Specific sugar fragment for research and development of vibrio cholerae vaccines

Abstract

The present invention relates to a specific sugar fragment for the research and development of Vibrio cholerae vaccines. By means of a chemical synthesis method and sugar fragments related to the Vibrio cholerae O100 serotype O antigen trisaccharide, combined with sugar chip technology, the structure-activity relationship between different sugar fragments and the antigenicity thereof is evaluated at the molecular level. Sugar chip screening shows that 3-hydroxybutyryl is a key structural feature of the O antigen. The non-reducing terminal disaccharide carrying 3-hydroxybutyryl is the smallest potential antigen epitope. The disaccharide has a strong ability to bind to an antibody, has a simple structure, and can be used as a specific sugar fragment for the research and development of vaccines. Sugar-conjugated vaccines designed on the basis of the specific sugar fragment can solve challenges such as difficult culture of pathogenic bacteria and inhomogeneous sugar antigens in naturally extracted polysaccharide vaccines. The present invention has good application prospects in the research and development of Vibrio cholerae sugar-conjugated vaccines, the detection of infections, the development of novel drugs, etc.

Description

Specific sugar fragment for development of Vibrio cholerae vaccine TECHNICAL FIELD

[0001] The present application relates to the development of a specific sugar fragment for the development of a Vibrio cholerae vaccine, belonging to the field of medicine. BACKGROUND

[0002] Cholera is a severe, life-threatening acute intestinal infectious disease caused by Vibrio cholerae, and cholera is a legal class A infectious disease in China. According to the introduction of the World Health Organization, in 2023, 30 countries reported cholera cases, causing more than 1 million diarrhea cases and a large number of deaths worldwide each year.

[0003] Vibrio cholerae usually exists in aquatic environments and multiplies in the human intestine, and this high-risk infection can cause watery diarrhea within a few hours, accompanied by dehydration, vomiting, coma, and even death.

[0004] According to the difference of cell surface lipopolysaccharide (LPS) O antigen, Vibrio cholerae strains are divided into more than 200 serotypes. Due to the serious drug resistance of Vibrio cholerae and the shortage of oral vaccines, it is urgent to provide a new cholera vaccine. In recent years, sugar conjugate vaccines have shown promising prospects, especially for protection against young children. Developing a multivalent sugar conjugate vaccine covering all important serotypes of cholera is considered an important technical direction for the eradication of this disease.

[0005] SUMMARY TECHNICAL PROBLEM

[0006] In view of the deficiencies of the prior art, the present application relates to the development of a specific sugar fragment for the development of a Vibrio cholerae vaccine.

[0007] TECHNICAL SCHEME

[0008] The present application obtains a sugar fragment related to the O100 serotype O antigen trisaccharide repeat unit of Vibrio cholerae based on chemical synthesis, fixes the synthesized sugar fragment on the surface of a chip to prepare a sugar chip; then incubates the sugar chip with antiserum, so that IgG antibodies in the antiserum specifically recognize the sugar fragment; then labels the antibodies in the sugar chip with a fluorescently labeled secondary antibody, and obtains a specific sugar fragment that can be used for the development of a Vibrio cholerae vaccine through fluorescence scanning and quantitative analysis.

[0009] The first object of the present application is to provide a specific sugar fragment for the preparation of a Vibrio cholerae vaccine, which has the structure R2-[U1] a -[U2]-[U3] b -O-Linker, and the structures of U1, U2 and U3 are as follows:

[0010] Formula I wherein, a, b, c respectively represent the number of U1 (D-quinovosamine), U3 (L-fucosamine), a, b are respectively 0 or 1; 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 application, the group at position 4 of monosaccharide U2 in the specific sugar fragment is (R)-3-hydroxybutyryl amino or (S)-3-hydroxybutyryl amino.

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

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

[0014] In one embodiment of the present application, n = 2 ~ 40; further specifically, 5 can be selected.

[0015] In one embodiment of the present application, the synthesis of the sugar fragments in the sugar library is obtained by three monosaccharide building blocks and five carboxylic acid derivatives, through a series of reductive acylation and amide condensation, catalytic hydrogenation and the like.

[0016] In one embodiment of the present application, the reducing agent used in the reductive acylation is one of zinc powder, triphenyl phosphine, 1, 3-propanedithiol, lithium aluminum hydride, trimethyl phosphine, stannous chloride dihydrate, sodium borohydride, sodium cyanoborohydride.

[0017] In one embodiment of the present application, the condensing agent used in the amide condensation is one of DCC (dicyclohexyl carbodiimide), DIC (diisopropyl carbodiimide), EDC (1- (3-dimethyl aminopropyl) -3-ethyl carbodiimide), DPPA (diphenyl azide phosphate), DPPCl (diphenyl phosphor chloride), DECP (cyanophosphoric acid diphenyl ester), HATU (2- (7-azabenzotriazole) -N, N, N', N'-tetramethyl urea hexafluorophosphate), HBTU (benzotriazole-N, N, N', N'-tetramethyl urea hexafluorophosphate), HCTU (6-chlorobenzotriazole-1, 1, 3, 3-tetramethyl urea hexafluorophosphate).

[0018] In one embodiment of the present application, the catalyst used in the catalytic hydrogenation can be 10% palladium carbon catalyst or palladium hydroxide, etc.

[0019] In one embodiment of the present application, the solvent used in the catalytic hydrogenation can be one of water / methanol / dichloromethane / acetic acid mixture, water / tert-butyl alcohol / dichloromethane mixture, water / tert-butyl alcohol / ethyl acetate mixture, water / tert-butyl alcohol / tetrahydrofuran mixture.

[0020] In one embodiment of the present application, the reaction temperature used in the catalytic hydrogenation can be between 0 and 40℃.

[0021] The present application also provides the use of the specific sugar fragments in the preparation of a vaccine against Vibrio cholerae.

[0022] The present application also provides the use of the specific sugar fragments in the preparation of a medicament for preventing or treating Vibrio cholerae infection.

[0023] The present application also provides a pharmaceutical composition comprising the specific sugar fragments.

[0024] The present application also provides a pharmaceutical composition comprising any one or a combination of the above five specific sugar fragments.

[0025] In one embodiment of the present application, the specific sugar fragments are preferably

[0026] The present application also provides a method for preparing the sugar chip, which combines the linker structure of the specific sugar fragments with the sugar chip.

[0027] In one embodiment of the present application, the method for preparing the sugar chip specifically comprises the following steps:

[0028] Step 1: Dissolve the obtained sugar fragments in 50mM phosphate solution (pH = 8.5), print on the chip using a chip spotting instrument, incubate overnight at room temperature and 65% humidity, so that the sugar fragments are covalently bound to the chip; after incubation, use a mixture of 100nM ethanolamine and 50nM sodium phosphate (pH = 9) to treat at 50℃ for 1 hour to quench the chip.

[0029] Step 2: Take the diluted rabbit antiserum and add it to the sugar chip for incubation, so that the IgG antibodies in the serum specifically bind to the sugar fragments, and then wash away the unbound serum antibodies; subsequently, use a fluorescently labeled secondary antibody (anti-IgG antibody) to bind to the IgG antibodies on the chip, incubate, and then wash away the unbound secondary antibody.

[0030] Step 3: Perform fluorescence scanning on a microarray scanner, and according to the scanning results, screen for sugar fragments with better antigenicity, which are the specific sugar fragments.

[0031] In one embodiment of the present application, the phosphate concentration used to dilute the sugar fragment can be 10 mM to 100 mM, preferably 50 mM; the pH can be 8 to 9, preferably the pH is 8.5.

[0032] In one embodiment of the present application, the concentration of the specific sugar fragment is 0.01 to 10 mM, preferably 0.05 mM.

[0033] In one embodiment of the present application, the temperature of the incubation is room temperature (20 to 30℃); the humidity is 50% to 70%, preferably 65%.

[0034] In one embodiment of the present application, the degree of serum dilution is 1:10 to 1:500, preferably 1:200.

[0035] In one embodiment of the present application, the fluorescently labeled secondary antibody refers to a Cy3-labeled goat anti-human or goat anti-rabbit IgG antibody. The dilution degree is 1:30 to 1:1000, preferably 1:400.

[0036] The present application also provides a Vibrio cholerae O100 infection detection device, which comprises the following components: a chip, a specific sugar fragment, a serum diluent, and a fluorescently labeled secondary antibody.

[0037] In one embodiment of the present application, after the specific sugar fragment is fixed and quenched, the infection detection device can be separated into multiple small spaces by different specifications of molds, allowing simultaneous detection of multiple samples, which is convenient and efficient.

[0038] In one embodiment of the present application, the mold can be a 16-well, 64-well, or 128-well mold.

[0039] In one embodiment of the present application, after the specific sugar fragment is fixed and quenched, it can be stored for later use. When used, the diluted serum is directly added to the surface of the chip, which is more convenient and efficient.

[0040] The present application also provides a Vibrio cholerae sugar protein conjugate for vaccine development, which is conjugated by the Linker structure of the specific sugar fragment and a protein.

[0041] In one embodiment of the present application, the carrier protein used in the sugar protein conjugate includes one of 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). Advantages:

[0042] The sugar conjugate vaccine based on natural extraction of polysaccharide antigens in the prior art has many defects, for example, some pathogenic bacteria are difficult to culture, sufficient extract cannot be obtained, and impurities are easily mixed in; compared with the prior art, the sugar conjugate vaccine based on synthetic oligosaccharide antigens can not only avoid the mixing of pathogenic bacterial virulence factors, but also obtain the minimum effective antigen epitope; in addition, the application of specific oligosaccharide antigens with clear structures also makes the production of the sugar vaccine more repeatable.

[0043] The application provides specific sugar fragments for cholera vaccine research and development by constructing a sugar library by a chemical synthesis method and combining a sugar chip technology. The non-reducing end disaccharide The non-reducing end disaccharide

[0044] Fig. 1 is a schematic diagram of the method involved in the application.

[0045] Fig. 2 is the structure of 11 sugar fragments in the sugar library of the application.

[0046] Fig. 3 is a synthesis route diagram of compound 10.

[0047] Fig. 4 is a synthesis route diagram of compounds 5, 6, 8, 9 and 11.

[0048] Fig. 5 is a synthesis route diagram of compounds 2 and 3.

[0049] Fig. 6 is a synthesis route diagram of compounds 1, 4 and 7.

[0050] Fig. 7 is a nuclear magnetic resonance spectrum of the O-antigen of the lipopolysaccharide of the O100 serotype of Vibrio cholerae. 1 H and 13 C spectrum.

[0051] Fig. 8 is an ELISA detection of IgG antibody titers in rabbit serum.

[0052] Fig. 9 is a sugar chip screening result; A is a structure schematic diagram of 11 sugar fragments, B is a spotting mode diagram, C is a chip scanning result, and D is a quantitative result diagram of average fluorescence, and the error line is from the standard deviation between two points of two uniform concentrations. DETAILED DESCRIPTION

[0053] All the commercial reagents used in the experiment were used directly without treatment. The anhydrous solvents used in the reaction were prepared by the MBraun MB-SPS 800 solvent drying system. The solvents used in the silica gel column chromatography were all analytical pure and were used after vacuum distillation. The silica gel plates used in the thin layer chromatography (TLC) were 60-F254 silica gel glass-based or aluminum foil-based silica gel plates, and the silica gel used in the normal phase silica gel column chromatography was 200-300 mesh silica gel.

[0054] The yield of each reaction step was calculated respectively, and the yield calculation method was: (the amount of substance of the target product / the amount of substance of the raw material) x 100%. The structure of the product was identified by nuclear magnetic spectrum, infrared spectrum, optical rotation, and high resolution mass spectrum, and the purity of the product was analyzed by nuclear magnetic spectrum. The hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum were measured by Bruker Ascend 600M, 400M nuclear magnetic resonance instrument at 25°C. The high resolution mass spectrum was measured by Agilent 6220 electrospray ion source-time of flight mass spectrometer. The infrared spectrum was measured by Thermo Fisher Scientific Nicolet iS5 infrared instrument, and the optical rotation was measured by Schmidt & Haensch UniPol L 10000 full-automatic polarimeter at 589 nm. The concentration (c) unit is g / 100mL.

[0055] In the present specification, certain embodiments can be disclosed in a format that is a range. It is to be understood that such a "range" format is merely used to simplify the discussion and is understood and intended to include individual (discrete) values of the range. Although the present application has been disclosed in the above with reference to preferred embodiments, it is not intended to limit the present application to the preferred embodiments. Any modification, equivalent, and alternative (including all the modifications, equivalents, and alternatives) that can be made by those skilled in the art without departing from the principles and scope of the present application should be included in the scope of the present application, which should be defined by the appended claims.

[0056] Example 1:

[0057] The synthesis of compound 10 is shown in Figure 3.

[0058] Compound 12 (selenoglycoside, can be prepared according to the literature: Codée. et al, Organic & Biomolecular Chemistry 2020, 18(15), 2834-2837) and N-benzyl-N-benzyloxy carbonyl-5-aminopentanol were subjected to glycosylation under the condition of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and iodosuccinimide (NIS) to obtain compound 13; then reduction acylation was carried out using zinc powder, acetic acid and acetic anhydride, and the deprotected target compound 10 was obtained after catalytic hydrogenation.

[0059] Specific experimental procedures and steps:

[0060] Compound 13: Selenoglycoside 12 (170 mg, 0.28 mmol) and N-benzyl-N- benzyloxycarbonyl-5-aminopentanol (136 mg; 0.42 mmol) were mixed, azeotropically evaporated with toluene (3 x 15 ml), and dried under vacuum with a freshly activated molecular sieve for 2 h using an oil pump; then it was dissolved in a DCM (10 ml) solution, and NIS (94 mg; 0.42 mmol) and TMSOTf (20 μl; 0.11 mmol) were added slowly at 0 °C. After stirring for 4 h, the reaction mixture was neutralized with 1 drop of Et3N at 0 °C, warmed to room temperature, and the molecular sieve was filtered off. The filtrate was washed with 10% aqueous Na2S203, saturated aqueous NaHC03, and saturated brine. The combined organic layers were dried over Na2S04, 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%). 25 D = +30.2 ° (c = 0.47, CHCl3); IR vmax(film) 3029, 2943, 2872, 2109, 1697, 1525, 1423, 1361, 1230, 1082, 821, 758, 698 cm -1 ; 1 H NMR (400 MHz, Chloroform-d) δ 7.88 - 7.79 (m, 4H, Ar), 7.50 (d, J = 7.6 Hz, 3H, Ar), 7.43 - 7.11 (m, 11H, Ar), 5.18 (d, J = 20.4 Hz, 2H, Ar-CH2), 5.01 (d, J = 10.8 Hz, 1H, Ar-CH2), 4.88 (d, J = 10.1 Hz, 2H, 1-H, Ar-CH2), 4.50 (s, 2H, Ar-CH2), 4.22 (dt, J = 22.3, 9.7 Hz, 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 = 6.1 Hz, 3H, 6-H), 1.38 - 1.23 (m, 2H, linker-CH2). 13C NMR (101 MHz, Chloroform-d) δ 162.1 (NHAc-C=0) 156.7 (Cbz-C=0) 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) + : 804.2093 found: 804.210

[0061] Compound 10: Compound 13 (50 mg, 64 pmol) was dissolved in THF / Ac20 / AcOH (3 / 2 / 1, v / v / v, 3 mL) and freshly activated Zn (1 g) was added. After stirring overnight at room temperature, the mixture was diluted and filtered. The filtrate was washed with saturated NaHC03solution and saturated sodium chloride solution. The organic layer was dried with anhydrous Na2S04, filtered, evaporated in vacuum and then dried in vacuum for 2 hours in an oil pump. It was then dissolved in DCM / t-BuOH / H20 (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 on a Sep-Pak cartridge C18 (Macherey-Nagel, Duren, Germany) using water and methanol as eluents to give compound 10 (13.8 mg, 41.6 pmol, 65% yield over two steps). 1H NMR (600 MHz, Deuterium Oxide) δ 4.37 (d, J = 8.5 Hz, 1H, 1-H), 3.76 (dt, J = 11.5, 6.2 Hz, 1H, linker-OCH2), 3.58 (t, J = 9.0 Hz, 1H, 2-H), 3.51 - 3.42 (m, 4H, 3-H, 4-H, 5-H, linker-OCH2), 2.87 (t, J = 7.7 Hz, 2H, linker-NCH2), 1.92 (d, J = 3.3 Hz, 6H, NHAc), 1.55 (m, J = 7.7 Hz, linker-CH2), 1.48 (p, J = 6.7 Hz, 2H, linker-CH2), 1.28 (dt, J = 9.1, 5.5 Hz, 2H, linker-CH2), 1.11 (d, J = 5.0 Hz, 3H, 6-CH3). 13 C NMR (151 MHz, Deuterium Oxide) δ 174.6 (NHAc-C=0), 174.5 (NHAc-C=0), 101.0 (1-C), 71.6, 71.0, 70.1 (linker-OCH2), 57.1, 56.3 (2-C), 39.3 (linker-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 for C 15 H 30 O5N3 + (M+H) + :332.2180 found:332.2219.

[0062] Example 2:

[0063] Synthesis of compounds 5, 6, 8, 9, 11, as shown in Figure 4.

[0064] Compound 14 (reference from literature: Cai J T, Ph.D. Dissertation, Jiangnan University, 2020) was hydrolyzed by NIS, then esterified with trifluoro-N-phenylacetimidate, followed by dissolving in DCM solution in the presence of Ph3OP and TMSI to obtain compound 15; the 2-naphthaldehyde group was removed by DDQ to obtain acceptor 16; the glycosylation reaction of acceptor 16 and donor selenoglycoside 12 was carried out to obtain disaccharide 17; compound 5 and 8 were synthesized by reducing and acylating disaccharide 17 and compound 16 with zinc powder and acetic anhydride respectively, and then directly deprotecting; the azido group in compound 15 was reduced by 1,3-propanedithiol, and then amide condensation was carried out with butyric acid 18 and 19 (reference from literature: Tanasova. et al, Angew. Chem. Int. Ed. 2015, 54(14), 4274-4278) in the presence of EDC and HOBt to obtain compounds 20, 21; compounds 9 and 11 were obtained by deprotecting compounds 20 and 21 respectively; the 2-naphthaldehyde group in compound 20 was selectively removed to obtain acceptor 22; the glycosylation reaction of acceptor 22 and donor selenoglycoside 12 was carried out to obtain disaccharide 23, and then deprotecting disaccharide 23 to obtain compound 6.

[0065] Specific experimental operations and steps:

[0066] 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, stirred uniformly, then NIS (171.4 mg, 0.7 mmol) was added, and stirred for 1 hour. TLC showed that the reaction was complete, the mixture was diluted with ethyl acetate, and washed with 10% (w / v) Na2S2O3. The organic layer was dried with Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate 20 / 1→1 / 1 v / v) to obtain an intermediate compound. The intermediate compound was dissolved in DCM (4.8 mL) solution at 0°C, then 2,2,2-trifluoro-N-phenylacetimidate chloride (171 μL, 1.14 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (171 μL, 1.14 mmol) were added. The reaction was stirred at 0°C for 3 hours, the mixture was concentrated in vacuum, 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 for 3 times, then dissolved in anhydrous DCM (1 mL), and pre-activated dry molecular sieves and Ph3OP (848 mg, 3.05 mmol). Then TMSI (56.5 μL, 0.38 mmol, 1.0 eq) was slowly added to the mixture. The reaction was stirred at room temperature until TLC analysis indicated that the reaction was complete, the solution was diluted and the reaction was quenched with saturated Na2S203. The organic phase was washed with water and brine, dried over anhydrous Na2S04, filtered and concentrated in vacuo. Then it was purified by column chromatography on silica gel (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, CHCI3); IR vmax(film) 3029, 2944, 2903, 2108, 1697, 1454, 1361, 1279, 1227, 1127, 1096, 1044, 820, 755, 698 cm-1. -1 ; 1 H NMR (400 MHz, Chloroform-d) δ 7.87 - 7.76 (m, 4H, Ar), 7.49 (ddd, J = 16.3, 7.5, 2.5 Hz, 3H, Ar), 7.35 - 7.14 (m, 17H, Ar), 5.17 (d, J = 9.5 Hz, 2H, Ar-CH2), 5.00 (d, J = 11.9 Hz, 1H, Ar-CH2), 4.88 (d, J = 11.9 Hz, 1H, Ar-CH2), 4.81 (d, J = 12.0 Hz, 1H, Ar-CH2), 4.70 - 4.60 (m, 2H, Ar-CH2, 1-H), 4.48 (d, J = 8.0 Hz, 2H, Ar-CH2), 4.05 (s, 1H, 3-H), 3.85 (dd, J = 9.8, 3.8 Hz, 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 = 6.4 Hz, 3H, 6-CH3). 13C NMR (101 MHz, Chloroform-d) δ 138.4 (Ar), 135.8 (Ar), 133.3 (Ar), 133.0 (Ar), 128.6 (Ar), 128.5 (Ar), 128.4 (Ar), 128.2 (Ar), 128.0 (Ar), 127.9 (Ar), 127.8 (Ar), 127.77, 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): calcd for C 44 H 48 O6N4Na + (M+Na) + : 751.3466 found: 751.3501

[0067] 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 h, after which TLC showed that the reaction was complete. The mixture was diluted with DCM (2 x 10 mL) and washed with saturated NaHC03(20 mL). The organic layer was dried over anhydrous Na2S04and the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether: acetone = 1 : 1) to give compound 16 (11.9 mg, 20.2 μmol, 88%) [a] 25 D = +30.8° (c = 0.3, CHCl3); IR vmax(film) 3029, 2944, 2903, 2108, 1697, 1454, 1422, 1361, 1279, 1227, 1127, 1096, 1044, 820, 755, 698 cm -1 ; 1HNMR (400 MHz, Chloroform-d) δ 7.44 - 7.10 (m, 17H, Ar), 5.17 (d, J = 10.3 Hz, 2H, Ar-CH2), 4.74 - 4.56 (m, 3H, Ar-CH2, 1-H), 4.48 (d, J = 7.5 Hz, 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 = 6.5 Hz, 5H, Linker-CH2, 6-CH3). 13 C NMR (101 MHz, Chloroform-d) δ 137.9 (Ar), 137.8 (Ar), 128.6 (Ar), 128.6 (Ar), 128.5 (Ar), 128.2 (Ar), 128.1 (Ar), 128.0 (Ar), 127.8 (Ar), 127.3 (Ar), 96.5 (1-C), 77.0 (2-C), 72.7 (Ar-CH2), 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 (Linker-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) + : 611.2840 found: 611.2895.

[0068] Compound 17: Se-glycoside 12 (49 mg, 0.08 mmol) and acceptor 16 (56.5 mg, 0.10 mmol) were mixed, azeotropically dried with toluene (3 x 5 mL) and dried under vacuum with an oil pump for 2 h. The mixed donor-acceptor was dissolved in DCM (8 mL) at 0 °C and freshly activated 4 A molecular sieves were added followed by slow addition of NIS (27 mg; 0.12 mmol) and TfOH (14.2 μL; 0.16 mmol). After stirring for 4 h, the reaction was neutralized with a drop of triethylamine (Et3N), filtered ​Molecular sieves. The filtrate was washed with 10% Na2S2O3 solution, 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 give compound 17 (63.6 mg, 0.061 mmol, 76%).[α] 25 D = +43.7° (c = 0.6, CHCl3); IR vmax(film) 3032, 2947, 2903, 2108, 1678, 1454, 1422, 1361, 1279, 1226, 1127, 1096, 1044, 820, 755, 698 cm -1 ; 1 H NMR (600 MHz, Chloroform-d) δ 7.81 (dd, J = 10.7, 8.3 Hz, 4H, Ar), 7.47 (dd, J = 8.7, 5.0 Hz, 3H, Ar), 7.40 - 7.13 (m, 17H, Ar), 6.99 (d, J = 7.8 Hz, 1H, NHAc-H), 5.23 (d, J = 8.2 Hz, 1H, 1'-H), 5.18 (d, J = 18.2 Hz, 2H, Ar-CH2), 4.97 (d, J = 10.8 Hz, 1H, Ar-CH2), 4.86 (d, J = 10.7 Hz, 1H, Ar-CH2), 4.77 (d, J = 12.2 Hz, 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 = 7.9 Hz, 1H, 4-H), 3.74 (dd, J = 10.2, 3.7 Hz, 1H, 2-H), 3.58 (dt, J = 18.0, 9.0 Hz, 1H, 2'-H), 3.41 (d, J = 23.7 Hz, 2H, 5'-H, Linker-OCH2), 3.30 - 3.10 (m, 4H, 4'-H Linker-OCH2, Linker-NCH2), 1.51 (dt, J = 27.9, 6.9 Hz, 4H, Linker-CH2), 1.39 (d, J = 5.6 Hz, 3H, 6'-H), 1.26 (d, J = 10.0 Hz, 2H, Linker-CH2), 1.17 (d, J = 6.2 Hz, 3H, 6-CH3). 13C NMR (151 MHz, Chloroform-d) δ 161.7 (NH-C=0), 138.5 (Ar), 137.9 (Ar), 134.7 (Ar), 133.3 (Ar), 133.1 (Ar), 128.6 (Ar), 128.5 (Ar), 128.3 (Ar), 128.0 (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 (1-H), 78.6 (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 (5-C), 59.6 (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 for C 52 H 57 O9N8Cl3Na + (M+Na) + : 1065.3206 found: 1065.3227.

[0069] Compound 5: Compound 17 (40 mg, 38.38 pmol) was dissolved in a mixture solution of THF / Ac20 / 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 with DCM and filtered. The filtrate was washed with saturated aqueous NaHC03and saturated brine; the combined organic layers were dried over anhydrous Na2S04, filtered, evaporated in vacuo and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to give an intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added to the solution. After stirring under hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water, concentrated. The crude was purified with a Sep-Pak cartridge C18 (Macherey-Nagel, Duren, Germany) using water and methanol as eluents to give compound 5 (11.9 mg, 23.03 pmol, 60% yield over two steps). 1 H NMR (600 MHz, Deuterium Oxide) δ 4.84 (d, J = 4.2 Hz, 1H, 1-H), 4.65 (d, J = 8.5 Hz, 1H, 1'-H), 4.31 (d, J = 4.7 Hz, 1H, 4-H), 4.13 (q, J = 6.6 Hz, 1H, 5-H), 3.98 (dd, J = 10.6, 4.7 Hz, 1H, 3-H), 3.80 (dd, J = 10.6, 4.0 Hz, 1H, 2-H), 3.63 (q, J = 7.8, 6.9 Hz, 2H, 2', Linker-OCH2), 3.57 - 3.45 (m, 4H, 3'-H, 4'-H, 5'-H, Linker-OCH2), 2.96 (t, J = 7.7 Hz, 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, 6.0 Hz, 4H, Linker-CH2), 1.41 (dh, J = 14.1, 6.8 Hz, 2H, Linker-CH2), 1.15 (d, J = 5.6 Hz, 3H, 6'-H), 1.04 (d, J = 6.5 Hz, 3H, 6-H). 13C NMR (151 MHz, Deuterium Oxide) d 174.8 (NH-C=0), 174.6 (NH-C=0), 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) + : 519.3025 found: 519.3052.

[0070] Compound 8: Compound 16 (30 mg, 51.0 pmol) was dissolved in a mixture solution of THF / Ac20 / 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 with DCM and filtered. The filtrate was washed with saturated aqueous NaHC03and saturated brine; the combined organic layers were dried over anhydrous Na2S04, filtered, evaporated in vacuum and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to give the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added to the solution. After stirring under hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water, concentrated. The crude was purified with a Sep-Pak cartridge C18 (Macherey-Nagel, Duren, Germany) using water and methanol as eluents to give compound 8 (7.5 mg, 26.0 pmol, 51% yield over two steps). 1H NMR (600 MHz, Deuterium Oxide) δ 4.87 - 4.83 (m, 1H, 1-H), 4.19 (d, J = 4.6 Hz, 1H, 4-H), 4.16 (q, J = 6.6 Hz, 1H, 5-H), 3.95 (ddd, J = 10.6, 4.9, 1.8 Hz, 1H, 3-H), 3.67 - 3.60 (m, 2H, 2-H, Linker-OCH2), 3.46 (m, J = 11.4, 7.7, 3.7 Hz, 1H, Linker-OCH2), 2.95 (t, J = 7.7 Hz, 2H, Linker-NCH2), 2.04 (s, 3H, NHAc-CH3), 1.62 (dp, J = 20.4, 6.9 Hz, 4H, Linker-CH2), 1.48 - 1.33 (m, 2H, Linker-CH2), 1.05 (dd, J = 6.6, 1.9 Hz, 3H, 6-CH3). 13 C NMR (151 MHz, Deuterium Oxide) δ 175.6 (NH-C=0), 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.

[0071] Compound 20: Compound 15 (180 mg, 0.25 mmol) was dissolved in pyridine (8 mL) under nitrogen protection, then water (2 mL), Et3N (1.51 ml, 10.87 mmol) and 1,3-propanedithiol (1.48 ml, 14.8 mmol) were added to the reaction system; 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 amino sugar. Then sodium bicarbonate (62 mg, 0.74 mmol) was added to a solution of (R)-3-O-benzylbutyric acid 18 (96 mg, 0.49 mmol) and amino sugar in acetonitrile (20 mL) and stirred at room temperature. After 10 minutes, HOBt (6.7 mg, 49.4 μmol), EDC.HC1 (57 mg, 0.30 mmol) were added in turn, and stirred at the same temperature for 6 hours. After TLC detection, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution, the separated organic layer was dried over anhydrous Na2S04, concentrated and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 20 (188.9 mg, 0.22 mmol, yield 87%).[α] 25 D = +114.5° (c = 0.3, CHCl3); IR vmax(film) 3029, 2938, 2109, 1697, 1539, 1454, 1361, 1217, 1102, 1044, 819, 756, 698 cm -1 ; 1H NMR (400 MHz, Chloroform-d) δ 7.88 - 7.73 (m, 4H, Ar-H), 7.55 - 7.14 (m, 24H, Ar-H), 6.73 (d, J = 10.3 Hz, 1H, NHAc-H), 5.20 (d, J = 7.9 Hz, 2H, Ar-CH2), 4.99 (d, J = 11.3 Hz, 1H, Ar-CH2), 4.66 (td, J = 13.8, 12.6, 3.0 Hz, 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), 2.66 - 2.47 (m, 2H, RHb-2), 1.65 - 1.50 (m, 4H, Linker-CH2), 1.27 (d, J = 6.3 Hz, 5H, Linker-CH2, RHb-4), 1.13 (d, J = 6.4 Hz, 3H, 6-CH3). 13 C NMR (101 MHz, Chloroform-d) δ 171.6 (NH-C=0), 138.7 (Ar), 138.1 (Ar), 137.9 (Ar), 136.8 (Ar), 136.1 (Ar), 133.3 (Ar), 132.9 (Ar), 128.5 (Ar), 128.5 (Ar), 128.4 (Ar), 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), 126.6 (Ar), 126.3 (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), 64.5 (5-H), 50.4 (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 (m / z): calcd for C55 H 63 O8N2 + (M+H) + :879.4579found:879.4635

[0072] Compound 21 : Compound 15 (150 mg, 0.21 mmol) was dissolved in pyridine (8 mL) under nitrogen protection, then water (2 mL), Et3N (1.26 ml, 9.06 mmol) and 1,3-propanedithiol (1.24 mL, 12.36 mmol) were added to the reaction system; 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 amino sugar. Then sodium bicarbonate (52 mg, 0.62 mmol) was added to a solution of (S)-3-O-benzylbutyric acid 19 (80 mg, 0.41 mmol) and amino sugar in acetonitrile (17 mL) and stirred at room temperature. After 10 minutes, HOBt (5.6 mg, 41.2 μmol), EDC (64 mg, 0.33 mmol) were added in turn, and stirred at the same temperature for 6 hours. After TLC detection, 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%).[α] 25 D = +80.1° (c = 0.75, CHCl3); IR vmax(film) 3029, 2942, 2868, 2109, 1697, 1532, 1454, 1361, 1217, 1104, 1045, 818, 755, 698 cm -1 ; 1HNMR (400 MHz, Chloroform-d) δ 7.85 - 7.70 (m, 4H Ar-H), 7.54 - 7.13 (m, 24H Ar-H), 6.59 (d, J = 10.1 Hz, 1H, NHAc-H), 5.17 (d, J = 8.5 Hz, 2H, Ar-CH2), 4.95 (d, J = 11.3 Hz, 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 = 9.4 Hz, 1H, Linker-OCH2), 3.40 - 3.13 (m, 4H, Linker-OCH2, Linker-NCH2, 2-H), 2.54 (m, J = 5.4, 2.1 Hz, 2H, RHb-2), 1.54 (s, 4H, Linker-CH2), 1.37 - 1.19 (m, 5H, Linker-CH2,, RHb-4), 1.15 (d, J = 6.4 Hz, 3H, 6-CH3). 13 C NMR (101 MHz, Chloroform-d) δ 171.7 (NH-C=0), 138.7 (Ar), 138.2 (Ar), 137.9 (Ar), 136.1 (Ar), 133.3 (Ar), 132.9 (Ar), 128.5 (Ar), 128.4 (Ar), 128.3 (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), 125.9 (Ar), 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 (Ar-CH2), 64.5 (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 / z): calcd for C 55 H 62 O8N2Na + (M+Na)+ 901.4398 found: 901.4446.

[0073] Compound 9: Compound 20 (30 mg, 34.15 pmol) was dissolved in a mixture of DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v) and then 10% Pd / C (50 mg) was added. After stirring under an atmosphere of hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water. The residue was then purified on a Sep-Pak cartridge C18 (Macherey-Nagel, Germany) using water and methanol as eluents to give product 9 (10.2 mg, 30.4 pmol, 89%). 1 H NMR (400 MHz, Deuterium Oxide) d 4.79 (d, J = 3.9 Hz, 1H, 1-H), 4.12 (m, J = 20.1, 13.5, 5.7 Hz, 3H, 4-H, 5-H, RHb-3), 3.90 (dd, J = 10.5, 4.3 Hz, 1H, 3-H), 3.58 (m, J = 8.9, 5.8, 4.9 Hz, 2H, 2-H, Linker-OCH2), 3.40 (dt, J = 10.6, 6.2 Hz, 1H, Linker-OCH2), 2.89 (t, J = 7.6 Hz, 2H, Linker-NCH2), 2.46 - 2.36 (m, 2H, RHb-2), 1.57 (dq, J = 12.6, 6.7, 5.5 Hz, 4H, Linker-CH2), 1.33 (dp, J = 13.5, 6.6 Hz, 2H, Linker-CH2), 1.13 (d, J = 6.1 Hz, 3H, RHb-4), 1.00 (d, J = 6.3 Hz, 3H, 6-CH3). 13 C NMR (101 MHz, Deuterium Oxide) d 98.3 (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) + :335.2177 found: 335.2219.

[0074] Compound 11: Compound 21 (30 mg, 34.15 pmol) was dissolved in a mixture solution of DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added. After stirring under hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water. The residue was purified with a Sep-Pak cartridge C18 (Macherey-Nagel, Germany) using water and methanol as eluents to give compound 11 (10.4 mg, 31.08 pmol, 91%). 1 H NMR (400 MHz, Deuterium Oxide) d 4.92 (d, J = 4.0 Hz, 1 H, 1-H), 4.26 (m, J = 19.2, 5.4 Hz, 3H, 4-H, 5-H, RHb-3), 4.02 (dd, J = 10.5, 4.5 Hz, 1 H, 3-H), 3.78 - 3.65 (m, 2H, 2-H, Linker-OCH2), 3.53 (dt, J = 9.9, 6.2 Hz, 1 H, Linker-OCH2), 3.02 (t, J = 7.6 Hz, 2H, Linker-NCH2), 2.61 - 2.48 (m, 2H, RHb-2), 1.76 - 1.65 (m, 4H, Linker-CH2), 1.56 - 1.39 (m, 2H, Linker-CH2), 1.25 (d, J = 6.2 Hz, 3H, RHb-4), 1.13 (d, J = 6.4 Hz, 3H, 6-CH3). 13 C NMR (101 MHz, Deuterium Oxide) d 175.3 (NH-C=0), 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) + :335.2177 found:335.2214.

[0075] 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 h, after which TLC showed that 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 Na2SO4and the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography on silica gel (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); IR vmax(film) 3030, 2923, 1697, 1540, 1453, 1361, 1216, 1100, 1037, 819, 736, 697 cm -1 ; 1 H NMR (600 MHz, Chloroform-d) δ 7.39 - 7.13 (m, 20H, Ar-H), 6.72 (d, J = 9.4 Hz, 1H, NH-H), 5.17 (d, J = 16.9 Hz, 2H, Ar-CH2), 4.59 (d, J = 11.3 Hz, 1H, Ar-CH2), 4.55 - 4.42 (m, 6H, Ar-CH2, 1-H), 4.30 (d, J = 8.1 Hz, 1H, 4-H), 4.14 - 4.07 (m, 1H, 3-H), 4.03 (d, J = 12.2 Hz, 1H, 5-H), 3.97 (qd, J = 6.4, 3.8 Hz, 1H, RHb-3), 3.52 (d, J = 25.7 Hz, 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 = 6.4 Hz, 3H, 6-CH3). 13C NMR (151 MHz, Chloroform-d) δ 172.9 (NH-C=0), 156.8 (Cbz-C=0), 156.2 (Cbz-C=0), 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 (Ar), 127.72 (Ar), 127.3 (Ar), 127.2 (Ar), 97.0 (1-C), 77.1 (2-C) 72.5 (RHa-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 (RHa-2-CH2), 29.2 (linker-CH2), 28.0 (linker-CH2), 27.5 (linker-CH2), 23.5 (linker-CH2), 19.0 (RHa-4), 16.7 (6-CH3). HR-ESI-MS (m / z): calcd for C 44 H 54 O8N2Na + (M+Na) + : 761.3772 found: 761.3807.

[0076] Compound 23: Selenoglycoside 12 (156 mg, 0.26 mmol) and acceptor 22 (94.3 mg, 0.13 mmol) were mixed, azeotropically dried with toluene (3 x 5 mL) and dried under vacuum with an oil pump for 2 h. The mixed donor-acceptor was dissolved in DCM (8 mL) at 0 °C and freshly activated 4 A molecular sieves were added, followed by slow addition of NIS (57.4 mg; 0.26 mmol) and TfOH (22.6 ul; 0.26 mmol). After stirring for 4 h, the reaction was neutralized with a drop of triethylamine (Et3N) and filtered ​Molecular sieves. The filtrate was washed with 10% Na2S2O3 solution, saturated NaHCO3 solution respectively. The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated, the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) to give compound 23 (109.7 mg, 0.09 mmol, yield 72%).[α] 25 D = +60.9° (c = 1.1, CHCl3); IR vmax(film) 3029, 2938, 2870, 2107, 1681, 1525, 1454, 1361, 1217, 1093, 1042, 819, 755, 698 cm -1 ; 1 H NMR (400 MHz, Chloroform-d) δ 7.85 - 7.73 (m, 4H), 7.51 - 7.12 (m, 27H, Ar), 6.73 (d, J = 10.1 Hz, 1H, NHAc), 6.64 (d, J = 7.7 Hz, 1H, NHAc), 5.17 (m, 2H, Ar-CH2), 4.96 (d, J = 7.5 Hz, 1H, 1'-H), 4.90 (d, J = 10.8 Hz, 1H, Ar-CH2), 4.78 (d, J = 10.9 Hz, 1H, Ar-CH2), 4.60 (d, J = 11.1 Hz, 1H, Ar-CH2), 4.47 (m, 2H, Ar-CH2), 4.43 (m, 2H, 1-H, 4-H), 4.35 (d, J = 11.9 Hz, 1H, Ar-CH2), 4.24 (d, J = 12.0 Hz, 1H, Ar-CH2), 4.09 (dd, J = 10.2, 4.6 Hz, 1H, 2-H), 3.97 (td, J = 6.6, 3.6 Hz, 2H, RHb-3, 5-H), 3.72 (m, 2H, 2'-H, 4'-H), 3.49 (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, 3.6 Hz, 1H, RHb-2), 2.47 (dd, J = 15.3, 7.0 Hz, 1H, RHb-2), 1.52 (s, 4H, Linker-CH2), 1.35 (dd, J = 9.9, 5.9 Hz, 6H, 6'-CH3, RHb-4), 1.27 (s, 2H, Linker-CH2), 1.06 (d, J = 6.4 Hz, 3H, 6-CH3). 13C NMR (101 MHz, Chloroform-d) δ 171.5 (NH-C=0), 161.4 (NH-C=0), 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), 128.4 (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-CH2), 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 O 11 N6Cl3Na + (M+Na) + : 1215.4139 found: 1215.4149.

[0077] Compound 6: Compound 23 (50 mg, 41.9 pmol) was dissolved in a mixture solution of THF / Ac20 / 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 NaHC03and saturated brine; the combined organic layers were dried over anhydrous Na2S04, filtered, evaporated in vacuo and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to give an intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added to the solution. After stirring under hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water, concentrated. The crude was purified with a Sep-Pak cartridge C18 (Macherey-Nagel, Duren, Germany) using water and methanol as eluents to give compound 6 (12 mg, 21.4 pmol, 51% yield over two steps). 1 H NMR (400 MHz, Deuterium Oxide) δ 4.70 (d, 1 H, 1-H), 4.60 (d, J = 8.2 Hz, 1 H, 1'-H), 4.27 (d, J = 4.7 Hz, 1 H, 4-H), 4.08 (p, J = 6.5, 6.0 Hz, 2 H, 5-H, RHb-3), 3.93 (dd, J = 10.4, 4.7 Hz, 1 H, 3-H), 3.72 (dd, J = 10.4, 3.9 Hz, 1 H, 2-H), 3.57 (dt, J = 9.6, 6.5 Hz, 2 H, 2'-H, Linker-OCH2), 3.53 - 3.48 (m, 2 H, 3'-H, Linker-OCH2, 4'-H, 5'-H), 2.90 (t, J = 7.6 Hz, 2 H, Linker-NCH2), 2.38 (h, J = 8.7, 8.1 Hz, 2 H, RHb-2), 1.82 (m, 6 H, NHAc-CH3), 1.58 (m, J = 9.1, 8.5 Hz, 4 H, Linker-CH2), 1.36 (m, J = 7.5, 7.1 Hz, 2 H, Linker-CH2), 1.15 (d, J = 6.1 Hz, 3 H, RHb-4), 1.08 (d, J = 4.5 Hz, 3 H, 6'-CH3), 0.98 (d, J = 6.4 Hz, 3 H, 6-CH3). 13C NMR (101 MHz, Deuterium Oxide) d 174.6 (NH-C=0), 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-CH2), 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) + :563.3287 found:563.3349.

[0078] Example 3:

[0079] Synthesis of compounds 2 and 3 as shown in Figure 5:

[0080] Compound 24 was synthesized by known method (Cai J, Doctoral Dissertation of Jiangnan University, 2020), and compound 24 was selectively removed 2-naphthalene methyl under the action of DDQ to obtain disaccharide acceptor 25; donor selenoglycoside 12 and acceptor 25 were catalyzed by trimethylsilyl trifluoromethanesulfonate and iodobutyrylimide to obtain trisaccharide 26; then compound 26 and 25 were subjected to reductive acylation and catalytic hydrogenation using zinc powder, acetic acid and acetic anhydride, respectively, to obtain the deprotected target compounds 2 and 3

[0081] Specific experimental operations and steps

[0082] 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 h, after which TLC showed that 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 Na2SO4and the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography on silica gel (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); IR vmax(film) 3029, 2939, 2876, 2108, 1697, 1540, 1454, 1361, 1228, 1089, 1045, 830, 755, 699 cm -1 ; 1 H NMR (600 MHz, Chloroform-d) δ 7.46 - 7.09 (m, 24 H, Ar), 6.36 (s, 1 H, NHAc-H), 5.18 - 5.13 (m, 2 H, Ar-CH2), 5.04 (d, J = 3.5 Hz, 1 H, 1'-H), 4.96 (s, 1 H, 1-H), 4.84 (d, J = 11.8 Hz, 1 H, Ar-CH2), 4.70 (s, 2 H, Ar-CH2), 4.64 (d, J = 11.7 Hz, 1 H, Ar-CH2), 4.47 (d, J = 9.3 Hz, 2 H, Ar-CH2), 4.39 (s, 1 H, 2'-H), 4.19 - 4.11 (m, 2 H, 3-H, 5-H), 3.82 (m, 2 H, 3'-H, 5'-H), 3.77 (dd, J = 9.8, 3.3 Hz, 1 H, 2-H), 3.71 (dd, J = 3.8, 1.5 Hz, 1 H, 4-H), 3.57 (m, 2 H, 4'-H, Linker-OCH2), 3.38 (m, 1 H, Linker-OCH2), 3.19 (m, 2 H, Linker-NCH2), 1.62 (s, 3 H, NHAc-CH3), 1.55 (s, 4 H, Linker-CH2), 1.34 - 1.24 (m, 2 H, Linker-CH2), 1.15 (d, J = 6.4 Hz, 3 H, 6-H), 1.13 - 1.08 (m, 3 H, 6'-H). 13C NMR (101 MHz, Chloroform-d) δ 170.5 (NH-C=0), 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), 97.1 (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 N5Na + (M+Na) + :888.4154found:888.4215.

[0083] Compound 26: Se-glycoside 12 (122.4 mg, 0.2 mmol) and acceptor 25 (86.5 mg, 0.1 mmol) were mixed, azeotropically dried with toluene (3 x 5 mL) and dried under vacuum with an oil pump for 2 h. The mixed donor-acceptor was dissolved in DCM (8 mL) at 0 °C and freshly activated 4 A molecular sieves were added, followed by slow addition of NIS (45 mg; 0.2 mmol) and TfOH (17.7 μΐ; 0.2 mmol). After stirring for 4 h, the reaction was neutralized by the addition of one drop of triethylamine (Et3N) and filtered molecular sieves. The filtrate was washed with 10% Na2S2O3solution, saturated NaHCO3solution, 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 give compound 26 (95 mg, 0.072 mmol, 72%). [a] 25 D ​= -7.8 ° (c = 0.9, CHCI3); IR vmax(film) 3029, 2938, 2880, 2108, 1697, 1658, 1525, 1454, 1361, 1231, 1096, 1048, 822, 757, 699 cm -1 ; 1 H NMR (400 MHz, Methanol-d4) δ 7.85 - 7.78 (m, 4H, Ar), 7.51 - 7.42 (m, 7H, Ar), 7.40 - 7.17 (m, 16H, Ar), 5.14 (d, J = 12.3 Hz, 2H, Ar-CH2), 4.98 (d, J = 13.4 Hz, 3H, Ar-CH2, 1"-H), 4.94 - 4.82 (m, 4H, Ar-CH2, 1'-H, 1-H), 4.75 (dd, J = 29.5, 11.7 Hz, 2H, Ar-CH2), 4.64 (d, J = 12.0 Hz, 1H, Ar-CH2), 4.43 (s, 1H, 2-H), 4.24 (dd, J = 10.1, 3.7 Hz, 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, 3.6 Hz, 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, 5.9 Hz, 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 (101 MHz, Methanol-d4) δ 172.1 (NH-C=0), 162.6 (NH-C=0), 138.9 (Ar), 138.0 (Ar), 135.2 (Ar), 133.2 (Ar), 128.3 (Ar), 128.2 (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) + : 1342.4520 found: 1342.4535.

[0084] Compound 2: Compound 26 (30 mg, 22.7 pmol) was dissolved in a mixture of THF / Ac20 / 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 NaHC03and saturated brine; the combined organic layers were dried over anhydrous Na2S04, filtered, evaporated in vacuo and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to give the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added to the solution. After stirring under hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water, concentrated. The crude was purified with a Sep-Pak cartridge C18 (Macherey-Nagel, Duren, Germany) using water and methanol as eluents to give compound 2 (8 mg, 11.35 pmol, 50% over two steps). 1H NMR (600 MHz, Deuterium Oxide) δ 4.88 (d, J = 4.4 Hz, 1 H, 1'-H), 4.76 (d, 1-H), 4.55 (d, J = 8.5 Hz, 1 H, 1"-H), 4.24 (dd, J = 4.8, 1.7 Hz, 1 H, 4'-H), 4.17 (dd, J = 11.1, 3.8 Hz, 1 H, 2-H), 4.12 (tt, J = 6.6, 3.7 Hz, 1 H, 5'-H), 4.02 - 3.95 (m, 2H, 3'-H, 5-H), 3.79 (dd, J = 11.1, 3.2 Hz, 1 H, 3-H), 3.70 (d, J = 3.2 Hz, 1 H, 4-H), 3.66 - 3.62 (m, 1 H, 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 = 7.7 Hz, 2H, Linker-NCH2), 1.94 (s, 3H, NHAc-CH3), 1.90 (d, J = 7.5 Hz, 9H, NHAc-CH3), 1.60 - 1.47 (m, 4H, Linker-CH2), 1.32 (tq, J = 14.2, 7.5, 6.4 Hz, 2H, Linker-CH2), 1.08 (dd, J = 6.6 Hz, 3H, 6-H), 1.08 (dd, J = 5.8 Hz, 3H, 6"-H), 0.94 (d, J = 6.5 Hz, 3H, 6'-H). 13C NMR (151 MHz, Deuterium Oxide) d 174.8 (NH-C=0), 174.7 (NH-C=0), 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) + : 706.3869 found: 706.3881.

[0085] Compound 3: Compound 25 (50 mg, 57.8 pmol) was dissolved in a mixture solution of THF / Ac20 / AcOH (3 / 2 / 1, v / v / v, 3 mL) and fresh 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 NaHC03and saturated brine; the combined organic layers were dried over anhydrous Na2S04, filtered, evaporated in vacuum and purified by silica gel column chromatography (DCM / MeOH: 50 / 1 → 10 / 1 v / v) to give the intermediate compound. The intermediate compound was then dissolved in DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added to the solution. After stirring under hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water, concentrated. The crude was purified with a Sep-Pak cartridge C18 (Macherey-Nagel, Duren, Germany) using water and methanol as eluents to give compound 3 (14.9 mg, 31.2 pmol, 54% yield over two steps). 1H NMR (400 MHz, Deuterium Oxide) δ 4.95 (d, J = 4.1 Hz, 1H, 1'-H), 4.73 (d, J = 3.4 Hz, 1H, 1-H), 4.25 - 4.14 (m, 3H, 2-H, 4'-H, 5'-H), 3.99 (dq, J = 19.9, 5.2, 4.1 Hz, 2H, 5-H, 3'-H), 3.83 (dd, J = 11.0, 3.1 Hz, 1H, 3-H), 3.74 (m, J = 3.0 Hz, 1H, 4-H), 3.64 - 3.48 (m, 2H, Linker-OCH2, 2'-H), 3.38 (dt, J = 10.0, 6.2 Hz, 1H, Linker-OCH2), 2.90 (t, J = 7.6 Hz, 2H, Linker-NCH2), 1.96 (d, J = 30.7 Hz, 6H, NHAc), 1.59 (m, J = 15.4, 7.7 Hz, 4H, Linker-CH2), 1.34 (m, J = 12.1, 7.5, 3.5 Hz, 2H, Linker-CH2), 1.13 (d, J = 6.6 Hz, 3H, 6-H), 1.00 (d, J = 6.2 Hz, 3H, 6'-H). 13 C NMR (101 MHz, Deuterium Oxide) δ 175.5 (NH-C=0), 174.6 (NH-C=0), 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) + : 478.2759 found: 478.2819.

[0086] Example 4:

[0087] Synthesis of compounds 1, 4 and 7 as shown in Figure 6.

[0088] Compounds 27, 28 and 29 were synthesized using known methods (Cai, J. Ph. D. Thesis, Jiangnan University, 2020), and deprotection of compounds 27, 28 and 29 by catalytic hydrogenation gave the target compounds 1, 4 and 7, respectively.

[0089] Detailed experimental procedures and steps:

[0090] Compound 1: Tri-saccharide 27 (30 mg; 20.41 pmol) was dissolved in a mixture of tetrahydrofuran and acetic anhydride, acetic acid (3 / 2 / 1, v / v / v, 3 mL), and freshly activated Zn (1 g) was added and stirred at room temperature overnight. After TLC detection of the complete reaction of the starting material, the reaction 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. 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. After stirring under an atmosphere of hydrogen (4 atm) for 36 hours, the mixture was filtered over celite and washed with water, repeating the washing 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 a linear gradient of 10% to 30% solvent B (acetonitrile) in solvent A (ultrapure water containing 0.1% formic acid) over 30 minutes, to give compound 1 (9.9 mg, 13.27 pmol, 65% overall yield for two steps). 1H NMR (600 MHz, Deuterium Oxide) δ 4.93 (t, J = 3.3 Hz, 1H, 1'-H), 4.76 (d, J = 3.3 Hz, 1H, 1-H), 4.60 (dd, J = 7.8, 2.0 Hz, 1H, 1"-H), 4.33 (d, J = 4.7 Hz, 1H, 4'-H), 4.23 (d, J = 11.3, 3.1 Hz, 1H, 2-H), 4.18 (q, J = 6.1 Hz, 1H, RHb-3), 4.08 - 4.00 (m, 2H, 3'-H, 5'-H), 3.83 (dt, J = 11.1, 2.9 Hz, 1H, 3-H), 3.75 (d, J = 3.0 Hz, 1H, 4-H), 3.68 (dt, J = 10.6, 3.2 Hz, 1H, 2'-H), 3.60 (m, J = 7.9, 7.4 Hz, 2H, 2"-H, Linker-OCH2), 3.44 (m, J = 28.2, 9.7, 4.4 Hz, 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, 2.2 Hz, 9H, NHAc-CH3), 1.66 - 1.54 (m, 4H, Linker-CH2), 1.37 (q, J = 7.4 Hz, 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 (151 MHz, Deuterium Oxide) d 174.6 (NH-C=0), 171.0 (NH-C=0), 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)+: 772.3951 found: 772.3968.

[0091] Compound 4: Compound 28 (30 mg, 29.5 pmol) was dissolved in a mixture of DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added to the reaction. After stirring under hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water. The residue was purified on a Sep-Pak cartridge C18 (Macherey-Nagel, Duren, Germany) using water and methanol as eluents to give product 4 (13.1 mg, 25.11 pmol, 85% yield). 1H NMR (600 MHz, Deuterium Oxide) δ 5.03 (d, J = 4.0 Hz, 1H, 1-H), 4.81 (s, 1H, 1'-H), 4.28 (d, J = 12.9 Hz, 3H, 4-H, 5-H, 2'-H), 4.19 (q, J = 6.3 Hz, 1H, RHb-3), 4.08 (dq, J = 22.1, 5.2, 4.0 Hz, 2H, 3-H, 5'-H), 3.91 (dd, J = 11.0, 3.0 Hz, 1H, 3'-H), 3.79 (d, J = 3.2 Hz, 1H, 4'-H), 3.67 (dt, J = 12.6, 6.7 Hz, 1H, linker-OCH2), 3.63 - 3.58 (m, 1H, 2-H), 3.47 (dt, J = 11.0, 6.3 Hz, 1H, linker-OCH2), 2.99 (t, J = 7.7 Hz, 2H, linker-NCH2), 2.55 - 2.45 (m, 2H, RHb-2), 2.00 (s, 2H, NHAc), 1.65 (dq, J = 23.2, 7.6 Hz, 4H, linker-CH2), 1.42 (tq, J = 14.6, 7.7, 7.1 Hz, 2H, linker-CH2), 1.22 (dd, J = 13.0, 6.2 Hz, 6H, RHb-4, 6'-H), 1.09 (s, 3H, 6-H). 13 C NMR (151 MHz, Deuterium Oxide) δ 101.1 (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)+: 522.3021 found: 522.3083.

[0092] Compound 7: Compound 29 (20 mg, 33.10 pmol) was dissolved in a mixture of DCM / t-BuOH / H20 (2 / 1 / 1, v / v / v, 2 mL) and 10% Pd / C (50 mg) was added to the reaction system. After stirring in hydrogen gas (4 atm) for 36 h, the mixture was filtered and washed with water. The residue was purified with a Sep-Pak cartridge C18 (Macherey-Nagel, Germany) using water and methanol as eluents to give compound 7 (9.1 mg, 31.5 pmol, 95%). 1 H NMR (400 MHz, Deuterium Oxide) δ 4.85 (d, J = 3.3 Hz, 1H, 1-H), 4.09 (q, J = 7.9 Hz, 2H, 2-H, 5-H), 3.91 (d, J = 11.1 Hz, 1H, 3-H), 3.81 (s, 1H, 4-H), 3.73 - 3.59 (m, 1H, linker-OCH2), 3.47 (dt, J = 11.1, 6.6 Hz, 1H, linker-OCH2), 3.00 (t, J = 7.8 Hz, 2H, linker-NCH2), 2.04 (s, 3H, NHAc), 1.66 (dp, J = 21.8, 8.0 Hz, 4H, linker-CH2), 1.44 (p, J = 7.9 Hz, 2H, linker-CH2), 1.23 (d, J = 6.6 Hz, 3H, 6-H). 13 C NMR (101 MHz, Deuterium Oxide) δ 174.6 (NHAc-C=0), 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): calcd for C 13 H 27 O5N2 + (M+H) + :291.1914found:291.1946.

[0093] Example 5:

[0094] Extraction of Vibrio cholerae O100 serotype lipopolysaccharide (LPS) and O-antigen (OPS); NMR spectra of OPS 1 H and 13 C spectra are shown in Figure 7.

[0095] The inactivated strain of Vibrio cholerae O100 serotype was provided by Nankai University, and LPS was extracted by the hot phenol water method according to previous reports; the bacterial suspension 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 the mixture was shaken at 68°C for 30 minutes. The mixture was cooled and centrifuged again, and the aqueous phase was separated; the two aqueous phases were combined and dialyzed against distilled water overnight, and then lyophilized to obtain crude LPS. The crude LPS was further treated with DNase I, RNase A and proteinase K in a Tris buffer solution (0.1 M, pH = 8). Then the solution was heated at 100°C for 10 minutes, then 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.

[0096] The lipopolysaccharide was delipidated with 2% aqueous acetic acid at 100°C until the lipid A precipitated (3 hours); the precipitate was removed by centrifugation (13000 rpm, 20 minutes), and the resulting product was purified by G50 gel column to obtain OPS.

[0097] Example 6:

[0098] The effective titer of antibodies in rabbit serum was evaluated by ELISA, as shown in Figure 8.

[0099] 8 New Zealand rabbits (male, 1.8-2.2 kg, Hengtai Experimental Animal Breeding Co., Ltd. of Wuxi) were randomly divided into a control group and an experimental group. Experimental group: 4 New Zealand rabbits, subcutaneous injection of Vibrio cholerae O100 lipopolysaccharide mixed with Freund's adjuvant 1:1 at multiple points on the back every fourteen days; at the same time, the rabbits in this group were bled from the marginal ear vein; LPS: 0.4 mg per rabbit, immunized three times (day 0, day 14, day 28); control group: 4 New Zealand rabbits, subcutaneous injection of PBS mixed with Freund's adjuvant 1:1 at multiple points on the back every fourteen days (day 0, day 14, day 28); the antisera were aliquoted and stored at -80°C. The serum was collected on days 0, 7, 14, 28 and 35, and the IgG antibodies in the serum were detected by enzyme-linked immunosorbent assay (ELISA). The P / N value represents the ratio of the absorbance of the experimental group to the control group, and when the P / N value of the immunized group / control group is ≥2.1, it is considered that the IgG antibodies have a positive immune response to Vibrio cholerae O100 serotype LPS.

[0100] ELISA specific operation and steps

[0101] (1) Coating and washing the plate: coat the ELISA plate with 20 μg / ml antigen, 100 μL / well, 4°C for 24 h. Wash with PBST 3 times and pat dry on absorbent paper.

[0102] (2) Blocking and washing: add blocking solution (5% skim milk powder in PBST) to the coated ELISA plate at 300 μl / well, block at 4°C overnight, and wash 3 times with PBST and dry.

[0103] (3) Add serum to be tested: add serum diluted with 1% BSA-PBS to the ELISA plate at 100 μl / well, with a dilution ratio of 1:12800; and add a blank control (containing only 1% BSA-PBS), and incubate at 4°C overnight. Then wash 4 times and dry.

[0104] (4) Add enzyme-labeled secondary antibody: dilute the anti-rabbit HRP secondary antibody with 1% BSA-PBS at a dilution ratio of 1:2000, add 100 μl / well, and incubate at 37°C for 1 h; then wash 4 times and dry.

[0105] (5) Color development: add 200 μl / well of TMB color developing solution, incubate in the dark for 10 min, then immediately quench with 1 M dilute H2SO4 at 50 μl / well, and then read the absorbance at 450 nm using an enzyme-labeled instrument.

[0106] (6) Clean up: after the samples are processed, neutralize the sulfuric acid in the 96-well plate with ammonium bicarbonate solution.

[0107] The results are shown in Figure 8, where 1-1, 1-2, 1-3, and 1-4 refer to the four parallel experimental rabbit serum samples in the experimental group. The results show that after 7 days of immunization of the rabbits, the P / N value began to increase significantly, and after 14 days, the P / N value of all samples was greater than 2.1, i.e., it can be considered that the IgG antibody has a positive immune response to the LPS of the Vibrio cholerae O100 serotype.

[0108] Example 7:

[0109] Glycan microarray screening of specific glycoantigens, as shown in Figure 9.

[0110] Specific experimental operations and steps:

[0111] Synthetic oligosaccharides and LPS were dissolved in coupling buffer (50 mM sodium phosphate, pH 8.5) for printing onto "CodeLink" slides (SurModics Inc.) using RMA-Arrayer 96 (Rayme China). The slides were then incubated at 26 °C in a humidified chamber with 55% humidity overnight. 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 at room temperature for 1 hour. The slides were washed once with PBST (0.1% Tween in PBS) and twice with PBS. After centrifugation, the slides were placed into incubation chambers (ProPlate). Rabbit serum was diluted 1:200 in 1% PBS-BSA (w / v) and added to the incubation chambers. There were at least four replicates for each sample. The microarrays were incubated at 4 °C in a dark humidified chamber overnight. The slides were washed 3 times with PBST, goat anti-rabbit IgG (Thermo) secondary antibody was added to each well, the secondary antibody was diluted 1:400 in 1% PBS-BSA (w / v), and the slides were incubated at 37 °C in a dark humidified chamber for 60 minutes. Then, the slides were washed 3 times with PBST, washed 3 times with water over 15 minutes, and centrifuged. Finally, the chips were scanned using LuxScan 10K / B (CapitalBio Technology). Image analysis was performed using GenePix Pro 7 software (Molecular Devices).

[0112] The results are shown in Figure 9. Compounds 1, 4, 6, 9, 11 containing 3-hydroxybutanoyl group have significant antigenic activity, and compounds 2, 3, 5, 7, 8, 10 without 3-hydroxybutanoyl group cannot be recognized by the antibody, highlighting the key role of 3-hydroxybutanoyl group in antibody recognition. Non-reducing end disaccharide 6 exhibits strong antibody recognition ability and is considered the smallest antigen epitope. This finding provides an important reference for the development of Vibrio cholerae glycoconjugate vaccine.

[0113] The above provided examples are not intended to limit the scope of the present application, nor are the described steps intended to limit the order of their execution. Those skilled in the art, in combination with existing common knowledge, make obvious improvements to the present application, which also fall within the protection scope defined by the claims of the present application.

Claims

1. A specific glycofragment for the preparation of a vaccine against Vibrio cholerae, characterized in that, Structure of R2-[U1] a -[U2]-[U3] b -O-Linker, Structure of U1, U2, U3 are as follows: Wherein, a, b represent the number of U1, U3 respectively, a, b are 0 or 1 respectively; R1 is one of 3, 5 dihydroxyhexanoyl or acetyl; R2 is H, or H-U3-, or H-U2-U3-, or H-U1-U2-U3-; Linker represents -(CH2) n -NH2or -(CH) n SH, n = 2-40.

2. The specific glycofragment according to claim 1, characterized in that, The group at position 4 of U2 in the specific sugar fragment is (R)-3-hydroxybutyryl amino, or (S)-3-hydroxybutyryl amino.

3. The specific glycofragment of claim 1, wherein, said specific sugar fragment is selected from the group consisting of: n＝2～40。 4. The specific sugar fragment of any one of claims 1-3 for use in preparation of a vaccine against Vibrio cholerae.

5. The specific sugar fragment of any one of claims 1-3 for use in preparation of a medicament for preventing or treating Vibrio cholerae infection.

6. A pharmaceutical composition, characterized by, Comprising the specific sugar fragment of any one of claims 1-2.

7. A pharmaceutical composition, characterized by, Comprising any one or more of the five specific sugar fragments of claim 3 in combination.

8. A sugar chip, characterized by, Is prepared by binding the Linker structure in the specific sugar fragment of any one of claims 1-3 to a sugar chip.

9. The sugar chip of claim 8 for use in preparation of a device for detecting Vibrio cholerae O100 infection.

10. A Vibrio cholerae glycoprotein conjugate for vaccine development, characterized in that, Is prepared by conjugating the Linker structure in the specific sugar fragment of any one of claims 1-3 to a protein.

Images