Specific Saccharide Fragment for Development of Vibrio Cholerae Vaccines

Abstract

The present disclosure discloses a specific saccharide fragment for development of Vibrio cholerae vaccines, and belongs to the field of medicine. In the present disclosure, a saccharide fragment related to a trisaccharide of V. cholerae O100 serotype O-antigen is chemically synthesized. Combined with a glycan microarray technology, the structure-activity relationship between different saccharide fragments and antigenicity thereof is evaluated at a molecular level. Glycan microarray screening indicates that 3-hydroxybutyryl is an essential structural feature of the O-antigen. A non-reducing end disaccharide carrying 3-hydroxybutyryl is a potential minimal antigenic epitope, and the disaccharide has strong binding capacity to antibodies and a simple structure, and may serves as a specific saccharide fragment for vaccine development. A glycoconjugate vaccine designed based on the specific saccharide fragment may solve the challenges of difficulty in culturing pathogenic bacteria and heterogeneity of saccharide antigens in naturally extracted polysaccharide vaccines. The present disclosure has bright application prospects in the development of glycoconjugate V. cholerae vaccine, infection detection, and new drug development.

Claims

1. A specific saccharide fragment for development of Vibrio cholerae vaccines, having a structure of R.sub.2[U1].sub.a-[U2][U3].sub.b-O-Linker, the structures of U1, U2, and U3 being as follows: ##STR00009## wherein a and b represent the quantities of U1 and U3, respectively, and a and b are 0 or 1, respectively; R.sub.1 represents one of 3,5-dihydroxyhexanoyl or acetyl groups; R.sub.2 represents H, or H-U3-, or H-U2-U3-, or H-U1-U2-U3-; and Linker represents (CH.sub.2).sub.nNH.sub.2 or (CH).sub.nSH, wherein n=2-40.

2. The specific saccharide fragment according to claim 1, wherein a group at position 4 of U2 in the specific saccharide fragment is (R)-3-hydroxybutyrylamino or(S)-3-hydroxybutyrylamino.

3. The specific saccharide fragment according to claim 1, wherein the specific saccharide fragment is selected from: ##STR00010## n=240.

4. A pharmaceutical composition, comprising the specific saccharide fragment according to claim 1 and pharmaceutical excipients.

5. A pharmaceutical composition, containing any one or a combination of more of the five types of specific saccharide fragments according to claim 3, and pharmaceutical excipients.

6. A glycan microarray, prepared by binding a Linker structure of the specific saccharide fragment according to claim 1 with the glycan microarray.

7. A Vibrio cholerae glycoprotein conjugate for vaccine development, obtained by conjugating a Linker structure of the specific saccharide fragment according to claim 1 with a protein.

Description

BRIEF DESCRIPTION OF FIGURES

[0043] FIG. 1 shows a schematic diagram of a method according to the present disclosure.

[0044] FIG. 2 shows structures of 11 saccharide fragments in a glycan library of the present disclosure.

[0045] FIG. 3 shows a synthetic route of a compound 10.

[0046] FIG. 4 shows synthetic routes of compounds 5, 6, 8, 9, and 11.

[0047] FIG. 5 shows synthetic routes of compounds 2 and 3.

[0048] FIG. 6 shows synthetic routes of compounds 1, 4 and 7.

[0049] FIG. 7A shows 1H-NMR spectra of Vibrio cholerae O100 serotype lipopolysaccharide O-antigen, and FIG. 7B shows 13C-NMR spectra of Vibrio cholerae O100 serotype lipopolysaccharide O-antigen.

[0050] FIG. 8 shows IgG antibody titers in rabbit sera detected via ELISA.

[0051] FIG. 9 shows a screening result using a glycan microarray, including: a schematic structural diagram of 11 saccharide fragments; a microarray printing pattern; a microarray scan result; and quantification of the mean fluorescence intensity. Error bars represent the standard error of the mean of two spots at uniform concentration.

DETAILED DESCRIPTION

[0052] Commercial agents used in experiments are used as received without further treatment, and anhydrous solvents used in reactions are prepared by an MBraun MB-SPS 800 type solvent drying system. Solvents used for silica gel column chromatography are all analytically pure and are used after reduced pressure distillation. A silica gel plate used for thin layer chromatography (TLC) is a glass-based or aluminum foil-based silica gel plate prepared from 60-F254 silica gel, and the silica gel used for normal-phase silica gel column chromatography is 200-300 mesh silica gel.

[0053] The yield of each reaction step is calculated as follows: (amount of target product substance/amount of raw material substance)*100%. The structures of products are identified via nuclear magnetic resonance (NMR) spectra, infrared spectra, optical rotation, and high-resolution mass spectra. The purity of products is analyzed by NMR spectra. A proton NMR spectrum, a carbon-13 NMR spectrum, and two-dimensional NMR spectrum are measured by Bruker Ascend 600 M and 400 M NMR spectrometers at 25 C. High-resolution mass spectra are obtained by an Agilent 6220 electrospray ionization time-of-flight mass spectrometer. Infrared spectra are obtained by a Thermo Fisher Scientific Nicolet iS5 FT-IR spectrometer. The optical rotation is measured by a Schmidt & Haensch UniPol L 10000 fully-automatic polarimeter at 589 nm. The unit of a measured concentration (c) is g/100 mL.

[0054] In this specification, certain implementations may be disclosed in a format that falls within a certain range. It should be understood that the description of being within a certain range is only for convenience and conciseness, and should not be interpreted as a rigid limitation on the disclosed scope. Although the present disclosure has been disclosed above with preferred examples, they are not intended to limit the present disclosure. Anyone familiar with the technology can make various changes and modifications without departing from the principle and scope of the present disclosure. Therefore, the protection scope of the present disclosure should be defined by claims.

Example 1

##STR00005##

[0055] Compound 10 was synthesized, as shown in FIG. 3.

[0056] Compound 12 (selenoglycoside, self-made by reference to Code. et al, Organic & Biomolecular Chemistry 2020,18 (15), 2834-2837) and N-benzyl-N-benzyloxycarbonyl-5-aminopentanol underwent a glycosylation reaction in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and N-iodosuccinimide (NIS), resulting in a compound 13. Subsequently, zinc powder, acetic acid, and acetic anhydride were used for reductive acylation, followed by catalytic hydrogenation to obtain deprotected target compound 10.

Specific Experimental Operations and Steps:

[0057] 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 (315 ml), and spin dried. Then a newly activated 4 molecular sieve was added, and dried under vacuum for 2 h using an oil pump. Subsequently, the resulting mixture was dissolved in a DCM (10 mL) solution, and NIS (94 mg, 0.42 mmol) and TMSOTf (20 l, 0.11 mmol) were added slowly at 0 C. After being stirred for 4 h, the reaction mixture was neutralized with one drop of Et.sub.3N at 0 C. and heated to room temperature, and the 4 molecular sieve was filtered out. A filtrate was washed with a 10% Na.sub.2S.sub.2O.sub.3 aqueous solution, a saturated NaHCO.sub.3 aqueous solution, and saturated brine. A combined organic layer was dried over Na.sub.2SO.sub.4, filtered, and evaporated under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether::ethyl acetate=3:1) to obtain compound 13 (202 mg, 0.26 mmol, 93%). [].sup.25.sub.D=+30.2 (c=0.47, CHCl.sub.3); IR vmax (film) 3029, 2943, 2872, 2109, 1697, 1525, 1423, 1361, 1230, 1082, 821, 758, 698 cm.sup.1; .sup.1H NMR (400 MHZ, Chloroform-d) 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, ArCH.sub.2), 5.01 (d, J=10.8 Hz, 1H, ArCH.sub.2), 4.88 (d, J=10.1 Hz, 2H, 1-H, ArCH.sub.2), 4.50 (s, 2H, ArCH.sub.2), 4.22 (dt, J=22.3, 9.7 Hz, 1H, 3-H), 3.87 (s, 1H, linker-OCH.sub.2), 3.53-3.33 (m, 3H, linker-OCH.sub.2, 2-H, 5-H), 3.32-3.10 (m, 3H,4-H, linker-NCH.sub.2), 1.58 (s, 4H, linker-CH.sub.2), 1.41 (d, J=6.1 Hz, 3H, 6-H), 1.38-1.23 (m, 2H, linker-CH.sub.2). .sup.13C NMR (101 MHZ, Chloroform-d) 162.1 (NHAc-CO) 156.7 (Cbz-CO) 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 (ArCH.sub.2), 70.7 (5-H), 70.0 (linker-OCH.sub.2), 68.9 (4-C), 67.2 (ArCH.sub.2), 59.6 (2-C), 50.3 (ArCH.sub.2), 47.2 (linker-NCH.sub.2), 29.2 (linker-CH.sub.2), 28.8 (linker-CH.sub.2), 23.5 (linker-CH.sub.2), 18.5 (6-C). HR-ESI-MS (m/z): calcd for C.sub.39H.sub.42O.sub.6N.sub.5Cl.sub.3Na.sup.+ (M+Na).sup.+: 804.2093 found: 804.210.

[0058] Compound 10: Compound 13 (50 mg, 64 umol) was dissolved in THF/Ac.sub.2O/AcOH (3/2/1, v/v/v, 3 mL), and freshly activated Zn (1 g) was added. After being stirred overnight at room temperature, the mixture was diluted and filtered. A filtrate was washed with a saturated NaHCO.sub.3 solution and a saturated sodium chloride solution. An organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and evaporated under vacuum, and then dried under vacuum using an oil pump for 2 h. Subsequently, the organic layer was dissolved in DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL), and 10% Pd/C (50 mg) was added. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water. Subsequently, a residue was purified using a Sep-Pak column C18 (Macherey-Nagel, Dren, Germany) with water and methanol as eluents to obtain compound 10 (13.8 mg, 41.6 mol, two-step yield: 65%). .sup.1H NMR (600 MHZ, Deuterium Oxide) 4.37 (d, J=8.5 Hz, 1H, 1-H), 3.76 (dt, J=11.5, 6.2 Hz, 1H, linker-OCH.sub.2), 3.58 (t, J=9.0 Hz, 1H, 2-H), 3.51-3.42 (m, 4H, 3-H, 4-H, 5-H, linker-OCH.sub.2), 2.87 (t, J=7.7 Hz, 2H, linker-NCH.sub.2), 1.92 (d, J=3.3 Hz, 6H, NHAc), 1.55 (m, J=7.7 Hz, linker-CH.sub.2), 1.48 (p, J=6.7 Hz, 2H, linker-CH.sub.2), 1.28 (dt, J=9.1, 5.5 Hz, 2H, linker-CH.sub.2), 1.11 (d, J=5.0 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (151 MHZ, Deuterium Oxide) 174.6 (NHAc-CO), 174.5 (NHAc-CO), 101.0 (1-C), 71.6, 71.0, 70.1 (linker-OCH.sub.2), 57.1, 56.3 (2-C), 39.3 (linker-NCH.sub.2), 28.1 (linker-CH.sub.2), 26.4 (linker-CH.sub.2), 22.2 (NHAc-CH.sub.3, linker-CH.sub.2), 16.8 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.15H.sub.30O.sub.5N.sub.3.sup.+ (M+H).sup.+: 332.2180 found: 332.2219.

Example 2

##STR00006##

[0059] Compounds 5, 6, 8, 9, and 11 were synthesized, as shown in FIG. 4.

[0060] Compound 14 (self-made by reference to doctoral thesis of Cai Juntao, Jiangnan University, 2020) was hydrolyzed with NIS, esterified with trifluoro-N-phenylacetimidoyl chloride, and then dissolved in a DCM solution in the presence of triphenylphosphine oxide (Ph.sub.3OP) and trimethylsilyl iodide (TMSI) to obtain compound 15. 2-Naphthalene methylene was removed by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to obtain acceptor 16. Acceptor 16 and the donor selenoglycoside 12 underwent a glycosylation reaction to obtain disaccharide 17. Disaccharide 17 and compound 16 were reduced and acylated with zinc powder and acetic anhydride, respectively, followed by direct deprotection to synthesize compounds 5 and 8 respectively. 1,3-Propanedithiol was used to reduce an azide group in compound 15, and amide condensation was carried out with butyric acids 18 and 19 (self-made by reference to Tanasova. et al., Angew. Chem. Int. Ed. 2015, 54 (14), 4274-4278), respectively in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt) to obtain compounds 20 and 21. Compounds 20 and 21 were deprotected respectively to obtain compounds 9 and 11. 2-Naphthalene methylene was selectively removed from compound 20 to obtain acceptor 22. Acceptor 22 and the donor selenoglycoside 12 underwent a glycosylation reaction to obtain disaccharide 23, followed by deprotection to obtain compound 6.

Specific Experimental Operations and Steps:

[0061] Compound 15: Compound 14 (200 mg, 0.38 mmol) was dissolved in acetone and H.sub.2O (10:1, v/v, 5.5 mL) at room temperature and stirred uniformly. Then NIS (171.4 mg, 0.7 mmol) was added and stirred for 1 h. After TLC indicated that the reaction was complete, the mixture was diluted with ethyl acetate and washed with 10% (w/v) Na.sub.2S.sub.2O.sub.3. An organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum. A residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=20/1 to 1/1, v/v) to obtain a compound intermediate. The compound intermediate was dissolved in a DCM solution (4.8 mL) at 0 C., and then 2,2,2-trifluoro-N-phenylacetimidoyl chloride (171 L, 1.14 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (171 L, 1.14 mmol) were added. A reaction was carried out at 0 C. for 3 h, the mixture was concentrated under vacuum, and then purified by silica gel column chromatography (petroleum ether/ethyl acetate=20/1 to 5/1, v/v) to obtain trifluoroacetamide ester. The trifluoroacetamide ester and N-benzyl-N-benzyloxycarbonyl-5-aminopentanol (187.3 mg, 0.572 mmol) were co-evaporated with toluene three times, and then dissolved in anhydrous DCM (1 mL). A preactivated dry molecular sieve 4 and Ph.sub.3OP (848 mg, 3.05 mmol) were added. Then TMSI (56.5 L, 0.38 mmol, 1.0 eq) was slowly added to the mixture. The mixture was stirred for reaction at room temperature until TLC analysis indicates complete reaction. The solution was diluted, and the reaction was quenched with saturated Na.sub.2S.sub.2O.sub.3. The organic phase was washed with water and brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, concentrated under vacuum, and purified by silica gel column chromatography (petroleum ether/ethyl acetate=20/1 to 1/1, v/v) to obtain compound 15 (203 mg, 0.28 mmol, three-step yield: 73%). [].sup.25.sub.D=+11.1 (c=1.0, CHCl.sub.3); IR vmax (film) 3029, 2944, 2903, 2108, 1697, 1454, 1361, 1279, 1227, 1127, 1096, 1044, 820, 755, 698 cm.sup.1; .sup.1H NMR (400 MHZ, Chloroform-d) 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, ArCH.sub.2), 5.00 (d, J=11.9 Hz, 1H, ArCH.sub.2), 4.88 (d, J=11.9 Hz, 1H, ArCH.sub.2), 4.81 (d, J=12.0 Hz, 1H, ArCH.sub.2), 4.70-4.60 (m, 2H, ArCH.sub.2, 1-H), 4.48 (d, J=8.0 Hz, 2H, ArCH.sub.2), 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-OCH.sub.2), 3.34 (s, 1H, Linker-OCH.sub.2), 3.27-3.14 (m, 2H, Linker-NCH.sub.2), 1.57 (m, 4H, Linker-CH.sub.2) 1.25 (m, 2H, Linker-CH.sub.2), 1.19 (d, J=6.4 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (101 MHZ, Chloroform-d) 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 V, 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 (ArCH.sub.2), 73.3 (ArCH.sub.2), 68.2 (ArCH.sub.2), 67.2 (ArCH.sub.2), 65.2 (4-H), 64.3 (5-H), 50.2 (ArCH.sub.2) 29.1 (Linker-CH.sub.2), 23.5 (Linker-CH.sub.2), 17.3 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.44H.sub.48O.sub.6N.sub.4Na.sup.+ (M+Na).sup.+: 751.3466 found: 751.3501.

[0062] Compound 16: H.sub.2O (1 mL) was added to a solution of compound 15 (16.75 mg, 23.0 mol) in DCM (2.0 mL), followed by addition of DDQ (7.7 mg, 35.0 mol). The reaction mixture was stirred at room temperature for 5 h, after which TLC indicated that the reaction was complete. The mixture was diluted with DCM (210 mL) and washed with saturated NaHCO.sub.3 (20 mL). An organic layer was dried over anhydrous Na.sub.2SO.sub.4, and the reaction mixture was concentrated under vacuum. A residue was purified by silica gel column chromatography (petroleum ether: acetone=1:1) to obtain compound 16 (11.9 mg, 20.2 mol, 88%). [].sup.25.sub.D=+30.8 (c=0.3, CHCl.sub.3); IR vmax (film) 3029, 2944, 2903, 2108, 1697, 1454, 1422, 1361, 1279, 1227, 1127, 1096, 1044, 820,755, 698 cm.sup.1; .sup.1H NMR (400 MHZ, Chloroform-d) 7.44-7.10 (m, 17H, Ar), 5.17 (d, J=10.3 Hz, 2H, ArCH.sub.2), 4.74-4.56 (m, 3H, ArCH.sub.2, 1-H), 4.48 (d, J=7.5 Hz, 2H, ArCH.sub.2), 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- OCH.sub.2), 3.31-3.12 (m, 3H, Linker-OCH.sub.2, Linker-NCH.sub.2), 2.52 (s, 1H, 3-OH), 1.51 (m, 4H, Linker-CH.sub.2) 1.23 (d, J=6.5 Hz, 5H, Linker-CH.sub.2, 6-CH.sub.3). .sup.13C NMR (101 MHZ, Chloroform-d) 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 (ArCH.sub.2), 69.9 (3-H), 68.2 (ArCH.sub.2), 67.2 (ArCH.sub.2), 65.9 (4-C), 64.6 (5-C), 50.3 (ArCH.sub.2), 47.1 (Linker-OCH.sub.2), 29.2 (Linker-CH.sub.2), 23.5 (Linker-CH.sub.2), 17.3 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.33H.sub.40O.sub.6N.sub.4Na.sup.+ (M+Na).sup.+: 611.2840 found: 611.2895.

[0063] Compound 17: Selenoglycoside 12 (49 mg, 0.08 mmol) and acceptor 16 (56.5 mg, 0.10 mmol) were mixed, azeotropically evaporated with toluene (35 mL) to remove water, and dried under vacuum using an oil pump for 2 h. The donor-acceptor mixture was dissolved in DCM (8 mL) at 0 C., a freshly activated 4 molecular sieve was added, and then NIS (27 mg, 0.12 mmol) and TfOH (14.2 L, 0.16 mmol) were slowly added. After stirring for 4 h, a drop of triethylamine (Et.sub.3N) was added to the reaction system for neutralization, and the 4 molecular sieve was filtered. A filtrate was washed with a 10% Na.sub.2S.sub.2O.sub.3 solution and a saturated NaHCO.sub.3 solution, respectively. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. A crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to obtain compound 17 (63.6 mg, 0.061 mmol, 76% yield). [].sup.25.sub.D=+43.7 (c=0.6, CHCl.sub.3); IR vmax (film) 3032, 2947, 2903, 2108, 1678, 1454, 1422, 1361, 1279, 1226, 1127, 1096, 1044, 820,755, 698 cm.sup.1; .sup.1H NMR (600 MHZ, Chloroform-d) 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, ArCH.sub.2), 4.97 (d, J=10.8 Hz, 1H, ArCH.sub.2), 4.86 (d, J=10.7 Hz, 1H, ArCH.sub.2), 4.77 (d, J=12.2 Hz, 1H, ArCH.sub.2), 4.53-4.38 (m, 4H, 1-H, ArCH.sub.2), 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-OCH.sub.2), 3.30-3.10 (m, 4H, 4-H Linker-OCH.sub.2, Linker-NCH.sub.2), 1.51 (dt, J=27.9, 6.9 Hz, 4H, Linker-CH.sub.2), 1.39 (d, J=5.6 Hz, 3H, 6-H), 1.26 (d, J=10.0 Hz, 2H, Linker-CH.sub.2), 1.17 (d, J=6.2 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (151 MHZ, Chloroform-d) 161.7 (NHCO), 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 (ArCH.sub.2), 73.3 (ArCH.sub.2), 70.8 (5-H), 68.7 (4-H), 68.2 (Linker-OCH.sub.2), 67.2 (ArCH.sub.2), 66.5 (4-C), 63.9 (5-C), 59.6 (2-H), 50.3 (ArCH.sub.2), 46.1 (Linker-NCH.sub.2), 29.1 (Linker-CH.sub.2), 27.5 (Linker-CH.sub.2), 23.4 (Linker-CH.sub.2), 18.5 (6-C), 17.2 (6-C). HR-ESI-MS (m/z): calcd for C.sub.52H.sub.57O.sub.9N.sub.8Cl.sub.3Na.sup.+ (M+Na).sup.+: 1065.3206 found: 1065.3227.

[0064] Compound 5: Compound 17 (40 mg, 38.38 mol) was dissolved in a mixed solution of THF/Ac.sub.2O/AcOH (3/2/1, v/v/v, 3 mL) and freshly activated Zn (1 g) was added. After being stirred overnight at room temperature, the mixture was diluted with DCM and filtered. A filtrate was washed with a saturated NaHCO.sub.3 aqueous solution and saturated brine. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, evaporated under vacuum, and purified by silica gel column chromatography (DCM/MeOH=50/1 to 10/1, v/v) to obtain a compound intermediate. Then the compound intermediate was dissolved in a DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL) solution and 10% Pd/C (50 mg) was added to the solution. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered, washed with water, and concentrated. A crude product was purified using a Sep-Pak column C18 (Macherey-Nagel, Dren, Germany) with water and methanol as eluents to obtain compound 5 (11.9 mg, 23.03 mol, two-step yield: 60%). .sup.1H NMR (600 MHZ, Deuterium Oxide) 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-OCH.sub.2), 3.57-3.45 (m, 4H, 3-H, 4-H, 5-H, Linker-OCH.sub.2), 2.96 (t, J=7.7 Hz, 2H, Linker-NCH.sub.2), 2.03 (s, 3H, NHAc-CH.sub.3), 2.00-1.96 (m, 6H, NHAc-CH.sub.3), 1.64 (tq, J=14.1, 6.9, 6.0 Hz, 4H, Linker-CH.sub.2), 1.41 (dh, J=14.1, 6.8 Hz, 2H, Linker-CH.sub.2), 1.15 (d, J=5.6 Hz, 3H, 6-H), 1.04 (d, J=6.5 Hz, 3H, 6-H). .sup.13C NMR (151 MHz, Deuterium Oxide) 174.8 (NHCO), 174.6 (NHCO), 101.0 (1-C), 98.2 (1-C), 77.0 (3-C), 71.5-71.1 (3-C, 5-C), 68.1 (Linker-OCH.sub.2), 67.1 (2-C), 65.5 (5-C), 57.1 (4-H), 56.5 (2-H), 52.5 (4-H), 39.4 (Linker-NCH.sub.2), 28.1 (Linker-CH.sub.2), 26.5 (Linker-CH.sub.2), 22.3 (NHAc-CH.sub.3), 22.2 (Linker-CH.sub.2), 22.1 (NHAc-CH.sub.3), 21.9 (NHAc-CH.sub.3), 16.9 (6-CH.sub.3), 15.4 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.23H.sub.43O.sub.9N.sub.4.sup.+ (M+H).sup.+: 519.3025 found: 519.3052.

[0065] Compound 8: Compound 16 (30 mg, 51.0 mol) was dissolved in a mixed solution of THF/AC.sub.2O/AcOH (3/2/1, v/v/v, 3 mL) and freshly activated Zn (1 g) was added. After being stirred overnight at room temperature, the mixture was diluted with DCM and filtered. A filtrate was washed with a saturated NaHCO.sub.3 aqueous solution and saturated brine. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, evaporated under vacuum, and purified by silica gel column chromatography (DCM/MeOH=50/1 to 10/1, v/v) to obtain a compound intermediate. Then the compound intermediate was dissolved in a DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL) solution and 10% Pd/C (50 mg) was added to the solution. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered, washed with water, and concentrated. A crude product was purified using a Sep-Pak column C18 (Macherey-Nagel, Dren, Germany) with water and methanol as eluents to obtain compound 8 (7.5 mg, 26.0 mol, two-step yield: 51%). .sup.1H NMR (600 MHZ, Deuterium Oxide) 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-OCH.sub.2), 3.46 (m, J=11.4, 7.7, 3.7 Hz, 1H, Linker-OCH.sub.2), 2.95 (t, J=7.7 Hz, 2H, Linker-NCH.sub.2), 2.04 (s, 3H, NHAc-CH.sub.3), 1.62 (dp, J=20.4, 6.9 Hz, 4H, Linker-CH.sub.2), 1.48-1.33 (m, 2H, Linker-CH.sub.2), 1.05 (dd, J=6.6, 1.9 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (151 MHZ, Deuterium Oxide) 175.6 (NHCO), 98.3 (1-H), 68.7 (2-H), 68.4 (3-H), 68.1 (Linker-OCH.sub.2), 65.3 (5-H), 54.0 (4-H), 39.4 (Linker-NCH.sub.2), 28.1 (Linker-CH.sub.2), 26.5 (Linker-CH.sub.2), 22.4 (Linker-CH.sub.2), 21.9 (NHAc-CH.sub.3), 15.6 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.13H.sub.27O.sub.5N.sub.2+ (M+H).sup.+: 291.1914 found: 291.1952.

[0066] Compound 20: Under nitrogen protection, compound 15 (180 mg, 0.25 mmol) was dissolved in a solution of pyridine (8 mL). Subsequently, water (2 mL), Et.sub.3N (1.51 ml, 10.87 mmol), and 1,3-propanedithiol (1.48 ml, 14.8 mmol) were added to the reaction system, and stirred at room temperature for 6 h. The reaction mixture was concentrated and a residue was purified by silica gel column chromatography (DCM:MeOH=20:1, v/v) to obtain an amino sugar. Subsequently, sodium bicarbonate (62 mg, 0.74 mmol) was added to a solution of (R)-3-O-benzyl butyric acid 18 (96 mg, 0.49 mmol) and the amino sugar in acetonitrile (20 mL), and stirred at room temperature. After 10 min, HOBt (6.7 mg, 49.4 mol) and EDC.HCl (57 mg, 0.30 mmol) were sequentially added and stirred at the same temperature for 6 h. After TLC detected that the reaction was completed, a crude product was dissolved in ethyl acetate and washed with a saturated sodium chloride solution. A separated organic layer was dried over anhydrous Na.sub.2SO.sub.4, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate=1:1) to obtain compound 20 (188.9 mg, 0.22 mmol, 87% yield). [].sup.25.sub.D=+114.5 (c=0.3, CHCl.sub.3); IR vmax (film) 3029, 2938, 2109, 1697, 1539, 1454, 1361, 1217, 1102, 1044, 819, 756, 698 cm.sup.1; .sup.1H NMR (400 MHZ, Chloroform-d) 7.88-7.73 (m, 4H, ArH), 7.55-7.14 (m, 24H, ArH), 6.73 (d, J=10.3 Hz, 1H, NHAc-H), 5.20 (d, J=7.9 Hz, 2H, ArCH.sub.2), 4.99 (d, J=11.3 Hz, 1H, ArCH.sub.2), 4.66 (td, J=13.8, 12.6, 3.0 Hz, 3H, ArCH.sub.2,4-H), 4.59-4.42 (m, 6H, ArCH.sub.2, 1-H), 4.09-3.93 (m, 3H, 5-H, 3-H, RHb-3), 3.63-3.47 (m, 1H, Linker-OCH.sub.2), 3.42-3.14 (m, 4H, Linker-OCH.sub.2, Linker-NCH.sub.2, 2-H), 2.66-2.47 (m, 2H, RHb-2), 1.65-1.50 (m, 4H, Linker-CH.sub.2), 1.27 (d, J=6.3 Hz, 5H, Linker-CH.sub.2, RHb-4), 1.13 (d, J=6.4 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (101 MHZ, Chloroform-d) 171.6 (NHCO), 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 (ArCH.sub.2), 72.7, 71.5 (ArCH.sub.2), 70.7 (ArCH.sub.2), 68.2 (Linker-OCH.sub.2), 67.2 (ArCH.sub.2), 64.5 (5-H), 50.4 (ArCH.sub.2,4-H), 47.2 (Linker-NCH.sub.2), 43.7 (RHb-2), 29.1 (Linker-CH.sub.2), 27.6 (Linker-CH.sub.2), 23.5 (Linker-CH.sub.2), 18.9 (RHb-4), 16.8 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.55H.sub.63O.sub.8N.sub.2.sup.+ (M+H).sup.+: 879.4579 found: 879.4635

[0067] Compound 21: Under nitrogen protection, compound 15 (150 mg, 0.21 mmol) was dissolved in a solution of pyridine (8 mL). Subsequently, water (2 mL), Et.sub.3N (1.26 ml, 9.06 mmol), and 1,3-propanedithiol (1.24 mL, 12.36 mmol) were added to the reaction system, and stirred at room temperature for 6 h. The reaction mixture was concentrated and a residue was purified by silica gel column chromatography (DCM:MeOH=20:1, v/v) to obtain an amino sugar. Subsequently, sodium bicarbonate (52 mg, 0.62 mmol) was added to a solution of(S)-3-O-benzyl butyric acid 19 (80 mg, 0.41 mmol) and the amino sugar in acetonitrile (17 mL), and stirred at room temperature. After 10 min, HOBt (5.6 mg, 41.2 mol) and EDC (64 mg, 0.33 mmol) were added sequentially and stirred at the same temperature for 6 h. After TLC detected that the reaction was completed, a crude product was dissolved in ethyl acetate and washed with a saturated sodium chloride solution. A separated organic layer was dried over anhydrous Na.sub.2SO.sub.4, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate=1:1) to obtain compound 21 (150.2 mg, 0.17 mmol, 83% yield). [].sup.25.sub.D=+80.1 (c=0.75, CHCl.sub.3); IR vmax (film) 3029, 2942, 2868, 2109, 1697, 1532, 1454, 1361, 1217, 1104, 1045, 818, 755, 698 cm.sup.1; .sup.1H NMR (400 MHZ, Chloroform-d) 7.85-7.70 (m, 4H ArH), 7.54-7.13 (m, 24H ArH), 6.59 (d, J=10.1 Hz, 1H, NHAcH), 5.17 (d, J=8.5 Hz, 2H, ArCH.sub.2), 4.95 (d, J=11.3 Hz, 1H, ArCH.sub.2), 4.61 (m, 4H, 4-H.1-H. ArCH.sub.2), 4.43 (m, 5H, ArCH.sub.2), 4.10-3.84 (m, 3H, 5-H, 3-H, RHb-3), 3.54 (d, J=9.4 Hz, 1H, Linker-OCH.sub.2), 3.40-3.13 (m, 4H, Linker-OCH.sub.2, Linker-NCH.sub.2, 2-H), 2.54 (m, J=5.4, 2.1 Hz, 2H, RHb-2), 1.54 (s, 4H, Linker-CH.sub.2), 1.37-1.19 (m, 5H, Linker-CH.sub.2, RHb-4), 1.15 (d, J=6.4 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (101 MHZ, Chloroform-d) 171.7 (NHCO), 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 (ArCH.sub.2), 71.4 (ArCH.sub.2), 70.4 (ArCH.sub.2), 68.2 (Linker-OCH.sub.2), 67.1 (ArCH.sub.2), 64.5 (5-C), 50.2 (4-C, ArCH.sub.2), 46.2 (Linker-NCH.sub.2), 43.7 (RHb-2), 29.1 (Linker-CH.sub.2), 23.5 (Linker-CH.sub.2), 19.7 (RHb-4), 16.9 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.55H.sub.62O.sub.8N.sub.2Na.sup.+ (M+Na).sup.+: 901.4398 found: 901.4446.

[0068] Compound 9: Compound 20 (30 mg, 34.15 mol) was dissolved in a mixed solution of DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v), and then 10% Pd/C (50 mg) was added. After being stirred for 36 h under a hydrogen (4 atm) atmosphere, the mixture was filtered and washed with water. Then, a residue was purified using a Sep-Pak column C18 (Macherey-Nagel, Germany) with water and methanol as eluents to obtain compound 9 (10.2 mg, 30.4 mol, 89%). .sup.1H NMR (400 MHz, Deuterium Oxide) 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-OCH.sub.2), 3.40 (dt, J=10.6, 6.2 Hz, 1H, Linker-OCH.sub.2), 2.89 (t, J=7.6 Hz, 2H, Linker-NCH.sub.2), 2.46-2.36 (m, 2H, RHb-2), 1.57 (dq, J=12.6, 6.7, 5.5 Hz, 4H, Linker-CH.sub.2), 1.33 (dp, J=13.5, 6.6 Hz, 2H, Linker-CH.sub.2), 1.13 (d, J=6.1 Hz, 3H, RHb-4), 1.00 (d, J=6.3 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (101 MHZ, Deuterium Oxide) 98.3 (1-H), 68.6 (2-H), 68.4 (3-H), 68.0 (Linker-OCH.sub.2), 65.0 (5-C, RHb-3), 53.8 (4-C), 44.7 (RHb-2), 39.3 (Linker-NCH.sub.2), 28.0 (Linker-CH.sub.2), 26.5 (Linker-CH.sub.2), 22.4 (Linker-CH.sub.2), 22.1 (RHb-4), 15.5 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.15H.sub.31O.sub.6N.sub.2+ (M+H).sup.+: 335.2177 found: 335.2219.

[0069] Compound 11: Compound 21 (30 mg, 34.15 mol) was dissolved in a mixed solution of DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL), and 10% Pd/C (50 mg) was added. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water. A residue was purified using a Sep-Pak column C18 (Macherey-Nagel, Germany) with water and methanol as eluents to obtain compound 11 (10.4 mg, 31.08 mol, 91% yield). .sup.1H NMR (400 MHZ, Deuterium Oxide) 4.92 (d, J=4.0 Hz, 1H, 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, 1H, 3-H), 3.78-3.65 (m, 2H, 2-H, Linker-OCH.sub.2), 3.53 (dt, J=9.9, 6.2 Hz, 1H, Linker-OCH.sub.2), 3.02 (t, J=7.6 Hz, 2H, Linker-NCH.sub.2), 2.61-2.48 (m, 2H, RHb-2), 1.76-1.65 (m, 4H, Linker-CH.sub.2), 1.56-1.39 (m, 2H, Linker-CH.sub.2), 1.25 (d, J=6.2 Hz, 3H, RHb-4), 1.13 (d, J=6.4 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (101 MHz, Deuterium Oxide) 175.3 (NHCO), 98.3 (1-C), 68.6 (3-C), 68.4 (2-C), 68.0 (Linker-OCH.sub.2), 65.2-65.0 (5-C, RHb-3), 53.9 (4-C), 44.5 (RHb-2), 39.3 (Linker-NCH.sub.2), 28.0 (Linker-CH.sub.2), 26.5 (Linker-CH.sub.2), 22.4 (Linker-CH.sub.2), 21.9 (RHb-4), 15.6 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.15H.sub.31O.sub.6N.sub.2.sup.+ (M+H).sup.+: 335.2177 found: 335.2214.

[0070] Compound 22: H.sub.2O (1 mL) was added to a solution of compound 20 (150 mg, 0.17 mmol) in DCM (2.0 mL), followed by addition of DDQ (58 mg, 0.26 mmol). The reaction mixture was stirred at room temperature for 5 h, after which TLC indicated that the reaction was complete. The mixture was diluted with DCM (210 mL) and washed with saturated NaHCO.sub.3 (20 mL). An organic layer was dried over anhydrous Na.sub.2SO.sub.4, and the reaction mixture was concentrated under vacuum. A residue was purified by silica gel column chromatography (petroleum ether: acetone=1:1) to obtain compound 22 (116.7 mg, 0.16 mmol, 93% yield). [].sup.25.sub.D=+33 (c=1.2, CHCl.sub.3); IR vmax (film) 3030, 2923, 1697, 1540, 1453, 1361, 1216, 1100, 1037, 819,736, 697 cm.sup.1; .sup.1H NMR (600 MHZ, Chloroform-d) 7.39-7.13 (m, 20H, ArH), 6.72 (d, J=9.4 Hz, 1H, NHH), 5.17 (d, J=16.9 Hz, 2H, ArCH.sub.2), 4.59 (d, J=11.3 Hz, 1H, ArCH.sub.2), 4.55-4.42 (m, 6H, ArCH.sub.2, 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-OCH.sub.2), 3.30-3.14 (m, 4H, 2-H, linker-OCH.sub.2, linker-NCH.sub.2), 2.61-2.44 (m, 2H, RHb-2), 1.59-1.47 (m, 4H, linker-CH.sub.2), 1.36-1.25 (m, 5H, RHb-4, linker-CH.sub.2), 1.06 (d, J=6.4 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (151 MHz, Chloroform-d) 172.9 (NHCO), 156.8 (Cbz-CO), 156.2 (Cbz-CO), 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 (RHb-3), 72.4 (ArCH.sub.2), 70.7 (ArCH.sub.2), 69.8 (3-C), 68.2 (linker-OCH.sub.2), 67.2 (linker-OCH.sub.2), 64.2 (5-C), 53.7 (4-C), 50.3 (ArCH.sub.2), 47.2 (linker-NCH.sub.2), 46.2 (linker-NCH.sub.2), 43.5 (RHb-2-CH.sub.2), 29.2 (linker-CH.sub.2), 28.0 (linker-CH.sub.2), 27.5 (linker-CH.sub.2), 23.5 (linker-CH.sub.2), 19.0 (RHb-4), 16.7 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.44H.sub.54O.sub.8N.sub.2Na.sup.+ (M+Na).sup.+: 761.3772 found: 761.3807.

[0071] Compound 23: Selenoglycoside 12 (156 mg, 0.26 mmol) and acceptor 22 (94.3 mg, 0.13 mmol) were mixed, azeotropically evaporated with toluene (35 mL) to remove water, and dried under vacuum using an oil pump for 2 h. The donor-acceptor mixture was dissolved in DCM (8 mL) at 0 C., a freshly activated 4 molecular sieve was added, and then NIS (57.4 mg, 0.26 mmol) and TfOH (22.6 L, 0.26 mmol) were slowly added. After stirring for 4 h, a drop of triethylamine (Et.sub.3N) was added to the reaction system for neutralization, and the 4 molecular sieve was filtered. A filtrate was washed with a 10% Na.sub.2S.sub.2O.sub.3 solution and a saturated NaHCO.sub.3 solution, respectively. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. A 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). [] 25)=+60.9 (c=1.1, CHCl.sub.3); IR vmax (film) 3029, 2938, 2870, 2107, 1681, 1525, 1454, 1361, 1217, 1093, 1042, 819, 755, 698 cm.sup.1; .sup.1H NMR (400 MHZ, Chloroform-d) 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-CH.sub.2), 4.96 (d, J=7.5 Hz, 1H, 1-H), 4.90 (d, J=10.8 Hz, 1H, ArCH.sub.2), 4.78 (d, J=10.9 Hz, 1H, ArCH.sub.2), 4.60 (d, J=11.1 Hz, 1H, ArCH.sub.2), 4.47 (m, 2H, ArCH.sub.2), 4.43 (m, 2H, 1-H,4-H), 4.35 (d, J=11.9 Hz, 1H, ArCH.sub.2), 4.24 (d, J=12.0 Hz, 1H, ArCH.sub.2), 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-OCH.sub.2), 3.26 (m, 3H, Linker-OCH.sub.2, 3-H, 5-H), 3.17 (m, 3H, 3-H, Linker-NCH.sub.2), 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-CH.sub.2), 1.35 (dd, J=9.9, 5.9 Hz, 6H, 6-CH.sub.3, RHb-4), 1.27 (s, 2H, Linker-CH.sub.2), 1.06 (d, J=6.4 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (101 MHZ, Chloroform-d) 171.5 (NHCO), 161.4 (NHCO), 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 (ArCH.sub.2), 73.7 (2-C), 72.9 (5-C), 72.1 (ArCH.sub.2), 70.8 (5-C), 70.7 (ArCH.sub.2), 68.1 (ArCH.sub.2), 68.0 (3-C), 67.2 (ArCH.sub.2), 65.0 (RHb-3), 58.4 (2-H), 52.7 (4-H), 50.3 (ArCH.sub.2), 47.3 (Linker-NCH.sub.2), 43.3 (RHb-2), 29.1 (Linker-CH.sub.2), 27.6 (Linker-CH.sub.2), 23.4 (Linker-CH.sub.2), 19.3 (RHb-4), 18.5 (6-CH.sub.3), 16.5 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.63H.sub.71O.sub.11N.sub.6Cl.sub.3Na.sup.+ (M+Na).sup.+: 1215.4139 found: 1215.4149.

[0072] Compound 6: Compound 23 (50 mg, 41.9 mol) was dissolved in a mixed solution of THF/AC.sub.2O/AcOH (3/2/1, v/v/v, 3 mL) and freshly activated Zn (1 g) was added. After being stirred overnight at room temperature, the mixture was diluted with DCM and filtered. A filtrate was washed with a saturated NaHCO.sub.3 aqueous solution and saturated brine. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, evaporated under vacuum, and purified by silica gel column chromatography (DCM/MeOH=50/1 to 10/1, v/v) to obtain a compound intermediate. Then the compound intermediate was dissolved in a DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL) solution and 10% Pd/C (50 mg) was added to the solution. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered, washed with water, and concentrated. A crude product was purified using a Sep-Pak column C18 (Macherey-Nagel, Dren, Germany) with water and methanol as eluents to obtain compound 6 (12 mg, 21.4 mol, two-step yield: 51% yield). .sup.1H NMR (400 MHZ, Deuterium Oxide) 4.70 (d, 1H, 1-H), 4.60 (d, J=8.2 Hz, 1H, 1-H), 4.27 (d, J=4.7 Hz, 1H, 4-H), 4.08 (p, J=6.5, 6.0 Hz, 2H, 5-H, RHb-3), 3.93 (dd, J=10.4, 4.7 Hz, 1H, 3-H), 3.72 (dd, J=10.4, 3.9 Hz, 1H, 2-H), 3.57 (dt, J=9.6, 6.5 Hz, 2H, 2-H, Linker-OCH.sub.2), 3.53-3.48 (m, 2H, 3-H, Linker-OCH.sub.2, 4-H, 5-H), 2.90 (t, J=7.6 Hz, 2H, Linker-NCH.sub.2), 2.38 (h, J=8.7, 8.1 Hz, 2H, RHb-2), 1.82 (m, 6H, NHAc-CH.sub.3), 1.58 (m, J=9.1, 8.5 Hz, 4H, Linker-CH.sub.2), 1.36 (m, J=7.5, 7.1 Hz, 2H, Linker-CH.sub.2), 1.15 (d, J=6.1 Hz, 3H, RHb-4), 1.08 (d, J=4.5 Hz, 3H, 6-CH.sub.3), 0.98 (d, J=6.4 Hz, 3H, 6-CH.sub.3). .sup.13C NMR (101 MHz, Deuterium Oxide) 174.6 (NHCO), 101.2 (1-C), 98.1 (1-C), 76.8 (5-C), 71.7-70.9 (3-C, 5-C), 68.0 (Linker-OCH.sub.2), 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-NCH.sub.2), 28.0 (Linker-CH.sub.2), 26.4 (Linker-CH.sub.2), 22.3 (Linker-CH.sub.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.sub.25H.sub.47O.sub.10N.sub.4.sup.+(M+H).sup.+: 563.3287 found: 563.3349.

Example 3

##STR00007##

[0073] Compounds 2 and 3 were synthesized, as shown in FIG. 5.

[0074] Compound 24 was synthesized by a known method (Cai Juntao, doctoral thesis, Jiangnan University, 2020). 2-Naphthalene methylene was selectively removed from compound 24 by DDQ to obtain disaccharide acceptor 25. The donor selenoglycoside 12 and acceptor 25 were catalyzed by TMSOTf and NIS to obtain trisaccharide 26. Subsequently, zinc powder, acetic acid, and acetic anhydride were used for conducting reductive acylation and catalytic hydrogenation on compounds 26 and 25, respectively to obtain deprotected target compounds 2 and 3.

Specific Experimental Operations and Steps:

[0075] Compound 25: H.sub.2O (2 mL) was added to a solution of compound 24 (380 mg, 0.38 mmol) in DCM (10 mL), followed by addition of DDQ (126 mg, 0.57 mmol). The reaction mixture was stirred at room temperature for 5 h, after which TLC indicated that the reaction was complete. The mixture was diluted with DCM (210 mL) and washed with saturated NaHCO.sub.3 (20 mL). An organic layer was dried over anhydrous Na.sub.2SO.sub.4, and the reaction mixture was concentrated under vacuum. A residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=1:1) to obtain compound 25 (213 mg, 0.25 mmol, 65% yield). [].sup.25.sub.D=16.2 (c=0.8, CHCl.sub.3); IR vmax (film) 3029, 2939, 2876, 2108, 1697, 1540, 1454, 1361, 1228, 1089, 1045, 830, 755, 699 cm.sup.1; .sup.1H NMR (600 MHZ, Chloroform-d) 7.46-7.09 (m, 24H, Ar), 6.36 (s, 1H, NHAc-H), 5.18-5.13 (m, 2H, ArCH.sub.2), 5.04 (d, J=3.5 Hz, 1H, 1-H), 4.96 (s, 1H, 1-H), 4.84 (d, J=11.8 Hz, 1H, ArCH.sub.2), 4.70 (s, 2H, ArCH.sub.2), 4.64 (d, J=11.7 Hz, 1H, ArCH.sub.2), 4.47 (d, J=9.3 Hz, 2H, ArCH.sub.2), 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, 3.3 Hz, 1H, 2-H), 3.71 (dd, J=3.8, 1.5 Hz, 1H, 4-H), 3.57 (m, 2H, 4-H, Linker-OCH.sub.2), 3.38 (m, 1H, Linker-OCH.sub.2), 3.19 (m, 2H, Linker-NCH.sub.2), 1.62 (s, 3H, NHAc-CH.sub.3), 1.55 (s, 4H, Linker-CH.sub.2), 1.34-1.24 (m, 2H, Linker-CH.sub.2), 1.15 (d, J=6.4 Hz, 3H, 6-H), 1.13-1.08 (m, 3H, 6-H). .sup.13C NMR (101 MHZ, Chloroform-d) 170.5 (NHCO), 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 (ArCH.sub.2), 70.1, 68.0 (ArCH.sub.2), 67.2 (ArCH.sub.2), 66.6, 66.4 (4-C), 66.3, 49.7 (2-C), 47.1 (Linker-NCH.sub.2), 29.3 (Linker-CH.sub.2), 23.5 (Linker-CH.sub.2), 22.8 (NHAc-CH.sub.3), 17.2 (6-CH.sub.3), 16.8 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.48H.sub.59O.sub.10N.sub.5Na.sup.+ (M+Na).sup.+: 888.4154 found: 888.4215.

[0076] Compound 26: Selenoglycoside 12 (122.4 mg, 0.2 mmol) and acceptor 25 (86.5 mg, 0.1 mmol) were mixed, azeotropically evaporated with toluene (35 mL) to remove water, and dried under vacuum using an oil pump for 2 h. The donor-acceptor mixture was dissolved in DCM (8 mL) at 0 C., a freshly activated 4 molecular sieve was added, and then NIS (45 mg, 0.2 mmol) and TfOH (17.7 L, 0.2 mmol) were slowly added. After stirring for 4 h, a drop of triethylamine (Et.sub.3N) was added to the reaction system for neutralization, and the 4 molecular sieve was filtered. A filtrate was washed with a 10% Na.sub.2S.sub.2O.sub.3 solution and a saturated NaHCO.sub.3 solution, respectively. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. A 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%). [].sup.25.sub.D=7.8 (c=0.9, CHCl.sub.3); IR vmax (film) 3029, 2938, 2880, 2108, 1697, 1658, 1525, 1454, 1361, 1231, 1096, 1048, 822, 757, 699 cm.sup.1; .sup.1H NMR (400 MHZ, Methanol-d.sub.4) 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, ArCH.sub.2), 4.98 (d, J=13.4 Hz, 3H, ArCH.sub.2, 1-H), 4.94-4.82 (m, 4H, ArCH.sub.2, 1-H, 1-H), 4.75 (dd, J=29.5, 11.7 Hz, 2H, ArCH.sub.2), 4.64 (d, J=12.0 Hz, 1H, ArCH.sub.2), 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-OCH.sub.2), 3.36 (m, 1H, Linker-OCH.sub.2), 3.27 (dt, J=11.3, 5.9 Hz, 4H, Linker-OCH.sub.2, 4-H, 5-H), 1.83-1.72 (m, 3H, NHAc), 1.53 (s, 4H, Linker-CH.sub.2), 1.41-1.32 (m, 2H, Linker-CH.sub.2), 1.29-1.24 (m, 3H, 6-H), 1.23-1.16 (m, 3H, 6-H), 1.12 (s, 3H, 6-H). .sup.13C NMR (101 MHZ, Methanol-d.sub.4) 172.1 (NHCO), 162.6 (NHCO), 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 (ArCH.sub.2, 2-C), 74.4, 70.5, 68.1, 67.0, 66.7 (ArCH.sub.2), 65.3 (5-H), 58.3, 49.0 (2-C), 47.8 (Linker-NCH.sub.2), 28.5 (Linker-CH.sub.2), 21.9 (NHAc-CH.sub.3), 17.3 (6-CH.sub.3), 16.1 (6-CH.sub.3), 15.97 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.67H.sub.76O.sub.13N.sub.9Cl.sub.3Na.sup.+ (M+Na).sup.+: 1342.4520 found: 1342.4535.

[0077] Compound 2: Compound 26 (30 mg, 22.7 mol) was dissolved in a mixed solution of THF/AC.sub.2O/AcOH (3/2/1, v/v/v, 3 mL) and freshly activated Zn (0.5 g) was added. After being stirred overnight at room temperature, the mixture was diluted with DCM and filtered. A filtrate was washed with a saturated NaHCO.sub.3 aqueous solution and saturated brine. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, evaporated under vacuum, and purified by silica gel column chromatography (DCM/MeOH=50/1 to 10/1, v/v) to obtain a compound intermediate. Then the compound intermediate was dissolved in a DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL) solution and 10% Pd/C (50 mg) was added to the solution. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered, washed with water, and concentrated. A crude product was purified using a Sep-Pak column C18 (Macherey-Nagel, Dren, Germany) with water and methanol as eluents to obtain compound 2 (8 mg, 11.35 mol, two-step yield: 50%). .sup.1H NMR (600 MHZ, Deuterium Oxide) 4.88 (d, J=4.4 Hz, 1H, 1-H), 4.76 (d, 1-H), 4.55 (d, J=8.5 Hz, 1H, 1-H), 4.24 (dd, J=4.8, 1.7 Hz, 1H, 4-H), 4.17 (dd, J=11.1, 3.8 Hz, 1H, 2-H), 4.12 (tt, J=6.6, 3.7 Hz, 1H, 5-H), 4.02-3.95 (m, 2H, 3-H,5-H), 3.79 (dd, J=11.1, 3.2 Hz, 1H, 3-H), 3.70 (d, J=3.2 Hz, 1H, 4-H), 3.66-3.62 (m, 1H, 2-H), 3.62-3.52 (m, 2H, 2-H Linker-OCH.sub.2), 3.48-3.32 (m, 4H, 3-H, 4-H, 5-H, Linker-OCH.sub.2), 2.87 (t, J=7.7 Hz, 2H, Linker-NCH.sub.2), 1.94 (s, 3H, NHAc-CH.sub.3), 1.90 (d, J=7.5 Hz, 9H, NHAc-CH.sub.3), 1.60-1.47 (m, 4H, Linker-CH.sub.2), 1.32 (tq, J=14.2, 7.5, 6.4 Hz, 2H, Linker-CH.sub.2), 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). .sup.13C NMR (151 MHZ, Deuterium Oxide) 174.8 (NHCO), 174.7 (NHCO), 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-OCH.sub.2), 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-NCH.sub.2), 28.0 (Linker-CH.sub.2), 26.5 (Linker-CH.sub.2), 22.3 (Linker-CH.sub.2), 22.2 (NHAc-CH.sub.3), 22.1 (NHAc-CH.sub.3), 22.0 (NHAc-CH.sub.3), 21.9 (NHAc-CH.sub.3), 16.9 (6-CH.sub.3), 15.4 (6-CH.sub.3), 15.3 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.31H.sub.56O.sub.13N.sub.5.sup.+ (M+H).sup.+: 706.3869 found: 706.3881.

[0078] Compound 3: Compound 25 (50 mg, 57.8 mol) was dissolved in a mixed solution of THF/AC.sub.2O/AcOH (3/2/1, v/v/v, 3 mL) and freshly activated Zn (0.5 g) was added. After being stirred overnight at room temperature, the mixture was diluted with DCM and filtered. A filtrate was washed with a saturated NaHCO.sub.3 aqueous solution and saturated brine. A combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, evaporated under vacuum, and purified by silica gel column chromatography (DCM/MeOH=50/1 to 10/1, v/v) to obtain a compound intermediate. Then the compound intermediate was dissolved in a DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL) solution and 10% Pd/C (50 mg) was added to the solution. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered, washed with water, and concentrated. A crude product was purified using a Sep-Pak column C18 (Macherey-Nagel, Dren, Germany) with water and methanol as eluents to obtain compound 3 (14.9 mg, 31.2 mol, two-step yield: 54%). .sup.1H NMR (400 MHZ, Deuterium Oxide) 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-OCH.sub.2, 2-H), 3.38 (dt, J=10.0, 6.2 Hz, 1H, Linker-OCH.sub.2), 2.90 (t, J=7.6 Hz, 2H, Linker-NCH.sub.2), 1.96 (d, J=30.7 Hz, 6H, NHAc), 1.59 (m, J=15.4, 7.7 Hz, 4H, Linker-CH.sub.2), 1.34 (m, J=12.1, 7.5, 3.5 Hz, 2H, Linker-CH.sub.2), 1.13 (d, J=6.6 Hz, 3H, 6-H), 1.00 (d, J=6.2 Hz, 3H, 6-H). 13C NMR (101 MHz, Deuterium Oxide) 175.5 (NHCO), 174.6 (NHCO), 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-OCH.sub.2), 66.4 (5-C), 65.8 (5-C), 53.8 (4-C) 48.4 (2-C), 39.3 (Linker-NCH.sub.2), 28.0 (Linker-CH.sub.2), 26.4 (Linker-CH.sub.2), 22.2 (Linker-CH.sub.2), 21.9 (NHAc-CH.sub.3), 21.8 (NHAc-CH.sub.3), 15.4 (6-CH.sub.3), 15.3 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.21H.sub.40O.sub.9N.sub.3.sup.+ (M+H).sup.+: 478.2759 found: 478.2819.

Example 4

##STR00008##

[0079] Compounds 1, 4, and 7 were synthesized, as shown in FIG. 6.

[0080] Compounds 27, 28, and 29 were synthesized by known methods (Cai Juntao, doctoral thesis, Jiangnan University, 2020), and catalytic hydrogenation was conducted on compounds 27, 28, and 29, respectively to obtain deprotected target compounds 1, 4, and 7.

Specific Experimental Operations and Steps:

[0081] Compound 1: Trisaccharide 27 (30 mg, 20.41 mol) was dissolved in a mixed solution of tetrahydrofuran, acetic anhydride, and acetic acid (3/2/1, v/v/v, 3 mL), newly activated Zn (1 g) was added, and the mixture was stirred overnight at room temperature. After TLC detected that the reaction of raw materials was complete, the reaction solution was diluted with dichloromethane and filtered. A filtrate was washed with a saturated sodium bicarbonate solution and a saturated sodium chloride solution. Subsequently, a combined organic layer was dried over anhydrous sodium sulfate, filtered, evaporated under vacuum, and dried under vacuum using an oil pump. A crude product was dissolved in a mixed solution of dichloromethane, tert-butyl alcohol, and water (3/6/1, v/v/v, 2 mL), and an appropriate amount of 10% palladium on carbon was added to the solution. The solution was stirred for 36 h under a hydrogen (4 atm) atmosphere. Subsequently, the mixture was filtered with diatomite and washed with water three times, and the solvent was evaporated in vacuum. A residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL/min, and was eluted with ultrapure water (solvent A) containing 0.1% formic acid and acetonitrile (solvent B) in a linear gradient of the solvent B (10% to 30%) for 30 min to obtain compound 1 (9.9 mg, 13.27 mol, two-step yield: 65%). .sup.1H NMR (600 MHZ, Deuterium Oxide) 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 (m, J=6.8 Hz, 1H, 5-H), 4.12 (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-OCH.sub.2), 3.44 (m, J=28.2, 9.7, 4.4 Hz, 4H, 3-H, 4-H, 5-H, Linker-OCH.sub.2), 2.98-2.89 (m, 2H, Linker-NCH.sub.2), 2.45-2.36 (m, 2H, RHb-2), 1.94 (dt, J=6.4, 2.2 Hz, 9H, NHAc-CH.sub.3), 1.66-1.54 (m, 4H, Linker-CH.sub.2), 1.37 (q, J=7.4 Hz, 2H, Linker-CH.sub.2), 1.18 (m, 3H, RHb-4), 1.15 (m, 3H, 6-CH.sub.3), 1.11 (m, 3H, 6-CH.sub.3) 1.00 (m, 3H, 6-CH.sub.3). .sup.13C NMR (151 MHz, Deuterium Oxide) 174.6 (NHCO), 171.0 (NHCO), 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-OCH.sub.2), 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-NCH.sub.2), 28.0 (Linker-CH.sub.2), 26.5 (Linker-CH.sub.2), 22.3 (Linker-CH.sub.2), 22.2 (NHAc-CH.sub.3), 22.1 (NHAc-CH.sub.3), 22.0 (NHAc-CH.sub.3), 16.9 (6-H), 15.5 (6-H), 15.3 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.33H.sub.59N.sub.5O.sub.14Na.sup.+ (M+Na).sup.+: 772.3951 found: 772.3968.

[0082] Compound 4: Compound 28 (30 mg, 29.5 mol) was dissolved in a mixed solution of DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL), and 10% Pd/C (50 mg) was added to the reaction system. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water. A residue was purified using a Sep-Pak column C18 (Macherey-Nagel, Germany) with water and methanol as eluents to obtain compound 4 (13.1 mg, 25.11 mol, 85% yield). 1H NMR (600 MHz, Deuterium Oxide) 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-OCH.sub.2), 3.63-3.58 (m, 1H, 2-H), 3.47 (dt, J=11.0, 6.3 Hz, 1H, linker-OCH.sub.2), 2.99 (t, J=7.7 Hz, 2H, linker-NCH.sub.2), 2.55-2.45 (m, 2H, RHb-2), 2.00 (s, 2H, NHAc), 1.65 (dq, J=23.2, 7.6 Hz, 4H, linker-CH.sub.2), 1.42 (tq, J=14.6, 7.7, 7.1 Hz, 2H, linker-CH.sub.2), 1.22 (dd, J=13.0, 6.2 Hz, 6H, RHb-4, 6H), 1.09 (s, 3H, 6-H). .sup.13C NMR (151 MHz, Deuterium Oxide) 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-OCH.sub.2), 66.5 (5-C), 65.8, 65.0 (RHb-3), 53.8, 48.5, 44.7 (RHb-2), 39.4 (linker-CH.sub.2), 28.0 (linker-CH.sub.2), 26.4 (linker-CH.sub.2), 22.1-22.0 (NHAc, linker-CH.sub.2, RHb-4), 15.5 (6-H), 15.3 (6-H). HR-ESI-MS (m/z): calcd for C.sub.23H.sub.44O.sub.10N.sub.3.sup.+ (M+H).sup.+: 522.3021 found: 522.3083.

[0083] Compound 7: Compound 29 (20 mg, 33.10 mol) was dissolved in a mixed solution of DCM/t-BuOH/H.sub.2O (2/1/1, v/v/v, 2 mL), and 10% Pd/C (50 mg) was added to the reaction system. After being stirred in hydrogen (4 atm) for 36 h, the mixture was filtered and washed with water. A residue was purified using a Sep-Pak column C18 (Macherey-Nagel, Germany) with water and methanol as eluents to obtain compound 7 (9.1 mg, 31.5 mol, 95% yield). 1H NMR (400 MHZ, Deuterium Oxide) 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-OCH.sub.2), 3.47 (dt, J=11.1, 6.6 Hz, 1H, linker-OCH.sub.2), 3.00 (t, J=7.8 Hz, 2H, linker-NCH.sub.2), 2.04 (s, 3H, NHAc), 1.66 (dp, J=21.8, 8.0 Hz, 4H, linker-CH.sub.2), 1.44 (p, J=7.9 Hz, 2H, linker-CH.sub.2), 1.23 (d, J=6.6 Hz, 3H, 6-H). .sup.13C NMR (101 MHz, Deuterium Oxide) 174.6 (NHAc-CO), 96.9 (1-C), 71.1 (4-C), 67.8 (3-C, linker-OCH.sub.2), 66.6 (5-C), 49.8 (2-C), 39.4 (linker-NCH.sub.2), 28.0 (linker-CH.sub.2), 26.5 (linker-CH.sub.2), 22.3 (linker-CH.sub.2), 22.0 (NHAc-CH.sub.3), 15.5 (6-CH.sub.3). HR-ESI-MS (m/z): calcd for C.sub.13H.sub.27O.sub.5N.sub.2.sup.+ (M+H).sup.+: 291.1914 found: 291.1946.

Example 5

[0084] Lipopolysaccharide (LPS) and O-antigen (OPS) of V. cholerae O.sub.100 serotype were extracted. The 1H-NMR and 13C-NMR spectra of the OPS are shown in FIG. 7A-B.

[0085] An inactivated strain of V. cholerae O100 serotype was provided by Nankai University, and LPS was extracted by a hot phenol-water method as reported previously. The strain was suspended in sterile water, and after multiple freezing and thawing cycles, a bacterial suspension and 90% phenol were mixed and shaken at 68 C. for 30 min. The mixture was cooled and centrifuged, and an aqueous phase was collected. An equal volume of sterile water was added to an organic phase and shaken at 68 C. for 30 min. The mixture was cooled and centrifuged again to separate an aqueous phase. The two aqueous phases were combined, dialyzed overnight with distilled water, and freeze-dried 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 min, and then cooled and centrifuged. A supernatant was extracted with water-saturated phenol. Following centrifugation, an aqueous phase was collected, dialyzed with distilled water, and freeze-dried to obtain purified LPS.

[0086] The LPS was de-lipidated with a 2% acetic acid aqueous solution at 100 C. until lipid A precipitated (3 h). Centrifugation (13,000 revolutions, 20 min) was carried out to remove precipitate, and the resulting product was purified using a G50 gel column to obtain OPS.

Example 6

[0087] Effective titers of antibodies in rabbit sera were evaluated via ELISA, as shown in FIG. 8.

[0088] 8 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. Experimental group: 4 New Zealand rabbits were subcutaneously injected with a mixture of LPS of V. cholerae O100 and Freund's adjuvant at a ratio of 1:1 at different sites every 14 days (LPS: 0.4 mg/animal), and immunized three times (day 0, day 14, and day 28); and blood was collected from the ear vein of the rabbits. Control group: 4 New Zealand rabbits were subcutaneously injected with a mixture of PBS and Freund's adjuvant at a ratio of 1:1 at different sites every 14 days (day 0, day 14, and day 28). The anti-sera were aliquoted and stored at 80 C. Sera were collected on days 0, 7, 14, 28, and 35, and IgG antibodies in the sera were detected via enzyme-linked immunosorbent assay (ELISA). A P/N value represents a ratio of absorbance between the experimental group and the control group. When the P/N value of the immune group/control group is greater than or equal to 2.1, it is considered that the immune response of the IgG antibodies to the LPS of V. cholerae O100 serotype is positive.

Specific Operations and Steps of ELISA

[0089] (1) Coating and washing: An ELISA plate was coated with antigens (20 g/ml, 100 l/well) at 4 C. for 24 h, and the plate was washed with PBST three times, and blotted dry on absorbent paper.

[0090] (2) Blocking and washing: A blocking solution (PBST containing 5% skim milk powder) was added to the coated ELISA plate (300 l/well), and the plate was blocked overnight at 4 C., washed 3 times with PBST, and blotted dry.

[0091] (3) Addition of sera to be tested: Sera diluted with 1% BSA-PBS (at a dilution ratio of 1:12,800) were added to the ELISA plate (100 l/well); a blank control (containing only 1% BSA-PBS) was also added to the ELISA plate; and the plate was incubated overnight at 4 C., washed 4 times, and blotted dry.

[0092] (4) Addition of ELISA secondary antibodies: Anti-rabbit HRP secondary antibodies diluted with 1% BSA-PBS (at a dilution ratio of 1:2,000) were added (100 l/well), and the plate was incubated at 37 C. for 1 h, washed 4 times, and blotted dry.

[0093] (5) Color development: A TMB color solution was added (200 l/well); the plate was incubated in the dark for 10 min, and then immediately quenched with 1 M diluted H.sub.2SO.sub.4 (50 l/well); and the absorbance was read at 450 nm using an ELISA reader.

[0094] (6) Cleaning: After treatment of the sample, the sulfuric acid in the 96 well plate was neutralized with an ammonium bicarbonate solution.

[0095] The results are shown in FIG. 8, where 1-1, 1-2, 1-3, and 1-4 represent rabbit serum samples from four parallel experiments in the experimental group, respectively. The results indicated that after 7 days of immunization of rabbits, the P/N values began to increase significantly, and after 14 days, the P/N values of all samples were greater than 2.1, indicating that the immune response of the IgG antibodies to the LPS of V. cholerae O100 serotype was positive.

Example 7

[0096] A specific saccharide antigen was screened by a glycan microarray, as shown in FIG. 9.

Specific Experimental Operations and Steps:

[0097] Synthetic oligosaccharides and LPS were dissolved in a coupling buffer (50 mM sodium phosphate, pH=8.5) for printing onto a CodeLink slide (SurModics Co., Ltd.) using an RMA-Arrayer 96 (Rayme China). Then the slide was incubated overnight in a humidifying chamber at 26 C. and 55% humidity. The slide and the microarray were incubated in a quenching buffer (50 nM Na.sub.2HPO.sub.4, 100 nM ethanolamine) at 50 C. for 1 h. After being washed with distilled water and centrifuged, the quenched slide was blocked with 3% BSA (w/v) in PBS at room temperature for 1 h. The slide was washed with PBST (0.1% Tween in PBS) once and with PBS twice. After being centrifuged, the slide was placed in a culture chamber (ProPlate). Rabbit sera were diluted with 1% PBS-BSA (w/v) at a ratio of 1:200 and added to an incubation chamber. Each sample had at least four replicates. The microarray was incubated overnight in a dark and humid room at 4 C. Following washing 3 times with PBST, goat anti-rabbit IgG (Thermo) secondary antibodies were added per well, and incubated in a dark and humid room at 37 C. for 60 min, where the secondary antibodies were diluted with 1% PBS-BSA (w/v) at a ratio of 1:400. Then, the slide was rinsed with PBST three times, rinsed with water three times within 15 min, and centrifuged. Finally, the microarray was scanned using LuxScan 10K/B (CapitalBio Technology). Image analysis was carried out using GenePix Pro 7 software (Molecular Devices).

[0098] As shown in FIG. 9, compounds 1, 4, 6, 9, and 11 that contain 3-hydroxybutyryl have significant antigenicity, while compounds 2, 3, 5, 7, 8, and 10 that lack 3-hydroxybutyryl cannot be recognized by the antibodies, indicating that the 3-hydroxybutyryl plays a critical role in antibody recognition. The non-reducing end disaccharide 6 exhibits strong antibody recognition capacity, and is considered as the minimal antigenic epitope. These findings provide important reference for the development of glycoconjugate V. cholerae vaccine.

[0099] The examples provided above are not intended to limit the scope covered by the present disclosure, nor are the steps described to limit the execution order. Apparent improvements made to the present disclosure by those skilled in the art based on existing common knowledge also fall within the scope of protection defined in the claims of the present disclosure.