz,1H,3-H),4.16(d,J=7.8Hz,1H,5-H),3.58(d,J=3.4Hz,1H,3-OH),2.60(d ,J=4.4Hz,1H,5-OH),2.54–2.37(m,2H,2-CH2),1.65–1.55(m,2H,4-CH2),1.49(s,9H,tBu-CH3),1.26(d,J=6.3Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ172.5,81.5,65.9,65.8,65.1,65.0,43.5,43.5,42.2,42.1,42.0,28.2,28.1,23.6,23.5; HRMS(ESI) calculated for C 10 H 20 O4Na + [M+Na] + :227.1254,found:227.1271

[0105] Compound 12*: Triacetoxytetramethylborane (3.0 g, 11.5 mmol) was added to a mixed solution of dry acetonitrile (9.1 mL) and acetic acid (9.1 mL) at room temperature and stirred for 30 min. Then, compound 8* (370 mg, 1.83 mmol) was dissolved in acetonitrile (2.7 mL) and added dropwise to the reaction system at -40 °C, and stirred for 94 h. After the reaction was complete as determined by TLC, the mixture was quenched with saturated potassium sodium tartrate solution, diluted and extracted with dichloromethane; the organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and the product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 1:1, v / v) to give dihydroxy ester 12* (302 mg, 1.48 mmol, 81%). [α] 25 D =+8.5°(c=0.3,CHCl3); IR νmax(film)2975,2931,1728,1393,1367,1257,1152,1084,954,842,760,664cm -1 ; 1 H NMR(600MHz,Chloroform-d)δ4.31(ddd,J=12.1,8.4,3.4Hz,1H,3-H),4.14(ddp,J=9.4,6.1,3.1Hz,1H,5-H),3.57(s,1H,3-OH ),2.59(s,1H,5-OH),2.51–2.33(m,2H,2-CH2),1.65–1.53(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.24(d,J=6.3Hz,3H,6-CH3). 13 C NMR(151MHz,Chloroform-d)δ172.5,81.5,65.9,65.0,43.5,42.1,28.1,23.5; HRMS(ESI)calculated for C 10 H 20 O4Na + [M+Na] + :227.1254,found:227.1283

[0106] Compound 13*: Compound 9* (150 mg, 0.74 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (4:1, v / v, 7.5 mL) at -70 °C, followed by the addition of diethylmethoxyborane (0.8 mL, 0.8 mmol). After one hour, sodium borohydride (31 mg, 0.82 mmol) was added to the solution. The reaction was then continued at -70 °C for 22 hours. After the starting material was confirmed to be completely reacted by TLC, a saturated ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with a saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, concentrated under vacuum, and the product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 1:1, v / v) to give 13* (110.2 mg, 0.54 mmol, 73%). [α] 25 D =+26.2°(c=0.2,CHCl3);IR νmax(film)2978,2931,1728,1368,1324,1256,1154,1085,843,761,664cm -1 ; 1 H NMR(600MHz,Chloroform-d)δ4.23(dq,J=9.3,6.4,4.7Hz,1H,3-H),4.07(dqd,J=8.9,6.2,2.3Hz,1H,5-H),3.79(m,1H,3- OH),3.42(s,1H,5-OH),2.40(d,J=6.2Hz,2H,2-CH2),1.59–1.51(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.19(s,3H,6-CH3). 13 C NMR(151MHz,Chloroform-d)δ172.2,81.6(1-C),69.4,69.2,68.2,44.0,42.6,28.1,23.7.(ESI)calculated for C 10 H 20 O4Na + [M+Na] + :227.1254,found:227.1278

[0107] Compound 14*: Compound 10* (642.6 mg, 3.15 mmol) was dissolved in anhydrous dichloromethane (31.5 mL) under nitrogen protection, and benzyl bromide (3.74 mL, 31.5 mmol) and silver oxide (8.99 g, 37.8 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 5 h, and then stirred at room temperature for 28 h. After diatomaceous earth filtration and concentration, the organic layer was extracted with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 9:1, v / v) to give product 14* (726 mg, 1.89 mmol, 60%). [α] 25 D =-26.1°(c=1.1,CHCl3);IR νmax(film)3029,2931,1714,1453,1373,1271,1143,1094,1065,746,697cm -1 ; 1 H NMR(400MHz, Chloroform-d)δ7.33–7.11(m,10H,Ar),4.49(dd,J=11.5,3.2Hz,2H,Ar-CH2),4.39(dd,J=15 .7,11.5Hz,2H,Ar-CH2),3.94(ddd,J=12.6,6.9,5.6Hz,1H,3-H),3.59(h,J=6.2Hz,1H,5-H),2 .44(dd,J=15.0,7.2Hz,1H,2-CH2),2.34(dd,J=15.0,5.4Hz,1H,2-CH2),1.98(dt,J=13.6,6.7 Hz,1H,4-CH2),1.58–1.52(m,1H,4-CH4),1.37(s,9H,tBu-CH3),1.13(d,J=6.1Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ171.1,138.9,138.7,128.5,128.4,127.9,127.7,127. 7,127.6,80.7,73.9,71.9,71.5,70.4,41.6,41.3,28.3,19.8; HRMS(ESI)calculated for C 24 H 32 O4Na + [M+Na] + :407.2193,found:407.2232

[0108] Compound 15*: Compound 11* (764 mg, 3.65 mmol) was dissolved in anhydrous dichloromethane (36.5 mL) under nitrogen protection, and benzyl bromide (4.34 mL, 36.52 mmol) and silver oxide (10.4 g, 43.8 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 5 h, and then stirred at room temperature for 28 h. After diatomaceous earth filtration and concentration, the organic layer was extracted with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 9:1, v / v) to give product 15* (926 mg, 2.41 mmol, 66%). [α] 25 D =+44.6°(c=0.68,CHCl3); IR νmax(film)2974,2930,1728,1454,1367,1256,1152,1065,954,843,736,697cm -1 ; 1 H NMR(400MHz,Chloroform-d)δ7.35–7.26(m,10H,Ar),4.60(dd,J=11.4,3.5Hz,2H,Ar-CH2),4.36(dd,J=18.1,11.4Hz,2H,Ar-CH2),4.13(m,J=6.2 Hz,1H,3-H),3.78(m,J=6.1Hz,1H,5-H),2.60–2.42(m,2H,2-CH2),1.77–1.72(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.23(d,J=6.1Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ170.9,138.6,138.6,128.3,127.8,127.7,127.5,127.4,80.5, 73.4,71.9,71.6,71.5,70.3,43.3,41.7,28.1,28.1,28.1,20.0,19.9.HRMS(ESI)calculated for C 24 H 32 O4Na + [M+Na] + :407.2193,found:407.2233

[0109] Compound 16*: Compound 12* (950 mg, 4.65 mmol) was dissolved in anhydrous dichloromethane (46.5 mL) under nitrogen protection, and benzyl bromide (5.5 mL, 46.5 mmol) and silver oxide (13.3 g, 55.8 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 5 h, and then stirred at room temperature for 28 h. After diatomaceous earth filtration and concentration, the organic layer was extracted with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 9:1, v / v) to give product 16* (1.14 g, 0.025 mol, 64%). [α] 25 D =-48.8°(c=0.4,CHCl3); IR νmax(film)2931,1730,1455,1368,1257,1156,1116,1063,846,735,697cm -1 ; 1 H NMR(400MHz,Chloroform-d)δ7.35–7.20(m,10H,Ar),4.58(dd,J=11.4,3.3Hz,2H,Ar -CH2),4.34(dd,J=17.9,11.4Hz,2H,Ar-CH2),4.11(m,J=6.2Hz,1H,3-H),3.76(m,J= 6.2Hz,1H,5-H),2.54(dd,J=14.8,6.4Hz,1H,2-CH2),2.43(dd,J=14.8,5.8Hz,1H,2- CH2),1.76–1.68(m,2H,4-CH2),1.44(s,9H,tBu-CH3),1.21(d,J=6.1Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ170.9,139.0,138.6,128.3,127.8,127.7,127.5,127. 4,80.5,73.5,71.9,71.6,71.5,70.3,43.3,41.7,28.1,19.9.HRMS(ESI)calculated for C 24 H 32 O4Na + [M+Na] + :407.2193,found:407.2250

[0110] Compound 17*: Compound 13* (319 mg, 1.56 mmol) was dissolved in anhydrous dichloromethane (15.6 mL), and benzyl bromide (1.85 mL, 15.6 mmol) and silver oxide (4.45 g, 18.74 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 5 h, then stirred at room temperature for 28 h. After diatomaceous earth filtration and concentration, the organic layer was extracted with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 9:1, v / v) to give product 17* (347.6 mg, 0.905 mmol, 58%). [α] 25 D =-4.7°(c=0.2,CHCl3); IR νmax(film)2927,1729,1454,1368,1258,1157,1090,1064,844,760,697cm -1 ; 1 H NMR(600MHz,Chloroform-d)δ7.36–7.23(m,10H,Ar),4.56(dd,J=11.5,4.2Hz,2H,Ar-CH2),4 .46(dd,J=23.3,11.5Hz,2H,Ar-CH2),4.01(m,J=6.3Hz,1H,3-H),3.66(m,J=6.2Hz,1H,5-H),2 .51(dd,J=15.0,7.2Hz,1H,2-CH2),2.41(dd,J=15.0,5.4Hz,1H,2-CH2),2.05(dt,J=13.7,6.7 Hz,1H,4-CH2),1.67–1.60(m,1H,4-CH2),1.45(s,9H,tBu-CH3),1.20(d,J=6.1Hz,3H,6-CH3). 13 C NMR(151MHz,Chloroform-d)δ170.9,138.8,138.5,128.4,128.3,127.8,127.7,127. 5,127.5,80.5,73.8,71.7,71.3,70.2,41.4,41.1,28.1,19.7.HRMS(ESI)calculated for C 24 H 32 O4Na + [M+Na] + :407.2193,found:407.2221

[0111] Compound 18*: Under nitrogen protection, tert-butyl 3,5-dibenzyl hexanoate 14* (61 mg, 0.159 mmol) was dissolved in anhydrous DCM (1 mL), and then trifluoroacetic acid (1 mL) was added to the reaction system at 0 °C. The temperature was slowly increased to room temperature, and the reaction was carried out for 12 hours. After the reaction was complete as determined by TLC, dichloromethane was added for dilution, and the organic layer was extracted with ultrapure water and saturated sodium bicarbonate solution, respectively. The mixture was then dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound 18* (43.8 mg, 0.134 mmol, 84%). [α] 25 D =-10.8°(c=1.0,CHCl3);IR νmax(film)2963,2928,1727,1454,1367,1270,1154,1119,1069,794,696cm -1 ; 1 H NMR(400MHz,Chloroform-d)δ7.42–7.13(m,10H,Ar),4.64–4.48(m,3H,Ar-CH2),4.41(d,J=11.7Hz,1H,Ar-CH2),4.05(p,J=6.2Hz,1H,3-H),3.73–3.56(m,1H ,5-H),2.58(dd,J=6.1,1.4Hz,2H,2-CH2),2.06(ddd,J=13.7,7.6,5.7Hz,1H,4 -CH2), 1.67 (ddd, J=14.3, 6.8, 5.0Hz, 1H, 4-CH2), 1.21 (d, J=6.1Hz, 3H, 6-CH3). 13 C NMR(101MHz,Chloroform-d)δ176.2,138.6,138.1,128.6,128.0,127.9,127.9,127. 8,127.7,73.2,71.6,71.5,70.6,70.4,41.1,39.5,21.5,19.8.HRMS(ESI)calculated for C 20 H 24 O4Na + [M+Na] + :351.1567,found:351.1604

[0112] Compound 19*: Under nitrogen protection, tert-butyl 3,5-dibenzyl hexanoate 15* (74 mg, 0.19 mmol) was dissolved in anhydrous DCM (1 mL), and then trifluoroacetic acid (1 mL) was added to the reaction system at 0 °C. The temperature was slowly increased to room temperature, and the reaction was carried out for 12 hours. After the reaction was complete as determined by TLC, dichloromethane was added for dilution, and the organic layer was extracted with ultrapure water and saturated sodium bicarbonate solution, respectively. The extract was then dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound 19* (53.8 mg, 0.164 mmol, 85%). [α] 25 D =+102°(c=0.5,CHCl3);IR νmax(film)2927,1715,1451,1274,1176,1111,1069,1026,751,712cm -1 ; 1 H NMR(600MHz,Chloroform-d)δ7.36–7.23(m,10H,Ar),4.58(dd,J=11.4,9.0Hz,2H,Ar-CH2),4.38(d,J=11.2Hz,1H,Ar-CH2),4.31(d,J=11.5Hz, 1H,Ar-CH2),4.19–4.04(m,1H,3-H),3.94–3.68(m,1H,5-H),2.70–2.55(m,2H,2-CH2),1.84–1.65(m,2H,4-CH2),1.22(d,J=6.1Hz,3H,6-CH3). 13 C NMR(151MHz,Chloroform-d)δ138.64-,137.9,128.5,128.4,128.0,127.9,127.85,127.59,73.0,72.0,71.4,71.37,70.3,42.9,39.6,19.8; HRMS(ESI) calculated for C 20 H 24 O4Na + [M+Na] + :351.1567,found, 351.1589

[0113] Compound 20*: Under nitrogen protection, tert-butyl 3,5-dibenzyl hexanoate 16* (100 mg, 0.26 mmol) was dissolved in anhydrous DCM (1 mL), and then trifluoroacetic acid (1 mL) was added to the reaction system at 0 °C. The temperature was slowly increased to room temperature, and the reaction was carried out for 12 hours. After the reaction was complete as determined by TLC, dichloromethane was added for dilution, and the organic layer was extracted with ultrapure water and saturated sodium bicarbonate solution, respectively. The mixture was then dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 20* (62 mg, 0.19 mmol, 74%). [α] 25 D =-63.3°(c=0.75,CHCl3);IR νmax(film)3029,2966,2930,1709,1454,1373,1146,1109,1062,736,697cm -1 ; 1 H NMR(400MHz,Chloroform-d)δ7.39–7.24(m,10H,Ar),4.61(dd,J=11.4,5.8Hz,2H,Ar-CH2),4.40(d,J=11.2Hz,1H,Ar-CH2),4.34(d,J=11.6Hz,1 H,Ar-CH2),4.21–4.08(m,1H,3-H),3.87–3.74(m,1H,5-H),2.65(d,J=5.8Hz,2H,2-CH2),1.84–1.76(m,2H,3-CH2),1.25(d,J=6.1Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ176.4,138.7,138.0,128.4,128.42,127.99,127.87,127.79,127.61,73.0,72.0,71.4,70.3,43.0,39.8,19.8.HRMS(ESI)calculated for C 20 H 24 O4Na + [M+Na] + :351.1567,found,351.1607

[0114] Compound 21*: Under nitrogen protection, tert-butyl 3,5-dibenzyl hexanoate 17* (89 mg, 0.23 mmol) was dissolved in anhydrous DCM (1 mL), and then trifluoroacetic acid (1 mL) was added to the reaction system at 0 °C. The temperature was slowly increased to room temperature, and the reaction was carried out for 12 hours. After the reaction was complete as determined by TLC, dichloromethane was added for dilution, and the organic layer was extracted with ultrapure water and saturated sodium bicarbonate solution, respectively. The mixture was then dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 21* (62.4 mg, 0.19 mmol, 82%). [α] 25 D =+28.6°(c=0.4,CHCl3); IR νmax(film)3029,2926,2857,1714,1496,1454,1373,1184,1091,1061,749,697,608cm -1 ; 1 H NMR(400MHz,Chloroform-d)δ7.36–7.26(m,10H,Ar),4.64–4.51(m,3H,Ar-CH2),4.41(d,J=11.6Hz,1H,Ar-CH2),4.05(p,J=6.1Hz,1H,3-H),3.68(q,J=6.2H z,1H,5-H),2.59(d,J=6.0Hz,2H,2-CH2),2.07(ddd,J=13.7,7.9,5.4Hz,1H,4- CH2), 1.68 (ddd, J=14.2, 7.0, 4.7Hz, 1H, 4-CH2), 1.22 (d, J=6.1Hz, 3H, 6-CH3). 13 C NMR(101MHz,Chloroform-d)δ174.5,138.4,137.8,128.5,127.9,127.8,127.7,71.4,70.3,40.8,39.1,19.7,19.7.HRMS(ESI)calculated for C 20 H 24 O4Na + [M+Na] + :351.1567,found:351.1588.

[0115] Compound 22*: Under nitrogen protection, tert-butyl 3,5-dibenzyl hexanoate 18* (89 mg, 0.23 mmol) was dissolved in anhydrous DCM (1 mL). Oxaloyl chloride was then added to the reaction system at 0 °C, followed by a catalytic amount of DMF. The reaction was carried out at 0 °C for 30 minutes. After the reaction was completed by TLC, the mixture was quenched with an appropriate amount of methanol to quantitatively obtain the acyl chloride compound 22*. Acyl chlorides are highly reactive and readily hydrolyzed; therefore, the reaction mixture was prepared and used immediately in the experiment.

[0116] Example 2

[0117] Synthesis of disaccharide receptor 28*

[0118] As shown in Figure 3: L-fucosamine building blocks 23* and D-fucosamine building blocks 24* (Cai Juntao, doctoral dissertation, Jiangnan University, 2020) were used as raw materials for glycosylation to obtain compound 25*. The non-reducing azide group at the disaccharide 25* was reduced to an amino group using pre-activated zinc powder. Then, under the action of condensing agent HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), (R)-3-O-benzylbutyric acid 26* (which can be prepared by reference: Tanasova. et al, Angew. Chem. Int. Ed. 2015, 54(14), 4274-4278) was condensed with aminoglycoamide, successfully introducing a butyryl group at the C4' position of the disaccharide to obtain disaccharide 27*. The naphthyl methylene group in 27* was selectively removed using DDQ to obtain compound 28*.

[0119] Experimental procedures and steps:

[0120] Compound 25*: The synthesis of disaccharide 25* initially utilized a pre-activation method, but failed to yield the desired result. Subsequently, the donor and acceptor were dissolved in dichloromethane solution, and glycosylation was carried out under the conditions of trimethylsilyl trifluoromethanesulfonate and iodosuccinimide, successfully yielding compound 25*, but with poor selectivity (α:β = 1.5:1). Following this, the solvent effect of diethyl ether was utilized to further optimize the glycosylation reaction conditions, ultimately yielding compound 25* with good selectivity (α:β = 6:1). The specific glycosylation method is as follows:

[0121] Method 1: Under argon protection, glycosyl donor 24* (53.2 mg, 0.104 mmol) was dissolved in anhydrous dichloromethane (2.0 mL) and cooled to -60°C. Then, pre-activated [reagent / material] was added. Molecular sieves, 1-(phenylsulfonyl)piperidine (BSP, 26.1 mg, 0.125 mmol), 2,4,6-tri-tert-butylpyrimidine (TTBP, 38.8 mg, 0.156 mmol), and trifluoromethanesulfonic anhydride (Tf₂O, 22.7 μL, 0.135 mmol) were reacted at -60 °C with stirring for 30 min. After confirming complete reaction of the starting materials by TLC monitoring, 1-octene (16.3 μL, 0.104 mmol) was added, and the reaction was continued at -60 °C with stirring for another 15 min. Subsequently, the reaction solution was cooled to -78 °C, and glycosyl acceptor 23* (72.4 mg, 0.120 mmol) dissolved in anhydrous dichloromethane (2.0 mL) was added dropwise to the reaction solution, and the reaction was continued at -78 °C with stirring for 3 h. After TLC monitoring confirmed the complete reaction of the starting materials, triethyl phosphite (53.5 μL, 0.312 mmol) was added to terminate the reaction, and the mixture was then stirred at -78°C for 1 hour. TLC analysis revealed a complex composition of the reaction system, and the target compound was not detected by liquid chromatography-mass spectrometry.

[0122] Method 2: D-fucose donor 24* (80.6 mg, 0.158 mmol) and L-fucose acceptor 23* (115 mg, 0.19 mmol) were dissolved in toluene (5 ml), azeotropically removed three times, concentrated under vacuum until dry, and then pre-activated [reagent / concentrate] was added. Molecular sieves were used, and the mixture was evacuated overnight using an oil pump. Under argon protection, anhydrous dichloromethane (6.0 mL) and iodosuccinimide (43 mg, 0.19 mmol) were added sequentially. The mixture was cooled to 0 °C and stirred for 30 minutes. Trimethylsilyl trifluoromethanesulfonate (5.7 μL, 0.032 mmol) was added dropwise, and the mixture was stirred at 0 °C for 5 hours. After the reaction of the starting materials was confirmed to be complete by TLC, triethylamine was added to quench the reaction. Diatomaceous earth was added to a sintered glass funnel, and the molecular sieves were removed by filtration. The mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered through filter paper to remove sodium sulfate, and concentrated by vacuum distillation using a rotary evaporator. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate, 1:1, v / v) to give α-configuration disaccharide 25* (128.7 mg, 0.128 mmol, 81%, α:β = 1.5:1).

[0123] Method 3: D-fucose donor 24* (100 mg, 0.196 mmol) and L-fucose acceptor 23* (141.6 mg, 0.235 mmol) were dissolved in toluene (5 ml), azeotropically removed three times, concentrated under vacuum until dry, and then pre-activated [reagent / concentration] was added. Molecular sieves were used, and the mixture was evacuated overnight using an oil pump. Under argon protection, anhydrous dichloromethane (4.5 mL), diethyl ether (3.0 mL), and iodosuccinimide (52.9 mg, 0.235 mmol) were added sequentially. The mixture was cooled to 0 °C and stirred for 30 minutes. Trimethylsilyl trifluoromethanesulfonate (3.45 μL, 0.039 mmol) was added dropwise, and the mixture was stirred at 0 °C for 5 hours. After the reaction was confirmed to be complete by TLC, triethylamine was added to quench the reaction. Diatomaceous earth was added to a sintered funnel, and the molecular sieves were removed by filtration. The mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered through filter paper to remove sodium sulfate, and concentrated by vacuum distillation using a rotary evaporator. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate, 1:1, v / v) to obtain α-configuration disaccharide 25* (177 mg, 0.176 mmol, 90%, α:β = 6:1, α configuration: 3 J H1 / H2 =3.4Hz, 1 J H1 / C1 =166Hz). [α] 25 D =+11.67°(c=1.0,CHCl3); IR νmax(film)3366,2934,2108,1732,1700,1538,1498,1456,1423,1362,1278,1228,1177,1127,1092,1047,858,823,752,735,700cm -1 ; α: 1H NMR(400MHz,CDCl3)δ=7.96-7.01(m,27H,Ar),6.61(d,J=5.7Hz,1H,NHAc),5.15(d,J=6.2Hz,2H,ArCH2),5.12(d,J=3.5Hz,1H,1-H),4.95-4.78(m,5H,1'-H,ArCH2),4.71(dd,J=11.3,5.1Hz,1H,ArCH2),4.63(d,J=11.8Hz,1H,ArCH2),4.47(d,J=6.9Hz,2H,ArCH2),4.32(s,1H,2-H),4.16-4.05(m,2H,3'-H,5'-H),3.98(dd,J=9.8,3.4Hz,1H,2'-H),3.91(t,J=11.6Hz,1H,3-H),3.85-3.74(m,2H,4'-H,5-H),3.64-3.46(m,2H,4-H,linker-OCH2),3.45-3.14(m,3H,linker-OCH2,linker-NCH2),1.55(ddt,J=17.5,13.8,6.5Hz,4H,linker-CH2),1.39(d,J=4.5Hz,3H,Ac),1.35-1.25(m,2H,linker-CH2),1.13(d,J=6.4Hz,3H,6'-CH3),1.09(m,3H,6-CH3); 13 C NMR(100MHz,CDCl3)δ=170.5,156.8,156.3,138.6,137.9,137.3,136.9,136.8,135.2,133.3,133.2,129.1,128.8,128.7,128.6,128.4,128.1,128.0,127.9,127.7,127.4,127.3,126.7,126.5,126.3,125.7,99.0,97.0,79.1,78.9,77.7,75.3,74.6,73.3,68.1,67.3,66.4,66.2,64.5,50.5,50.2,50.0,47.2,46.2,29.5,28.1,27.6,23.6,22.5,17.5,16.9.HR-ESI-MS(m / z):calcd for C 59 H 67 O 10 N5Na+(M+Na)+:1028.4780found:1028.4786。

[0124] Compound 27*: Under nitrogen protection, disaccharide 25* (20.5 mg, 20.4 μmol) was dissolved in a mixture of tetrahydrofuran (0.8 mL) and acetic acid (0.2 mL), followed by the addition of pre-activated zinc powder (0.5 g), and stirred at room temperature for 12 hours. The reaction mixture was then filtered and concentrated to obtain crude trisaccharide, which was then directly used for the next reaction. (R)-3-O-benzylbutyric acid 26* (6 mg, 30.6 μmol) and trisaccharide were dissolved in DMF (1 mL) at room temperature, followed by the addition of HATU (9.3 mg, 22.48 μmol) and DIPEA (6.8 μL, 40.8 μmol), and stirred at room temperature for 6 hours. After TLC detection showed complete reaction of the starting material, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution. The separated organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by column chromatography (petroleum ether:ethyl acetate, 1:1, v / v) to give compound 27* (18.8 mg, 16.3 μmol, 80%). [α] 25 D =+34.6°(c=1.2,CHCl3);IR νmax(film)3029,2924,2853,1681,1543,1507,1455,1361,1217,1092,1045,758,698cm -1 ; 1H NMR(400MHz,CDCl3)δ=7.96-7.01(m,32H,Ar),6.80(d,J=10.2Hz,1H,NHRHb),6.60(d,J=5.5Hz,1H,NHAc),5.15(s,2H,ArCH2),5.10(d,J=3.5Hz,1H,1-H),5.01(d,J=11.1Hz,1H,ArCH2),4.86(d,J=11.7Hz,1H,ArCH2),4.71-4.58(m,5H,1'-H,4'-H,ArCH2),4.55-4.43(m,5H,ArCH2),4.25(m,2H,2-H,5'-H),4.01(dt,J=6.4,3.2Hz,1H,RHb-CH),3.94(dd,J=10.1,4.2Hz,1H,3'-H),3.89-3.74(m,2H,3-H,5-H),3.60-3.44(m,2H,4-H,linker-OCH2),3.36-3.14(m,3H,linker-OCH2,linker-NCH2),2.65(dd,J=15.2,3.7Hz,1H,RHb-CH2),2.52(dd,J=15.2,6.7Hz,1H,RHb-CH2),1.62-1.46(m,4H,linker-CH2),1.35-1.25(m,8H,RHb-CH3,Ac,linker-CH2),1.10(d,J=6.4Hz,3H,6-CH3),1.03(d,J=6.4Hz,3H,6'-CH3); 13 C NMR(100MHz,CDCl3)δ=171.7,170.5,138.7,138.3,138.0,137.5,135.7,133.4,133.24,133.15,132.3,132.3,132.2,132.1,132.0,128.8,128.69,128.67,128.6,128.54,128.48,128.4,128.2,128.1,128.03,128.00,127.94,127.89,127.8,127.7,127.4,127.1,126.4,126.2,126.1,98.8,97.0,78.9,77.8,75.2,74.5,72.9,71.6,70.9,68.1,67.3,66.8,66.3,50.2,50.0,43.6,29.5,23.5,22.4,19.1,17.0,16.9.HR-ESI-MS(m / z):calcd for C 70 H81 O 12 N3Na+(M+Na)+:1178.5712found:1178.5704.

[0125] Compound 28*: Compound 27* (26.8 mg, 23.0 μmol) was dissolved in dichloromethane (5.0 mL) under argon protection, followed by the addition of deionized water (1.0 mL) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (7.7 mg, 35.0 μmol). The mixture was stirred at room temperature for 5 hours. After the reaction was complete as monitored by TLC, the mixture was extracted with dichloromethane, washed with 10% (w / w) sodium thiosulfate solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered through filter paper to remove sodium sulfate, concentrated by rotary evaporation under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate, 1:1, v / v) to give compound 28* (21 mg, 20.7 μmol, 90%). [α] 25 D =-14.4°(c=1.0,CHCl3);IR νmax(film)3342,3030,2936,1670,1541,1496,1453,1422,1362,1304,1216,1180,1092,1043,827,754,698cm -1 ; 1H NMR(400MHz,CDCl3)δ=7.42-7.13(m,25H,Ar),6.81(d,J=8.6Hz,1H,NHRHb),6.61(d,J=5.8Hz,1H,NHAc),5.15(d,J=4.9Hz,2H,ArCH2),5.08(d,J=3.5Hz,1H,1-H),4.84(d,J=11.8Hz,1H, ArCH2),4.77(d,J=11.0Hz,1H,ArCH2),4.67-4.53(m,4H,1'-H,ArCH2),4.51-4.43(m,3H,ArCH2),4.35-4.20(m,3H,2-H,4'-H,5'-H),4.14(m,1H,3'-H),4.00(dt,J=6.2,3.5Hz,1H,RHb-CH),3.86(d,J=11.2Hz,1H,3-H),3.80(d,J=7.3Hz,1H,5-H),3.60-3.44(m,2H,4-H,linker-OCH2),3.38(dd,J=9.8,3.5Hz,1H,2'-H),3.36-3.18(m,3H,linker-OCH2,linker-NCH2),2.63(dd,J=15.0,3.8Hz,1H,RHb-CH2),2.50(dd,J=15.2,6.6Hz,1H,RHb-CH2),1.57(s,3H,Ac),1.56-1.46(m,4H,linker-CH2),1.37(d,J=6.2Hz,3H,RHb-CH3),1.33-1.24(m,2H,linker-CH2),1.11(d,J=6.4Hz,3H,6-CH3),0.95(d,J=6.4Hz,3H,6'-CH3); 13 C NMR(100MHz,CDCl3)δ=173.8,170.7,138.7,138.04,137.97,137.8,136.9,128.71,128.67,128.6,128.44,128.36,128.1,127.94,127.90,127.88,127.7,127.4,127.3,98.6,97.1,79.2,78.6,74.9,74.5,72.5,71.7,70.9,68.0,67.3,66.3,66.1,54.7,49.9,47.2,46.3,43.5,29.8,29.4,28.1,27.6,23.5,22.8,19.1,17.0,16.7.HR-ESI-MS(m / z):calcd for C59 H 73 O 12 N3Na + (M+Na) + :1038.5086found:1038.5117.

[0126] Example 3

[0127] Synthesis of trisaccharide 31*

[0128] As shown in Figure 4: The known D-quinoline amine building block 29* (which can be prepared by referring to existing literature Codée et al, Organic & Biomolecular Chemistry. 2020, 18(15), 2834-2837) was dissolved in a mixed solution of acetone and H2O and hydrolyzed under the catalysis of iodosuccinimide (NIS). Subsequently, under the action of 2,2,2-trifluoro-N-phenylacetylimide acyl chloride (171 μL, 1.14 mmol) and 1,8-diazabicycloundec-7-ene (DBU), trifluoroacetylimide ester 30* was obtained. Trifluoroacetylimide ester 30* and disaccharide acceptor 28* were glycosylated under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to successfully obtain a single β-configuration trisaccharide compound 31* (yield 13%).

[0129] Due to the low yield of 31*, the glycosylation conditions were optimized in this invention. After replacing compound 30* with compound 29* as the glycosyl donor, glycosylation was carried out under the catalysis of trimethylsilyl trifluoromethanesulfonate and iodosuccinimide, yielding trisaccharide 31* in 28% yield. Subsequently, using compound 29* as the glycosyl donor, the glycosylation yield was successfully increased to 54% by changing the catalyst stoichiometry. Further optimization of the glycosylation yield by changing the temperature revealed that slowly increasing from 0°C to room temperature yielded a better glycosylation yield. Finally, with the combined effect of temperature and catalyst, the target trisaccharide was successfully obtained in a relatively high yield of 81%. The specific optimization process and reaction conditions are shown in Table 1.

[0130] Table 1

[0131] Specific experimental procedures and steps:

[0132] Compound 30*: Compound 29* (232.5 mg, 0.38 mmol) was dissolved in acetone and H2O (10:1, v / v, 5.5 mL) at room temperature and stirred until homogeneous. Then, NIS (171.4 mg, 0.7 mmol) was added and the mixture was stirred for 1 hour. After TLC showed complete reaction, the mixture was diluted with ethyl acetate and washed with 10% (w / v) Na2S2O3. The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate 20 / 1 → 1 / 1 v / v) to give the intermediate compound. At 0 °C, the intermediate compound was dissolved in DCM (4.8 mL) and then 2,2,2-trifluoro-N-phenylacetylimine chloride (171 μL, 1.14 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (171 μL, 1.14 mmol) were added. The mixture was stirred at 0°C for 3 hours, then concentrated under vacuum and purified by silica gel column chromatography (petroleum ether / ethyl acetate: 30 / 1→10 / 1v / v) to obtain trifluoroacetylimine ester 30* (223.4 mg, 0.34 mmol, two-step yield 90%). 1 HNMR(400MHz,Chloroform-d)δ=7.96–7.76(m,5H),7.59– 7.33(m,6H),7.30–7.05(m,1H),6.27(d,J=7.4,1H),4.94(q,J=11.9,2H),4.38(dd,J=7.4,4.1,1H) ,3.85(dd,J=5.7,4.1,1H), 3.50(dq,J=9.6,6.1,1H), 3.34(dd,J=9.7,5.7,1H), 1.38(d,J=6.1,3H). 13 C NMR(101MHz,Chloroform-d)δ=162.9,135.1,134.2,133.2,133.2,129.4,128.5,128.0,127.7,12 7.6126.4,126.3,126.2,126.0,105.6,78.9,72.3,67.9,66.7,64.6,19.1, HR-ESI-MS (m / z): calcd for C 28 H 25 Cl3F3N5O4Na + (M+Na) + :680.0924, found:680.1030

[0133] Compound 31*: Two representative glycosylation reaction procedures are as follows.

[0134] Method 1: Under argon protection, trifluoroacetylimine ester 30* (24.3 mg, 37 μmol) and disaccharide acceptor 28* (25.3 mg, 25 μmol) were dissolved in toluene and azeotropically evaporated three times to remove water. The mixture was then evacuated under vacuum for 2 hours using an oil pump. Pre-activated [material / material] was then added. Molecular sieves and anhydrous dichloromethane (1.0 mL) were added to the reaction solution, which was cooled to 0 °C and stirred for 30 minutes. Trimethyl trifluoromethanesulfonate (0.9 μL, 5.0 μmol) was added dropwise, and the reaction was stirred at 0 °C for 5 hours. After the reaction of the starting materials was confirmed to be complete by TLC, triethylamine was added to quench the reaction. The molecular sieves were removed by diatomaceous earth filtration, and the product was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. The product was dried over anhydrous sodium sulfate and concentrated by vacuum distillation using a rotary evaporator. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1, v / v) to obtain syrup 31* (4.78 mg, 3.25 μmol, 13%).

[0135] Method 2: Under argon protection, selenoside 29* (110 mg, 0.18 mmol) and disaccharide receptor 28* (122 mg, 0.12 mmol) were dissolved in toluene and azeotropically evaporated three times to remove water. The mixture was then evacuated under vacuum for 2 hours using an oil pump. Pre-activated [material / material] was then added. Molecular sieves and anhydrous dichloromethane (6.0 mL) were added to the reaction solution. The solution was cooled to 0 °C and stirred for 30 minutes. Trimethyl trifluoromethanesulfonate (8.7 μL, 0.048 mmol) and iodosuccinimide (40.5 mg, 0.18 mmol) were added dropwise. The mixture was slowly heated from 0 °C to room temperature and stirred for 5 hours. After the reaction of the starting materials was confirmed to be complete by TLC, triethylamine was added to quench the reaction. The molecular sieves were removed by diatomaceous earth filtration. The mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate and concentrated by vacuum distillation using a rotary evaporator. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1, v / v) to obtain syrup 31* (142.8 mg, 97.2 μmol, 81%). [α] 25 D =+15.2°(c=0.7,CHCl3); IR νmax(film)3343,3029,2979,2940,2877,2108,1659,1532,1496,1454,1423,1360,1309,1216,1167,1092,1047,822,756,698cm -1 ; 1H NMR(600MHz,Methanol-d4)δ7.85–7.72(m,4H,Ar),7.47–7.14(m,28H,Ar),5.13(d,J=12.9Hz,2H,Ar-CH2),5.06–4.96(m,1H,1-H'),4.94–4.86(m,3H,Ar-CH2),4.82(d,J=8.0Hz,2H,1-H,1"-H),4.69–4.63(m,2H,Ar-CH2),4.60(d,J=11.3Hz,2H,Ar-CH2),4.51–4.47(m,3H,Ar-CH2),4.45–4.38(m,2H,4'-H,2-H),4.24–4.15(m,2H,3'-H,5'-H),4.10–4.05(m,1H,RHb-3),3.99(m,1H,3-H),3.87(m,2H,2"-H,5-H),3.74(m,2H,2'-H,3"-H),3.66(d,J=9.1Hz,1H,4-H),3.54(s,1H,Linker-OCH2),3.33(m,1H,Linker-OCH2),3.28–3.18(m,3H,4"-H,Linker-NCH2),3.11–3.03(m,1H,5"-H),2.60–2.49(m,2H,RHb-2),1.66(d,J=10.2Hz,3H,NHAc-CH3),1.51(s,4H,Linker-CH2),1.33(d,J=6.2Hz,3H,RHb-4),1.29–1.24(m,2H,Linker-CH2),1.18(d,J=6.1Hz,6H,6"-CH3,6-CH3),0.96(d,J=6.4Hz,3H,6'-CH3). 13C NMR (151MHz, Methanol-d4) δ173.1(NH-C=O), 162.4(NH-C=O), 138.8(Ar), 138.6(Ar), 138.1(Ar), 135.2(Ar), 133.3(Ar), 133.1(Ar), 128.3(Ar), 126.2(Ar), 125.7(Ar),125.5(Ar),100.2(1"-H),98.1(1'-H),97.0(1-H),80.0(3"-C),78 .5(4-C),76.4(2'-C),75.3(3-C),75.1(Ar-CH2),74.0(3'-C),72.9(Ar-CH2,R Hb-3),70.6(5"-C),70.4(Ar-CH2),67.6(4"-C),67.4(Ar-CH2),66.6(5-C,Ar- CH2),66.2(5'-C),58.0(2"-C),53.8(4'-C),50.1(2-C,Ar-CH2),46.6(Linker -NCH2)43.2(RHb-2),28.8(Linker-CH2),23.04(Linker-CH2),21.6(NHAc-CH3),18.9(RHb-4),17.2(6"-CH3),15.7(6'-CH3,6-CH3).HR-ESI-MS(m / z):calcd for C 78 H 90 Cl3N7O 15 Na+(M+Na)+:1492.5453found:1492.5469.

[0136] Example 4

[0137] Synthesis of trisaccharides 32* and 33*, 34*, 35*

[0138] As shown in Figure 5: The non-reducing azido group at the non-reducing end of the trisaccharide 31* is reduced using mild 1,3-propanedithiol to obtain an aminotrisaccharide. Subsequently, in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole, the aminotrisaccharide undergoes an amide condensation reaction with 3,5-dibenzylhexanoic acid 18* or 19*, 20*, and 21*, respectively, to obtain fully protected trisaccharides 32* to 35*. The above amide condensation method was optimized through a series of trial and error steps.

[0139] Using compound 31* as the starting material, the reduction of the azide group by triphenylphosphine was first attempted, followed by amide coupling using freshly prepared acyl chloride 22*, but only trace amounts of the target compound 32* were obtained. Subsequently, a combination of 1,3-propanedithiol and HATU was used, yielding compound 32* in 18% yield. Further optimization of the azide group reduction conditions revealed that 1,3-propanedithiol significantly improved the reduction yield in the presence of pyridine, water, and triethylamine. Further optimization of the condensing reagent led to the determination of EDC as the optimal condensing reagent. In summary, the combination of 1,3-propanedithiol and EDC significantly improved the overall yield of amide condensation. The specific optimization process and reaction conditions are shown in Table 2.

[0140] Table 2

[0141] Specific experimental procedures and steps

[0142] Compound 32*: Under nitrogen protection, trisaccharide 31* (30 mg, 20.4 μmol) was dissolved in a mixture of water (1 mL), Et3N (124.8 μl, 0.90 mmol), and pyridine (4 mL), followed by the addition of 1,3-propanedithiol (122 μl, 1.22 mmol), and stirred at room temperature for 6 hours. The reaction mixture was then concentrated to obtain crude aminotrisaccharide, which was directly used for the next reaction. 3,5-Dibenzylhexanoic acid 18* (10 mg, 30.6 μmol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 μmol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 μmol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After TLC analysis confirmed the reaction was complete, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution. The separated organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by column chromatography (DCM:MeOH = 20:1) to give compound 32* (20.5 mg, 13.67 μmol, 67%). [α] 25 D =+7.2°(c=0.36,CHCl3); IR νmax(film)2925,2857,1748,1660,1543,1496,1454,1374,1240,1081,1046,811,757,698cm -1 ; 1H NMR(600MHz,Methanol-d4)δ7.80–7.67(m,4H,Ar),7.46–7.11(m,39H,Ar),5.9(s,1H,DCA-CH),5.1(d,J=11.8Hz,2H,Ar-CH2),4.97(s,1H,1'-H),4.90(d,J=11.8Hz,1H,Ar-CH2),4.85(d,J=8.3Hz,2H,1-H,1"-H),4.75(d,J=11.3Hz,1H,Ar-CH2),4.72–4.64(m,3H,Ar-CH2),4.59(d,J=11.4Hz,2H,Ar-CH2),4.51–4.39(m,6H,Ar-CH2,4'-H,2-H),4.37–4.31(m,2H,Ar-CH2),4.28(d,J=11.6Hz,1H,Ar-CH2),4.16(dd,J=10.2,4.9Hz,2H,3'-H,5'-H),4.09–4.03(m,1H,RHb-3),4.02–3.96(m,1H,3-H),3.89(dt,J=12.3,5.4Hz,3H,dHh-3,4-H,5-H),3.77(dd,J=10.2,3.8Hz,2H,2"-H,2-H),3.69(m,1H,4"-H),3.64(m,1H,3"-H),3.58(h,J=6.0Hz,2H,dHh-5-H,Linker-OCH2),),3.40(m,1H,5"-H)),3.34(m,1H,Linker-OCH2)3.23(s,2H,Linker-NCH2),2.61–2.49(m,2H,RHb-2),2.33–2.21(m,2H,dHh-2),1.90(dt,J=13.6,6.7Hz,1H,dHh-4),1.72(d,J=9.4Hz,3H,NHAc-CH3),1.53(dt,J=14.0,5.8Hz,5H,dHh-4,Linker-CH2),1.35(d,J=6.1Hz,3H,RHb-4),1.30–1.26(m,2H,Linker-CH2),1.17(d,J=6.4Hz,3H,6-CH3),1.08(dd,J=6.1,3.5Hz,6H,6"-CH3,dHh-4),0.95(d,J=6.5Hz,3H,6'-CH3). 13C NMR(151MHz,Methanol-d4)δ=172.9(NH-C=O),172.1(NH-C=O),164.7(NH-C=O),138.8(Ar),1 38.7(Ar),138.6(Ar),138.3(Ar),138.2(Ar),135.9(Ar),133.3(Ar),133.0(Ar),128.3(Ar), 127.8(Ar),127.7–127.5(Ar),127.4(Ar),127.7(Ar),127.1(Ar),125.9(Ar),125.6 (Ar),125.4(Ar),100.7(1"-C),98.2(1'-C),97.1(1-C),78.5(3"-C),76.2(3-C),74. 9(Ar-CH2),74.8(3'-C),73.4(dHh-3),72.9(Ar-CH2,RHb-3),72.4(Ar-CH2),71.8(dHh-5),70.5(Ar-CH2,5"-C),69.8(Ar-CH2),67.5(Linker-OCH2),67.0(Ar-CH2),66.6 (Ar-CH2),66.5(5'-C),66.4(DCA-CH2),57.1(2"-C,4"-C),53.6(4'-C),50.1(2-C,A r-CH2),46.5(Linker-NCH2)43.1(RHb-2),40.9(dHh-4,dHh-2),31.4(1-C),28.8(Lin ker-CH2),27.1(Linker-CH2),23.2(1-C),22.3(Linker-CH2),21.8(NHAc-CH3),18.8(RHb-4),18.5(dHh-6),17.0(6"-C),15.7(6-C),15.7(6'-C).HR-ESI-MS(m / z):calcd for C 98 H 115 Cl2N5O 18 Na+(M+Na)+:1742.7506found:1742.7550。

[0143] Compound 33*: Under nitrogen protection, trisaccharide 31* (30 mg, 20.4 μmol) was dissolved in a mixture of water (1 mL), Et3N (124.8 μl, 0.90 mmol), and pyridine (4 mL), followed by the addition of 1,3-propanedithiol (122 μl, 1.22 mmol), and stirred at room temperature for 6 hours. The reaction mixture was then concentrated to obtain crude aminotrisaccharide, which was directly used for the next reaction. 3,5-Dibenzylhexanoic acid 19* (10 mg, 30.6 μmol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 μmol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 μmol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After TLC analysis confirmed the reaction was complete, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution. The separated organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by column chromatography (DCM:MeOH = 20:1) to give compound 33* (20.7 mg, 12.04 μmol, 59%). [α] 25 D =+11.6°(c=0.42,CHCl3); IR νmax(film)2926,2857,1749,1660,1543,1499,1454,1374,1240,1081,1046,819,757,698cm -1 ; 1H NMR(600MHz,Methanol-d4)δ7.80–7.67(m,4H,Ar),7.50–7.08(m,38H,Ar),5.95(s,1H,DCA-H),5.13(d,J=10.8Hz,2H,Ar-CH2),5.02–4.84(m,4H,1'-H,Ar-CH2,1-H,1"-H),4.75–4.64(m,4H,Ar-CH2),4.62–4.54(m,2H,Ar-CH2),4.49–4.38(m,7H,Ar-CH2,4"-H,2-H),4.25–4.12(m,4H,3'-H,5'-H,Ar-CH2),4.07–3.86(m,5H,RHb-3,dHh-3,3-H,4-H,5-H),3.83–3.74(m,2H,2"-H,2'-H),3.72(s,1H,3"-H),3.64(dtd,J=9.1,6.2,3.7Hz,2H,dHh-5,4"-H),3.61–3.49(m,1H,Linker-OCH2),3.37(d,J=33.8Hz,2H,Linker-OCH2,5"-H),3.25(d,J=27.4Hz,2H,Linker-NCH2),2.61–2.48(m,2H,RHb-2),2.36(dd,J=14.2,5.7Hz,1H,dHh-2),2.30–2.21(m,1H,dHh-2),1.77–1.69(m,3H,NHAc-CH3),1.64–1.44(m,6H,dHh-4,Linker-CH2),1.34(d,J=6.1Hz,3H,RHb-4),1.33–1.21(m,2H,Linker-CH2),1.16(dd,J=9.9,6.1Hz,3H,6-CH3),1.10(d,J=6.1Hz,3H,6"-CH3),1.06(d,J=6.1Hz,3H,dHh-6),0.95(d,J=6.4Hz,3H,6'-CH3). 13C NMR(151MHz,Methanol-d4)δ172.9(NH-C=O),172.0(NH-C=O),164.7(NH-C=O),141.8–137.4(Ar),136.3(Ar),133.1(Ar),126.0–123.8(Ar),100.7(1"-C),98.2(1) '-C),97.0(1-C),78.5(4-C,dHh-5),76.0(3-C,3"-C),74.9(4'-C),73.4(dHh-3) ,73.1(RHb-3,Ar-CH2),72.1(Ar-CH2),71.4(4"-H),70.9(Ar-CH2),70.5(5"-H),6 9.9(Ar-CH2),67.6(Ar-CH2),67.1(Ar-CH2)66.5(5'-C,5-C),56.9(2"-C),56.2(4"-C),53.6(4'-C),50.1(Ar-CH2,2-C),46.4(Linker-NCH2),43.1(RHb-2),42.9(dHh-4),41.4(dHh-2),28.8(Linker-CH2),23.2(Linker-CH2),21.8(NHAc-CH3),18.8(RHb-4,dHh-6),17.1(6"-C),15.7(6-CH3,6'-CH3,),HR-ESI-MS(m / z):calcd for C 98 H 115 Cl2N5O 18 Na+(M+Na)+:1742.7506found:1742.7547。

[0144] Compound 34*: Under nitrogen protection, trisaccharide 31* (30 mg, 20.4 μmol) was dissolved in a mixture of water (1 mL), Et3N (124.8 μl, 0.90 mmol), and pyridine (4 mL), followed by the addition of 1,3-propanedithiol (122 μl, 1.22 mmol), and stirred at room temperature for 6 hours. The reaction mixture was then concentrated to obtain crude aminotrisaccharide, which was directly used for the next reaction. 3,5-Dibenzylhexanoic acid 20* (10 mg, 30.6 μmol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 μmol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 μmol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After the reaction of the starting material was confirmed to be complete by TLC, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution. The separated organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 34* (21.4 mg, 12.4 μmol, 61%). [α] 25 D =+8.5°(c=0.3,CHCl3); IR νmax(film)2956,2857,1739,1660,1543,1499,1464,1374,1240,1081,1046,819,757,698cm -1 ; 1H NMR(600 MHz,Methanol-d4)δ7.80–7.65(m,4H,Ar),7.54–7.12(m,38H,Ar),5.95(s,1H,DCA-H),5.18–5.07(m,2H,Ar-CH2),4.97–4.84(m,4H,Ar-CH2,1'-H,1-H,1"-H),4.77–4.64(m,4H,Ar-CH2),4.61–4.55(m,2H,Ar-CH2),4.53–4.36(m,7H,Ar-CH2,4'-H,2-H),4.30(d,J=11.3Hz,1H,Ar-CH2),4.16(dd,J=11.4,6.2Hz,3H,Ar-CH2,3'-H,5'-H),4.05(ddd,J=14.4,7.1,5.5Hz,2H,RHb-3,dHh-3,),4.00(d,J=23.2Hz,1H,3-H),3.93–3.84(m,2H,4-H,5-H),3.82–3.71(m,3H,2"-H,4"-H,2'-H),3.70–3.50(m,3H,3"-H,dHh-5,Linker-OCH2),3.36(s,2H,Linker-OCH2,5"-H),3.23(s,2H,Linker-NCH2),2.62–2.46(m,2H,RHb-2),2.37–2.22(m,2H,dHh-2),1.72(d,J=7.8Hz,3H,NHAc-CH3),1.64–1.46(m,6H,dHh-4,Linker-CH2),1.35(d,J=6.2Hz,3H,RHb-4),1.28(s,2H,Linker-CH2),1.16(t,J=7.9Hz,3H,6-CH3),1.13–1.02(m,3H,6"-CH3),0.97(dd,J=25.3,6.3Hz,6H,dHh-CH3,6'-CH3). 13C NMR(151MHz,Methanol-d4)δ172.9(NH-C=O),172.2(NH-C=O),171.9(NH-C=O),164.7(NH-C=O),1 38.8(Ar),138.5(Ar),138.2(Ar),135.8(Ar),133.3(Ar),133.0(Ar),128.3(Ar),128.2(Ar),127 .9(Ar),127.3(Ar),127.1(Ar),125.8(Ar),125.6(Ar),125.4(Ar),100.6(1"-C),98.2(1'-C),97 .1(1-C),78.5(3"-C),76.2(3-C,2'-C),74.9(3'-C),73.6(Ar-CH2),72.9(RHb-3,dHh-3),71.4(A r-CH2),71.3(dHh-5,Ar-CH2),70.5(5"-C,Ar-CH2),69.8(Ar-CH2),67.6(Ar-CH2),66.6(5-C,Ar-CH2),66.4(5'-C)57.07(2"-C),55.9(4"-C),53.6(4'-C),50.1(Ar-CH2,2-C),46.6(Linker-NCH 2),43.1(RHb-2),42.4(dHh-4),41.8(dHh-2),28.7(Linker-CH2),23.1(Linker-CH2),21.8(NHAc-CH3),18.8(RHb-4),18.7(dHh-6),17.0(6"-C),15.8(6-C),15.7(6"-C).HR-ESI-MS(m / z):calcd for C 98 H 115 Cl2N5O 18 Na+(M+Na)+:1742.7506found:1742.7512。

[0145] Compound 35*: Under nitrogen protection, trisaccharide 31* (30 mg, 20.4 μmol) was dissolved in a mixture of water (1 mL), Et3N (124.8 μl, 0.90 mmol), and pyridine (4 mL), followed by the addition of 1,3-propanedithiol (122 μl, 1.22 mmol), and stirred at room temperature for 6 hours. The reaction mixture was then concentrated to obtain crude aminotrisaccharide, which was directly used for the next reaction. 3,5-Dibenzylhexanoic acid 21* (10 mg, 30.6 μmol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 μmol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 μmol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After TLC analysis confirmed the reaction was complete, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution. The separated organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by column chromatography (DCM:MeOH = 20:1) to give compound 35* (23.86 mg, 13.87 μmol, 68%). [α] 25 D =-7.8°(c=0.3,CHCl3); IR νmax(film)2989,2851,1749,1660,1545,1499,1454,1384,1240,1081,1046,819,757,698cm -1 ; 1H NMR(600MHz,Methanol-d4)δ7.77–7.66(m,4H,Ar),7.45–7.13(m,39H,Ar),5.95(s,1H,DCA-CH),5.17–5.07(m,2H,Ar-CH2),4.98–4.84(m,4H,1'-H,1-H,1"-H,Ar-CH2),4.75(d,J=11.1Hz,1H,Ar-CH2),4.71–4.64(m,3H,Ar-CH2),4.59(d,J=11.3Hz,2H,Ar-CH2),4.49(d,J=12.0Hz,3H,Ar-CH2),4.45–4.35(m,5H,Ar-CH2,4'-H,2-H),4.29(d,J=11.5Hz,1H,Ar-CH2),4.19–4.10(m,2H,3'-H,5'-H),4.06(h,J=6.4Hz,1H,RHb-3),3.98(p,J=6.3Hz,2H,dHh-3,3-H),3.88(dt,J=21.2,8.7Hz,2H,4-H,5-H),3.77(dd,J=10.3,3.9Hz,3H,2"-H,2'-H,4"-H),3.62(dd,J=28.1,7.7Hz,2H,3"-H,Linker-OCH2),3.49(p,J=6.2Hz,1H,dHh-5),3.35(m,2H,5"-H,Linker-OCH2),3.23(m,2H,Linker-NCH2),2.55(ddd,J=47.7,14.3,6.3Hz,2H,RHb-2),2.26(pd,J=15.3,14.4,8.6Hz,2H,dHh-2),1.88(dq,J=13.4,6.2Hz,1H,dHh-4),1.78–1.66(m,3H,NHAc-CH3),1.65–1.42(m,5H,Linker-CH2,dHh-4),1.35(d,J=6.1Hz,3H,RHb-4),1.28(m,2H,Linker-CH2),1.17(d,J=6.1Hz,3H,6-CH3),1.04(dd,J=13.5,6.1Hz,6H,6"-CH3,dHh-6),0.95(d,J=6.4Hz,3H,6'-CH3). 13C NMR (151MHz, Methanol-d4) δ172.9(NH-C=O), 172.3(NH-C=O), 164.8(NH-C=O), 139.6–137.7(Ar),135.9(Ar),133.1(Ar),126.2–125.1(Ar),100.6(1"-H),98 .3(1'-H),97.1(1-H),78.6(3"-C),76.2(3-C),74.9(3'-C,2'-C),73.7(dHh-3 ),72.9(RHb-3),71.7(dHh-5),70.8(5"-H,Ar-CH2),70.5(Ar-CH2),69.9(Ar-C H2),67.6(Ar-CH2),66.8(5-C)66.5(5'-C),57.1(2"-C),55.8(4"-C),53.6(4' -C),50.1(Ar-CH2,2-C),46.2(Linker-NCH2),43.1(RHb-2),41.4(dHh-2),40. 7(dHh-4),28.8(Linker-CH2),23.0(Linker-CH2)21.8(NHAc-CH3),18.8(RHb-4),18.6(dHh-6),17.0(6"-C),15.7(6-C),15.7(6'-C).HR-ESI-MS(m / z):calcd for C 98 H 115 Cl2N5O 18 Na+(M+Na)+:1742.7506found:1742.7526.

[0146] Example 5

[0147] Synthesis of compounds 1* and 2*, 3*, 4*, 5*

[0148] As shown in Figure 6, the fully protected trisaccharides 32*, 33*, 34*, and 35* were deprotected under palladium on carbon hydrogenation to synthesize four target trisaccharides 1*, 2*, 3*, and 4* with different configurations. Trisaccharide 31* was deprotected by converting its azide group to NHAc under zinc powder, acetic acid, and acetic anhydride conditions, followed by direct deprotection using palladium on carbon hydrogenation to obtain trisaccharide 5*.

[0149] The two-dimensional HSQC NMR spectra of compounds 1* and 2*, 3*, 4*, and 5* are shown in Figure 10-14.

[0150] Specific experimental procedures and steps:

[0151] Compound 1*: Trisaccharide 32* (12 mg, 6.0 μmol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v / v / v, 3 mL). Air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred under hydrogen for 24 hours. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL / min. Elution was performed for 30 minutes with ultrapure water (solvent A) containing 0.1% formic acid and acetonitrile (solvent B) in a linear gradient of 10% to 30% of solvent B to give compound 1* (4.67 mg, 5.58 μmol, 80%). 1 H NMR(600MHz, Deuterium Oxide)δ5.00(d,J=4.3Hz,1H,1'-H),4.83(d,1H,1-H),4.68(d,J=8.4Hz,1H,1"-H),4.40(d, J=4.8Hz,1H,4'-H),4.33–4.23(m,2H,2-H,5'-H),4.23–4.08(m,4H,RHb-3,dHh-3,3'-H,5-H) ,3.98(h,J=6.5Hz,1H,dHh-5),3.91(dd,J=11.0,3.1Hz,1H,3-H),3.82(d,J=3.2Hz,1H,4-H), 3.75(dd,J=10.6,4.3Hz,1H,2'-H),3.68(td,J=10.0,5.5Hz,2H,2"-H,Linker-OCH2),3.57(q ,J=7.3,6.2Hz,3H,3"-H,4"-H,5"-H),3.48(dt,J=10.1,6.4Hz,1H,Linker-OCH2),3.02–2.96 (m,2H,Linker-NCH2),2.54–2.41(m,4H,RHb-2,dHh-2),2.01(d,J=4.1Hz,6H,NHAc-CH3),1.7 8–1.55(m,6H,dHh-4,Linker-CH2),1.45(dq,J=14.9,7.4,7.0Hz,2H,Linker-CH2),1.26(d,J =6.3Hz,3H,RHb-4),1.24–1.15(m,9H,6-CH3,6"-CH3,dHh-6),1.08(d,J=6.5Hz,3H,6'-CH3). 13C NMR (151MHz,Deuterium Oxide)δ174.7(NH-C=O),174.2(NH-C=O),171.0(NH-C=O),101.7(1"-H),101.1(1'-H),97.1(1-H),76.7(3-C),76.6(3'-C),71.7(5"-C), 71.4(4-C),70.9(3"-C),67.8(2'-C,Linker-CH2),66.6(dHh-3),66.5(5-C),66.0(5'-C),65.5(dHh-5),65.2(RHb-3),57.0(4"-C),56.4 (2"-C),53.0(4'-C),48.5(2-C),44.8(RHb-2),44.5(dHh-4),43.8(dHh-2),39.4(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2) ,22.3(NHAc-CH3),22.2(Linker-CH2),22.0(RHb-4),21.7(6"-CH3),17.0(dHh-6),15.5(6'-CH3),15.3(6-CH3).HR-ESI-MS(m / z):calcd for C 37 H 67 N5O 16 Na+(M+Na)+:860.4475found:860.4478

[0152] Compound 2*: Trisaccharide 33* (17 mg, 9.89 μmol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v / v / v, 3 mL). The air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred under hydrogen for 24 hours. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL / min. Elution was performed for 30 minutes with ultrapure water (solvent A) containing 0.1% formic acid and acetonitrile (solvent B) in a linear gradient of 10% to 30% of solvent B to give compound 2* (7 mg, 8.40 μmol, 85%). 1HNMR(600MHz,Deuterium Oxide)δ5.03(d,J=4.3Hz,1H,1'-H),4.85(d,J=3.8Hz,1H,1-H),4.70(d,J=8.4Hz,1H,1"-H),4.44–4.40(m,1H,4'-H),4.32(dd,J=11.1,3.8Hz,1H,2-H),4.30–4.26(m,1H,5'-H),4.22(p,J=6.8Hz,2H,RHb-3,dHh-3),4.14(m,J=13.5,12.0,5.6Hz,2H,3'-H,5-H),4.02(q,J=6.3Hz,1H,dHh-5),3.93(dd,J=11.0,3.1Hz,1H,3-H),3.85(d,J=3.2Hz,1H,4-H),3.77(dd,J=10.6,4.2Hz,1H,2'-H),3.70(m,J=8.1Hz,2H,2"-H,Linker-OCH2),3.59(d,J=6.0Hz,3H,3"-H,4"-H,5"-H),3.51(m,J=10.3,6.2Hz,1H,Linker-OCH2),3.02(t,J=7.7Hz,2H,Linker-NCH2),2.54–2.43(m,4H,RHb-2,dHh-2),2.03(d,J=4.4Hz,6H,NHAc-CH3),1.70(td,J=15.4,7.7Hz,4H,Linker-CH2),1.63(dt,J=7.9,3.9Hz,2H,dHh-4),1.47(q,J=7.1Hz,2H,Linker-CH2),1.28(d,J=6.3Hz,3H,RHb-4),1.25(d,J=6.5Hz,3H,6-CH3),1.22(d,J=6.0Hz,6H,dHh-6,6"-CH3),1.10(d,J=6.5Hz,3H,6'-CH3). 13C NMR (151MHz,Deuterium Oxide)δ174.7(NH-C=O),174.6(NH-C=O),174.5(NH-C=O),174.2(NH-C=O),101.7(1"-C),101.1(1'-C),97.1(1-C),76.7(3-C),76.6 (3'-C),71.7(5"-C),71.4(4-C),70.9(3"-C),67.8(2'-C,Linker-OCH2),66.5(5-C),66.0(5'-C),65.5(RHb-3),65.2(dHh-3),64.3 (dHh-5),57.0(4"-C),56.4(2"-C),53.0(4'-C),48.5(2-C),44.8-44.8(RHb-2,dH h-4),44.1(dHh-2),39.4(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2), 22.7(dHh-6-CH3),22.3(Linker-CH2),22.2(NHAc-CH3),22.0(NHAc-CH3),22.02(RHb-4-CH3),17.0(6"-CH3),15.5(6'-CH3),15.3(6-CH3).HR-ESI-MS(m / z):calcd for C 37 H 67 N5O 16 Na+(M+Na)+:860.4475found:860.4490

[0153] Compound 3*: Trisaccharide 34* (9 mg, 3.1 μmol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v / v / v, 3 mL). The air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred for 24 hours under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL / min. Elution was performed for 30 minutes with ultrapure water (solvent A) containing 0.1% formic acid and acetonitrile (solvent B) in a linear gradient of 10% to 30% of solvent B, yielding compound 3* (2.2 mg, 2.64 μmol, 85%). 1H NMR(600MHz,Deuterium Oxide)δ5.01(d,J=4.2Hz,1H,1'-H),4.84(d,1H,1-H),4.69(d,J=8.6Hz,1H,1"-H),4.41(m,1H,4'-H),4.31(dd,J=11.1,3.6Hz,1H,2-H),4.27(t,J=6.8Hz,1H,5'-H),4.20(m,J=6.6Hz,2H,RHb-3,dHh-3),4.13(dq,J=14.2,7.1,5.8Hz,2H,3'-H,5-H),4.01(q,J=6.4Hz,1H,dHh-5),3.92(d,J=11.3Hz,1H,3-H),3.84(m,1H,4-H),3.76(dd,J=10.7,4.1Hz,1H,2'-H),3.70(t,J=9.5Hz,2H,2"-H,Linker-OCH2),3.62(t,J=9.9Hz,1H,4"-H),3.56(t,J=9.8Hz,2H,3"-H,5"-H),3.50(m,1H,Linker-OCH2),3.01(t,J=7.9Hz,2H,Linker-NCH2),2.53–2.38(m,4H,RHb-2,dHh-2),2.02(d,J=3.3Hz,6H,NHAc),1.74–1.59(m,6H,Linker-CH2,dHh-4),1.46(dt,J=15.0,7.5Hz,2H,Linker-CH2),1.27(m,J=6.3Hz,3H,RHb-4),1.22(m,J=15.0,6.3Hz,9H,6-CH3,6"-CH3,dHh-6),1.09(d,J=6.5Hz,3H,6'-CH3). 13C NMR (151MHz,Deuterium Oxide)δ174.6(NH-C=O),174.6(NH-C=O),174.4(NH-C=O),101.7(1"-H),101.1(1'-H),97.1(1-H),76.7(3-C),76.6(3'-C),71.8(5" -C),71.4(4-C),70.1(3"-C),67.82(2'-C,Linker-OCH2),66.5(5-C),66.0(5'-C),65.6-65.2(RHb-3,dHh-3),64.3(dHh-5),56.9(4" -C),56.4(2"-C),53.0(4'-C),48.5(2-C),45.0(dHh-4),44.8(RHb-2),44.3(dHh-2),39.3(Linker-NCH2),28.0(2-C),26.5(2-C),22 .7(dHh-6),22.3(Linker-CH2),22.2(NHAc-CH3),22.0(RHb-4),17.0(6"-CH3),15.5(6'-CH3),15.3(6-CH3).HR-ESI-MS(m / z):calcd for C 37 H 67 N5O 16 Na+(M+Na)+:860.4475found:860.4476

[0154] Compound 4*: Trisaccharide 35* (7.8 mg, 4.536 μmol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v / v / v, 3 mL). The air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred under hydrogen for 24 hours. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL / min. Elution was performed for 30 minutes with ultrapure water (solvent A) containing 0.1% formic acid and acetonitrile (solvent B) in a linear gradient of 10% to 30% of solvent B, yielding compound 4* (3.15 mg, 3.765 μmol, 83%). 1H NMR(600MHz,Deuterium Oxide)δ5.03(d,J=4.3Hz,1H,1'-H),4.86(s,1H,1-H),4.71(d,J=8.4Hz,1H,1"-H),4.43(d,J=4.7Hz,1H,4'-H),4.33(dd,J=11.2,3.8Hz,1H,2-H),4.29(d,J=6.6Hz,1H,5'-H),4.22(q,J=6.4Hz,1H,RHb-3),4.18–4.11(m,3H,dHh-3,3'-H,5-H),4.01(q,J=6.4Hz,1H,dHh-5),3.94(dd,J=11.2,3.2Hz,1H,3-H),3.85(d,J=3.2Hz,1H,4-H),3.78(dd,J=10.6,4.2Hz,1H,2'-H),3.71(q,J=8.7,8.0Hz,2H,2"-H,Linker-OCH2),3.65–3.61(m,1H,4"-H),3.60–3.54(m,2H,3"-H,5"-H),3.52(m,J=10.4,6.4,5.2Hz,1H,Linker-OCH2),3.07–2.97(m,2H,Linker-NCH2),2.55–2.49(m,3H,RHb-2,dHh-2),2.43(dd,J=14.2,8.5Hz,1H,dHh-2),2.04(d,J=3.7Hz,6H,NHAc-CH3),1.78–1.63(m,6H,dHh-4,Linker-CH2),1.47(m,J=7.2Hz,2H,Linker-CH2),1.29(d,J=6.3Hz,3H,RHb-4),1.25(d,J=6.6Hz,3H,6-CH3),1.23(d,J=6.1Hz,6H,6"-CH3,dHh-6),1.11(d,J=6.5Hz,3H,6'-CH3). 13C NMR(151MHz,Deuterium Oxide)δ174.7(NH-C=O),174.6(NH-C=O),174.6(NH-C=O),174.3(NH-C=O),101.7(1"-C),101.2(1'-C),97.1(1-C),76.7(3-C),76.6(3'-C),71.8(5"-C),71.4(4-C),71.0(3"-C),67.9(2'-C,Linker-OCH2),66.7(dHh-3),66.5(C-5),66.1(5'-C),65.5(dHh-5),65.2(RHb-3),56.9(4"-C),56.5(2"-C),53.0(4'-C),48.5(2-C),44.9(dHh-4),44.7(RHb-2),44.0(dHh-2),39.5(Linker-NCH2),28.1(Linker-CH2),26.8(Linker-CH2),22.3(Linker-CH2),22.2(NHAc-CH3),22.1(RHb-4),21.8(dHh-6),17.0(6"-CH3),15.5(6'-CH3),15.3 (6-CH3).HR-ESI-MS(m / z):calcd for C 37 H 67 N5O 16 Na+(M+H)+:838.4656found:838.4647

[0155] Compound 5*: The trisaccharide 31* (30 mg; 20.41 μmol) was dissolved in a mixed solution of tetrahydrofuran and acetic anhydride, acetic acid (3 / 2 / 1, v / v / v, 3 mL), freshly activated Zn (1 g) was added, and the mixture was stirred overnight at room temperature. After the reaction was confirmed to be complete by TLC, the reaction solution was diluted with dichloromethane and filtered. The filtrate was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution. The combined organic layers were then dried with anhydrous sodium sulfate, filtered, evaporated under vacuum, and dried under vacuum on an oil pump. The crude product was dissolved in dichloromethane and tert-butanol and water (3 / 6 / 1, v / v / v, 2 mL), and an appropriate amount of 10% palladium-carbon was added to the solution. The mixture was stirred for 36 hours under a hydrogen atmosphere (4 atm), then filtered through diatomaceous earth and washed with water, repeated three times, and the solvent was evaporated under vacuum. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL / min, eluted with ultrapure water (solvent A) containing 0.1% formic acid and acetonitrile (solvent B) in a linear gradient of 10% to 30% of solvent B for 30 min, to give compound 5* (9.9 mg, 13.27 μmol, overall yield of 65% in both steps). 1HNMR(600MHz,Deuterium Oxide)δ4.93(t,J=3.3Hz,1H,1'-H),4.76(d,J=3.3Hz,1H,1-H),4.60(dd,J=7.8,2.0Hz,1H,1"-H),4.33(d,J=4.7Hz,1H,4'-H),4.23(d,J=11.3,3.1Hz,1H,2-H),4.18(m,J=6.8Hz,1H,5-H),4.12(q,J=6.1Hz,1H,RHb-3),4.08–4.00(m,2H,3'-H,5'-H),3.83(dt,J=11.1,2.9Hz,1H,3-H),3.75(d,J=3.0Hz,1H,4-H),3.68(dt,J=10.6,3.2Hz,1H,2'-H),3.60(m,J=7.9,7.4Hz,2H,2"-H,Linker-OCH2),3.44(m,J=28.2,9.7,4.4Hz,4H,3"-H,4"-H,5"-H,Linker-OCH2),2.98–2.89(m,2H,Linker-NCH2),2.45–2.36(m,2H,RHb-2),1.94(dt,J=6.4,2.2Hz,9H,NHAc-CH3),1.66–1.54(m,4H,Linker-CH2),1.37(q,J=7.4Hz,2H,Linker-CH2),1.18(m,3H,RHb-4),1.15(m,3H,6'-CH3),1.11(m,3H,6"-CH3)1.00(m,3H,6-CH3). 13C NMR (151MHz,Deuterium Oxide)δ174.6(NH-C=O),171.0(NH-C=O),101.7(1"-H),101.1(1'-H),97.1(1'-H),76.7(3'-C,3-C),71.8(4-C ),71.4(3"-C),70.9(5"-C),67.8(2'-H,Linker-OCH2),66.5(5'-C),66.0(5-C),65.2(RHb-3)57.0(4"-C),56.3 (2"-H),44.8(RHb-2),39.3(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2),22.3(Linker-CH2),22.2( NHAc-CH3),22.1(NHAc-CH3),22.0(NHAc-CH3),16.9(6"-H),15.5(6-H),15.3(6'-CH3).HR-ESI-MS(m / z): calcd for C 33 H 59 N5O 14 Na+(M+Na)+:772.3951found:772.3968.

[0156] Example 6

[0157] The absolute configuration of dHh was elucidated by NMR analysis of four synthesized oligosaccharides, as shown in Figure 7-8.

[0158] Detailed analysis process: To minimize errors, the NMR spectra of the synthesized oligosaccharides (1*~4*) were all performed using 600M instruments and at the same temperature (25℃). The NMR spectra of the four synthesized oligosaccharides were compared with those of natural OPS to attempt to determine the possible absolute configuration of the dHh side chain. Notably, none of the four synthesized trisaccharides (1*-4*) showed the same NMR repeating unit as the naturally extracted O-antigen (OPS) trisaccharide. 1 H and NMR- 13 C data. We speculate that this phenomenon is mainly due to the difference in length between the synthetic oligosaccharide and the natural OPS, as well as the presence of the reduced-terminal amino linker and the non-reduced-terminal D-quinoline hydroxyl group at position 3 (D-Qui-C3-OH), which also increases the error. Since the RHb side chain located in the middle of the trisaccharide structure is presumably less affected by the linker and D-Qui-C3-OH, we strategically chose the RHb side chain as a control while comparing the differences in dHh. NMR- 13C-analysis showed that the four synthetic trisaccharides of RHb were correlated with the natural trisaccharides, while the NMR of dHh was... 13 The C-chemical shifts are significantly different from those of natural trisaccharides. Compared with synthetic sugars 2* and 3*, the dHh-NMR shifts of synthetic sugars 1* and 4* are significantly different. 13 The C-position chemical shifts are relatively similar to those of the natural trisaccharides, as shown in Figure 7. Preliminary analysis confirms that synthetic sugars 1 and 4* are the most likely natural configurations. Further analysis through integration and processing of the NMR spectra revealed, as shown in Figure 8, that the four isomers have NMR shifts at positions 2 and 4 of the dHh-2 column. 13 The significant difference in C indicates that the synthetic sugar 4* exhibits a good match with the native configuration. Therefore, the absolute configuration of the dHh modified group in Vibrio cholerae O100 serotype OPS is identified as 3S,5S.

[0159] Example 7

[0160] Immunological evaluation was performed using the five synthesized oligosaccharides, as shown in Figure 9.

[0161] The specific steps and methods are as follows:

[0162] Sugar chip fabrication: Five synthesized oligosaccharides (1*~5*, Figure 9A) and an unrelated synthetic sugar (6*, D-FucNAc) were dissolved in 50 mM phosphate solution (pH 8.5) to prepare 0.1 mM and 0.5 mM sugar solutions, respectively. Correspondingly, 1 mg / mL and 5 mg / mL solutions of Vibrio cholerae O100 serum-type lipopolysaccharide (LPS1) and Proteus mirabilis lipopolysaccharide (LPS2) were prepared. The chips were then printed onto the chip (9 mm long × 9 mm wide) using a chip spotting instrument according to the grid shown in Figure 9B. The chips were incubated overnight at room temperature and 65% humidity to allow the sugar fragments to covalently bind to the chip. After incubation, the chips were treated with a mixed solution of 100 nM ethanolamine and 50 nM sodium phosphate (pH = 9) at 50°C for 1 hour, followed by washing with ultrapure water.

[0163] The sugar chip was blocked with PBS solution containing 3% BSA at room temperature for 1 hour. It was then washed once with 0.1% Tween 20 PBS solution (PBST solution), followed by two washes with PBS solution. After centrifugation, the sugar chip was loaded into a 16-well incubator (ProPlate). 120 μL of rabbit serum sample diluted 1:200 in PBS solution containing 1% BSA was added to each well. The incubator was then placed in a humidified chamber at 4°C in the dark overnight to allow rabbit serum IgG antibodies that recognize the synthetic sugar fragments to bind to the synthetic oligosaccharides on the sugar chip. The sample was then removed, and the chip was washed three times with 200 μL of PBST solution to remove unbound serum IgG antibodies. Subsequently, using Cy3 fluorescently labeled goat anti-rabbit serum IgG antibody as secondary antibody, 120 μL of secondary antibody diluted 1:400 in PBS solution containing 1% BSA was added to each well. The mixture was incubated in a humidified chamber at room temperature in the dark for 45 min, and the secondary antibody solution was removed. The mixture was then washed three times with 200 μL of PBST solution to remove unbound secondary antibody. The 16-well incubator was disassembled, and the cells were washed once with ultrapure water, followed by washing with ultrapure water for 15 min to obtain the final detection chip.

[0164] Scanning with a microarray scanner revealed fluorescence signals, and the results (Figures 9C-D) showed that the IgG antibody exhibited excellent binding ability to all synthetic sugars 1*-5*. No antibody binding was observed for synthetic sugar 6* and LPS2, consistent with expectations. Compared to trisaccharide 4*, synthetic sugars 1*-3* and 5* did not show a loss of binding ability or a significant reduction in binding capacity.

[0165] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the principles and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A chemical synthesis method for oligosaccharides containing cholerae O100 serotype O antigen, characterized in that, The method uses three monosaccharide building blocks and five carboxylic acid derivatives as raw materials; The structure of the cholerae arc O100 serum type O antigen oligosaccharide is shown in formulas (1) to (5) below: Where Linker* is -(CH2) n NH2 or -(CH) n SH, where n = 1 to 25; The structures of the three monosaccharide building blocks are shown in formulas (6) to (8), and the structures of the five carboxylic acid derivatives are shown in formulas (9) to (13). PG2, PG3, PG4, PG6, and PG7 are temporary hydroxyl protecting groups, each independently selected from benzyl, 2-naphthylmethyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and triethylsilyl, respectively. PG8, PG9, PG 11 PG 12 PG 14 PG 15 PG 17 PG 18 PG 20 The hydroxyl temporary protecting group is independently selected from benzyl, 2-naphthylmethyl, acetyl, benzoyl, neopentanoyl, 9-pentanemethoxycarbonyl, and 2-p-methoxybenzyl; PG 10 PG 13 PG 16 PG 19 PG 21 Each is independently selected from one of hydroxyl, chlorine, bromine, fluorine, or C1-4 alkoxy groups; PG1 is a temporary amino protecting group, selected from trichloroacetyl, dichloroacetyl, and chloroacetyl. PG5 is an amino temporary protecting group selected from acetyl, trichloroacetyl, dichloroacetyl, chloroacetyl, trichloroethoxycarbonyl, phthaloyl, 9-fluorenylmethoxycarbonyl, and tert-butyloxycarbonyl. The linker is -(CH2). n N-Y1Y2 or -(CH) n S-Y1, where n = 1 to 25, and Y1 and Y2 are one of hydrogen, acyl, benzyl, 2-naphthylmethyl, and benzylmethoxycarbonyl; The leaving group LG1 is N-phenyltrifluoroacetylimide; The leaving group LG2 is selected from one of the following: trichloroacetylimine ester, N-phenyltrifluoroacetylimine ester, methylthio, selenylphenyl, ethylthio, phenylthio, p-toluenethio, and dibutylphosphonic acid. The synthesis method includes the following steps: (1) Construction of disaccharide receptor: The monosaccharide building block 8 was de-protected by the hydroxyl protecting group PG6 at position 3 to obtain receptor 14; receptor 14 and monosaccharide building block 7 were subjected to glycosylation reaction in a mixture of anhydrous dichloromethane and diethyl ether to obtain disaccharide 15; the azide group in disaccharide 15 was reduced to an amino group by a reducing agent, and compound 13 was added for amidation to obtain compound 16; the hydroxyl protecting group PG4 at position 3 on monosaccharide building block 7 was removed from compound 16 to obtain disaccharide receptor 17; (2) Constructing the target trisaccharide: Disaccharide acceptor 17 and monosaccharide building block 6 undergo glycosylation under the action of an activator, and the reaction temperature is gradually increased from 0°C to room temperature to obtain trisaccharide 18; then the azide group of trisaccharide 18 is reduced by a reducing agent, and any one of the carboxylic acid derivatives in formulas (9) to (12) is added for amidation to obtain compounds 19 to 22; compounds 19 to 22 are subjected to catalytic hydrogenation and deprotection to obtain target compounds 1 to 4; Alternatively, trisaccharide 18 is reduced and acylated to convert the azide group to an acetamino group, followed by catalytic hydrogenation and deprotection to give target compound 5; PG a PG b The hydroxyl temporary protecting group is independently selected from benzyl, 2-naphthylmethyl, acetyl, benzoyl, neopentanoyl, 9-pentomethoxycarbonyl, and 2-p-methoxybenzyl.

2. The chemical synthesis method according to claim 1, characterized in that, In step (1), the concentration of the glycosylation reaction is 0.01-0.1M; the glycosylation reaction is carried out using an activating reagent, which is one of TMSOTf, NIS / TMSOTf, or NIS / TfOH; the molar ratio of monosaccharide building block 7 to acceptor 14 is (1-3):1 or 1:(1-3).

3. The chemical synthesis method according to claim 1, characterized in that, In step (1), the specific conditions for the glycosylation reaction are as follows: the monosaccharide building block 7 and the acceptor 14 are dissolved in a mixed solvent of dichloromethane and diethyl ether, stirred under the protection of argon, molecular sieves are added, the reaction temperature is -20℃ to 0℃, an activating reagent of 0.1 to 0.3 molar equivalents relative to the monosaccharide building block 7 is added, and the reaction time is 2 to 8 hours.

4. The chemical synthesis method according to claim 1, characterized in that, In step (1), the reducing agent used to reduce the azide group in the disaccharide 15 is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.

5. The chemical synthesis method according to claim 1, characterized in that, In step (2), the concentration of the glycosylation reaction is 0.01-0.1M; the activator is one of TMSOTf, NIS / TMSOTf, and NIS / TfOH.

6. The chemical synthesis method according to claim 1, characterized in that, In step (2), the glycosylation reaction is carried out in a solvent, which is one or more of anhydrous dichloromethane, diethyl ether, toluene, methanol, tetrahydrofuran, acetonitrile, N,N-dimethylformamide or water; the molar ratio of the monosaccharide building block 6 and the disaccharide acceptor 17 is (1-3):1 or 1:(1-3).

7. The chemical synthesis method according to claim 1, characterized in that, In step (2), the conditions for the glycosylation reaction include: dissolving disaccharide acceptor 17 and monosaccharide building block 6 in dichloromethane solvent, adding molecular sieves, adding 0.2 to 1 molar equivalent of activating reagent relative to disaccharide acceptor 17, controlling the reaction temperature to gradually increase to room temperature at 0°C, and the reaction time to be 2 to 8 hours.

8. The chemical synthesis method according to claim 1, characterized in that, In step (2), the reducing agent is 1,3-propanedithiol; the reduction process also includes the addition of a condensing agent, which is selected from HATU and EDC.

9. The chemical synthesis method according to claim 1, characterized in that, In step (2), the catalyst used for catalytic hydrogenation can be a 10% palladium on carbon catalyst or palladium hydroxide.

10. The application of the method according to any one of claims 1-9 in the preparation of sugar chips or Vibrio cholerae glycoprotein conjugates, the application comprising the following steps: S1: Prepare a cholerae arc O100 serotype O antigen oligosaccharide fragment with a connecting arm using the method described in any one of claims 1-9; S2: Subsequently, the linker arm of the obtained oligosaccharide fragment is used to bind to the chip or carrier protein to obtain the corresponding sugar chip or Vibrio cholerae glycoprotein conjugate.

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