A specific sugar fragment for the development of Vibrio cholerae vaccine

Abstract

The present invention discloses a specific sugar fragment for the development of Vibrio cholerae vaccines, belonging to the field of medicine. The present invention uses a chemical synthesis method to synthesize sugar fragments related to the trisaccharide of the O antigen of the Vibrio cholerae serotype O100; and combined with sugar chip technology, the structure-activity relationship between different sugar fragments and their antigenicity is evaluated at the molecular level. Sugar chip screening shows that 3-hydroxybutyryl is the key structural feature of the O antigen; the non-reducing end disaccharide carrying 3-hydroxybutyryl is a potential minimal antigenic epitope. The disaccharide has a strong binding ability with antibodies and a simple structure, and can be used as a specific sugar fragment for vaccine development; the sugar conjugate vaccine designed based on this specific sugar fragment can solve the challenges of difficult cultivation of pathogens and heterogeneity of sugar antigens in naturally extracted polysaccharide vaccines. The present invention will have good application prospects in the development of Vibrio cholerae sugar conjugate vaccines, infection detection, and new drug development.

Description

Technical Field

[0001] The present invention relates to the development of a specific sugar fragment for the research and development of Vibrio cholerae vaccines, and belongs to the field of medicine. Background Art

[0002] Vibrio cholerae typically persists in aquatic environments and multiplies in the human intestine. This high-risk infection can cause watery diarrhea within hours, accompanied by dehydration, vomiting, coma, and even death.

[0003] Vibrio cholerae strains are classified into over 200 serotypes based on differences in their cell surface lipopolysaccharide (LPS) O antigens. Due to the severe drug resistance of V. cholerae and the shortage of oral vaccines, there is an urgent need for a new cholera vaccine. In recent years, glycoconjugate vaccines have shown promising results, particularly for the protection of young children. The development of a multivalent glycoconjugate vaccine covering all major cholera serotypes is considered a key technological advancement for eradicating the disease. Summary of the Invention

[0004] Technical issues:

[0005] In view of the deficiencies of the existing technology, the present invention relates to the development of a specific sugar fragment for the research and development of Vibrio cholerae vaccine.

[0006] Technical solution:

[0007] The present invention obtains sugar fragments related to the trisaccharide repeating unit of the Vibrio cholerae O100 serotype O antigen through chemical synthesis, and immobilizes the synthesized sugar fragments on the chip surface to prepare a sugar chip. The sugar chip is then incubated with antiserum to allow the IgG antibodies in the antiserum to specifically recognize the sugar fragments. The antibodies in the sugar chip are then labeled with a fluorescently labeled secondary antibody, and through fluorescence scanning and quantitative analysis, specific sugar fragments that can be used in the development of Vibrio cholerae vaccines are obtained.

[0008] The first object of the present invention is to provide a specific sugar fragment for the preparation of Vibrio cholerae vaccine, which has the structure of R2-[U1] a -[U2]-[U3] b -O-Linker, the structures of U1, U2, and U3 are as follows:

[0009]

[0010] Formula I wherein a, b, and c represent the number of U1 (D-quinosamine) and U3 (L-fucosamine), respectively, and a and b are 0 or 1, respectively; R1 is one of 3, 5-dihydroxyhexanoyl or acetyl, R2 is H (hydrogen) or H-U3- (monosaccharide) or H-U2-U3- (disaccharide) or H-U1-U2-U3- (trisaccharide); Linker represents -(CH2)n -NH2 or -(CH) n SH, n = 2–40;

[0011] In one embodiment of the present invention, the group at position 4 of the monosaccharide U2 in the specific sugar fragment is a (R)-3-hydroxybutyrylamino group or a (S)-3-hydroxybutyrylamino group.

[0012] In one embodiment of the present invention, the (R)-3-hydroxybutyryl modifying group is a key component of the specific sugar fragment.

[0013] In one embodiment of the present invention, the specific sugar fragment is further selected from:

[0014]

[0015] In one embodiment of the present invention, n=2-40; more specifically, 5 can be selected.

[0016] In one embodiment of the present invention, the sugar fragments in the sugar library are synthesized from three monosaccharide building blocks and five carboxylic acid derivatives through a series of reductive acylation and amide condensation, catalytic hydrogenation, etc.

[0017] In one embodiment of the present invention, the reducing agent used in the reductive acylation is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.

[0018] In one embodiment of the present invention, the condensing agent used in the amide condensation is one of DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), DPPA (diphenylphosphoryl azide), DPPC1 (diphenylphosphoryl chloride), DECP (diphenylphosphorocyanide), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), and HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate).

[0019] In one embodiment of the present invention, the catalyst used for the catalytic hydrogenation can be a 10% palladium-carbon catalyst or palladium hydroxide.

[0020] In one embodiment of the present invention, the solvent used for the catalytic hydrogenation can be one of a water / methanol / dichloromethane / acetic acid mixture, a water / tert-butanol / dichloromethane mixture, a water / tert-butanol / ethyl acetate mixture, and a water / tert-butanol / tetrahydrofuran mixture.

[0021] In one embodiment of the present invention, the reaction temperature used for the catalytic hydrogenation may be between 0 and 40°C.

[0022] The present invention also provides the use of the specific sugar fragment in the preparation of Vibrio cholerae vaccine.

[0023] The present invention also provides use of the specific sugar fragment in preparing a drug for preventing or treating Vibrio cholerae infection.

[0024] The present invention also provides a pharmaceutical composition comprising the above-mentioned specific sugar fragment.

[0025] The present invention also provides a pharmaceutical composition comprising any one or more combinations of the above five specific sugar fragments.

[0026] In one embodiment of the present invention, the specific sugar fragment is preferably

[0027] The present invention also provides a method for preparing the sugar chip, which comprises combining the linker structure of the specific sugar fragment with the sugar chip.

[0028] In one embodiment of the present invention, the method for preparing the sugar chip comprises the following steps:

[0029] Step 1: Dissolve the obtained sugar fragments in 50mM phosphate solution (pH=8.5), print them on the chip using a chip spotter, and incubate them overnight at room temperature and 65% humidity to allow the sugar fragments to covalently bind to the chip. After incubation, treat the chip with a mixed solution of 100nM ethanolamine and 50nM sodium phosphate (pH=9) at 50°C for 1 hour to quench the chip.

[0030] Step 2: Add the diluted rabbit antiserum to the sugar chip and incubate them together to allow the IgG antibodies in the serum to specifically bind to the sugar fragments, and wash away the unbound serum antibodies; then, use a fluorescently labeled secondary antibody (anti-IgG antibody) to bind to the IgG antibodies on the chip, incubate, and wash away the unbound secondary antibody.

[0031] Step 3: Perform fluorescence scanning on a microarray scanner, and screen sugar fragments with better antigenicity based on the scanning results, which are the specific sugar fragments.

[0032] In one embodiment of the present invention, the concentration of phosphate used to dilute the sugar fragments may be 10 mM to 100 mM, preferably 50 mM; the pH may be 8 to 9, preferably pH 8.5;

[0033] In a specific embodiment of the present invention, the concentration of the specific sugar fragment is 0.01-10 mM, preferably 0.05 mM.

[0034] In a specific embodiment of the present invention, the incubation temperature is room temperature (20-30° C.); the humidity is 50%-70%, preferably 65%.

[0035] In a specific embodiment of the present invention, the serum is diluted to a degree of 1:10 to 1:500, preferably 1:200.

[0036] In one embodiment of the present invention, the fluorescently labeled secondary antibody is a Cy3-labeled goat anti-human or goat anti-rabbit IgG antibody, and its dilution range is 1:30 to 1:1000, preferably 1:400.

[0037] The present invention also provides a Vibrio cholerae O100 infection detection device, which comprises the following components: a chip, a specific sugar fragment, a diluent for a sample to be tested, and a fluorescently labeled secondary antibody.

[0038] In one embodiment of the present invention, after the infection detection device completes the fixation and quenching of specific sugar fragments, it can be divided into a large number of small spaces using molds of different specifications, allowing for the simultaneous detection of multiple samples, which is convenient and efficient.

[0039] In a specific embodiment of the present invention, the mold can be a 16-hole, 64-hole, or 128-hole mold.

[0040] In a specific embodiment of the present invention, after the fixation and quenching of the specific sugar fragments, they can be retained for future use. When used, the diluted serum can be directly dripped onto the chip surface, which is more convenient and efficient.

[0041] The present invention also provides a Vibrio cholerae glycoprotein conjugate for vaccine development, which is formed by conjugating the linker structure of the above-mentioned specific sugar fragment to a protein.

[0042] In one embodiment of the present invention, the carrier protein used in the glycoprotein conjugate includes: diphtheria toxin non-toxic mutant protein (CRM197), hemocyanin (KLID), bovine serum albumin (BSA), meningitis outer membrane protein (OMPC), tetanus toxoid (TT) or diphtheria toxoid (DT).

[0043] Beneficial effects:

[0044] The existing saccharide conjugate vaccines based on naturally extracted polysaccharide antigens have many disadvantages, such as the difficulty in culturing some pathogens, the inability to obtain sufficient amounts of extracts, and the easy incorporation of impurities. In contrast, the saccharide conjugate vaccines based on synthetic oligosaccharide antigens of the present invention can not only avoid the incorporation of virulence factors of pathogens, but also obtain the smallest effective antigen epitope. In addition, the use of specific oligosaccharide antigens with clear structures also makes the production of saccharide vaccines more reproducible.

[0045] The present invention constructs a sugar library through chemical synthesis and combines it with sugar chip technology to provide specific sugar fragments for the development of Vibrio cholerae vaccines. The sugar chip screening results show that monosaccharides, disaccharides, and trisaccharides modified with 3-hydroxyhexanoyl groups can be specifically recognized by antisera immunized with lipopolysaccharide of Vibrio cholerae O100 serotype. Monosaccharides, disaccharides, and trisaccharides that do not contain 3-hydroxyhexanoyl modifications cannot be recognized, indicating that 3-hydroxybutyryl modification is a typical feature of the O antigen and a key modification for designing synthetic sugar conjugate vaccines for Vibrio cholerae; non-reducing end disaccharides The simple structure and good antigenicity make it a potential minimal antigenic epitope, which can be used as a specific sugar fragment in the development of Vibrio cholerae vaccines. In addition, the invention will also provide a theoretical basis for infection detection and new drug development of Vibrio cholerae. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a schematic diagram of the method involved in the present invention.

[0047] Figure 2 The structures of 11 sugar fragments in the sugar library of the present invention are shown in FIG.

[0048] Figure 3 This is the synthetic route of compound 10.

[0049] Figure 4 This is the synthetic route of compounds 5, 6, 8, 9, and 11.

[0050] Figure 5 The synthetic route of compounds 2 and 3.

[0051] Figure 6 The synthetic routes of compounds 1, 4 and 7 are shown in FIG.

[0052] Figure 7 NMR analysis of lipopolysaccharide O-antigen of Vibrio cholerae O100 serotype 1 H and 13 C spectrum.

[0053] Figure 8 ELISA was used to detect the IgG antibody titer in rabbit serum.

[0054] Figure 9The results of sugar chip screening; A is a schematic diagram of the 11 sugar fragment structures, B is a spotting pattern diagram, C is the chip scanning result, and D is a quantitative result diagram of the average fluorescence. The error bar comes from the standard deviation between two points of the same concentration. DETAILED DESCRIPTION

[0055] All commercial reagents used in the experiments were used directly without treatment. Anhydrous solvents were prepared using an MBraun MB-SPS 800 solvent drying system. Solvents used in silica gel column chromatography were analytical grade and distilled under reduced pressure. Thin-layer chromatography (TLC) used glass- or aluminum-foil-based silica gel plates prepared from 60-F254 silica gel. Normal-phase silica gel column chromatography used 200-300 mesh silica gel.

[0056] The yield of each reaction step was calculated as follows: (amount of target product / amount of starting material) × 100%. The product's structure was identified using NMR, IR, optical rotation, and high-resolution mass spectrometry. Its purity was analyzed using NMR. H, C, and 2D NMR spectra were obtained on Bruker Ascend 600M and 400M NMR instruments at 25°C. High-resolution mass spectra were obtained on an Agilent 6220 electrospray ionization-time-of-flight mass spectrometer. IR spectra were obtained on a Thermo Fisher Scientific Nicolet iS5 infrared spectrometer, and optical rotations were measured at 589 nm on a Schmidt & Haensch UniPol L10000 fully automated polarimeter. Concentrations (c) are reported in g / 100 mL.

[0057] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed scope. Although the present invention has been disclosed above with preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the principles and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0058] Example 1:

[0059]

[0060] The synthesis of compound 10 was as follows Figure 3 shown.

[0061] Compound 12 (selenoglycoside, which can be prepared by referring to the literature: Codée. et al, Organic & Biomolecular Chemistry 2020, 18 (15), 2834-2837) and N-benzyl-N-benzyloxycarbonyl-5-aminopentanol were glycosylated under the conditions of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and iodosuccinimide (NIS) to obtain compound 13; then, zinc powder, acetic acid and acetic anhydride were used for reductive acylation, and then catalytic hydrogenation was performed to obtain the deprotected target compound 10.

[0062] Specific experimental operations and steps:

[0063] Compound 13: Selenoglucosides 12 (170 mg, 0.28 mmol) and N-benzyl-N-benzyloxycarbonyl-5-aminopentanol (136 mg; 0.42 mmol) were mixed, evaporated with toluene (3×15 ml), and freshly activated Molecular sieves were dried under vacuum using an oil pump for 2 hours; then dissolved in DCM (10 ml) solution, and NIS (94 mg; 0.42 mmol) and TMSOTf (20 μl; 0.11 mmol) were slowly added at 0°C. After stirring for 4 hours, the reaction mixture was neutralized with 1 drop of Et3N at 0°C, warmed to room temperature, and filtered. The filtrate was washed with 10% aqueous Na2S2O3 solution, saturated aqueous NaHCO3 solution, and saturated brine. The combined organic layers were dried over Na2SO4, filtered, and evaporated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether::ethyl acetate = 3:1) to give compound 13 (202 mg, 0.26 mmol, 93%). [α] 25 D =+30.2°(c=0.47,CHCl3);IRνmax(film)3029,2943,2872,2109,1697,1525,1423,1361,1230,1082,821,758,698cm -1 ; 1H NMR(400MHz,Chloroform-d)δ7.88–7.79(m,4H,Ar),7.50(d,J=7.6Hz,3H,Ar),7.43–7.11(m,11H,Ar),5.18(d,J=20.4Hz,2H,Ar-CH2),5.01(d,J=10.8Hz,1H,Ar-CH2),4.88(d,J=10.1Hz,2H,1-H,Ar-CH2),4.50(s,2H,Ar-CH2),4.22(dt,J=22.3,9.7Hz,1H,3-H),3.87(s,1H,linker-OCH2),3.53–3.33(m,3H,linker-OCH2,2-H,5-H),3.32–3.10(m,3H,4-H,linker-NCH2),1.58(s,4H,linker-CH2),1.41(d,J=6.1Hz,3H,6-H),1.38–1.23(m,2H,linker-CH2). 13 C NMR(101MHz,Chloroform-d)δ162.1(NHAc-C=O)156.7(Cbz-C=O)137.9(Ar),133.2(Ar),128.6(Ar),128.5(Ar),128.4(Ar),128.0(Ar),127.95(Ar),127.8(Ar),127.7(Ar),127.3(Ar),127.2(Ar),127.1(Ar),126.2(Ar),126.0(Ar),98.6(1-H),78.4(3-H),75.2(Ar-CH2),70.7(5-H),70.0(linker-OCH2),68.9(4-C),67.2(Ar-CH2),59.6(2-C),50.3(Ar-CH2),47.2(linker-NCH2),29.2(linker-CH2),28.8(linker-CH2),23.5(linker-CH2),18.5(6-C).HR-ESI-MS(m / z):calcd for C 39 H 42 O6N5Cl3Na + (M+Na) + :804.2093found:804.210

[0064] Compound 10: Compound 13 (50 mg, 64 μmol) was dissolved in THF / Ac2O / AcOH (3 / 2 / 1, v / v / v, 3 mL) and fresh activated Zn (1 g) was added. After stirring at room temperature overnight, the mixture was diluted and filtered. The filtrate was washed with saturated NaHCO3 solution and saturated sodium chloride solution. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated in vacuo, then dried in an oil pump vacuum for 2 hours. It was then dissolved in DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added. After stirring under hydrogen (4 atm) for 36 hours, the mixture was filtered and washed with water. The residue was then purified using a Sep-Pak column C18 (Macherey-Nagel, Düren, Germany) using water and methanol as eluents to obtain compound 10 (13.8 mg, 41.6 μmol, 65% yield over two steps). 1 H NMR(600MHz, Deuterium Oxide)δ4.37(d,J=8.5Hz,1H,1-H),3.76(dt,J=11.5,6.2Hz,1H,linker-OCH2),3.58(t,J =9.0Hz,1H,2-H),3.51–3.42(m,4H,3-H,4-H,5-H,linker-OCH2),2.87(t,J=7.7Hz,2H,lin ker-NCH2),1.92(d,J=3.3Hz,6H,NHAc),1.55(m,J=7.7Hz,linker-CH2),1.48(p,J=6.7Hz ,2H,linker-CH2),1.28(dt,J=9.1,5.5Hz,2H,linker-CH2),1.11(d,J=5.0Hz,3H,6-CH3). 13 C NMR (151MHz,Deuterium Oxide)δ174.6(NHAc-C=O),174.5(NHAc-C=O),101.0(1-C),71.6,71.0,70.1(linker-OCH2),57.1,56.3(2-C),39.3(lin ker-NCH2),28.1(linker-CH2),26.4(linker-CH2),22.2(NHAc-CH3,linker-CH2),16.8(6-CH3).HR-ESI-MS(m / z):calcd forC 15 H 30 O5N3 + (M+H) + :332.2180found:332.2219.

[0065] Example 2:

[0066]

[0067] The synthesis of compounds 5, 6, 8, 9, and 11, such as Figure 4 shown.

[0068] Compound 14 (self-made by Cai Juntao, doctoral dissertation of Jiangnan University, 2020) was hydrolyzed with NIS and esterified with trifluoro-N-phenylacetimidyl chloride, and then dissolved in DCM solution in the presence of triphenylphosphine (Ph3OP) and trimethylsilyl iodide (TMSI) to obtain compound 15; 2-naphthylidene was removed by dichlorodicyanoquinone (DDQ) to obtain acceptor 16; acceptor 16 and donor selenoglycoside 12 were glycosylated to obtain disaccharide 17; disaccharide 17 and compound 16 were reductively acylated by zinc powder and acetic anhydride, respectively, and then directly deprotected to synthesize compounds 5 and 8, respectively; the azide group in compound 15 was reduced by 1,3-propanedithiol and reacted with butyric acid 18 and 19 (self-made by Tanasova.et al.) in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt), respectively. al, Angew. Chem. Int. Ed. 2015, 54(14), 4274-4278) amide condensation was performed to obtain compounds 20 and 21; compounds 20 and 21 were deprotected to obtain compounds 9 and 11 respectively; the 2-naphthylidene group in compound 20 was selectively removed to obtain the acceptor 22; the acceptor 22 was glycosylated with the donor selenoglycoside 12 to obtain the disaccharide 23, which was then deprotected to obtain compound 6.

[0069] Specific experimental operations and steps:

[0070] Compound 15: Compound 14 (200 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 stirred for 1 hour. After TLC indicated completion of the 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 in vacuo. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate 20 / 1 → 1 / 1 v / v) to provide the intermediate compound. The intermediate compound was dissolved in DCM (4.8 mL) at 0°C, followed by the addition of 2,2,2-trifluoro-N-phenylacetimidoyl chloride (171 μL, 1.14 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (171 μL, 1.14 mmol). The reaction was stirred at 0°C for 3 hours, the mixture was concentrated in vacuo, and then purified by silica gel column chromatography (petroleum ether / ethyl acetate: 20 / 1→5 / 1 v / v) to obtain trifluoroacetimidate; trifluoroacetimidate and N-benzyl-N-benzyloxycarbonyl-5-aminopentanol (187.3 mg, 0.572 mmol) were co-evaporated with toluene 3 times, and then dissolved in anhydrous DCM (1 mL), and pre-activated dry molecular sieves were added. and Ph3OP (848 mg, 3.05 mmol). TMSI (56.5 μL, 0.38 mmol, 1.0 eq) was then slowly added to the mixture. The reaction was stirred at room temperature until TLC analysis indicated completion of the reaction. The solution was diluted and quenched with saturated Na2S2O3. The organic phase was washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. It was then purified by silica gel column chromatography (petroleum ether / ethyl acetate 20 / 1 → 1 / 1 v / v) to give compound 15 (203 mg, 0.28 mmol, 73% yield over three steps). [α] 25 D =+11.1°(c=1.0,CHCl3);IRνmax(film)3029,2944,2903,2108,1697,1454,1361,1279,1227,1127,1096,1044,820,755,698cm -1 ; 1<h2 style=";text-align:left;direction:ltr">H NMR (400MHz, Chloroform-d) δ7.87–7.76 (m, 4H, Ar), 7.49 (ddd, J = 16.3, 7.5, 2.5Hz, 3H, Ar), 7.35–7.14 (m, 17H, Ar), 5.17 (d, J = 9.5Hz, 2H, Ar-CH2), 5.00 (d, J = 11.9Hz, 1H, Ar-CH2), 4.88 (d, J = 11.9Hz, 1H, Ar-CH2), 4.81 (d, J = 12.0Hz, 1H, Ar-CH2), 4.70–4.60 (m, 2H, Ar-CH2, 1-H), 4.48(d,J=8.0Hz,2H,Ar-CH2),4.05(s,1H,3-H),3.85(dd,J=9.8,3.8Hz,2H,5-H,2-H),3.71(s,1H,4-H),3.51(s,1H,Linker-OCH2),3.34( s,1H,,Linker-OCH2),3.27–3.14(m,2H,,Linker-NCH2),1.57(m,4H,Linker-CH2)1.25(m,2H,Linker-CH2),1.19(d,J=6.4Hz,3H,6-CH3).<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR(101MHz,Chloroform-d)δ138.4(Ar),135.8(Ar),133.3(Ar),133.0(Ar),128.6(Ar),128.5(Ar),128.4(Ar),1 28.2(Ar),128.0(Ar),127.9(Ar),127.8(Ar),127.77V,127.7(Ar),127.2(Ar),126.4(Ar),126.1(Ar),125.9(Ar), 125.7(Ar),97.4(1-H),78.1(3-H),76.1(2-H),73.5(Ar-CH2),73.3(Ar-CH2),68.2(Ar-CH2),67.2(Ar-CH2),65.2 (4-H),64.3(5-H),50.2(Ar-CH2)29.1(Linker-CH2),23.5(Linker-CH2),17.3(6-CH3).HR-ESI-MS(m / z):calcdfor C<h2 style=";text-align:left;direction:ltr"> 44 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 48 <h2 style=";text-align:left;direction:ltr"> O6N4Na<h2 style=";text-align:left;direction:ltr"> + <h2 style=";text-align:left;direction:ltr"> (M+Na)<h2 style=";text-align:left;direction:ltr"> + <h2 style=";text-align:left;direction:ltr"> :751.3466found:751.3501<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0071] Compound 16: To a solution of compound 15 (16.75 mg, 23.0 μmol) in DCM (2.0 mL) was added H2O (1 mL), followed by DDQ (7.7 mg, 35.0 μmol). The reaction mixture was stirred at room temperature for 5 hours, after which TLC indicated the reaction was complete. The mixture was diluted with DCM (2×10 mL) and washed with saturated NaHCO3 (20 mL). The organic layer was dried over anhydrous Na2SO4, the reaction mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether:acetone = 1:1) to give compound 16 (11.9 mg, 20.2 μmol, 88%) [α] 25 D =+30.8°(c=0.3,CHCl3);IRνmax(film)3029,2944,2903,2108,1697,1454,1422,1361,1279,1227,1127,1096,1044,820,755,698cm -1 ; 1 HNMR(400MHz,Chloroform-d)δ7.44–7.10(m,17H,Ar),5.17(d,J=10.3Hz,2H,Ar-CH2),4.74–4 .56(m,3H,Ar-CH2,1-H),4.48(d,J=7.5Hz,2H,Ar-CH2),4.17(s,1H,3-H),3.94(s,1H,5-H),3. 77–3.65(m,2H,4-H,2-H),3.53(s,1H,Linker-OCH2),3.31–3.12(m,3H,Linker-OCH2,Linker- NCH2),2.52(s,1H,3-OH),1.51(m,4H,Linker-CH2)1.23(d,J=6.5Hz,5H,Linker-CH2,6-CH3). 13C NMR(101MHz,Chloroform-d)δ137.9(Ar),137.8(Ar),128.6(Ar),128.6(Ar),128.5(Ar),12 8.2(Ar),128.1(Ar),128.0(Ar),127.8(Ar),127.3(Ar),96.5(1-C),77.0(2-C),72.7(Ar-CH 2),69.9(3-H),68.2(Ar-CH2),67.2(Ar-CH2),65.9(4-C),64.6(5-C),50.3(Ar-CH2),47.1(L inker-OCH2),29.2(Linker-CH2),23.5(Linker-CH2),17.3(6-CH3).HR-ESI-MS(m / z):calcd for C 33 H 40 O6N4Na + (M+Na) + :611.2840found:611.2895.

[0072] Compound 17: Selenoglucosides 12 (49 mg, 0.08 mmol) and acceptor 16 (56.5 mg, 0.10 mmol) were mixed, azeotropically dehydrated with toluene (3×5 mL), and dried under vacuum using an oil pump for 2 hours. The mixed donor-acceptor was dissolved in DCM (8 mL) at 0°C and freshly activated Molecular sieves, then slowly add NIS (27 mg; 0.12 mmol) and TfOH (14.2 μL; 0.16 mmol). After stirring for 4 hours, add a drop of triethylamine (Et3N) to the reaction system to neutralize it, filter The filtrate was washed with a 10% Na2S2O3 solution and a saturated NaHCO3 solution. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to yield compound 17 (63.6 mg, 0.061 mmol, 76%). [α] 25 D =+43.7°(c=0.6,CHCl3);IRνmax(film)3032,2947,2903,2108,1678,1454,1422,1361,1279,1226,1127,1096,1044,820,755,698cm -1 ; 1H NMR(600MHz,Chloroform-d)δ7.81(dd,J=10.7,8.3Hz,4H,Ar),7.47(dd,J=8.7,5.0Hz,3H,Ar),7.40–7.13(m,17H,Ar),6.99(d,J=7.8Hz,1H,NHAc-H),5.23(d,J=8.2Hz,1H,1'-H),5.18(d,J=18.2Hz,2H,Ar-CH2),4.97(d,J=10.8Hz,1H,Ar-CH2),4.86(d,J=10.7Hz,1H,Ar-CH2),4.77(d,J=12.2Hz,1H,Ar-CH2),4.53–4.38(m,4H,1-H,Ar-CH2),4.23(m,1H,3-H),4.09(m,1H,3'-H),3.91(m,1H,5-H),3.81(d,J=7.9Hz,1H,4-H),3.74(dd,J=10.2,3.7Hz,1H,2-H),3.58(dt,J=18.0,9.0Hz,1H,2'-H),3.41(d,J=23.7Hz,2H,5'-H,Linker-OCH2),3.30–3.10(m,4H,4'-H Linker-OCH2,Linker-NCH2),1.51(dt,J=27.9,6.9Hz,4H,Linker-CH2),1.39(d,J=5.6Hz,3H,6'-H),1.26(d,J=10.0Hz,2H,Linker-CH2),1.17(d,J=6.2Hz,3H,6-CH3). 13CNMR(151MHz,Chloroform-d)δ161.7(NH-C=O),138.5(Ar),137.9(Ar),134.7(Ar),133.3(Ar),133.1(Ar),128.6(Ar),128.5(Ar),128.3(Ar),128.8(Ar),128.8(Ar). ,127.97(Ar),127.94(Ar),127.8(Ar),127.7(Ar),127.4(Ar),127.2(Ar),127.0(Ar),126.1(Ar),126.1(Ar),125.9(Ar),99.1(1'-H),97.0(H),97.7(H),6.8(H). 3'-C),76.4,76.3(3-C,2-C),75.1(Ar-CH2),73.3(Ar-CH2),70.8(5'-H),68.7(4'-H),68.2(Linker-OCH2),67.2(Ar-CH2),66.5(4-C),63.9(C),63.9(9). 2'-H),50.3(Ar-CH2),46.1(Linker-NCH2),29.1(Linker-CH2),27.5(Linker-CH2),23.4(Linker-CH2),18.5(6'-C),17.2(6-C).HR-ESI-MS(m / z):calcd: forC 52 H 57 O9N8Cl3Na + (M+Na) + :1065.3206found:1065.3227.

[0073] Compound 5: Compound 17 (40 mg, 38.38 μmol) was dissolved in a mixture of THF / Ac2O / AcOH (3 / 2 / 1, v / v / v, 3 mL) and freshly activated Zn (1 g) was added. After stirring at room temperature overnight, the mixture was diluted with DCM and filtered. The filtrate was washed with saturated aqueous NaHCO3 and saturated brine; the combined organic layers were dried over anhydrous Na2SO4, filtered, evaporated in vacuo, and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to obtain the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added to the solution. The mixture was stirred under hydrogen (4 atm) for 36 hours, filtered, washed with water, and concentrated. The crude product was purified by Sep-Pak C18 column (Macherey-Nagel, Düren, Germany) using water and methanol as eluents to give compound 5 (11.9 mg, 23.03 μmol, 60% yield over two steps). 1 H NMR(600MHz, Deuterium Oxide)δ4.84(d,J=4.2Hz,1H,1-H),4.65(d,J=8.5Hz,1H,1'-H),4.31(d,J=4.7Hz,1H,4-H),4.13(q,J=6.6Hz,1H,5-H),3.98(dd,J =10.6,4.7Hz,1H,3-H),3.80(dd,J=10.6,4.0Hz,1H,2-H),3.63(q,J=7.8,6.9Hz,2H,2',Linker-OCH2),3.57–3.45(m,4H,3'-H,4'- H,5'-H,Linker-OCH2),2.96(t,J=7.7Hz,2H,Linker-NCH2),2.03(s,3H,NHAc-CH3),2.00–1.96(m,6H,NHAc-CH3),1.64(tq,J=14. 1,6.9,6.0Hz,4H,Linker-CH2),1.41(dh,J=14.1,6.8Hz,2H,Linker-CH2),1.15(d,J=5.6Hz,3H,6'-H),1.04(d,J=6.5Hz,3H,6-H). 13C NMR (151MHz,Deuterium Oxide)δ174.8(NH-C=O),174.6(NH-C=O),101.0(1'-C),98.2(1-C),77.0(3-C),71.5-71.1(3' -C,5'-C),68.1(Linker-OCH2),67.1(2-C),65.5(5-C),57.1(4'-H),56.5(2'-H),52.5(4-H), 39.4(Linker-NCH2),28.1(Linker-CH2),26.5(Linker-CH2),22.3(NHAc-CH3),22.2(Linker- CH2),22.1(NHAc-CH3),21.9(NHAc-CH3),16.9(6'-CH3),15.4(6-CH3).HR-ESI-MS(m / z): calcd for C 23 H 43 O9N4 + (M+H) + :519.3025found:519.3052.

[0074] Compound 8: Compound 16 (30 mg, 51.0 μmol) was dissolved in a mixture of THF / Ac2O / AcOH (3 / 2 / 1, v / v / v, 3 mL) and freshly activated Zn (1 g) was added. After stirring at room temperature overnight, the mixture was diluted with DCM and filtered. The filtrate was washed with saturated aqueous NaHCO3 and saturated brine; the combined organic layers were dried over anhydrous Na2SO4, filtered, evaporated in vacuo, and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to obtain the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added to the solution. The mixture was stirred under hydrogen (4 atm) for 36 hours, filtered, washed with water, and concentrated. The crude product was purified by Sep-Pak column C18 (Macherey-Nagel, Düren, Germany) using water and methanol as eluents to give compound 8 (7.5 mg, 26.0 μmol, 51% yield over two steps). 1H NMR(600MHz,Deuterium Oxide)δ4.87–4.83(m,1H,1-H),4.19(d,J=4.6Hz,1H,4-H),4.16(q,J=6.6Hz,1H,5-H),3.95(ddd,J=10.6,4.9,1.8Hz,1H,3-H),3.67–3.60(m,2H,2-H,Linker-OCH2),3.46(m,J=11.4,7.7,3.7Hz,1H,Linker-OCH2),2.95(t,J=7.7Hz,2H,Linker-NCH2),2.04(s,3H,NHAc-CH3),1.62(dp,J=20.4,6.9Hz,4H,Linker-CH2),1.48–1.33(m,2H,Linker-CH2),1.05(dd,J=6.6,1.9Hz,3H,6-CH3). 13 C NMR(151MHz,Deuterium Oxide)δ175.6(NH-C=O),98.3(1-H),68.7(2-H),68.4(3-H),68.1(Linker-OCH2),65.3(5-H),54.0(4-H),39.4(Linker-NCH2),28.1(Linker-CH2),26.5(Linker-CH2),22.4(Linker-CH2),21.9(NHAc-CH3),15.6(6-CH3).HR-ESI-MS(m / z):calcd for C 13 H 27 O5N2 + (M+H) + :291.1914found:291.1952.

[0075] Compound 20: Under nitrogen, compound 15 (180 mg, 0.25 mmol) was dissolved in pyridine (8 mL). Water (2 mL), Et3N (1.51 mL, 10.87 mmol), and 1,3-propanedithiol (1.48 mL, 14.8 mmol) were then added to the reaction system and stirred at room temperature for 6 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (DCM:MeOH = 20:1, v / v) to obtain the aminosugar. Sodium bicarbonate (62 mg, 0.74 mmol) was then added to a solution of (R)-3-O-benzylbutyric acid 18 (96 mg, 0.49 mmol) and the aminosugar in acetonitrile (20 mL), and stirred at room temperature. After 10 minutes, HOBt (6.7 mg, 49.4 μmol) and EDC-HCl (57 mg, 0.30 mmol) were added sequentially, and the mixture was stirred at the same temperature for 6 hours. After the reaction was completed 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 Na2SO4, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound 20 (188.9 mg, 0.22 mmol, yield 87%). 25 D =+114.5°(c=0.3,CHCl3);IRνmax(film)3029,2938,2109,1697,1539,1454,1361,1217,1102,1044,819,756,698cm -1 ; 1H NMR(400MHz,Chloroform-d)δ7.88–7.73(m,4H,Ar-H),7.55–7.14(m,24H,Ar-H),6.73(d,J=10.3Hz,1H,NHAc-H),5.20(d,J=7.9.9). Hz,2H,Ar-CH2),4.99(d,J=11.3Hz,1H,Ar-CH2),4.66(td,J=13.8,12.6,3.0Hz,3H,Ar-CH2,4-H),4.59–4.42(m,6H,Ar-CH2,1-H ),4.09–3.93(m,3H,5-H,3-H,RHb-3),3.63–3.47(m,1H,Linker-OCH2),3.42–3.14(m,4H,Linker-OCH2,Linker-NCH2,2-H),2.6 6–2.47(m,2H,RHb-2),1.65–1.50(m,4H,Linker-CH2),1.27(d,J=6.3Hz,5H,Linker-CH2,RHb-4),1.13(d,J=6.4Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ171.6(NH-C=O),138.7(Ar),138.1(Ar),137.9(Ar),136.8(Ar),136.1(Ar),133.3(Ar),132.9(Ar),128.8(Ar),128.5(Ar),128.8(Ar),128.8(Ar). r),128.3(Ar),128.0(Ar),127.9(Ar),127.8(Ar),127.8(Ar),127.8(Ar),127.7(Ar),127.6(Ar),127.6(Ar),127.5(Ar),127.3(Ar),127.2(Ar),127.6(Ar),127.6(Ar),127.6(Ar). 3(Ar),125.8(Ar),125.6(Ar),97.4(1-H),75.9(2-C),73.0(Ar-CH2),72.7,71.5(Ar-CH2),70.7(Ar-CH2),68.2(Linker-OCH2),67.2(Ar-CH2),5.4(4),5(4). Ar-CH2,4-H),47.2(Linker-NCH2),43.7(RHb-2),29.1(Linker-CH2),27.6(Linker-CH2),23.5(Linker-CH2),18.9(RHb-4),16.8(6-CH3).HR-ESI-MS(mcal / cd): for C55 H 63 O8N2 + (M+H) + :879.4579found:879.4635

[0076] Compound 21: Under nitrogen, compound 15 (150 mg, 0.21 mmol) was dissolved in pyridine (8 mL). Water (2 mL), Et3N (1.26 mL, 9.06 mmol), and 1,3-propanedithiol (1.24 mL, 12.36 mmol) were then added to the reaction system and stirred at room temperature for 6 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (DCM:MeOH = 20:1, v / v) to obtain the aminosugar. Sodium bicarbonate (52 mg, 0.62 mmol) was then added to a solution of (S)-3-O-benzylbutyric acid 19 (80 mg, 0.41 mmol) and the aminosugar in acetonitrile (17 mL), and stirred at room temperature. After 10 minutes, HOBt (5.6 mg, 41.2 μmol) and EDC (64 mg, 0.33 mmol) were added sequentially, and the mixture was stirred at the same temperature for 6 hours. After the reaction was completed 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 Na2SO4, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound 21 (150.2 mg, 0.17 mmol, 83%). 25 D =+80.1°(c=0.75,CHCl3);IRνmax(film)3029,2942,2868,2109,1697,1532,1454,1361,1217,1104,1045,818,755,698cm -1 ; 1HNMR(400MHz,Chloroform-d)δ7.85–7.70(m,4HAr-H),7.54–7.13(m,24HAr-H),6.59(d,J=10.1Hz,1H,NHAc-H),5.17(d, J=8.5Hz,2H,Ar-CH2),4.95(d,J=11.3Hz,1H,Ar-CH2),4.61(m,4H,4-H.1-H.Ar-CH2),4.43(m,5H,Ar-CH2),4.10–3.84(m, 3H,5-H,3-H,RHb-3),3.54(d,J=9.4Hz,1H,Linker-OCH2),3.40–3.13(m,4H,Linker-OCH2,Linker-NCH2,2-H),2.54(m,J =5.4,2.1Hz,2H,RHb-2),1.54(s,4H,Linker-CH2),1.37–1.19(m,5H,Linker-CH2,,RHb-4),1.15(d,J=6.4Hz,3H,6-CH3). 13 C NMR(101MHz,Chloroform-d)δ171.7(NH-C=O),138.7(Ar),138.2(Ar),137.9(Ar),136.1(Ar),133.3(Ar),132.9(Ar),128.5(Ar),128.4(Ar),128.8(Ar),128.3(Ar),128.8(Ar),138.3(Ar). 128.2(Ar),128.0(Ar),127.9(Ar),127.88(Ar),127.82(Ar),127.6(Ar),127.5(Ar),127.45(Ar),127.3(Ar),127.2(Ar),126.6(Ar),126.3(Ar),126.3(Ar),126.5(Ar),126.5(Ar),126.5(Ar),126.5(Ar). r),125.7(Ar),97.3(1-C),76.0(3-C)75.98(2-C),72.8(RHb-3),72.5(Ar-CH2),71.4(Ar-CH2),70.4(Ar-CH2),68.2(Linker-OCH2),67.1(CH-2),64(Ar-CH2). 5-C),50.2(4-C,Ar-CH2),46.2(Linker-NCH2),43.7(RHb-2),29.1(Linker-CH2),23.5(Linker-CH2),19.7(RHb-4),16.9(6-CH3).HR-ESI-MS(m / zd):cal. forC 55 H 62 O8N2Na + (M+Na)+ :901.4398found:901.4446.

[0077] Compound 9: Compound 20 (30 mg, 34.15 μmol) was dissolved in a DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v) mixture, and then 10% Pd / C (50 mg) was added. After stirring for 36 hours under a hydrogen (4 atm) atmosphere, the mixture was filtered and washed with water. The residue was then purified using a Sep-Pak C18 column (Macherey-Nagel, Germany) using water and methanol as eluents to obtain product 9 (10.2 mg, 30.4 μmol, 89%). 1 H NMR(400MHz, Deuterium Oxide)δ4.79(d,J=3.9Hz,1H,1-H),4.12(m,J=20.1,13.5,5.7Hz,3H,4-H,5-H,RHb-3),3.90(dd,J=10.5,4. 3Hz,1H,3-H),3.58(m,J=8.9,5.8,4.9Hz,2H,2-H,Linker-OCH2),3.40(dt,J=10.6,6.2Hz,1H,Linker-OCH2 ),2.89(t,J=7.6Hz,2H,Linker-NCH2),2.46–2.36(m,2H,RHb-2),1.57(dq,J=12.6,6.7,5.5Hz,4H,Linker- CH2),1.33(dp,J=13.5,6.6Hz,2H,Linker-CH2),1.13(d,J=6.1Hz,3H,RHb-4),1.00(d,J=6.3Hz,3H,6-CH3). 13 C NMR(101MHz,DeuteriumOxide)δ98.3(1-H),68.6(2-H),68.4(3-H),68.0(Linker-OCH2),65.0(5-C,RHb-3),53.8(4-C),44.7(RHb-2), 39.3(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2),22.4(Linker-CH2),22.1(RHb-4),15.5(6-CH3).HR-ESI-MS(m / z):calcd for C 15 H 31 O6N2 + (M+H) + :335.2177found:335.2219.

[0078] Compound 11: Compound 21 (30 mg, 34.15 μmol) was dissolved in a mixed solution of DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added. After stirring for 36 hours under hydrogen (4 atm), the mixture was filtered and washed with water. The residue was purified with Sep-Pak column C18 (Macherey-Nagel, Germany) using water and methanol as eluents to afford compound 11 (10.4 mg, 31.08 μmol, 91%). 1 H NMR(400MHz, Deuterium Oxide)δ4.92(d,J=4.0Hz,1H,1-H),4.26(m,J=19.2,5.4Hz,3H,4-H,5-H,RHb-3),4.02(dd,J=10 .5,4.5Hz,1H,3-H),3.78–3.65(m,2H,2-H,Linker-OCH2),3.53(dt,J=9.9,6.2Hz,1H,Linker-OC H2),3.02(t,J=7.6Hz,2H,Linker-NCH2),2.61–2.48(m,2H,RHb-2),1.76–1.65(m,4H,Linker-C H2),1.56–1.39(m,2H,Linker-CH2),1.25(d,J=6.2Hz,3H,RHb-4),1.13(d,J=6.4Hz,3H,6-CH3). 13 C NMR (101MHz,Deuterium Oxide)δ175.3(NH-C=O),98.3(1-C),68.6(3-C),68.4(2-C),68.0(Linker-OCH2),65.2-65.0(5-C,RHb-3),53.9(4-C),44.5(RHb-2), 39.3(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2),22.4(Linker-CH2),21.9(RHb-4),15.6(6-CH3).HR-ESI-MS(m / z):calcd for C 15 H 31 O6N2 + (M+H) + :335.2177found:335.2214.

[0079] Compound 22: To a solution of compound 20 (150 mg, 0.17 mmol) in DCM (2.0 mL) was added H2O (1 mL) followed by DDQ (58 mg, 0.26 mmol). The reaction mixture was stirred at room temperature for 5 hours, after which TLC indicated the reaction was complete. The mixture was diluted with DCM (2 x 10 mL) and washed with saturated NaHCO3 (20 mL). The organic layer was dried over anhydrous Na2SO4, the reaction mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether:acetone = 1:1) to give compound 22 (116.7 mg, 0.16 mmol, 93% yield). [α] 25 D =+33°(c=1.2,CHCl3);IRνmax(film)3030,2923,1697,1540,1453,1361,1216,1100,1037,819,736,697cm -1 ; 1 H NMR(600MHz,Chloroform-d)δ7.39–7.13(m,20H,Ar-H),6.72(d,J=9.4Hz,1H,NH-H),5.17(d,J=16.9Hz,2H,Ar-CH2),4.59(d,J=1 1.3Hz,1H,Ar-CH2),4.55–4.42(m,6H,Ar-CH2,1-H),4.30(d,J=8.1Hz,1H,4-H),4.14–4.07(m,1H,3-H),4.03(d,J=12.2Hz,1H,5- H),3.97(qd,J=6.4,3.8Hz,1H,RHb-3),3.52(d,J=25.7Hz,1H,linker-OCH2),3.30–3.14(m,4H,2-H,linker-OCH2,linker-NCH2) ,2.61–2.44(m,2H,RHb-2),1.59–1.47(m,4H,linker-CH2),1.36–1.25(m,5H,RHb-4,linker-CH2),1.06(d,J=6.4Hz,3H,6-CH3). 13C NMR(151MHz,Chloroform-d)δ172.9(NH-C=O),156.8(Cbz-C=O),156.2(Cbz-C= O),138.3(Ar),138.0(Ar),137.9(Ar),128.6(Ar),128.5(Ar),128.45(Ar),128 .4(Ar),128.37(Ar),127.9(Ar),127.8(Ar),127.77(Ar),127.75(Ar),127.7(A r),127.72(Ar),127.3(Ar),127.2(Ar),97.0(1-C),77.1(2-C)72.5(RHb-3),72 .4(Ar-CH2),70.7(Ar-CH2),69.8(3-C),68.2(linker-OCH2),67.2(linker-OCH2),64.2(5-C),53.7(4-C),50.3(Ar-CH2),47.2(linker-NCH2),46.2(linker- NCH2),43.5(RHb-2-CH2),29.2(linker-CH2),28.0(linker-CH2),27.5(linker-CH2),23.5(linker-CH2),19.0(RHb-4),16.7(6-CH3).HR-ESI-MS(m / z): calcd for C 44 H 54 O8N2Na + (M+Na) + :761.3772found:761.3807.

[0080] Compound 23: Selenoglucosides 12 (156 mg, 0.26 mmol) and acceptor 22 (94.3 mg, 0.13 mmol) were mixed, azeotropically dehydrated with toluene (3×5 mL), and dried under vacuum using an oil pump for 2 hours. The mixed donor-acceptor was dissolved in DCM (8 mL) at 0°C and freshly activated Molecular sieves, then slowly add NIS (57.4 mg; 0.26 mmol) and TfOH (22.6 ul; 0.26 mmol). After stirring for 4 hours, add a drop of triethylamine (Et3N) to the reaction system to neutralize it, filter The filtrate was washed with a 10% Na2S2O3 solution and a saturated NaHCO3 solution. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain compound 23 (109.7 mg, 0.09 mmol, 72% yield). [α] 25 D =+60.9°(c=1.1,CHCl3);IRνmax(film)3029,2938,2870,2107,1681,1525,1454,1361,1217,1093,1042,819,755,698cm -1 ; 1 HNMR(400MHz,Chloroform-d)δ7.85–7.73(m,4H),7.51–7.12(m,27H,Ar),6.73(d,J=10.1Hz,1H,N HAc), 6.64 (d, J = 7.7Hz, 1H, NHAc), 5.17 (m, 2H, Ar-CH2), 4.96 (d, J = 7.5Hz, 1H, 1'-H), 4.90 (d, J = 10. 8Hz,1H,Ar-CH2),4.78(d,J=10.9Hz,1H,Ar-CH2),4.60(d,J=11.1Hz,1H,Ar-CH2),4.47(m,2H,Ar- CH2),4.43(m,2H,1-H,4-H),4.35(d,J=11.9Hz,1H,Ar-CH2),4.24(d,J=12.0Hz,1H,Ar-CH2),4.09( dd,J=10.2,4.6Hz,1H,2-H),3.97(td,J=6.6,3.6Hz,2H,RHb-3,5-H),3.72(m,2H,2'-H,4'-H),3.4 9(s,1H,Linker-OCH2),3.26(m,3H,,Linker-OCH2,3'-H,5'-H),3.17(m,3H,3-H,Linker-NCH2),2. 57(dd,J=15.3,3.6Hz,1H,RHb-2),2.47(dd,J=15.3,7.0Hz,1H,RHb-2),1.52(s,4H,Linker-CH2),1 .35(dd,J=9.9,5.9Hz,6H,6'-CH3,RHb-4),1.27(s,2H,Linker-CH2),1.06(d,J=6.4Hz,3H,6-CH3). 13C NMR(101MHz,Chloroform-d)δ171.5(NH-C=O),161.4(NH-C=O),138.4(Ar),138.1(Ar),137. 9(Ar),136.7(Ar),134.8(Ar),133.3(Ar),133.1(Ar),128.6(Ar),128.5(Ar),128.5(Ar),12 8.4(Ar),128.2(Ar),128.0(Ar),127.97(Ar),127.9(Ar),127.8(Ar),127.7(Ar),127.66(Ar ),127.3(Ar),127.2(Ar),127.0(Ar),126.1(Ar),126.0(Ar),99.3(1'-H),96.4(1-H),92.6, 79.8(4'-C),77.2(3-C),74.4(Ar-CH2),73.7(2-C),72.9(5-C),72.1(Ar-CH2),70.8(5'-C), 70.7(Ar-CH2),68.1(Ar-CH2),68.0(3'-C),67.2(Ar-CH2),65.0(RHb-3),58.4(2'-H),52.7( 4-H),50.3(Ar-CH2),47.3(Linker-NCH2),43.3(RHb-2),29.1(Linker-CH2),27.6(Linker-C H2),23.4(Linker-CH2),19.3(RHb-4),18.5(6'-CH3),16.5(6-CH3).HR-ESI-MS(m / z):calcd for C 63 H 71 THEIR 11 N6Cl3Na + (M+Na) + :1215.4139found:1215.4149.

[0081] Compound 6: Compound 23 (50 mg, 41.9 μmol) was dissolved in a mixture of THF / Ac2O / AcOH (3 / 2 / 1, v / v / v, 3 mL) and freshly activated Zn (1 g) was added. After stirring at room temperature overnight, the mixture was diluted with DCM and filtered. The filtrate was washed with saturated aqueous NaHCO3 and saturated brine; the combined organic layers were dried over anhydrous Na2SO4, filtered, evaporated in vacuo, and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to obtain the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added to the solution. The mixture was stirred under hydrogen (4 atm) for 36 hours, filtered, washed with water, and concentrated. The crude product was purified by Sep-Pak C18 column (Macherey-Nagel, Düren, Germany) using water and methanol as eluents to give compound 6 (12 mg, 21.4 μmol, 51% yield over two steps). 1 H NMR(400MHz, Deuterium Oxide)δ4.70(d,1H,1-H),4.60(d,J=8.2Hz,1H,1'-H),4.27(d,J=4.7Hz,1H,4-H),4.08(p,J=6.5,6.0Hz,2H,5-H,RHb-3),3.93(dd,J=10.4, 4.7Hz,1H,3-H),3.72(dd,J=10.4,3.9Hz,1H,2-H),3.57(dt,J=9.6,6.5Hz,2H,2'-H,Linker-OCH2),3.53–3.48(m,2H,3'-H,Linker-OCH2,4 '-H,5'-H),2.90(t,J=7.6Hz,2H,Linker-NCH2),2.38(h,J=8.7,8.1Hz,2H,RHb-2),1.82(m,6H,NHAc-CH3),1.58(m,J=9.1,8.5Hz,4H,Linke r-CH2),1.36(m,J=7.5,7.1Hz,2H,Linker-CH2),1.15(d,J=6.1Hz,3H,RHb-4),1.08(d,J=4.5Hz,3H,6'-CH3),0.98(d,J=6.4Hz,3H,6-CH3). 13C NMR (101MHz,Deuterium Oxide)δ174.6(NH-C=O),101.2(1'-C),98.1(1-C),76.8(5-C),71.7-70.9(3'-C,5'-C),68. 0(Linker-OCH2),67.6(2-C),65.3-65.1(5-C,RHb-3),57.0(4'-C),56.4(2'-C),52.7(4-C), 44.8(RHb-2),39.3(Linker-NCH2),28.0(Linker-CH2),26.4(Linker-CH2),22.3(Linker-CH 2),22.1(NHAc),22.1(NHAc),22.0(RHb-4),16.9(6'-C),15.4(6-C).HR-ESI-MS(m / z):calcd for C 25 H 47 O 10 N4 + (M+H) + :563.3287found:563.3349.

[0082] Example 3:

[0083]

[0084] The synthesis of compounds 2 and 3, such as Figure 5 As shown:

[0085] Compound 24 was synthesized using a known method (Cai Juntao, doctoral dissertation, Jiangnan University, 2020). Compound 24 selectively removed the 2-naphthylidene group under the action of DDQ to obtain the disaccharide acceptor 25; the donor selenoglycoside 12 and the acceptor 25 were catalyzed by trimethylsilyl trifluoromethanesulfonate and iodosuccinimide to obtain the trisaccharide 26; then, zinc powder, acetic acid, and acetic anhydride were used to reductively acylate and catalytically hydrogenate compounds 2 and 3, respectively, to obtain the deprotected target compounds 2 and 3.

[0086] Specific experimental operations and steps

[0087] Compound 25: To a solution of compound 24 (380 mg, 0.38 mmol) in DCM (10 mL) was added H2O (2 mL), followed by DDQ (126 mg, 0.57 mmol). The reaction mixture was stirred at room temperature for 5 hours, after which TLC indicated the reaction was complete. The mixture was diluted with DCM (2 x 10 mL) and washed with saturated NaHCO3 (20 mL). The organic layer was dried over anhydrous Na2SO4, the reaction mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to give compound 25 (213 mg, 0.25 mmol, 65% yield). [α] 25 D =-16.2°(c=0.8,CHCl3);IRνmax(film)3029,2939,2876,2108,1697,1540,1454,1361,1228,1089,1045,830,755,699cm -1 ; 1 HNMR(600MHz,Chloroform-d)δ7.46–7.09(m,24H,Ar),6.36(s,1H,NHAc-H),5. 18–5.13(m,2H,Ar-CH2),5.04(d,J=3.5Hz,1H,1'-H),4.96(s,1H,1-H),4.84(d ,J=11.8Hz,1H,Ar-CH2),4.70(s,2H,Ar-CH2),4.64(d,J=11.7Hz,1H,Ar-CH2), 4.47(d,J=9.3Hz,2H,Ar-CH2),4.39(s,1H,2'-H),4.19–4.11(m,2H,3-H,5-H), 3.82(m,2H,3'-H,5'-H),3.77(dd,J=9.8,3.3Hz,1H,2-H),3.71(dd,J=3.8,1.5 Hz,1H,4-H),3.57(m,2H,4'-H,Linker-OCH2),3.38(m,1H,Linker-OCH2),3.19 (m,2H,Linker-NCH2),1.62(s,3H,NHAc-CH3),1.55(s,4H,Linker-CH2),1.34–1.24(m,2H,Linker-CH2),1.15(d,J=6.4Hz,3H,6-H),1.13–1.08(m,3H,6'-H). 13CNMR(101MHz,Chloroform-d)δ170.5(NH-C=O),138.5(Ar),137.8(Ar),137.0(Ar),128.9(Ar),128.8(Ar),128.76(Ar), 128.6(Ar),128.5(Ar),128.3(Ar),128.0(Ar),127.8(Ar),127.7(Ar),127.6(Ar),127.4(Ar),127.2(Ar),98.0(1-H),9 7.1(1'-H),78.5,74.5(Ar-CH2),70.1,68.0(Ar-CH2),67.2(Ar-CH2),66.6,66.4(4-C),66.3,49.7(2'-C),47.1(Linker -NCH2),29.3(Linker-CH2),23.5(Linker-CH2),22.8(NHAc-CH3),17.2(6-CH3),16.8(6'-CH3).HR-ESI-MS(m / z):calcd for C 48 H 59 O 10 N5N + (M+Na) + :888.4154found:888.4215.

[0088] Compound 26: Selenoglucosides 12 (122.4 mg, 0.2 mmol) and acceptor 25 (86.5 mg, 0.1 mmol) were mixed, azeotropically dehydrated with toluene (3×5 mL), and dried under vacuum using an oil pump for 2 hours. The mixed donor-acceptor was dissolved in DCM (8 mL) at 0°C and freshly activated Molecular sieves, then slowly add NIS (45 mg; 0.2 mmol) and TfOH (17.7 μl; 0.2 mmol). After stirring for 4 hours, add a drop of triethylamine (Et3N) to the reaction system to neutralize it, filter The filtrate was washed with 10% Na2S2O3 solution and saturated NaHCO3 solution, respectively. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain compound 26 (95 mg, 0.072 mmol, 72%). [α] 25 D=-7.8°(c=0.9,CHCl3);IRνmax(film)3029,2938,2880,2108,1697,1658,1525,1454,1361,1231,1096,1048,822,757,699cm -1 ; 1 HNMR(400MHz,Methanol-d4)δ7.85–7.78(m,4H,Ar),7.51–7.42(m,7H,Ar),7.40–7.17(m,16H,Ar),5.14(d,J=12.3Hz,2H,Ar-CH2),4.98(d,J=13.4Hz,3H,Ar-CH2,1"-H),4.94–4.82(m,4H,Ar-CH2,1'-H,1-H),4.75(dd,J=29.5,11.7Hz,2H,Ar-CH2),4.64(d,J=12.0Hz,1H,Ar-CH2),4.43(s,1H,2-H),4.24(dd,J=10.1,3.7Hz,1H,3'-H),4.14–4.07(m,1H,5'-H),4.04–3.83(m,5H,3-H,5-H,2"-H,3"-H,4'-H),3.76(dd,J=10.0,3.6Hz,1H,2'-H),3.69–3.49(m,2H,4-H,Linker-OCH2),3.36(m,1H,Linker-OCH2),3.27(dt,J=11.3,5.9Hz,4H,Linker-OCH2,4"-H,5"-H),1.83–1.72(m,3H,NHAc),1.53(s,4H,Linker-CH2),1.41–1.32(m,2H,Linker-CH2),1.29–1.24(m,3H,6"-H),1.23–1.16(m,3H,6-H),1.12(s,3H,6'-H). 13C NMR(101MHz, Methanol-d4)δ172.1(NH-C=O),162.6(NH-C=O),138.9(Ar),138.0(Ar),135.2(Ar),133.2(Ar),128.3(Ar),1 28.2(Ar),127.9(Ar),127.6(Ar),127.3(Ar),127.0(Ar),126.2–124.6(Ar),101.0(1"-H),98.6(1'-H),97.1(1-H),79.8, 78.9(4-C),78.0(3'-C),75.0(Ar-CH2,2'-C),74.4,70.5,68.1,67.0,66.7(Ar-CH2),65.3(5'-H),58.3,49.0(2-C),47.8( Linker-NCH2),28.5(Linker-CH2),21.9(NHAc-CH3),17.3(6"-CH3),16.1(6'-CH3),15.97(6-CH3).HR-ESI-MS(m / z):calcd for C 67 H 76 O 13 N9Cl3Na + (M+Na) + :1342.4520found:1342.4535.

[0089] Compound 2: Compound 26 (30 mg, 22.7 μmol) was dissolved in a mixture of THF / Ac2O / AcOH (3 / 2 / 1, v / v / v, 3 mL), and freshly activated Zn (0.5 g) was added. After stirring at room temperature overnight, the mixture was diluted with DCM and filtered. The filtrate was washed with saturated aqueous NaHCO3 and saturated brine; the combined organic layers were dried over anhydrous Na2SO4, filtered, evaporated in vacuo, and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to obtain the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added to the solution. The mixture was stirred under hydrogen (4 atm) for 36 hours, filtered, washed with water, and concentrated. The crude product was purified by Sep-Pak column C18 (Macherey-Nagel, Düren, Germany) using water and methanol as eluents to give compound 2 (8 mg, 11.35 umol, 50% for two steps). 1H NMR(600MHz,Deuterium Oxide)δ4.88(d,J=4.4Hz,1H,1'-H),4.76(d,1-H),4.55(d,J=8.5Hz,1H,1"-H),4.24(dd,J=4.8,1.7Hz,1H,4'-H),4.17(dd,J=11.1,3.8Hz,1H,2-H),4.12(tt,J=6.6,3.7Hz,1H,5'-H),4.02–3.95(m,2H,3'-H,5-H),3.79(dd,J=11.1,3.2Hz,1H,3-H),3.70(d,J=3.2Hz,1H,4-H),3.66–3.62(m,1H,2'-H),3.62–3.52(m,2H,2"-H Linker-OCH2),3.48–3.32(m,4H,3"-H,4"-H,5"-H,Linker-OCH2),2.87(t,J=7.7Hz,2H,Linker-NCH2),1.94(s,3H,NHAc-CH3),1.90(d,J=7.5Hz,9H,NHAc-CH3),1.60–1.47(m,4H,Linker-CH2),1.32(tq,J=14.2,7.5,6.4Hz,2H,Linker-CH2),1.08(dd,J=6.6Hz,3H,6-H),1.08(dd,J=5.8Hz,3H,6"-H),0.94(d,J=6.5Hz,3H,6'-H). 13CNMR(151MHz,Deuterium Oxide)δ174.8(NH-C=O),174.7(NH-C=O),101.6(1"-H),101.1(1'-H),97.1(1-H),76.8(3-H,3'-H)71.7(4"-H),71.4 (4-C),71.0(5"-H),67.9(Linker-OCH2),67.7(2'-H),66.5(5-H),66.1(5'-C),57.0(4"-C),56.3(2"-C),52.9(4'-C) ,48.5(2-H),39.4(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),21.9(NHAc-CH3),16.9(6"-CH3),15.4(6'-CH3),15.3(6-CH3).HR-ESI-MS(m / z): calcd for C 31 H 56 O 13 N5 + (M+H) + :706.3869found:706.3881.

[0090] Compound 3: Compound 25 (50 mg, 57.8 μmol) was dissolved in a mixture of THF / Ac2O / AcOH (3 / 2 / 1, v / v / v, 3 mL) and freshly activated Zn (0.5 g) was added. After stirring at room temperature overnight, the mixture was diluted with DCM and filtered. The filtrate was washed with saturated aqueous NaHCO3 and saturated brine; the combined organic layers were dried over anhydrous Na2SO4, filtered, evaporated in vacuo, and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to obtain the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added to the solution. The mixture was stirred under hydrogen (4 atm) for 36 hours, filtered, washed with water, and concentrated. The crude product was purified by Sep-Pak C18 column (Macherey-Nagel, Düren, Germany) using water and methanol as eluents to give compound 3 (14.9 mg, 31.2 μmol, 54% yield over two steps). 1H NMR(400MHz, Deuterium Oxide)δ4.95(d,J=4.1Hz,1H,1'-H),4.73(d,J=3.4Hz,1H,1-H),4.25–4.14(m,3H,2-H,4'-H,5'-H),3.99(dq,J=19.9,5.2,4. 1Hz,2H,5-H,3'-H),3.83(dd,J=11.0,3.1Hz,1H,3-H),3.74(m,J=3.0Hz,1H,4-H),3.64–3.48(m,2H,Linker-OCH2,2'-H),3.3 8(dt,J=10.0,6.2Hz,1H,Linker-OCH2),2.90(t,J=7.6Hz,2H,Linker-NCH2),1.96(d,J=30.7Hz,6H,NHAc),1.59(m,J=15.4,7 .7Hz,4H,Linker-CH2),1.34(m,J=12.1,7.5,3.5Hz,2H,Linker-CH2),1.13(d,J=6.6Hz,3H,6-H),1.00(d,J=6.2Hz,3H,6'-H). 13 C NMR (101 MHz, Deuterium Oxide)δ175.5(NH-C=O),174.6(NH-C=O),101.1(1'-H),97.0(1-H),76.5(3-C),71.3(4-C ),68.5(2'-C),68.3(3'-C),67.8(Linker-OCH2),66.4(5-C),65.8(5'-C),53.8(4'-C)48. 4(2-C),39.3(Linker-NCH2),28.0(Linker-CH2),26.4(Linker-CH2),22.2(Linker-CH2) ,21.9(NHAc-CH3),21.8(NHAc-CH3),15.4(6'-CH3),15.3(6-CH3).HR-ESI-MS(m / z):calcd for C 21 H 40 O9N3 + (M+H) + :478.2759found:478.2819.

[0091] Example 4:

[0092]

[0093] The synthesis of compounds 1, 4 and 7 was as follows Figure 6 shown.

[0094] Compounds 27, 28 and 29 were synthesized using known methods (Cai Juntao, doctoral dissertation of Jiangnan University, 2020), and compounds 27, 28 and 29 were catalytically hydrogenated to obtain deprotected target compounds 1, 4 and 7, respectively.

[0095] Specific experimental operations and steps:

[0096] Compound 1: Trisaccharide 27 (30 mg; 20.41 μmol) was dissolved in a mixture of tetrahydrofuran and acetic anhydride and acetic acid (3 / 2 / 1, v / v / v, 3 mL). Freshly activated Zn (1 g) was added and stirred at room temperature overnight. After TLC showed that the reaction of the starting material was complete, 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 over anhydrous sodium sulfate, filtered, evaporated in vacuo, and dried on an oil pump vacuum. The crude product was dissolved in dichloromethane and tert-butyl alcohol 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 under a hydrogen atmosphere (4 atm) for 36 hours, then filtered through celite and washed with water, the washing was repeated three times, and the solvent was evaporated in vacuo. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL / min, eluting with ultrapure water (solvent A) containing 0.1% formic acid and acetonitrile (solvent B) with a linear gradient from 10% to 30% solvent B over 30 minutes to afford compound 1 (9.9 mg, 13.27 μmol, 65% overall yield over two 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.

[0097] Compound 4: Compound 28 (30 mg, 29.5 μmol) was dissolved in a mixture of DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added to the reaction system. After stirring for 36 hours under hydrogen (4 atm), the mixture was filtered and washed with water. The residue was purified on a Sep-Pak C18 column (Macherey-Nagel, Düren, Germany) using water and methanol as eluents to obtain product 4 (13.1 mg, 25.11 μmol, 85% yield). 1HNMR(600MHz,Deuterium Oxide)δ5.03(d,J=4.0Hz,1H,1-H),4.81(s,1H,1'-H),4.28(d,J=12.9Hz,3H,4-H,5-H,2'-H),4.19(q,J=6.3Hz,1H,RHb-3),4.08(dq,J=22.1,5.2,4.0Hz,2H,3-H,5'-H),3.91(dd,J=11.0,3.0Hz,1H,3'-H),3.79(d,J=3.2Hz,1H,4'-H),3.67(dt,J=12.6,6.7Hz,1H,linker-OCH2),3.63–3.58(m,1H,2-H),3.47(dt,J=11.0,6.3Hz,1H,linker-OCH2),2.99(t,J=7.7Hz,2H,linker-NCH2),2.55–2.45(m,2H,RHb-2),2.00(s,2H,NHAc),1.65(dq,J=23.2,7.6Hz,4H,linker-CH2),1.42(tq,J=14.6,7.7,7.1Hz,2H,linker-CH2),1.22(dd,J=13.0,6.2Hz,6H,RHb-4,6'-H),1.09(s,3H,6-H). 13 C NMR(151MHz,DeuteriumOxide)δ101.1(1-C),97.0(1'-C),76.5(3'-C),71.3(4'-C),68.6(2-C),68.4(3-C),67.8(linker-OCH2),66.5(5'-C),65.8,65.0(RHb-3),53.8,48.5,44.7(RHb-2),39.4(linker-CH2),28.0(linker-CH2),26.4(linker-CH2),22.1-22.0(NHAc,linker-CH2,RHb-4),15.5(6-H),15.3(6'-H).HR-ESI-MS(m / z):calcd for C 23 H 44 O 10 N3+(M+H)+:522.3021found:522.3083.

[0098] Compound 7: Compound 29 (20 mg, 33.10 μmol) was dissolved in a mixed solution of DCM / t-BuOH / H2O (2 / 1 / 1, v / v / v, 2 mL), and 10% Pd / C (50 mg) was added to the reaction system. After stirring for 36 hours in hydrogen (4 atm), the mixture was filtered and washed with water. The residue was purified using a Sep-Pak column C18 (Macherey-Nagel, Germany) using water and methanol as eluents to give compound 7 (9.1 mg, 31.5 μmol, 95%). 1 HNMR(400MHz,Deuterium Oxide)δ4.85(d,J=3.3Hz,1H,1-H),4.09(q,J=7.9Hz,2H,2-H,5-H),3.91(d,J=11.1Hz,1 H,3-H),3.81(s,1H,4-H),3.73–3.59(m,1H,linker-OCH2),3.47(dt,J=11.1,6.6Hz,1H, linker-OCH2),3.00(t,J=7.8Hz,2H,linker-NCH2),2.04(s,3H,NHAc),1.66(dp,J=21.8 ,8.0Hz,4H,linker-CH2),1.44(p,J=7.9Hz,2H,linker-CH2),1.23(d,J=6.6Hz,3H,6-H). 13 C NMR (101MHz,Deuterium Oxide)δ174.6(NHAc-C=O),96.9(1-C),71.1(4-C),67.8(3-C,linker-OCH2),66.6(5-C),49.8(2-C),39.4(linker-NCH2) ,28.0(linker-CH2),26.5(linker-CH2),22.3(linker-CH2),22.0(NHAc-CH3),15.5(6-CH3).HR-ESI-MS(m / z):calcdfor C 13 H 27 O5N2 + (M+H) + :291.1914found:291.1946.

[0099] Example 5:

[0100] Extraction of lipopolysaccharide (LPS) and O-antigen (OPS) of Vibrio cholerae O100 serotype; NMR analysis of OPS 1 H and 13 C spectrum Figure 7 shown.

[0101] Inactivated strains of Vibrio cholerae serotype O100 were provided by Nankai University. Lipopolysaccharide (LPS) were extracted using the hot phenol water method as previously reported. The strain was suspended in sterile water and, after multiple freezing and thawing, the bacterial suspension was mixed with 90% phenol and shaken at 68°C for 30 minutes. The mixture was cooled and centrifuged, and the aqueous phase was collected. An equal volume of sterile water was added to the organic phase and shaken at 68°C for 30 minutes. The mixture was cooled and centrifuged again to separate the aqueous phase. The two aqueous phases were combined, dialyzed against distilled water overnight, and lyophilized to obtain crude LPS. The crude LPS was further treated with DNase I, RNase A, and proteinase K in Tris buffer (0.1 M, pH 8). The solution was then heated at 100°C for 10 minutes, cooled, and centrifuged. The supernatant was extracted with water-saturated phenol. After centrifugation, the aqueous phase was collected, dialyzed against distilled water, and lyophilized to obtain purified LPS.

[0102] Lipopolysaccharide was defatted with a 2% aqueous acetic acid solution at 100° C. until lipid A was precipitated (3 hours); the precipitate was removed by centrifugation (13,000 rpm, 20 minutes), and the resulting product was purified by a G50 gel column to obtain OPS.

[0103] Example 6:

[0104] ELISA was used to evaluate the effective titer of antibodies in rabbit serum, such as Figure 8 shown.

[0105] Eight New Zealand rabbits (male, 1.8-2.2 kg, Wuxi Hengtai Experimental Animal Breeding Co., Ltd.) were randomly divided into a control group and an experimental group. Four rabbits in the experimental group received multiple subcutaneous injections of a 1:1 mixture of Vibrio cholerae O100 lipopolysaccharide and Freund's adjuvant at multiple sites on the back every fourteen days. Blood was collected from the ear vein of each rabbit. LPS (0.4 mg / rabbit) was administered three times (days 0, 14, and 28). Four rabbits in the control group received multiple subcutaneous injections of a 1:1 mixture of PBS and Freund's adjuvant at multiple sites on the back every fourteen days (days 0, 14, and 28). Antiserum was aliquoted and stored at -80°C. Serum was collected on days 0, 7, 14, 28, and 35, and IgG antibodies were detected by enzyme-linked immunosorbent assay (ELISA). The P / N value represents the ratio of the absorbance of the experimental group to that of the control group. When the P / N value of the immunization group / control group is ≥2.1, the immune response of IgG antibodies to LPS of Vibrio cholerae O100 serotype is considered positive.

[0106] ELISA specific operations and steps

[0107] (1) Coating and washing: Coat the ELISA plate with 20 μg / ml antigen, 100 μL / well, at 4°C for 24 h. Wash three times with PBST and pat dry on absorbent paper.

[0108] (2) Blocking and plate washing: Add blocking solution (5% skim milk powder in PBST) to the coated ELISA plate at 300 μl / well, block overnight at 4°C, and wash three times with PBST and pat dry.

[0109] (3) Add the test serum: Add serum diluted with 1% BSA-PBS to the ELISA plate at 100 μl / well, with a dilution ratio of 1:12800. Add a blank control (containing only 1% BSA-PBS) and incubate overnight at 4°C. Wash four times and pat dry.

[0110] (4) Add enzyme-labeled secondary antibody: Use anti-rabbit HRP secondary antibody diluted in 1% BSA-PBS at a dilution ratio of 1:2000, 100 μl / well, incubate at 37°C for 1 h; then wash 4 times and pat dry.

[0111] (5) Color development: Add 200 μl / well of TMB color development solution, incubate in the dark for 10 min, and then immediately quench with 1 M dilute H2SO4, 50 μl / well, and then read the absorbance at 450 nm using a microplate reader.

[0112] (6) Cleaning: After processing the sample, use ammonium bicarbonate solution to neutralize the sulfuric acid in the 96-well plate.

[0113] The results are as follows Figure 8 As shown in the figure, 1-1, 1-2, 1-3, and 1-4 refer to the rabbit serum samples of four parallel experiments in the experimental group. The results show that: 7 days after immunization of rabbits, the P / N value began to increase significantly. After 14 days, the P / N values ​​of all samples were greater than 2.1, which means that the immune response of IgG antibodies to Vibrio cholerae O100 serotype LPS is positive.

[0114] Example 7:

[0115] Glycan microarray screening for specific sugar antigens, such as Figure 9 shown.

[0116] Specific experimental operations and steps:

[0117] The synthetic oligosaccharides and LPS were dissolved in coupling buffer (50 mM sodium phosphate, pH 8.5) for printing onto "CodeLink" slides (SurModics Co., Ltd.) using an RMA-Arrayer96 (Rayme China). The slides were then incubated overnight in a humidified chamber at 26°C and 55% humidity. The slides were incubated with microarray quenching buffer (50 nM Na2HPO4, 100 nM ethanolamine) at 50°C for 1 hour. After washing with distilled water and centrifugation, the quenched slides were blocked with 3% BSA (w / v) in PBS for 1 hour at room temperature. The slides were washed once with PBST (0.1% Tween in PBS) and twice with PBS. After centrifugation, the slides were placed in a culture chamber (ProPlate). Rabbit serum was diluted 1:200 in 1% PBS-BSA (w / v) and added to the incubation chamber. There were at least four replicates for each sample. The microarrays were incubated overnight in a dark, humid chamber at 4°C. The slides were washed three times with PBST, and goat anti-rabbit IgG (Thermo) secondary antibody was added to each well at a dilution of 1:400 in 1% PBS-BSA (w / v) and incubated at 37°C in a dark, humid chamber for 60 minutes. The slides were then rinsed three times with PBST, rinsed three times with water over 15 minutes, and centrifuged. Finally, the slides were scanned using a LuxScan 10K / B (CapitalBio Technology). Images were analyzed using GenePix Pro 7 software (Molecular Devices).

[0118] The results are as follows Figure 9 As shown, compounds 1, 4, 6, 9, and 11 containing a 3-hydroxybutyryl group exhibited significant antigenic activity, while compounds 2, 3, 5, 7, 8, and 10, which lacked a 3-hydroxybutyryl group, were not recognized by antibodies, highlighting the key role of the 3-hydroxybutyryl group in antibody recognition. The non-reducing disaccharide 6 exhibited strong antibody recognition and is considered the smallest antigenic epitope. This finding provides an important reference for the development of glycoconjugate vaccines against Vibrio cholerae.

[0119] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A specific sugar segment for the preparation of Vibrio cholerae vaccine, characterized in that: The specific sugar fragment is: 、 、 、 or ; n=2~40,-(CH2) n -NH2 is the linker.

2. Use of the specific sugar fragment according to claim 1 in the preparation of Vibrio cholerae vaccine.

3. A pharmaceutical composition, characterized in that Comprising any one or more combinations of the five specific sugar fragments in claim 1.

4. A sugar chip, characterized in that: The sugar chip is prepared by combining the specific sugar fragments according to claim 1 with a chip via the linker structure therein.

5. Use of the sugar chip according to claim 4 in preparing a Vibrio cholerae O100 infection detection device.

6. A Vibrio cholerae glycoprotein conjugate for vaccine development, characterized in that: The specific sugar fragment according to claim 1 is conjugated to a protein via the linker structure therein.

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