WO2025260577A1 - 一种霍乱弧菌o100血清型o抗原寡糖的化学合成方法及应用 - Google Patents

一种霍乱弧菌o100血清型o抗原寡糖的化学合成方法及应用

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WO2025260577A1
WO2025260577A1 PCT/CN2024/127376 CN2024127376W WO2025260577A1 WO 2025260577 A1 WO2025260577 A1 WO 2025260577A1 CN 2024127376 W CN2024127376 W CN 2024127376W WO 2025260577 A1 WO2025260577 A1 WO 2025260577A1
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linker
reaction
dhh
compound
synthesis method
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尹健
胡静
陈国栋
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Jiangnan University
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention relates to a chemical synthesis method and application of oligosaccharides containing O antigen from Vibrio cholerae O100 serotype, belonging to the field of chemical technology.
  • Vibrio cholerae is the pathogen of cholera, which can cause cholera pandemics.
  • Cholera is an acute diarrheal disease characterized by watery diarrhea and potentially fatal dehydration (Qadri et al. Clin. Microbiol. Rev. 2022, 35(3), e00211-00221).
  • WHO's outbreak briefing 30 countries reported cholera cases in 2023, with 40,900 cases and 775 deaths reported in January 2024 alone.
  • OCVs oral cholera vaccines
  • glycoprotein conjugate vaccines have shown promising potential, with several marketed glycoprotein conjugate vaccines already available for disease prevention and treatment, such as the pneumococcal 13-valent glycoconjugate vaccine, the meningococcal glycoconjugate vaccine, and the Salmonella conjugate vaccine.
  • glycoprotein conjugate vaccines already available for disease prevention and treatment, such as the pneumococcal 13-valent glycoconjugate vaccine, the meningococcal glycoconjugate vaccine, and the Salmonella conjugate vaccine.
  • Using structurally well-defined synthetic oligosaccharides conjugated to protein carriers can enhance the immunogenicity of oligosaccharides and induce T-cell-dependent immune responses, and has been proven safe and reliable (Seeberger, Chem. Rev. 2021, 121(7), 3598-3626).
  • Vibrio cholerae is classified into more than 200 serotypes.
  • Perepelov et al. isolated and identified the structure of the trisaccharide repeat unit of the O antigen in Vibrio cholerae serotype O100, which is [ ⁇ 3)- ⁇ -d-QuipNAc4N(dHh)-(1 ⁇ 3)- ⁇ -d-Fucp4N(RHb)-(1 ⁇ 3)- ⁇ -l-FucpNAc-(1 ⁇ ], where RHb and dHh represent (R)-3-hydroxybutyryl and 3,5-dihydroxyhexanoyl, respectively (Perepelov et al. Carbohydr. Res. 2019, 472, 98-102).
  • This O-antigen trisaccharide contains two difficult-to-construct 1,2-cis- ⁇ -fucoside bonds and one easily broken 1,2-trans- ⁇ -D-quinoside bond, with four nitrogen atoms coupled to two rare modifying groups. These factors make the synthesis of the repeating unit of this O-antigen trisaccharide extremely challenging, and it has not yet been totally synthesized. Notably, the bacterial surface glycan modifying group is considered a potential immune target. The absolute configuration of the two chiral centers in the unique dHh modifying group remains unclear, and its immunological function is also poorly understood.
  • this invention relates to a chemical synthesis method for an oligosaccharide of the O antigen of Vibrio cholerae O100 serotype; and to the application of the synthesized oligosaccharide to elucidate the absolute configuration and immunological function of the dHh modified group.
  • this invention utilizes three monosaccharide building blocks and five carboxylic acid derivatives, through a series of orthogonal protection, selective assembly, and amide coupling processes, to synthesize four possible trisaccharide isomers and one derivative.
  • One object of the present invention is to provide a chemical synthesis method for oligosaccharides of cholerae O100 serotype O antigen, wherein the method...
  • the method uses three monosaccharide building blocks and five carboxylic acid derivatives as raw materials;
  • PG 2 , PG 3 , PG 4 , PG 6 , and PG 7 are temporary hydroxyl protecting groups, which can be one of benzyl (Bn), 2-naphthylmethyl (Nap), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), or triethylsilyl (TES).
  • Bn benzyl
  • Nap 2-naphthylmethyl
  • TBS tert-butyldimethylsilyl
  • TDPS tert-butyldiphenylsilyl
  • TES triethylsilyl
  • PG 8 , PG 9 , PG 11 , PG 12 , PG 14 , PG 15 , PG 17 , PG 18 , and PG 20 are temporary hydroxyl protecting groups, which can be one of benzyl (Bn), 2-naphthylmethyl (Nap), acetyl (Ac), benzoyl (Bz), neopentyl, 9-pentylmethoxycarbonyl (Fmoc), or 2-p-methoxybenzyl (PMB).
  • PG 1 is a temporary amino protecting group, which can be one of trichloroacetyl (TCA), dichloroacetyl (DCA), or chloroacetyl (CIAc);
  • PG 5 is a temporary amino protecting group, which can be one of acetyl (Ac), trichloroacetyl (TCA), dichloroacetyl (DCA), chloroacetyl (CIAc), trichloroethoxycarbonyl (Troc), phthaloyl (Phth), 9-fluorenylmethoxycarbonyl (Fmoc), or tert-butyloxycarbonyl (Boc);
  • the synthesis method includes the following steps:
  • the monosaccharide building block 8 was deprotected by the hydroxyl protecting group PG6 at position 3 to obtain receptor 14; receptor 14 was glycosylated with monosaccharide building block 7 to obtain disaccharide 15; the azide group in disaccharide 15 was reduced to an amino group using a reducing agent, and then amidated by compound 13 to obtain compound 16; the monosaccharide building block 7 at position 3 was deprotected from compound 16.
  • the hydroxyl protecting group PG4 is used to obtain the disaccharide receptor 17;
  • Disaccharide acceptor 17 and monosaccharide building block 6 undergo glycosylation reaction under the action of an activator to construct trisaccharide 18; then the azide group of trisaccharide 18 is reduced by a reducing agent, and any one of the carboxylic acid derivatives in formulas (9) to (12) is added for amidation to obtain compounds 19 to 22; compounds 19 to 22 are subjected to catalytic hydrogenation and deprotection to obtain target compounds 1 to 4.
  • trisaccharide 18 is reduced and acylated to convert the azide group to an acetamino group, followed by catalytic hydrogenation and deprotection to obtain target compound 5;
  • PG a and PG b are temporary hydroxyl protecting groups, which are independently selected from benzyl, 2-naphthylmethyl, acetyl, benzoyl, neopentanoyl, 9-pentomethoxycarbonyl, and 2-p-methoxybenzyl, respectively.
  • C1-4 alkoxy groups include methyl (Me), ethyl (Et), and tert-butyl (t-Bu); when PG 10 , PG 13 , PG 16 , PG 19 , and PG 21 are C1-4 alkoxy groups, the protecting group must be removed before amide coupling.
  • the disaccharide acceptor 17 is synthesized by the following method: under the catalysis of an activator and the effect of a solvent, acceptor 14 and donor 7 undergo a glycosylation reaction to obtain disaccharide 15; then, under the action of a reducing agent, the azide group in disaccharide 15 is reduced to an amino group to obtain an aminobiose intermediate; (R)-3-hydroxybutyric acid derivative 13 is activated by a condensing agent or prepared into an acyl halide, and condensed with the aminobiose intermediate amide to obtain compound 16; the PG 4 protecting group in compound 16 is removed to obtain acceptor 17; the corresponding synthetic route is shown below:
  • the disaccharide 15 is synthesized by utilizing solvent effects, catalysis of activating reagents, and orthogonal protection of non-participating groups.
  • the concentration of the glycosylation reaction is 0.01 to 0.1 M.
  • the activating agent is one of TMSOTf, NIS/TMSOTf, and NIS/TfOH.
  • the solvent is one or more of anhydrous dichloromethane, diethyl ether, toluene, methanol, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, or water.
  • anhydrous dichloromethane, diethyl ether, and toluene is preferred.
  • the molar ratio of the donor to the acceptor is (1-3):1 or 1:(1-3).
  • the specific reaction conditions for synthesizing the disaccharide are as follows: the glycosyl donor and the glycosyl acceptor are dissolved in a mixed solvent of toluene, dichloromethane and diethyl ether, stirred under argon protection, molecular sieves are added, the reaction temperature is -20°C to 0°C, 0.1 to 0.3 equivalents (compared to the molar equivalents of the donor) of activating reagent are added, and the reaction time is 2 to 8 hours.
  • the reducing agent used to reduce the azide group in the disaccharide 15 is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.
  • the condensing agent is one of DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), DPPA (diphenyl azidophosphate), DPC1 (diphenyl phosphoric acid chloride), DECP (diphenyl cyanophosphate), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate).
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbod
  • the acyl halide includes one of acyl chloride, acyl bromide, and acyl fluoride.
  • the method for preparing the acyl chloride includes one of the following: synthesizing acyl chloride using sulfoxide, synthesizing acyl chloride using oxalyl chloride, or preparing acyl chloride using trichlorotriazine.
  • trisaccharide 18 is synthesized by: utilizing temperature effects and the neighboring group participation effect at the C2 position, acceptor 17 and donor 6 are glycosylated under the catalysis of an activator to construct a single configuration of trisaccharide 18; the corresponding synthetic route is shown below:
  • the construction of the 1,2-trans- ⁇ -glycosidic bond in the trisaccharide 18 is achieved by utilizing the temperature effect, the catalysis of the activating reagent, and the participation of the neighboring group of PG 1 .
  • the concentration of the glycosylation reaction is 0.01 to 0.1 M.
  • the activator is one of TMSOTf, NIS/TMSOTf, and NIS/TfOH.
  • the solvent is one or more of anhydrous dichloromethane, diethyl ether, toluene, methanol, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, or water.
  • the molar ratio of the donor to the acceptor is (1-3):1 or 1:(1-3).
  • the glycosylation reaction conditions of the trisaccharide 18 include: dissolving the disaccharide acceptor 17 and the donor 6 in dichloromethane solvent, adding molecular sieves, adding an activating reagent in an amount of 0.2 to 1 equivalent (compared to the molar equivalent of the acceptor), and controlling the reaction temperature to gradually increase to room temperature from 0°C for a reaction time of 2 to 8 hours.
  • the synthesis method of compound 19 is as follows: reducing the azide group at the non-reducing end of trisaccharide 18 with a reducing agent to obtain an aminotrisaccharide intermediate; activating compound 9 with a condensing agent or preparing it into an acyl halide, and then coupling it with the aminotrisaccharide intermediate amide to obtain compound 19; the corresponding synthetic route is as follows:
  • the reducing agent is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.
  • the condensing agent is one of DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide)), DPPA (diphenyl azidophosphate), DPPCl (diphenylphosphochloride), DECP (diphenyl cyanophosphate), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate).
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbod
  • the acyl halide includes one of acyl chloride, acyl bromide, and acyl fluoride.
  • the method for preparing the acyl chloride includes one of the following: synthesizing acyl chloride using sulfoxide, synthesizing acyl chloride using oxalyl chloride, or preparing acyl chloride using trichlorotriazine.
  • the synthesis methods of compounds 20, 21, and 22 are the same as or similar to those of compound 19. That is, the synthesis of compounds 20, 21, and 22 involves reducing the azide group at the non-reducing end 4 position of trisaccharide 18 with a reducing agent to obtain an aminotrisaccharide intermediate, and then coupling compounds 10, 11, or 12 with an aminotrisaccharide intermediate amide, respectively.
  • the method for removing the ester protecting group in the deprotection method can be potassium hydroxide/methanol/water, sodium hydroxide/ethanol/water, lithium hydroxide/methanol/water, sodium hydroxide/methanol/water, potassium hydroxide/ethanol/water, lithium hydroxide/ethanol/water, etc.
  • the method for removing the silyl ether protecting group in the deprotection method can be tetrabutylammonium fluoride, hydrofluoric acid, etc.
  • the solvent used in the deprotection reaction can be a mixture of water/methanol/dichloromethane/acetic acid, a mixture of water/tert-butanol/dichloromethane, a mixture of water/tert-butanol/tetrahydrofuran, etc., and the reaction temperature can be between 0 and 40°C.
  • the reducing agent is one of zinc powder, triphenylphosphine, 1,3-propanedithiol, lithium aluminum hydride, trimethylphosphine, stannous chloride dihydrate, sodium borohydride, and sodium cyanoborohydride.
  • the reductive acylation of compound 5 can also be obtained by directly adding compound 18 to a mixed solution of thioacetic acid and pyridine.
  • One application of this invention is to use the synthesized oligosaccharide to elucidate the absolute configuration of dHh.
  • the absolute configuration of dHh is determined by NMR error analysis using four synthesized oligosaccharide isomers (1-4) and extracted natural O-polysaccharide (OPS).
  • the OPS is extracted from inactivated Vibrio cholerae O100 serotype bacteria.
  • the NMR instruments used for the deprotected products (1-5) are all 600M, and the tests are conducted at the same temperature (25°C).
  • Another application of this invention is to elucidate the immunological role of dHh using the synthesized oligosaccharides.
  • the immunological function of dHh is demonstrated to be achieved through glycan chip technology.
  • the preparation process of the sugar chip includes: attaching the linker arms of the reducing ends of five oligosaccharide fragments to the chip, incubating with antiserum, labeling with secondary antibody, and fluorescence scanning.
  • the results of the sugar chip show that the deletion and conformational changes of dHh do not affect its binding ability with the antibody.
  • This invention also provides a Vibrio cholerae glycoprotein conjugate for vaccine development, which comprises the five compounds mentioned above. It is composed of oligosaccharide fragments (1-5) conjugated with proteins. Its general formula can be represented as sugar-linker-carrier protein.
  • the carrier protein comprises one of the following: diphtheria toxin nontoxic mutant protein (CRM197), hemocyanin (KLID), bovine serum albumin (BSA), meningitis perimeningeal protein (OMPC), tetanus toxoid (TT), or diphtheria toxoid (DT).
  • CCM197 diphtheria toxin nontoxic mutant protein
  • KLID hemocyanin
  • BSA bovine serum albumin
  • OMPC meningitis perimeningeal protein
  • TT tetanus toxoid
  • DT diphtheria toxoid
  • the present invention also provides the use of the glycoconjugate in the preparation of a vaccine for the prevention or treatment of diseases caused by Vibrio cholerae infection.
  • the main symptoms of the disease include one or more of the following: diarrhea, vomiting, sunken eyes, dry skin, altered consciousness, shock, and death.
  • This invention also provides the application of the above-mentioned chemical synthesis method in the preparation of sugar chips or Vibrio cholerae glycoprotein conjugates, the application comprising the following process:
  • the linker arm of the obtained oligosaccharide fragment is used to bind to the chip or carrier protein to obtain the corresponding sugar chip or Vibrio cholerae glycoprotein conjugate.
  • This invention utilizes three monosaccharide building blocks and five carboxylic acid derivatives, and by leveraging neighboring group participation effect, long-range participation effect, solvent effect, etc., through a series of orthogonal protection, stereoselective assembly and efficient amide coupling, to develop an efficient and concise method for synthesizing O antigen oligosaccharides. Five oligosaccharide fragments were successfully synthesized using this method.
  • this invention will provide a reliable theoretical basis for the design of Vibrio cholerae vaccines, infection diagnosis, and drug development.
  • Figure 1 shows five oligosaccharide fragments, the three required monosaccharide building blocks, and five carboxylic acid derivatives
  • Figure 2 shows the synthetic routes of four hexanoic acid derivatives 18* to 22*;
  • FIG. 3 shows the synthetic route of the disaccharide receptor 28*
  • FIG. 4 shows the synthetic route of trisaccharide 31*
  • Figure 5 shows the synthetic route of the fully protected trisaccharides 32* to 35*
  • Figure 6 shows the synthetic route of the target compounds (1* ⁇ 5*);
  • Figure 7 shows the full-spectrum error analysis of the target compounds (1* ⁇ 5*) using 13 C-NMR.
  • Figure 8 shows the error analysis of the processed 13 C-NMR spectra of the target compounds (1* ⁇ 5*); in the figure, 2' ⁇ 6' represents the NMR signals of different 13 C in dHh, and similarly 2" ⁇ 4" represents the NMR signals of different 13 C in RHb;
  • Figure 9 shows a comparison of the results of the sugar chip screening; where A is a schematic diagram of the oligosaccharide structure, B is a spotting pattern diagram, C is the chip scanning result, and D is the quantitative result of the average fluorescence.
  • the error bar comes from the standard deviation between two points of two uniform concentrations.
  • Figure 10 shows the NMR-HSQC structural identification of compound 1*
  • Figure 11 shows the NMR-HSQC structural identification of compound 2*
  • Figure 12 shows the NMR-HSQC structural identification of compound 3*
  • Figure 13 shows the NMR-HSQC structural identification of compound 4*
  • Figure 14 shows the NMR-HSQC structural identification of compound 5*.
  • the yields of each reaction step were calculated as follows: (amount of target product / amount of starting material) ⁇ 100%.
  • the product structure was identified using NMR spectroscopy, infrared spectroscopy, optical rotation, and high-resolution mass spectrometry. Purity analysis was performed using NMR spectroscopy. Proton, carbon, and two-dimensional NMR spectra were measured at 25°C using Bruker Ascend 600M and 400M NMR spectra. High-resolution mass spectrometry was performed using an Agilent 6220 electrospray ionization source-time-of-flight mass spectrometer.
  • Infrared spectroscopy was measured using a Thermo Fisher Scientific Nicolet iS5 infrared spectrometer, and optical rotation was measured at 589 nm using a Schmidt & Haensch UniPol L10000 fully automated polarimeter. Concentration (c) was measured in g/100 mL.
  • Compound 10* Compound 8* (150 mg, 0.74 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (4:1, v/v, 7.5 mL) at -70 °C, followed by the addition of diethylmethoxyborane (0.8 mL, 0.8 mmol). After one hour, sodium borohydride (31 mg, 0.82 mmol) was added to the solution. The reaction was then continued at -70 °C for 22 hours. After the starting material was confirmed to be completely reacted by TLC, a saturated ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate.
  • Compound 13* Compound 9* (150 mg, 0.74 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (4:1, v/v, 7.5 mL) at -70 °C, followed by the addition of diethylmethoxyborane (0.8 mL, 0.8 mmol). After one hour, sodium borohydride (31 mg, 0.82 mmol) was added to the solution. The reaction was then continued at -70 °C for 22 hours. After the reaction was confirmed to be complete by TLC, a saturated ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate.
  • Compound 17* Compound 13* (319 mg, 1.56 mmol) was dissolved in anhydrous dichloromethane (15.6 mL), and benzyl bromide (1.85 mL, 15.6 mmol) and silver oxide (4.45 g, 18.74 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 5 h, and then stirred at room temperature for 28 h.
  • L-fucosamine building blocks 23* and D-fucosamine building blocks 24* were used as raw materials for glycosylation to obtain compound 25*.
  • the non-reducing azide group at the disaccharide 25* was reduced to an amino group using pre-activated zinc powder.
  • HATU condensing agent
  • glycosyl donor 24* (53.2 mg, 0.104 mmol) was dissolved in anhydrous dichloromethane (2.0 mL) and cooled to -60°C. Then, pre-activated [material/material] was added. Molecular sieves, 1-(phenylsulfonyl)piperidine (BSP, 26.1 mg, 0.125 mmol), 2,4,6-tri-tert-butylpyrimidine (TTBP, 38.8 mg, 0.156 mmol), and trifluoromethanesulfonic anhydride ( Tf2O , 22.7 ⁇ L, 0.135 mmol) were reacted at -60 °C with stirring for 30 min.
  • BSP 1-(phenylsulfonyl)piperidine
  • TTBP 2,4,6-tri-tert-butylpyrimidine
  • Tf2O trifluoromethanesulfonic anhydride
  • Method 2 D-fucose donor 24* (80.6 mg, 0.158 mmol) and L-fucose acceptor 23* (115 mg, 0.19 mmol) were dissolved in toluene (5 ml), azeotropically removed three times, concentrated under vacuum until dry, and then pre-activated [reagent/concentrate] was added. Molecular sieves were used, and the mixture was evacuated overnight using an oil pump. Under argon protection, anhydrous dichloromethane (6.0 mL) and iodosuccinimide (43 mg, 0.19 mmol) were added sequentially. The mixture was cooled to 0 °C and stirred for 30 minutes.
  • Trimethylsilyl trifluoromethanesulfonate (5.7 ⁇ L, 0.032 mmol) was added dropwise, and the mixture was stirred at 0 °C for 5 hours.
  • triethylamine was added to quench the reaction.
  • Diatomaceous earth was added to a sintered glass funnel, and the molecular sieves were removed by filtration.
  • the mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered through filter paper to remove sodium sulfate, and concentrated by vacuum distillation using a rotary evaporator.
  • Method 3 D-fucose donor 24* (100 mg, 0.196 mmol) and L-fucose acceptor 23* (141.6 mg, 0.235 mmol) were dissolved in toluene (5 ml), azeotropically removed three times, concentrated under vacuum until dry, and then pre-activated [reagent/concentration] was added. Molecular sieves were used, and the mixture was evacuated overnight using an oil pump. Under argon protection, anhydrous dichloromethane (4.5 mL), diethyl ether (3.0 mL), and iodosuccinimide (52.9 mg, 0.235 mmol) were added sequentially. The mixture was cooled to 0 °C and stirred for 30 minutes.
  • Trimethylsilyl trifluoromethanesulfonate (3.45 ⁇ L, 0.039 mmol) was added dropwise, and the mixture was stirred at 0 °C for 5 hours. After the reaction was confirmed to be complete by TLC, triethylamine was added to quench the reaction. Diatomaceous earth was added to a sintered funnel, and the molecular sieves were removed by filtration. The mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered through filter paper to remove sodium sulfate, and concentrated by vacuum distillation using a rotary evaporator.
  • D-quinoline amine building block 29* (which can be prepared by referring to existing literature Codée et al, Organic & Biomolecular Chemistry. 2020, 18(15), 2834-2837) was dissolved in a mixed solution of acetone and H2O and hydrolyzed under the catalysis of iodosuccinimide (NIS). Subsequently, under the action of 2,2,2-trifluoro-N-phenylacetylimide acyl chloride (171 ⁇ L, 1.14 mmol) and 1,8-diazabicycloundec-7-ene (DBU), trifluoroacetylimide ester 30* was obtained.
  • NIS iodosuccinimide
  • Trifluoroacetylimide ester 30* and disaccharide acceptor 28* were glycosylated under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to successfully obtain a single ⁇ -configuration trisaccharide compound 31* (yield 13%).
  • Compound 30* Compound 29* (232.5 mg, 0.38 mmol) was dissolved in acetone and H2O (10:1, v/v, 5.5 mL) at room temperature and stirred until homogeneous. Then, NIS (171.4 mg, 0.7 mmol) was added and the mixture was stirred for 1 hour. After TLC showed complete reaction, the mixture was diluted with ethyl acetate and washed with 10% (w/v) Na2S2O3. The organic layer was dried over Na2SO4 , filtered , and concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 20/1 ⁇ 1/1 v/v) to give the intermediate compound.
  • the intermediate compound was dissolved in DCM (4.8 mL) and then 2,2,2-trifluoro-N-phenylacetylimine chloride (171 ⁇ L, 1.14 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (171 ⁇ L, 1.14 mmol) were added.
  • DBU 1,8-diazabicycloundec-7-ene
  • the reaction was stirred at 0°C for 3 hours.
  • the mixture was then concentrated under vacuum and purified by silica gel column chromatography (petroleum ether/ethyl acetate: 30/1 ⁇ 10/1 v/v) to give trifluoroacetylimine ester 30* (223.4 mg, 0.34 mmol, two-step yield 90%).
  • Method 2 Under argon protection, selenoside 29* (110 mg, 0.18 mmol) and disaccharide receptor 28* (122 mg, 0.12 mmol) were dissolved in toluene and azeotropically evaporated three times to remove water. The mixture was then evacuated under vacuum for 2 hours using an oil pump. Pre-activated [material/material] was then added. Molecular sieves and anhydrous dichloromethane (6.0 mL) were added to the reaction solution. The solution was cooled to 0 °C and stirred for 30 minutes.
  • Trimethyl trifluoromethanesulfonate (8.7 ⁇ L, 0.048 mmol) and iodosuccinimide (40.5 mg, 0.18 mmol) were added dropwise.
  • the mixture was slowly heated from 0 °C to room temperature and stirred for 5 hours.
  • triethylamine was added to quench the reaction.
  • the molecular sieves were removed by diatomaceous earth filtration.
  • the mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate and concentrated by vacuum distillation using a rotary evaporator.
  • 3,5-Dibenzylhexanoic acid 18* (10 mg, 30.6 ⁇ mol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 ⁇ mol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 ⁇ mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 ⁇ mol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After TLC detection showed that the reaction of the starting material was complete, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution.
  • 3,5-Dibenzylhexanoic acid 19* (10 mg, 30.6 ⁇ mol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 ⁇ mol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 ⁇ mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 ⁇ mol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After TLC detection showed that the reaction of the starting material was complete, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution.
  • 3,5-Dibenzylhexanoic acid 20* (10 mg, 30.6 ⁇ mol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 ⁇ mol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 ⁇ mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 ⁇ mol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After the reaction of the starting material was completed by TLC, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution.
  • 3,5-Dibenzylhexanoic acid 21* (10 mg, 30.6 ⁇ mol) and aminotrisaccharide were dissolved in acetonitrile (5 mL) at room temperature, followed by the addition of sodium bicarbonate (5.1 mg, 61.2 ⁇ mol). After 10 minutes, 1-hydroxybenzotriazole (0.55 mg, 4.08 ⁇ mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (6.25 mg, 32.6 ⁇ mol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. After TLC detection showed that the reaction of the starting material was complete, the crude product was dissolved in ethyl acetate and washed with saturated sodium chloride solution.
  • Trisaccharide 31* was deprotected by converting its azide group to NHAc under zinc powder, acetic acid, and acetic anhydride conditions, followed by direct deprotection using palladium on carbon hydrogenation to obtain trisaccharide 5*.
  • Trisaccharide 32* (12 mg, 6.0 ⁇ mol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v/v/v, 3 mL). Air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred under hydrogen for 24 hours. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL/min.
  • Trisaccharide 33* (17 mg, 9.89 ⁇ mol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v/v/v, 3 mL). Air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred under hydrogen for 24 hours. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL/min.
  • Trisaccharide 34* (9 mg, 3.1 ⁇ mol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v/v/v, 3 mL). Air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred for 24 hours under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL/min.
  • Trisaccharide 35* (7.8 mg, 4.536 ⁇ mol) was dissolved in a mixture of dichloromethane, tert-butanol, and water (3:6:1, v/v/v, 3 mL). Air in the reaction flask was purged with nitrogen, and an appropriate amount of 10% palladium-carbon was added. The solution was purged with hydrogen for 5 minutes, then stirred under hydrogen for 24 hours. The mixture was filtered through diatomaceous earth and concentrated. The residue was purified by HPLC using a semi-preparative (Thermo Scientific Hypercarb) column at a flow rate of 1 mL/min.
  • the crude product was dissolved in dichloromethane and tert-butanol and water (3/6/1, v/v/v, 2 mL), and an appropriate amount of 10% palladium-carbon was added to the solution.
  • the mixture was stirred for 36 hours under a hydrogen atmosphere (4 atm), then filtered through diatomaceous earth and washed with water, repeated three times, and the solvent was evaporated under vacuum.
  • the NMR spectra of the synthesized oligosaccharides (1* ⁇ 4*) were all measured at 600M and at the same temperature (25°C).
  • the NMR spectra of the four synthesized oligosaccharides were compared with those of natural OPS to attempt to determine the possible absolute configuration of the dHh side chain.
  • none of the four synthesized trisaccharides (1*-4*) showed the same NMR- 1H and NMR- 13C data as the repeating unit of the naturally extracted O-antigen (OPS) trisaccharide.
  • OPS O-antigen
  • the sugar chip was blocked with PBS solution containing 3% BSA for 1 hour at room temperature. It was then washed once with 0.1% Tween 20 PBS solution (PBST solution), twice with PBS solution, centrifuged, and the sugar chip was loaded into a 16-well incubator (ProPlate). 120 ⁇ L of rabbit serum sample diluted 1:200 in PBS solution containing 1% BSA was added to each well, and the incubator was kept overnight in a humidified chamber at 4°C in the dark to allow the rabbit serum IgG antibody recognizing the synthetic sugar fragments to bind to the synthetic oligosaccharides on the sugar chip. The sample was then removed, and the chip was washed three times with 200 ⁇ L of PBST solution to remove unbound serum IgG antibodies.

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Abstract

本发明公开了一种霍乱弧菌O100血清型O抗原寡糖的化学合成方法及应用，属于化学技术领域。本发明利用三个单糖砌块和五种羧酸衍生物，在溶剂效应、温度效应、邻基参与效应等作用下，经正交保护、选择性组装和酰胺偶联，合成得到五种霍乱弧菌O100血清型O抗原寡糖片段。利用合成的寡糖片段，结合NMR分析和糖芯片技术，明确了该O抗原三糖中3，5-二羟基己酰基的绝对构型和免疫学作用，为进一步的构效研究和最小抗原表位筛选提供了理论基础。本发明将在霍乱弧菌合成糖缀合疫苗和新型药物开发等方面具有良好的应用前景。

Description

一种霍乱弧菌O100血清型O抗原寡糖的化学合成方法及应用 技术领域
本发明涉及一种霍乱弧菌O100血清型O抗原寡糖的化学合成方法及应用,属于化学技术领域。
背景技术
霍乱弧菌是霍乱的病原体，可导致霍乱大流行。霍乱是一种急性腹泻疾病，其特征是水样腹泻和可能致命的脱水(Qadri et al.Clin.Microbiol.Rev.2022,35(3),e00211-00221)。根据世卫组织的疫情简报，2023年，30个国家报告了霍乱病例，仅在2024年1月份就报告了40900例病例和775例死亡病例。
抗生素和口服霍乱疫苗(OCVs)已经被广泛用于治疗霍乱弧菌感染。研究表明，霍乱弧菌对所有的这些抗生素药物普遍产生了抗药性。虽然OCVs已被证明有效，但它们也有许多局限性，包括对幼儿的免疫原性差、保护期短、免疫诱导延迟等(Waldor et al.Annu.Rev.Microbiol.2022,76(1),681-702)。目前仍需要一种能够在全球霍乱控制中发挥关键作用的霍乱疫苗。
糖蛋白缀合疫苗展现了诱人的前景，己有多个上市的糖蛋白缀合疫苗用于疾病的预防和治疗，如肺炎链球菌十三价糖缀合物疫苗、脑膜炎糖缀合物疫苗、沙门氏菌缀合物疫苗等。使用结构明确的合成寡糖与蛋白质载体偶联，可以提高寡糖的免疫原性和诱导T细胞产生依赖性免疫反应，已被证明是安全可靠的(Seeberger,Chem.Rev.2021,121(7),3598-3626)。
根据细菌表面脂多糖O抗原的不同，霍乱弧菌被分为200多种血清型。2019年，Perepelov等人分离并鉴定了霍乱弧菌O100血清型O抗原三糖重复单元的结构，其结构为[→3)-β-d-QuipNAc4N(dHh)-(1→3)-α-d-Fucp4N(RHb)-(1→3)-α-l-FucpNAc-(1→]，其中RHb和dHh分别表示(R)-3-羟基丁酰基和3，5-二羟基己酰基(Perepelov.et al.Carbohydr.Res.2019,472,98-102)。
该O-抗原三糖含有两个难以构建的1,2-顺式-α-岩藻糖苷键和一个易断裂的1-2-反式-β-D-奎诺糖苷键，四个氮原子偶联两种罕见的修饰基团。上述因素的存在使得该O抗原三糖重复单元的合成极具挑战性，且至今尚未被全合成得到。值得注意的是，细菌表面聚糖修饰基团被认为是潜在的免疫靶点。独特的dHh修饰基团中2个手性中心的绝对构型尚未明确，并且对其免疫学作用也缺乏了解。化学合成结构均一O抗原寡糖，完成绝对构型的归属和初步的免疫学研究，对霍乱弧菌O100血清型糖缀合物疫苗和相关药物的开发具有重要的意义。
发明内容
技术问题：
针对上述问题，本发明涉及开发一种霍乱弧菌O100血清型O抗原寡糖的化学合成方法；以及应用合成的寡糖阐明dHh修饰基团的绝对构型和免疫学作用。
技术方案：
由于dHh中两个手性中心的绝对构型尚未确定，因此理论上该O抗原三糖存在四种可能的异构体。同时为便于研究dHh的免疫学作用，有必要合成一种不含dHh修饰的三糖衍生物。在合成寡糖还原端引入相应的连接臂，可以为进一步的免疫学探究提供基础。因此，本发明利用三个单糖砌块和五种羧酸衍生物，经一系列的正交保护、选择性组装和酰胺偶联，合成得到四种可能的三糖异构体和一种衍生物。利用NMR技术，将四种三糖异构体与提取的脂多糖O抗原进行误差分析，以此阐明dHh的绝对构型；将五种寡糖片段与芯片结合制成糖芯片，通过糖芯片筛选评估dHh的免疫学作用。
本发明的一个目的是提供一种霍乱弧O100血清型O抗原寡糖的化学合成方法，所述方 法是利用三个单糖砌块和五种羧酸衍生物作为原料；
所述霍乱弧O100血清型O抗原寡糖的结构如下式(1)～(5)所示：
所述的三个单糖砌块的结构分别如式(6)～(8)所示，五种羧酸衍生物的结构分别如式(9)～(13)所示，
PG2，PG3，PG4，PG6，PG7为羟基临时保护基，可以是苄基(Bn)、2-萘甲基(Nap)、叔丁基二甲基硅烷基(TBS)、叔丁基二苯基硅烷基(TBDPS)、三乙基硅烷基(TES)中的一种；
PG8，PG9，PG11，PG12，PG14，PG15，PG17，PG18，PG20为羟基临时保护基，可以是苄基(Bn)、2-萘甲基(Nap)、乙酰基(Ac)、苯甲酰基(Bz)、新戊酰基、9-戊甲氧羰基(Fmoc)、2-对甲氧基苄基(PMB)中的一种；
PG10，PG13，PG16，PG19，PG21分别独立的选自羟基(OH)、氯(Cl)、溴(Br)、氟(F)、C1-4烷氧基中的一种；
PG1为氨基临时保护基，可以是三氯乙酰基(TCA)、二氯乙酰基(DCA)、氯乙酰基(CIAc)中的一种；
PG5为氨基临时保护基，可以是乙酰基(Ac)、三氯乙酰基(TCA)、二氯乙酰基(DCA)、氯乙酰基(CIAc)、三氯乙氧羰基(Troc)、邻苯二甲酰基(Phth)、9-芴甲氧羰基(Fmoc)、叔丁基氧羰基(Boc)中的一种；
Linker为-(CH2)nN-Y1Y2或者-(CH)nS-Y1,其中n＝1～25，Y1和Y2为氢、酰基、苄基(Bn)、2-萘甲基(Nap)、苄甲氧羰基(Cbz)中的一种；Linker*为-(CH2)nNH2或者-(CH)nSH,其中n＝1～25；
离去基团LG1为N-苯基三氟乙酰亚胺酯(CF3C(＝NPh)O-)；
离去基团LG2选自三氯乙酰亚胺酯(CCl3C(＝NH)O-)、N-苯基三氟乙酰亚胺酯(CF3C(＝NPh)O-)、甲硫基(SMe)、硒苯基(SePh)、乙硫基(SEt)、苯硫基(SPh)、对甲苯硫基(STol)、二丁基膦酸基(-P(＝O)-(OBu)2)中的一种；
所述合成方法包括如下步骤：
(1)构建二糖受体：单糖砌块8脱除3号位的羟基保护基团PG6，得到受体14；受体14与单糖砌块7进行糖基化反应，得到二糖15；利用还原剂将二糖15中的叠氮基团还原为氨基，并加入化合物13进行酰胺化，得到化合物16；脱除化合物16中单糖砌块7上3号位 的羟基保护基PG4，得到二糖受体17；
(2)构建目标三糖：
二糖受体17和单糖砌块6在活化剂的作用进行糖基化反应，构建得到三糖18；再利用还原剂还原三糖18的叠氮基团，并加入式(9)～(12)中任意一种羧酸衍生物进行酰胺化，相应得到化合物19～22；将化合物19～22进行催化氢化，脱保护得到目标化合物1～4；
或者，三糖18经还原酰化、将叠氮基团转化为乙酰氨基，随后催化氢化，脱保护得到目标化合物5；
PGa、PGb为羟基临时保护基，分别独立地选自苄基、2-萘甲基、乙酰基、苯甲酰基、新戊酰基、9-戊甲氧羰基、2-对甲氧基苄基。
在本发明的一种实施方式中，C1-4烷氧基包括甲基(Me)、乙基(Et)、叔丁基(t-Bu)；当所述PG10、PG13、PG16、PG19、PG21为C1-4烷氧基时，酰胺偶联前，需先脱除该保护基。
在本发明的一种实施方式中，二糖受体17的合成是通过以下方式得到：在活化剂催化和溶剂效应的作用下，受体14和供体7进行糖基化反应得到二糖15；然后在还原剂的作用下，将二糖15中的叠氮基团还原为氨基，得到氨基二糖中间体；(R)-3-羟基丁酸衍生物13经缩合剂活化或制备成酰卤，与氨基二糖中间体酰胺缩合得到化合物16，脱除化合物16中的PG4保护基得到受体17；相应合成路线如下所示：
在本发明的一种实施方式中，所述二糖15的合成是通过利用溶剂效应、活化试剂的催化和非参与基团的正交保护。
在本发明的一种实施方式中，所述的糖基化反应浓度为0.01～0.1M。
在本发明的一种实施方式中，所述的活化试剂为TMSOTf，NIS/TMSOTf，NIS/TfOH中的一种。
在本发明的一种实施方式中，所述的溶剂为无水二氯甲烷、乙醚、甲苯、甲醇、四氢呋喃、乙腈、N，N—二甲基甲酰胺或水其中的一种或多种。优选无水二氯甲烷、乙醚、甲苯的混合体系。
在本发明的一种实施方式中，所述供体和受体的摩尔比为(1～3):1或者1：(1～3)。
在本发明的一种实施方式中，合成二糖的具体反应条件为：将糖基供体和糖基受体溶于甲苯、二氯甲烷、乙醚的混合溶剂中，在氩气的保护下进行搅拌，加入分子筛，反应温度为-20℃～0℃，加入活化试剂为0.1～0.3当量(相比供体的摩尔当量)，反应时间为2～8h。
在本发明的一种实施方式中，所述二糖15中还原叠氮基团使用的还原剂为锌粉、三苯基磷、1，3-丙二硫醇、氢化铝锂、三甲基磷、二水氯化亚锡、硼氢化钠、氰基硼氢化钠中的一种。
在本发明的一种实施方式中，所述缩合剂为DCC(二环己基碳二亚胺)、DIC(二异丙基碳二亚胺)、EDC(1-(3-二甲胺基丙基)-3-乙基碳二亚胺)、DPPA(叠氮化磷酸二苯酯)、DPPCl(二苯基磷酰氯)、DECP(氰代磷酸二苯酯)、HATU(2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯)、HBTU(苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐)、HCTU(6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯)中的一种。
在本发明的一种实施方式中，所述酰卤包括酰氯、酰溴、酰氟中的一种。
在本发明的一种实施方式中，所述酰氯的制备方法包括使用二氯亚砜合成酰氯、草酰氯合成酰氯、三氯均三嗪制备酰氯中的一种。
在本发明的一种实施方式中，三糖18的合成是通过以下方式得到：利用温度效应、C2号位的邻基参与效应，受体17和供体6在活化剂的催化下，经糖基化反应构建单一构型的三糖18；相应合成路线如下所示：
在本发明的一种实施方式中，所述三糖18中1，2-反式-β-糖苷键的构建是利用温度效应和活化试剂的催化、PG1的邻基参与作用。
在本发明的一种实施方式中，所述的糖基化反应浓度为0.01～0.1M。
在本发明的一种实施方式中，所述的活化剂为TMSOTf，NIS/TMSOTf，NIS/TfOH中的一种。
在本发明的一种实施方式中，所述的溶剂为无水二氯甲烷、乙醚、甲苯、甲醇、四氢呋喃、乙腈、N，N—二甲基甲酰胺或水其中的一种或多种。
在本发明的一种实施方式中，所述供体和受体的摩尔比为(1～3):1或者1：(1～3)。
在本发明的一种实施方式中，所述三糖18的糖基化反应条件包括：将二糖受体17和供体6溶于二氯甲烷溶剂中，加入分子筛，加入活化试剂为0.2～1当量(相比受体的摩尔当量)，并控制反应的温度为0℃逐步升温至室温，反应时间为2～8h。
在本发明的一种实施方式中，化合物19的合成方法为：利用还原剂还原三糖18非还原端4号位的叠氮基团，得到氨基三糖中间体；化合物9经缩合剂活化或制备成酰卤，随后与氨基三糖中间体酰胺偶联得到化合物19；相应合成路线如下所述：
在本发明的一种实施方式中，所述还原剂为锌粉、三苯基磷、1，3-丙二硫醇、氢化铝锂、三甲基磷、二水氯化亚锡、硼氢化钠、氰基硼氢化钠中的一种。
在本发明的一种实施方式中，所述缩合剂为DCC(二环己基碳二亚胺)、DIC(二异丙基碳二亚胺)、EDC(1-(3-二甲胺基丙基)-3-乙基碳二亚胺))、DPPA(叠氮化磷酸二苯酯)、DPPCl(二苯基磷酰氯)、DECP(氰代磷酸二苯酯)、HATU(2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯)、HBTU(苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐)、HCTU(6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯)中的一种。
在本发明的一种实施方式中，所述酰卤包括酰氯、酰溴、酰氟中的一种。
在本发明的一种实施方式中，所述酰氯的制备方法包括使用二氯亚砜合成酰氯、草酰氯合成酰氯、三氯均三嗪制备酰氯中的一种。
在本发明的一种实施方式中，化合物20，21，22的合成方法与化合物19相同或相似。即化合物20，21，22的合成，是利用还原剂还原三糖18中非还原端4号位的叠氮基团，得到氨基三糖中间体，化合物10或11或12分别与氨基三糖中间体酰胺偶联得到。
在本发明的一种实施方式中，所述化合物1～5的制备，是通过以下方式合成得到：化合物19、20、21、22(PGa和PGb代表相应的羟基保护基)分别在钯碳加氢的条件下，催化氢化，脱保护得到目标化合物1～4；三糖18经还原剂还原和乙酰化，非还原端4号位的叠氮基团转化为乙酰氨基，随后催化氢化，脱保护得到目标化合物5；相应合成路线如下所示：
在本发明的一种实施方式中，所述脱保护的方法中酯类保护基的脱除方法可以是氢氧化钾/甲醇/水，氢氧化钠/乙醇/水，氢氧化锂/甲醇/水，氢氧化钠/甲醇/水，氢氧化钾/乙醇/水，氢氧化锂/乙醇/水等。
在本发明的一种实施方式中，所述脱保护的方法中硅醚类保护基的脱除方法可以是四丁基氟化铵，氢氟酸等：
在本发明的一种实施方式中，所述脱保护的方法中碳醚类保护基的脱除可以是催化氢化，即在催化剂存在的条件下，通入氢气进行反应。
在本发明的一种实施方式中，所述催化氢化所用的催化剂可以是10％钯碳催化剂或氢氧化钯等。
在本发明的一种实施方式中，所述脱保护反应所用的溶剂可以是水/甲醇/二氯甲烷/醋酸混合液，水/叔丁醇/二氯甲烷混合液，水/叔丁醇/四氢呋喃混合液等，反应温度可以是0至40℃之间。
在本发明的一种实施方式中，所述化合物5的合成中，所述的还原剂为锌粉、三苯基磷、1，3-丙二硫醇、氢化铝锂、三甲基磷、二水氯化亚锡、硼氢化钠、氰基硼氢化钠中的一种。
在本发明的一种实施方式中，所述化合物5的合成中，乙酰化可以利用吡啶、乙酸酐；或甲醇、乙酸酐；或乙酸、乙酸酐等。
在本发明的一种实施方式中，所述化合物5的还原酰化，还可以是通过将化合物18直接加入硫代乙酸和吡啶混合溶液中反应得到。
本发明的一个应用是利用合成得到的寡糖阐明dHh的绝对构型。
在本发明的一种实施方式中，dHh绝对构型的归属是利用合成得到的四种寡糖异构体(1～4)与提取的天然O-多糖(OPS)进行NMR误差分析。
在本发明的一种实施方式中，所述OPS是从霍乱弧菌O100血清型灭活细菌中提取得到。
在本发明的一种实施方式中，为了尽可能的减少误差，脱保护产物(1～5)所使用的核磁测试仪器均为600M，并且在相同的温度(25℃)下测试。
本发明另一个应用是利用合成得到的寡糖阐明dHh的免疫学作用。
在本发明的一种实施方式中，阐明dHh的免疫学作用是通过糖芯片技术实现的。
在本发明的一种实施方式中，所述的糖芯片的制备流程包括：将五种寡糖片段还原端的连接臂与芯片结合，使用抗血清孵育，二抗标记，荧光扫描。
在本发明的一种实施方式中，所述抗血清来自霍乱弧菌O100血清型脂多糖(LPS)免疫的动物血清或者感染霍乱弧菌O100血清型的人血清。
在本发明的一种实施方式中，糖芯片结果表明，dHh的缺失和构型改变，均不会影响其与抗体的结合能力。
本发明还提供一种用于疫苗研发的霍乱弧菌糖蛋白缀合物，该缀合物由上述化合物五种 寡糖片段(1～5)与蛋白质缀合而成。其通式可表示为糖—Linker—载体蛋白。
在本发明的一种实施方式中，所述载体蛋白包括：白喉毒素无毒突变蛋白(CRM197)，血蓝蛋白(KLID)、牛血清蛋白(BSA)、脑膜炎炎外膜蛋白(OMPC)，破伤风类毒素(TT)或者白喉类毒素(DT)中的一种
本发明还提供所述糖缀合物在制备疫苗中的应用，所述疫苗应用于预防或治疗霍乱弧菌感染导致的疾病。
进一步，所述疾病主要症状为腹泻、呕吐、眼窝凹陷、皮肤干燥、意识障碍、休克、死亡中的一种或多种。
本发明还提供上述化学合成方法在糖芯片或者霍乱弧菌糖蛋白缀合物的制备中的应用，所述应用包括如下过程：
S1：利用上述化学合成方法制备具有连接臂的霍乱弧O100血清型O抗原寡糖片段；
S2：随后利用所得寡糖片段的连接臂与芯片或者载体蛋白质结合，得到相应的糖芯片或者霍乱弧菌糖蛋白缀合物。
有益效果
本发明利用三个单糖砌块和五种羧酸衍生物，借助邻基参与效应、远程参与效应、溶剂效应等，通过一系列的正交保护、立体选择性组装和高效的酰胺偶联，开发了一种高效简洁的O抗原寡糖的合成方法，应用该方法成功合成得到了五种寡糖片段。
应用合成得到的四种寡糖异构体(1～4)，结合NMR技术，阐明了dHh的绝对构型(3S,5S)，绝对构型的确定为后续构效关系的探究奠定了基础。将五种寡糖片段(1～5)制成糖芯片，糖芯片筛选表明，dHh的不是关键的抗原表位组分，接下来可以参考本发明所述的方法，继续合成其它寡糖片段，从而筛选最小抗原表位。
总而言之，本发明将会为霍乱弧菌疫苗设计、感染诊断和药物的研发提供了可靠的理论依据。
附图说明
图1为五种寡糖片段及所需的三个单糖砌块和五种羧酸衍生物；
图2为四种己酸衍生物18*～22*的合成路线图；
图3为二糖受体28*的合成路线图；
图4为三糖31*的合成路线图；
图5为全保护三糖32*～35*的合成路线图；
图6为目标化合物(1*～5*)合成路线图；
图7为目标化合物(1*～5*)的13C-NMR的全谱误差分析图；
图8为目标化合物(1*～5*)的13C-NMR的处理谱图误差分析图；图中2'～6'表示dHh中不同13C的NMR信号，类似的2"～4"表示RHb中不同13C的NMR信号；
图9为糖芯片筛选结果对比图；其中，A为寡糖结构示意图，B为点样模式图，C为芯片扫描结果，D为平均荧光的定量结果图，误差线来自两个统一浓度的两个点之间的标准差；
图10为化合物1*的NMR-HSQC结构鉴定图；
图11为化合物2*的NMR-HSQC结构鉴定图；
图12为化合物3*的NMR-HSQC结构鉴定图；
图13为化合物4*的NMR-HSQC结构鉴定图；
图14为化合物5*的NMR-HSQC结构鉴定图。
具体实施方式
实验中所用商品化试剂均未经处理直接使用，反应所用无水溶剂由MBraun MB-SPS 800型溶剂干燥系统制备。硅胶柱层析所用溶剂均为分析纯且经减压蒸馏后使用。薄层层析(TLC)所用硅胶板为60-F254硅胶制备的玻璃基或铝箔基硅胶板，正相硅胶柱层析所用硅胶为 200-300目硅胶。
分别对各反应步骤产率进行计算，产率计算方式为：(目标产物物质的量/原料物质的量)×100％。利用核磁图谱，红外图谱，旋光度，高分辨质谱对于产品进行结构鉴定，利用核磁图谱对于产品进行纯度分析，氢谱、碳谱以及二维核磁谱由BrukerAscend 600M、400M核磁共振仪在25℃下测得。高分辨质谱由Agilent 6220电喷雾离子源-飞行时间质谱仪测得。红外图谱由Thermo Fisher Scientific Nicolet iS5红外仪测得,旋光度由Schmidt&Haensch UniPol L10000全自动旋光仪在589nm下测得，测定浓度(c)单位为g/100mL。
实施例1
3，5-二苄基己酸的合成
如图2所示：分别以(R)-3-羟基丁酸甲酯6*和(S)-3-羟基丁酸甲酯7*为起始原料，经克莱森酯缩合得到(R)-5-羟基己酸叔丁酯8*和(S)-5-羟基己酸叔丁酯9*。进一步利用Narasaka和Evans的方法，选择性构建四种不同构型的3，5-二羟基己酸叔丁酯10*～13*。通过氧化银介导的选择性苄基化，得到化合物14*～17*。使用三氟乙酸脱除叔丁基，得到3，5-二苄基己酸18*～21*。
具体实验操作和步骤：
化合物8*：在-78℃的条件下，向四氢呋喃(1mL)溶液中加入二异丙胺锂(1.25mL,2.5mmol)和乙酸叔丁基酯(0.34mL，2.51mmol)，搅拌20min。将化合物6*(0.1g,0.85mmol)溶于THF(15mL)溶液，随后滴加到反应体系中。在-50℃的温度下搅拌2h，TLC检测原料反应完全后，加入去离水，用用乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，过滤除硫酸钠，用旋转蒸发仪减压蒸馏浓缩，粗品用硅胶柱层析(石油醚：乙酸乙酯，4:1，v/v)分离纯化得到化合物9*(134mg,0.66mmol,78％)。[α]25 D＝-13.7°(c＝0.2,CHCl3)；IR νmax(film)2973,2931,1731,1715,1456,1368,1252,1145,959,839,754cm-1；1H NMR(600MHz,Chloroform-d)δ4.26(ddq,J＝9.6,6.4,3.1Hz,1H,5-H),3.37(d,J＝2.2Hz,2H,2-CH2),2.85(d,J＝3.4Hz,1H,5-OH),2.76–2.60(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.21(d,J＝6.3Hz,3H,6-CH3).13C NMR(151MHz,Chloroform-d)δ204.2,166.1,63.8,51.1,28.0,22.4；HRMS(ESI)calculated for C10H18O4Na+[M+Na]+:225.1097,found:225.1083
化合物9*：在-78℃的条件下，向四氢呋喃(1mL)溶液中加入二异丙胺锂(1.25mL,2.5mmol)和乙酸叔丁基酯(0.34mL,2.51mmol)，搅拌20min。将化合物7*(0.1g,0.85mmol)溶于THF(15mL)溶液，随后滴加到反应体系中。在-50℃的温度下搅拌2h，TLC检测原料反应完全后，加入去离水，用用乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，过滤除硫酸钠，用旋转蒸发仪减压蒸馏浓缩，粗品用硅胶柱层析(石油醚：乙酸乙酯，4:1，v/v)分离纯化得到化合物9*(137mg,0.68mmol,80％)。[α]25 D＝+9.9°(c＝0.3,CHCl3)；IR νmax(film)2971,2925,2853,1731,1714,1456,1368,1257,1145,958,939,754cm-1；1H NMR(400MHz,Chloroform-d)δ4.27(dtd,J＝12.6,6.3,3.0Hz,1H,5-H),3.39(d,J＝1.1Hz,2H,2-CH2),2.79–2.60(m,2H,4-CH2),1.49(s,9H,tBu-CH3),1.23(d,J＝6.4Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ204.3,166.1,63.8,51.1,50.9,28.0,22.3；HRMS(ESI)calculated for C10H18O4Na+[M+Na]+:225.1097,found:225.1083
化合物10*：在-70℃下，将化合物8*(150mg,0.74mmol)溶于四氢呋喃和甲醇(4:1,v/v,7.5mL)的混合溶液中，随后加入二乙基甲氧基硼烷(0.8mL,0.8mmol)。一个小时后，向溶液中加入硼氢化钠(31mg,0.82mmol)。随后在-70℃的条件下，继续反应22小时。TLC检测原料反应完全后，加入饱和氯化铵溶液，用乙酸乙酯萃取三次。用饱和碳酸氢钠溶液和盐水洗涤有机层，然后使用无水硫酸钠干燥，真空浓缩，产物经硅胶柱层析(石油醚：乙酸乙酯，1:1，v/v)纯化得到10*(105.7mg,0.52mmol,70％)。[α]25 D＝-165.5°(c＝0.1,CHCl3)；IR νmax(film)3028,2976,2933,1715,1368,1272,1154,1069,843,760,605cm-1；1H NMR(600MHz,Chloroform-d)δ4.23(dq,J＝9.0,5.6Hz,1H,3-H),4.07(dqd,J＝8.9,6.2,2.5Hz,1H,5-H),3.80 (s,1H,3-OH),3.43(s,1H,5-OH),2.40(d,J＝6.1Hz,2H,2-CH2),1.61-1.49(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.20(d,J＝6.2Hz,3H,6-CH3).13C NMR(151MHz,Chloroform-d)δ172.2,81.6,69.2,68.2,44.0,42.6,28.1,23.7；HRMS(ESI)calculated for C10H20O4Na+[M+Na]+:227.1254,found:227.1283
化合物11*：在室温下，将三乙酰氧基四甲基硼烷(3.0g,11.5mmol)加入到干燥乙腈(9.1mL)和醋酸(9.1mL)的混合溶液中，搅拌30min。随后将化合物9*(370mg,1.83mmol)溶于乙腈(2.7mL)溶液中，在-40℃下将其滴加到反应体系中，搅拌94h。TLC检测原料反应完全后，然后用饱和酒石酸钾钠溶液淬灭，用二氯甲烷稀释和萃取；用饱和碳酸氢钠溶液洗有机层，然后使用无水硫酸钠干燥，真空浓缩，产物经硅胶柱层析(石油醚：乙酸乙酯，1:1，v/v)纯化得到二羟基酯11*(317mg,1.56mmol,85％)。[α]25 D＝+63.1°(c＝0.9,CHCl3)；IR νmax(film)2974,2931,1727,1393,1367,1257,1152,1084,954,842,760,664cm-1；1H NMR(400MHz,Chloroform-d)δ4.33(dq,J＝8.4,4.0Hz,1H,3-H),4.16(d,J＝7.8Hz,1H,5-H),3.58(d,J＝3.4Hz,1H,3-OH),2.60(d,J＝4.4Hz,1H,5-OH),2.54–2.37(m,2H,2-CH2),1.65–1.55(m,2H,4-CH2),1.49(s,9H,tBu-CH3),1.26(d,J＝6.3Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ172.5,81.5,65.9,65.8,65.1,65.0,43.5,43.5,42.2,42.1,42.0,28.2,28.1,23.6,23.5；HRMS(ESI)calculated for C10H20O4Na+[M+Na]+:227.1254,found:227.1271
化合物12*：在室温下，将三乙酰氧基四甲基硼烷(3.0g,11.5mmol)加入到干燥乙腈(9.1mL)和醋酸(9.1mL)的混合溶液中，搅拌30min。随后将化合物8*(370mg,1.83mmol)溶于乙腈(2.7mL)溶液中，在-40℃下将其滴加到反应体系中，搅拌94h。TLC检测原料反应完全后，然后用饱和酒石酸钾钠溶液淬灭，用二氯甲烷稀释和萃取；用饱和碳酸氢钠溶液洗有机层，然后使用无水硫酸钠干燥，真空浓缩，产物经硅胶柱层析(石油醚：乙酸乙酯，1:1，v/v)纯化得到二羟基酯12*(302mg,1.48mmol 81％)。[α]25 D＝+8.5°(c＝0.3,CHCl3)；IR νmax(film)2975,2931,1728,1393,1367,1257,1152,1084,954,842,760,664cm-1；1H NMR(600MHz,Chloroform-d)δ4.31(ddd,J＝12.1,8.4,3.4Hz,1H,3-H),4.14(ddp,J＝9.4,6.1,3.1Hz,1H,5-H),3.57(s,1H,3-OH),2.59(s,1H,5-OH),2.51–2.33(m,2H,2-CH2),1.65–1.53(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.24(d,J＝6.3Hz,3H,6-CH3).13C NMR(151MHz,Chloroform-d)δ172.5,81.5,65.9,65.0,43.5,42.1,28.1,23.5；HRMS(ESI)calculated for C10H20O4Na+[M+Na]+:227.1254,found:227.1283
化合物13*：在-70℃下，将化合物9*(150mg,0.74mmol)溶于四氢呋喃和甲醇(4:1,v/v,7.5mL)的混合溶液中，随后加入二乙基甲氧基硼烷(0.8mL,0.8mmol)。一个小时后，向溶液中加入硼氢化钠(31mg,0.82mmol)。随后在-70℃的条件下，继续反应22小时。TLC检测原料反应完全后，加入饱和氯化铵溶液，用乙酸乙酯萃取三次。用饱和碳酸氢钠溶液和盐水洗涤有机层，然后使用无水硫酸钠干燥，真空浓缩，产物经硅胶柱层析(石油醚：乙酸乙酯，1:1，v/v)纯化得到13*(110.2mg,0.54mmol,73％)。[α]25 D＝+26.2°(c＝0.2,CHCl3)；IR νmax(film)2978,2931,1728,1368,1324,1256,1154,1085,843,761,664cm-1；1H NMR(600MHz,Chloroform-d)δ4.23(dq,J＝9.3,6.4,4.7Hz,1H,3-H),4.07(dqd,J＝8.9,6.2,2.3Hz,1H,5-H),3.79(m,1H,3-OH),3.42(s,1H,5-OH),2.40(d,J＝6.2Hz,2H,2-CH2),1.59–1.51(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.19(s,3H,6-CH3).13C NMR(151MHz,Chloroform-d)δ172.2,81.6(1-C),69.4,69.2,68.2,44.0,42.6,28.1,23.7.(ESI)calculated for C10H20O4Na+[M+Na]+:227.1254,found:227.1278
化合物14*：在氮气的保护下，将化合物10*(642.6mg,3.15mmol)溶解于无水二氯甲烷(31.5mL)中，并在0℃下添加溴化苄(3.74mL,31.5mmol)和氧化银(8.99g,37.8mmol)。将反应混合物在0℃搅拌5h，然后在室温下搅拌28h。硅藻土过滤并浓缩后，用饱和碳酸氢钠溶液萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，通过硅胶柱层析(石油醚：乙酸乙酯，9:1，v/v)纯化得到产物14*(726mg,1.89mmol,60％)。[α]25 D＝-26.1°(c＝1.1,CHCl3)；IR νmax(film)3029,2931,1714,1453,1373,1271,1143,1094,1065,746,697cm-1；1H NMR(400MHz, Chloroform-d)δ7.33–7.11(m,10H，Ar),4.49(dd,J＝11.5,3.2Hz,2H,Ar-CH2),4.39(dd,J＝15.7,11.5Hz,2H,Ar-CH2),3.94(ddd,J＝12.6,6.9,5.6Hz,1H,3-H),3.59(h,J＝6.2Hz,1H,5-H),2.44(dd,J＝15.0,7.2Hz,1H,2-CH2),2.34(dd,J＝15.0,5.4Hz,1H,2-CH2),1.98(dt,J＝13.6,6.7Hz,1H,4-CH2),1.58–1.52(m,1H,4-CH4),1.37(s,9H,tBu-CH3),1.13(d,J＝6.1Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ171.1,138.9,138.7,128.5,128.4,127.9,127.7,127.7,127.6,80.7,73.9,71.9,71.5,70.4,41.6,41.3,28.3,19.8；HRMS(ESI)calculated for C24H32O4Na+[M+Na]+:407.2193,found:407.2232
化合物15*：在氮气的保护下，将化合物11*(764mg,3.65mmol)溶解于无水二氯甲烷(36.5mL)中，并在0℃下添加溴化苄(4.34mL,36.52mmol)和氧化银(10.4g,43.8mmol)。将反应混合物在0℃搅拌5h，然后在室温下搅拌28h。硅藻土过滤并浓缩后，用饱和碳酸氢钠溶液萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，通过硅胶柱层析(石油醚：乙酸乙酯，9:1，v/v)纯化得到产物15*(926mg,2.41mmol,66％)。[α]25 D＝+44.6°(c＝0.68,CHCl3)；IR νmax(film)2974,2930,1728,1454,1367,1256,1152,1065,954,843,736,697cm-1；1H NMR(400MHz,Chloroform-d)δ7.35–7.26(m,10H,Ar),4.60(dd,J＝11.4,3.5Hz,2H,Ar-CH2),4.36(dd,J＝18.1,11.4Hz,2H,Ar-CH2),4.13(m,J＝6.2Hz,1H,3-H),3.78(m,J＝6.1Hz,1H,5-H),2.60–2.42(m,2H,2-CH2),1.77–1.72(m,2H,4-CH2),1.47(s,9H,tBu-CH3),1.23(d,J＝6.1Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ170.9,138.6,138.6,128.3,127.8,127.7,127.5,127.4,80.5,73.4,71.9,71.6,71.5,70.3,43.3,41.7,28.1,28.1,28.1,20.0,19.9.HRMS(ESI)calculated for C24H32O4Na+[M+Na]+:407.2193,found:407.2233
化合物16*：在氮气的保护下，将化合物12*(950mg,4.65mmol)溶解于无水二氯甲烷(46.5mL)中，并在0℃下添加溴化苄(5.5mL,46.5mmol)和氧化银(13.3g,55.8mmol)。将反应混合物在0℃搅拌5h，然后在室温下搅拌28h。硅藻土过滤并浓缩后，用饱和碳酸氢钠溶液萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，通过硅胶柱层析(石油醚：乙酸乙酯，9:1，v/v)纯化得到产物16*(1.14g,0.025mol,64％)。[α]25 D＝-48.8°(c＝0.4,CHCl3)；IR νmax(film)2931,1730,1455,1368,1257,1156,1116,1063,846,735,697cm-1；1H NMR(400MHz,Chloroform-d)δ7.35–7.20(m,10H,Ar),4.58(dd,J＝11.4,3.3Hz,2H,Ar-CH2),4.34(dd,J＝17.9,11.4Hz,2H,Ar-CH2),4.11(m,J＝6.2Hz,1H,3-H),3.76(m,J＝6.2Hz,1H,5-H),2.54(dd,J＝14.8,6.4Hz,1H,2-CH2),2.43(dd,J＝14.8,5.8Hz,1H,2-CH2),1.76–1.68(m,2H,4-CH2),1.44(s,9H,tBu-CH3),1.21(d,J＝6.1Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ170.9,139.0,138.6,128.3,127.8,127.7,127.5,127.4,80.5,73.5,71.9,71.6,71.5,70.3,43.3,41.7,28.1,19.9.HRMS(ESI)calculated for C24H32O4Na+[M+Na]+:407.2193,found:407.2250
化合物17*：将化合物13*(319mg,1.56mmol)溶解于无水二氯甲烷(15.6mL)中，并在0℃下添加溴化苄(1.85mL,15.6mmol)和氧化银(4.45g,18.74mmol)。将反应混合物在0℃搅拌5h，然后在室温下搅拌28h。硅藻土过滤并浓缩后，用饱和碳酸氢钠溶液萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，通过硅胶柱层析(石油醚：乙酸乙酯，9:1，v/v)纯化得到产物17*(347.6mg,0.905mmol,58％)。[α]25 D＝-4.7°(c＝0.2,CHCl3)；IR νmax(film)2927,1729,1454,1368,1258,1157,1090,1064,844,760,697cm-1；1H NMR(600MHz,Chloroform-d)δ7.36–7.23(m,10H,Ar),4.56(dd,J＝11.5,4.2Hz,2H,Ar-CH2),4.46(dd,J＝23.3,11.5Hz,2H,Ar-CH2),4.01(m,J＝6.3Hz,1H,3-H),3.66(m,J＝6.2Hz,1H,5-H),2.51(dd,J＝15.0,7.2Hz,1H,2-CH2),2.41(dd,J＝15.0,5.4Hz,1H,2-CH2),2.05(dt,J＝13.7,6.7Hz,1H,4-CH2),1.67–1.60(m,1H,4-CH2),1.45(s,9H,tBu-CH3),1.20(d,J＝6.1Hz,3H,6-CH3).13C NMR(151MHz,Chloroform-d)δ170.9,138.8,138.5,128.4,128.3,127.8,127.7,127.5,127.5,80.5,73.8,71.7,71.3,70.2,41.4,41.1,28.1,19.7.HRMS(ESI)calculated for C24H32O4Na+[M+Na]+:407.2193,found:407.2221
化合物18*：在氮气保护下，将3,5-二苄基己酸叔丁酯14*(61mg,0.159mmol)溶于无水DCM(1mL)中，然后在0℃下向反应体系加入三氟乙酸(1mL)。温度缓慢上升至室温，反应12 小时。TLC检测原料反应完全后，加入二氯甲烷稀释，用超纯水和饱和碳酸氢钠溶液分别萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，粗产物经硅胶柱层析(石油醚:乙酸乙酯＝1∶1)纯化得到化合物18*(43.8mg,0.134mmol,84％)。[α]25 D＝-10.8°(c＝1.0,CHCl3)；IR νmax(film)2963,2928,1727,1454,1367,1270,1154,1119,1069,794,696cm-1；1H NMR(400MHz,Chloroform-d)δ7.42–7.13(m,10H,Ar),4.64–4.48(m,3H,Ar-CH2),4.41(d,J＝11.7Hz,1H,Ar-CH2),4.05(p,J＝6.2Hz,1H,3-H),3.73–3.56(m,1H,5-H),2.58(dd,J＝6.1,1.4Hz,2H,2-CH2),2.06(ddd,J＝13.7,7.6,5.7Hz,1H,4-CH2),1.67(ddd,J＝14.3,6.8,5.0Hz,1H,4-CH2),1.21(d,J＝6.1Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ176.2,138.6,138.1,128.6,128.0,127.9,127.9,127.8,127.7,73.2,71.6,71.5,70.6,70.4,41.1,39.5,21.5,19.8.HRMS(ESI)calculated for C20H24O4Na+[M+Na]+:351.1567,found:351.1604
化合物19*：在氮气保护下，将3,5-二苄基己酸叔丁酯15*(74mg,0.19mmol)溶于无水DCM(1mL)中，然后在0℃下向反应体系加入三氟乙酸(1mL)。温度缓慢上升至室温，反应12小时。TLC检测原料反应完全后，加入二氯甲烷稀释，用超纯水和饱和碳酸氢钠溶液分别萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，粗产物经硅胶柱层析(石油醚:乙酸乙酯＝1∶1)纯化得到化合物19*(53.8mg,0.164mmol,85％)。[α]25 D＝+102°(c＝0.5,CHCl3)；IR νmax(film)2927,1715,1451,1274,1176,1111,1069,1026,751,712cm-1；1H NMR(600MHz,Chloroform-d)δ7.36–7.23(m,10H,Ar),4.58(dd,J＝11.4,9.0Hz,2H,Ar-CH2),4.38(d,J＝11.2Hz,1H,Ar-CH2),4.31(d,J＝11.5Hz,1H,Ar-CH2),4.19–4.04(m,1H,3-H),3.94–3.68(m,1H,5-H),2.70–2.55(m,2H,2-CH2),1.84–1.65(m,2H,4-CH2),1.22(d,J＝6.1Hz,3H,6-CH3).13C NMR(151MHz,Chloroform-d)δ138.64-,137.9,128.5,128.4,128.0,127.9,127.85,127.59,73.0,72.0,71.4,71.37,70.3,42.9,39.6,19.8；HRMS(ESI)calculated for C20H24O4Na+[M+Na]+:351.1567,found，351.1589
化合物20*：在氮气保护下，将3,5-二苄基己酸叔丁酯16*(100mg,0.26mmol)溶于无水DCM(1mL)中，然后在0℃下向反应体系加入三氟乙酸(1mL)。温度缓慢上升至室温，反应12小时。TLC检测原料反应完全后，加入二氯甲烷稀释，用超纯水和饱和碳酸氢钠溶液分别萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，粗产物经硅胶柱层析(石油醚:乙酸乙酯＝1∶1)纯化得到化合物20*(62mg,0.19mmol,74％)。[α]25 D＝-63.3°(c＝0.75,CHCl3)；IR νmax(film)3029,2966,2930,1709,1454,1373,1146,1109,1062,736,697cm-1；1H NMR(400MHz,Chloroform-d)δ7.39–7.24(m,10H,Ar),4.61(dd,J＝11.4,5.8Hz,2H,Ar-CH2),4.40(d,J＝11.2Hz,1H,Ar-CH2),4.34(d,J＝11.6Hz,1H,Ar-CH2),4.21–4.08(m,1H,3-H),3.87–3.74(m,1H,5-H),2.65(d,J＝5.8Hz,2H,2-CH2),1.84–1.76(m,2H,3-CH2),1.25(d,J＝6.1Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ176.4,138.7,138.0,128.4,128.42,127.99,127.87,127.79,127.61,73.0,72.0,71.4,70.3,43.0,39.8,19.8.HRMS(ESI)calculated for C20H24O4Na+[M+Na]+:351.1567,found,351.1607
化合物21*：在氮气保护下，将3,5-二苄基己酸叔丁酯17*(89mg,0.23mmol)溶于无水DCM(1mL)中，然后在0℃下向反应体系加入三氟乙酸(1mL)。温度缓慢上升至室温，反应12小时。TLC检测原料反应完全后，加入二氯甲烷稀释，用超纯水和饱和碳酸氢钠溶液分别萃取有机层，然后使用无水硫酸钠干燥，真空浓缩，粗产物经硅胶柱层析(石油醚:乙酸乙酯＝1∶1)纯化得到化合物21*(62.4mg,0.19mmol,82％)。[α]25 D＝+28.6°(c＝0.4,CHCl3)；IR νmax(film)3029,2926,2857,1714,1496,1454,1373,1184,1091,1061,749,697,608cm-1；1H NMR(400MHz,Chloroform-d)δ7.36–7.26(m,10H,Ar),4.64–4.51(m,3H,Ar-CH2),4.41(d,J＝11.6Hz,1H,Ar-CH2),4.05(p,J＝6.1Hz,1H,3-H),3.68(q,J＝6.2Hz,1H,5-H),2.59(d,J＝6.0Hz,2H,2-CH2),2.07(ddd,J＝13.7,7.9,5.4Hz,1H,4-CH2),1.68(ddd,J＝14.2,7.0,4.7Hz,1H,4-CH2),1.22(d,J＝6.1Hz,3H,6-CH3).13C NMR(101MHz,Chloroform-d)δ174.5,138.4,137.8,128.5,127.9,127.8,127.7,71.4,70.3,40.8,39.1,19.7,19.7.HRMS(ESI)calculated for C20H24O4Na+[M+Na]+:351.1567,found:351.1588。
化合物22*：在氮气保护下，将3,5-二苄基己酸叔丁酯18*(89mg,0.23mmol)溶于无水DCM(1mL)中，然后在0℃下向反应体系加入草酰氯，加入催化量的DMF，0℃下反应30分钟，TLC检测反应完成后，使用适量甲醇淬灭，定量得到的酰氯化合物22*。酰氯较活泼，易水解，实验中现做现用。
实施例2
二糖受体28*的合成
如图3所示：以L-岩藻糖胺砌块23*和D-岩藻糖砌块24*(蔡军涛，江南大学博士论文，2020)为原料，进行糖基化反应，得到化合物25*；利用预活化的锌粉将二糖25*非还原端的叠氮基团还原成氨基，随后在缩合剂HATU(2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯)的作用下，将(R)-3-O-苄基丁酸26*(可参考文献自制：Tanasova.et al,Angew.Chem.Int.Ed.2015,54(14),4274-4278)与氨基糖酰胺缩合，成功在二糖的C4'位引入丁酰基，得到二糖27*。利用DDQ选择性脱除27*中的萘亚甲基，得到化合物28*。
实验操作和步骤：
化合物25*：二糖25*的合成首先利用了预活化的方法，但未能顺利合成得到；随后将供体与受体溶于二氯甲烷溶液中，在三氟甲磺酸三甲基硅脂和碘代丁二酰亚胺的条件下进行糖苷化反应，成功得到化合物25*，但反应的选择性较差(α：β＝1.5：1)。随后利用乙醚的溶剂效应，再次进行糖基化反应条件的优化，最终以良好的选择性得到化合物25*(α：β＝6：1)。糖基化方法具体如下：
方法1：在氩气保护下，将糖基供体24*(53.2mg，0.104mmol)溶解于无水二氯甲烷(2.0mL)，并冷却至零下60℃。随后加入预活化的分子筛、1-(苯基磺酰基)哌啶(BSP，26.1mg，0.125mmol)、2,4,6-三叔丁基嘧啶(TTBP，38.8mg，0.156mmol)和三氟甲磺酸酐(Tf2O，22.7μL，0.135mmol)，在零下60℃下搅拌反应30分钟。进行TLC监测，确认原料反应完全后，加入1-辛烯(16.3μL，0.104mmol)，并在零下60℃下继续搅拌反应15分钟。随后，将反应液冷却至零下78℃后，将糖基受体23*(72.4mg，0.120mmol)溶解于无水二氯甲烷(2.0mL)逐滴加入到反应液中，继续在零下78℃下搅拌反应3小时。当TLC监测确认原料反应完全后，加入亚磷酸三乙酯(53.5μL，0.312mmol)来终止反应，然后在零下78℃下继续搅拌1小时。TLC检测反应，发现反应体系成分复杂，液相质谱也未检测到目标化合物。
方法2：将D-岩藻糖供体24*(80.6mg，0.158mmol)和L-岩藻糖受体23*(115mg，0.19mmol)，溶于甲苯(5ml)，共沸除水3次，真空浓缩旋干，随后加入预先活化的分子筛，放油泵上抽真空过夜。在氩气保护下，依次加入无水二氯甲烷(6.0mL)、碘代丁二酰亚胺(43mg，0.19mmol)，冷却至0℃，搅拌30分钟，逐滴加入三氟甲磺酸三甲基硅脂(5.7μL，0.032mmol)，在0℃下搅拌反应5小时。TLC检测原料反应完全后，加入三乙胺淬灭反应，在砂芯漏斗中加入硅藻土，过滤除去分子筛，用二氯甲烷萃取，用饱和碳酸氢钠溶液洗涤，收集有机相，无水硫酸钠干燥，用滤纸过滤除硫酸钠，用旋转蒸发仪减压蒸馏浓缩，粗品用硅胶柱层析分离纯化(石油醚：乙酸乙酯，1:1，v/v)得到α构型二糖25*(128.7mg，0.128mmol，81％，α：β＝1.5：1)。
方法3：将D-岩藻糖供体24*(100mg，0.196mmol)和L-岩藻糖受体23*(141.6mg，0.235mmol)，溶于甲苯(5ml)，共沸除水3次，真空浓缩旋干，随后加入预先活化的分子筛，放油泵上抽真空过夜。在氩气保护下，依次加入无水二氯甲烷(4.5mL)和乙醚(3.0mL)、碘代丁二酰亚胺(52.9mg，0.235mmol)，冷却至0℃，搅拌30分钟，逐滴加入三氟甲磺酸三甲基硅脂(3.45μL，0.039mmol)，在0℃下搅拌反应5小时。TLC检测原料反应完全后，加入三乙胺淬灭反应，在砂芯漏斗中加入硅藻土，过滤除去分子筛，用二氯甲烷萃取，用饱和碳酸氢钠溶液洗涤，收集有机相，无水硫酸钠干燥，用滤纸过滤除硫酸钠，用旋转蒸发仪减压蒸馏浓缩，粗品用硅胶柱层析分离纯化(石油醚：乙酸乙酯，1:1，v/v)得到α构型二糖 25*(177mg，0.176mmol，90％，α：β＝6：1，α构型：3JH1/H2＝3.4Hz，1JH1/C1＝166Hz)。[α]25 D＝+11.67°(c＝1.0,CHCl3)；IR νmax(film)3366,2934,2108,1732,1700,1538,1498,1456,1423,1362,1278,1228,1177,1127,1092,1047,858,823,752,735,700cm-1；α:1H NMR(400MHz,CDCl3)δ＝7.96-7.01(m,27H,Ar),6.61(d,J＝5.7Hz,1H,NHAc),5.15(d,J＝6.2Hz,2H,ArCH2),5.12(d,J＝3.5Hz,1H,1-H),4.95-4.78(m,5H,1'-H,ArCH2),4.71(dd,J＝11.3,5.1Hz,1H,ArCH2),4.63(d,J＝11.8Hz,1H,ArCH2),4.47(d,J＝6.9Hz,2H,ArCH2),4.32(s,1H,2-H),4.16-4.05(m,2H,3'-H,5'-H),3.98(dd,J＝9.8,3.4Hz,1H,2'-H),3.91(t,J＝11.6Hz,1H,3-H),3.85-3.74(m,2H,4'-H,5-H),3.64-3.46(m,2H,4-H,linker-OCH2),3.45-3.14(m,3H,linker-OCH2,linker-NCH2),1.55(ddt,J＝17.5,13.8,6.5Hz,4H,linker-CH2),1.39(d,J＝4.5Hz,3H,Ac),1.35-1.25(m,2H,linker-CH2),1.13(d,J＝6.4Hz,3H,6'-CH3),1.09(m,3H,6-CH3)；13C NMR(100MHz,CDCl3)δ＝170.5,156.8,156.3,138.6,137.9,137.3,136.9,136.8,135.2,133.3,133.2,129.1,128.8,128.7,128.6,128.4,128.1,128.0,127.9,127.7,127.4,127.3,126.7,126.5,126.3,125.7,99.0,97.0,79.1,78.9,77.7,75.3,74.6,73.3,68.1,67.3,66.4,66.2,64.5,50.5,50.2,50.0,47.2,46.2,29.5,28.1,27.6,23.6,22.5,17.5,16.9.HR-ESI-MS(m/z)：calcd for C59H67O10N5Na+(M+Na)+:1028.4780found:1028.4786。
化合物27*：在氮气的保护，将二糖25*(20.5mg,20.4μmol)溶于四氢呋喃(0.8mL)、和醋酸(0.2mL)的混合溶液中，随后加入预活化的锌粉(0.5g)，室温下搅拌12小时。之后过滤并浓缩反应混合物，得到粗品氨基三糖，随后直接进行下一步反应。在室温下将(R)-3-O-苄基丁酸26*(6mg，30.6μmol)和氨基三糖溶解在在DMF(1mL)溶液中，加入HATU(9.3mg，22.48μmol)和DIPEA(6.8μL，40.8μmol)，在室温下搅拌6小时。TLC检测原料反应完全后，将粗产物溶解在乙酸乙酯中，并用饱和氯化钠溶液洗涤，分离的有机层用无水硫酸钠干燥，真空浓缩，并通过柱色谱法(石油醚：乙酸乙酯，1:1，v/v)纯化，得到化合物27*(18.8mg，16.3μmol，80％)。[α]25 D＝+34.6°(c＝1.2,CHCl3)；IR νmax(film)3029,2924,2853,1681,1543,1507,1455,1361,1217,1092,1045,758,698cm-1；1H NMR(400MHz,CDCl3)δ＝7.96-7.01(m,32H,Ar),6.80(d,J＝10.2Hz,1H,NHRHb),6.60(d,J＝5.5Hz,1H,NHAc),5.15(s,2H,ArCH2),5.10(d,J＝3.5Hz,1H,1-H),5.01(d,J＝11.1Hz,1H,ArCH2),4.86(d,J＝11.7Hz,1H,ArCH2),4.71-4.58(m,5H,1'-H,4'-H,ArCH2),4.55-4.43(m,5H,ArCH2),4.25(m,2H,2-H,5'-H),4.01(dt,J＝6.4,3.2Hz,1H,RHb-CH),3.94(dd,J＝10.1,4.2Hz,1H,3'-H),3.89-3.74(m,2H,3-H,5-H),3.60-3.44(m,2H,4-H,linker-OCH2),3.36-3.14(m,3H,linker-OCH2,linker-NCH2),2.65(dd,J＝15.2,3.7Hz,1H,RHb-CH2),2.52(dd,J＝15.2,6.7Hz,1H,RHb-CH2),1.62-1.46(m,4H,linker-CH2),1.35-1.25(m,8H,RHb-CH3,Ac,linker-CH2),1.10(d,J＝6.4Hz,3H,6-CH3),1.03(d,J＝6.4Hz,3H,6'-CH3)；13C NMR(100MHz,CDCl3)δ＝171.7,170.5,138.7,138.3,138.0,137.5,135.7,133.4,133.24,133.15,132.3,132.3,132.2,132.1,132.0,128.8,128.69,128.67,128.6,128.54,128.48,128.4,128.2,128.1,128.03,128.00,127.94,127.89,127.8,127.7,127.4,127.1,126.4,126.2,126.1,98.8,97.0,78.9,77.8,75.2,74.5,72.9,71.6,70.9,68.1,67.3,66.8,66.3,50.2,50.0,43.6,29.5,23.5,22.4,19.1,17.0,16.9.HR-ESI-MS(m/z)：calcd for C70H81O12N3Na+(M+Na)+:1178.5712found:1178.5704。
化合物28*：在氩气保护下，将化合物27*(26.8mg，23.0μmol)溶于二氯甲烷(5.0mL)，加入去离子水(1.0mL)和2,3-二氯-5,6-二氰基-1,4-苯醌(DDQ)(7.7mg，35.0μmol)，室温下搅拌反应5小时。TLC监测原料反应完全后，用二氯甲烷萃取，用10％(w/w)硫代硫酸钠溶液洗涤，收集有机相，无水硫酸钠干燥，用滤纸过滤除硫酸钠，用旋转蒸发仪减压蒸馏浓缩，粗品用硅胶柱层析分离纯化(石油醚：乙酸乙酯，1:1，v/v)得到化合物28*(21mg，20.7μmol，90％)。[α]25 D＝-14.4°(c＝1.0,CHCl3)；IR νmax(film)3342,3030,2936,1670,1541,1496,1453,1422,1362,1304,1216,1180,1092,1043,827,754,698cm-1；1H NMR(400MHz,CDCl3)δ＝7.42-7.13(m,25H,Ar),6.81(d,J＝8.6Hz,1H,NHRHb),6.61(d,J＝5.8Hz,1H,NHAc),5.15(d,J＝4.9Hz,2H,ArCH2),5.08(d,J＝3.5Hz,1H,1-H),4.84(d,J＝11.8Hz,1H, ArCH2),4.77(d,J＝11.0Hz,1H,ArCH2),4.67-4.53(m,4H,1'-H,ArCH2),4.51-4.43(m,3H,ArCH2),4.35-4.20(m,3H,2-H,4'-H,5'-H),4.14(m,1H,3'-H),4.00(dt,J＝6.2,3.5Hz,1H,RHb-CH),3.86(d,J＝11.2Hz,1H,3-H),3.80(d,J＝7.3Hz,1H,5-H),3.60-3.44(m,2H,4-H,linker-OCH2),3.38(dd,J＝9.8,3.5Hz,1H,2'-H),3.36-3.18(m,3H,linker-OCH2,linker-NCH2),2.63(dd,J＝15.0,3.8Hz,1H,RHb-CH2),2.50(dd,J＝15.2,6.6Hz,1H,RHb-CH2),1.57(s,3H,Ac),1.56-1.46(m,4H,linker-CH2),1.37(d,J＝6.2Hz,3H,RHb-CH3),1.33-1.24(m,2H,linker-CH2),1.11(d,J＝6.4Hz,3H,6-CH3),0.95(d,J＝6.4Hz,3H,6'-CH3)；13C NMR(100MHz,CDCl3)δ＝173.8,170.7,138.7,138.04,137.97,137.8,136.9,128.71,128.67,128.6,128.44,128.36,128.1,127.94,127.90,127.88,127.7,127.4,127.3,98.6,97.1,79.2,78.6,74.9,74.5,72.5,71.7,70.9,68.0,67.3,66.3,66.1,54.7,49.9,47.2,46.3,43.5,29.8,29.4,28.1,27.6,23.5,22.8,19.1,17.0,16.7.HR-ESI-MS(m/z)：calcd for C59H73O12N3Na+(M+Na)+:1038.5086found:1038.5117。
实施例3
三糖31*的合成
如图4所示：将已知的D-奎诺糖胺砌块29*(可参考现有文献自制Codée.et al,Organic&Biomolecular Chemistry.2020,18(15),2834-2837)溶解在丙酮和H2O的混合溶液中，在碘代丁二酰亚胺(NIS)的催化下水解，随后在2,2,2-三氟-N-苯基乙酰亚胺酰氯(171μL，1.14mmol)和1，8-二氮杂二环十一碳-7-烯(DBU)的作用下，得到三氟乙酰亚胺酯30*；将三氟乙酰亚胺酯30*和二糖受体28*，在三氟甲磺酸三甲基硅酯(TMSOTf)催化的条件下进行糖苷化反应，成功得到单一β构型三糖化合物31*(产率13％)。
由于31*的合成产率较低，本发明还进行了糖基化条件的优化。将糖基供体由化合物30*替换为化合物29*后，在三氟甲磺酸三甲基硅酯和碘代丁二酰亚胺催化的条件下进行糖苷化反应，以28％的产率得到三糖31*；随后继续以化合物29*为糖基供体，通过改变催化剂的当量，成功将糖基化产率提高到54％；接着通过改变温度进一步优化糖基化产率，发现零摄氏度缓慢升至室温，可以取得较好的糖基化产率。最终在温度效应和催化剂的双重促进下，以81％的较高产率顺利得到目标三糖。具体优化过程及反应条件如表1所示：
表1
具体实验操作和步骤：
化合物30*：在室温下，将化合物29*(232.5mg，0.38mmol)溶解在丙酮和H2O(10：1，v/v，5.5mL)中，搅拌均匀，然后加入NIS(171.4mg，0.7mmol)，并搅拌1小时。TLC显示反应完全后，用乙酸乙酯稀释混合物，用10％(w/v)Na2S2O3洗涤。将有机层用Na2SO4干燥，过滤并真空浓缩。将残余物通过硅胶柱色谱法(石油醚/乙酸乙酯20/1→1/1v/v)纯化，得到中间体化合物。在0℃下，将中间体化合物溶解于DCM(4.8mL)溶液中，然后加入2,2,2-三氟-N-苯基乙酰亚胺酰氯(171μL，1.14mmol)和1，8-二氮杂二环十一碳-7-烯(DBU)(171μL，1.14mmol)。在0℃下，搅拌反应3小时，将混合物真空浓缩，然后通过硅胶柱色谱法(石油醚/乙酸乙酯：30/1→10/1v/v)纯化得到三氟乙酰亚胺酯30*(223.4mg，0.34mmol，两步产率90％)。1HNMR(400MHz,Chloroform-d)δ＝7.96–7.76(m,5H),7.59– 7.33(m,6H),7.30–7.05(m,1H),6.27(d,J＝7.4,1H),4.94(q,J＝11.9,2H),4.38(dd,J＝7.4,4.1,1H),3.85(dd,J＝5.7,4.1,1H),3.50(dq,J＝9.6,6.1,1H),3.34(dd,J＝9.7,5.7,1H),1.38(d,J＝6.1,3H).13C NMR(101MHz,Chloroform-d)δ＝162.9,135.1,134.2,133.2,133.2,129.4,128.5,128.0,127.7,127.6126.4,126.3,126.2,126.0,105.6,78.9,72.3,67.9,66.7,64.6,19.1，HR-ESI-MS(m/z)：calcd for C28H25Cl3F3N5O4Na+(M+Na)+:680.0924，found:680.1030
化合物31*：具有代表性的两种糖基化反应操作方法如下
方法1：在氩气保护下，将三氟乙酰亚胺酯30*(24.3mg,37μmol)和二糖受体28*(25.3mg,25umol)，用甲苯溶解共沸旋蒸除水3次，在油泵上抽真空2小时，随后加入预先活化的分子筛、无水二氯甲烷(1.0mL)，反应液冷却至0℃，搅拌30分钟，逐滴加入三氟甲磺酸三甲基硅脂(0.9μL,5.0μmol)，在0℃下搅拌反应5小时。TLC检测原料反应完全后，加入三乙胺淬灭反应，硅藻土过滤除去分子筛，用二氯甲烷萃取，饱和碳酸氢钠溶液洗涤，收集有机相，无水硫酸钠干燥，用旋转蒸发仪减压蒸馏浓缩，粗品用硅胶柱层析分离纯化(石油醚：乙酸乙酯，4:1，v/v)得到糖浆31*(4.78mg,3.25μmol,13％)。
方法2：在氩气保护下，将硒苷29*(110mg,0.18mmol)和二糖受体28*(122mg,0.12mmol)，用甲苯溶解共沸旋蒸除水3次，在油泵上抽真空2小时，随后加入预先活化的分子筛、无水二氯甲烷(6.0mL)，反应液冷却至0℃，搅拌30分钟，逐滴加入三氟甲磺酸三甲基硅脂(8.7μL,0.048mmol)和碘代丁二酰亚胺(40.5mg,0.18mmol)，从0℃缓慢升温至室温搅拌反应5小时。TLC检测原料反应完全后，加入三乙胺淬灭反应，硅藻土过滤除去分子筛，用二氯甲烷萃取，饱和碳酸氢钠溶液洗涤，收集有机相，无水硫酸钠干燥，用旋转蒸发仪减压蒸馏浓缩，粗品用硅胶柱层析分离纯化(石油醚：乙酸乙酯，4:1，v/v)得到糖浆31*(142.8mg,97.2μmol,81％)。[α]25 D＝+15.2°(c＝0.7,CHCl3)；IR νmax(film)3343,3029,2979,2940,2877,2108,1659,1532,1496,1454,1423,1360,1309,1216,1167,1092,1047,822,756,698cm-1；1H NMR(600MHz,Methanol-d4)δ7.85–7.72(m,4H,Ar),7.47–7.14(m,28H,Ar),5.13(d,J＝12.9Hz,2H,Ar-CH2),5.06–4.96(m,1H,1-H'),4.94–4.86(m,3H,Ar-CH2),4.82(d,J＝8.0Hz,2H,1-H,1"-H),4.69–4.63(m,2H,Ar-CH2),4.60(d,J＝11.3Hz,2H,Ar-CH2),4.51–4.47(m,3H,Ar-CH2),4.45–4.38(m,2H,4'-H,2-H),4.24–4.15(m,2H,3'-H,5'-H),4.10–4.05(m,1H,RHb-3),3.99(m,1H,3-H),3.87(m,2H,2"-H,5-H),3.74(m,2H,2'-H,3"-H),3.66(d,J＝9.1Hz,1H,4-H),3.54(s,1H,Linker-OCH2),3.33(m,1H,Linker-OCH2),3.28–3.18(m,3H,4"-H,Linker-NCH2),3.11–3.03(m,1H,5"-H),2.60–2.49(m,2H,RHb-2),1.66(d,J＝10.2Hz,3H,NHAc-CH3),1.51(s,4H,Linker-CH2),1.33(d,J＝6.2Hz,3H,RHb-4),1.29–1.24(m,2H,Linker-CH2),1.18(d,J＝6.1Hz,6H,6"-CH3,6-CH3),0.96(d,J＝6.4Hz,3H,6'-CH3).13C NMR(151MHz,Methanol-d4)δ173.1(NH-C＝O),162.4(NH-C＝O),138.8(Ar),138.6(Ar),138.1(Ar),135.2(Ar),133.3(Ar),133.1(Ar),128.3(Ar),126.2(Ar),125.7(Ar),125.5(Ar),100.2(1"-H),98.1(1'-H),97.0(1-H),80.0(3"-C),78.5(4-C),76.4(2'-C),75.3(3-C),75.1(Ar-CH2),74.0(3'-C),72.9(Ar-CH2,RHb-3),70.6(5"-C),70.4(Ar-CH2),67.6(4"-C),67.4(Ar-CH2),66.6(5-C,Ar-CH2),66.2(5'-C),58.0(2"-C),53.8(4'-C),50.1(2-C,Ar-CH2),46.6(Linker-NCH2)43.2(RHb-2),28.8(Linker-CH2),23.04(Linker-CH2),21.6(NHAc-CH3),18.9(RHb-4),17.2(6"-CH3),15.7(6'-CH3,6-CH3).HR-ESI-MS(m/z)：calcd for C78H90Cl3N7O15Na+(M+Na)+:1492.5453found:1492.5469。
实施例4
三糖32*和33*，34*，35*的合成
如图5所示：利用温和的1，3-丙二硫醇还原三糖31*中非还原端的叠氮基，得到氨基三糖，随后在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和1-羟基苯并三唑存在的条件下，氨基三糖分别与3，5-二苄基己酸18*或19*，20*，21*进行酰胺缩合反应，得到全保护的三糖32*～35*。上述酰胺缩合的方法，是通过一系列的试错法优化得到。
以化合物31*为原料，首先尝试了三苯基磷还原叠氮基团，并利用新制备的酰氯22*进行酰胺偶联，但只得到了痕量的目标化合物32*；随后尝试1，3-丙二硫醇和HATU组合使用，以18％的产率得到化合物32*；随后继续优化叠氮基团的还原条件，发现在吡啶和水、三乙胺存在的条件下，利用1，3-丙二硫醇可以较好的提高还原产率；进一步优化缩合试剂，最终确定EDC为较好的缩合试剂。总的来说，1，3-丙二硫醇和EDC的组合使用可以显著的提高酰胺缩合总产率。具体优化过程及反应条件如表2所示：
表2
具体实验操作和步骤
化合物32*：在氮气的保护，将三糖31*(30mg,20.4μmol)溶于水(1mL)、Et3N(124.8μl，0.90mmol)和吡啶(4mL)的混合溶液中，随后加入1，3-丙二硫醇(122μl，1.22mmol)，室温下搅拌6小时。之后浓缩反应混合物，得到粗品氨基三糖，直接进行下一步反应。在室温下将3，5-二苄基己酸18*(10mg，30.6μmol)和氨基三糖溶解在在乙腈(5mL)溶液中，加入碳酸氢钠(5.1mg，61.2μmol)，10分钟后，依次加入1-羟基苯并三唑(0.55mg，4.08μmol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(6.25mg，32.6μmol)，在室温下搅拌6小时。TLC检测原料反应完全后，将粗产物溶解在乙酸乙酯中，并用饱和氯化钠溶液洗涤，分离的有机层用无水硫酸钠干燥，真空浓缩，并通过柱色谱法(DCM:MeOH＝20:1)纯化，得到化合物32*(20.5mg,13.67μmol,67％)。[α]25 D＝+7.2°(c＝0.36,CHCl3)；IR νmax(film)2925,2857,1748,1660,1543,1496,1454,1374,1240,1081,1046,811,757,698cm-1；1H NMR(600MHz,Methanol-d4)δ7.80–7.67(m,4H,Ar),7.46–7.11(m,39H,Ar),5.9(s,1H,DCA-CH),5.1(d,J＝11.8Hz,2H,Ar-CH2),4.97(s,1H,1'-H),4.90(d,J＝11.8Hz,1H,Ar-CH2),4.85(d,J＝8.3Hz,2H,1-H,1"-H),4.75(d,J＝11.3Hz,1H,Ar-CH2),4.72–4.64(m,3H,Ar-CH2),4.59(d,J＝11.4Hz,2H,Ar-CH2),4.51–4.39(m,6H,Ar-CH2,4'-H,2-H),4.37–4.31(m,2H,Ar-CH2),4.28(d,J＝11.6Hz,1H,Ar-CH2),4.16(dd,J＝10.2,4.9Hz,2H,3'-H,5'-H),4.09–4.03(m,1H,RHb-3),4.02–3.96(m,1H,3-H),3.89(dt,J＝12.3,5.4Hz,3H,dHh-3,4-H,5-H),3.77(dd,J＝10.2,3.8Hz,2H,2"-H,2-H),3.69(m,1H,4"-H),3.64(m,1H,3"-H),3.58(h,J＝6.0Hz,2H,dHh-5-H,Linker-OCH2),),3.40(m,1H,5"-H)),3.34(m,1H,Linker-OCH2)3.23(s,2H,Linker-NCH2),2.61–2.49(m,2H,RHb-2),2.33–2.21(m,2H,dHh-2),1.90(dt,J＝13.6,6.7Hz,1H,dHh-4),1.72(d,J＝9.4Hz,3H,NHAc-CH3),1.53(dt,J＝14.0,5.8Hz,5H,dHh-4,Linker-CH2),1.35(d,J＝6.1Hz,3H,RHb-4),1.30–1.26(m,2H,Linker-CH2),1.17(d,J＝6.4Hz,3H,6-CH3),1.08(dd,J＝6.1,3.5Hz,6H,6"-CH3,dHh-4),0.95(d,J＝6.5Hz,3H,6'-CH3).13C NMR(151MHz,Methanol-d4)δ＝172.9(NH-C＝O),172.1(NH-C＝O),164.7(NH-C＝O),138.8(Ar),138.7(Ar),138.6(Ar),138.3(Ar),138.2(Ar),135.9(Ar),133.3(Ar),133.0(Ar),128.3(Ar), 127.8(Ar),127.7–127.5(Ar),127.4(Ar),127.7(Ar),127.1(Ar),125.9(Ar),125.6(Ar),125.4(Ar),100.7(1"-C),98.2(1'-C),97.1(1-C),78.5(3"-C),76.2(3-C),74.9(Ar-CH2),74.8(3'-C),73.4(dHh-3),72.9(Ar-CH2,RHb-3),72.4(Ar-CH2),71.8(dHh-5),70.5(Ar-CH2,5"-C),69.8(Ar-CH2),67.5(Linker-OCH2),67.0(Ar-CH2),66.6(Ar-CH2),66.5(5'-C),66.4(DCA-CH2),57.1(2"-C,4"-C),53.6(4'-C),50.1(2-C,Ar-CH2),46.5(Linker-NCH2)43.1(RHb-2),40.9(dHh-4,dHh-2),31.4(1-C),28.8(Linker-CH2),27.1(Linker-CH2),23.2(1-C),22.3(Linker-CH2),21.8(NHAc-CH3),18.8(RHb-4),18.5(dHh-6),17.0(6"-C),15.7(6-C),15.7(6'-C).HR-ESI-MS(m/z)：calcd for C98H115Cl2N5O18Na+(M+Na)+:1742.7506found:1742.7550。
化合物33*：在氮气的保护，将三糖31*(30mg,20.4μmol)溶于水(1mL)、Et3N(124.8μl，0.90mmol)和吡啶(4mL)的混合溶液中，随后加入1，3-丙二硫醇(122μl，1.22mmol)，室温下搅拌6小时。之后浓缩反应混合物，得到粗品氨基三糖，直接进行下一步反应。在室温下将3，5-二苄基己酸19*(10mg，30.6μmol)和氨基三糖溶解在在乙腈(5mL)溶液中，加入碳酸氢钠(5.1mg，61.2μmol)，10分钟后，依次加入1-羟基苯并三唑(0.55mg，4.08μmol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(6.25mg，32.6μmol)，在室温下搅拌6小时。TLC检测原料反应完全后，将粗产物溶解在乙酸乙酯中，并用饱和氯化钠溶液洗涤，分离的有机层用无水硫酸钠干燥，真空浓缩，并通过柱色谱法(DCM:MeOH＝20∶1)纯化，得到化合物33*(20.7mg，12.04μmol，59％)。[α]25 D＝+11.6°(c＝0.42,CHCl3)；IR νmax(film)2926,2857,1749,1660,1543,1499,1454,1374,1240,1081,1046,819,757,698cm-1；1H NMR(600MHz,Methanol-d4)δ7.80–7.67(m,4H,Ar),7.50–7.08(m,38H,Ar),5.95(s,1H,DCA-H),5.13(d,J＝10.8Hz,2H,Ar-CH2),5.02–4.84(m,4H,1'-H,Ar-CH2,1-H,1"-H),4.75–4.64(m,4H,Ar-CH2),4.62–4.54(m,2H,Ar-CH2),4.49–4.38(m,7H,Ar-CH2,4"-H,2-H),4.25–4.12(m,4H,3'-H,5'-H,Ar-CH2),4.07–3.86(m,5H,RHb-3,dHh-3,3-H,4-H,5-H),3.83–3.74(m,2H,2"-H,2'-H),3.72(s,1H,3"-H),3.64(dtd,J＝9.1,6.2,3.7Hz,2H,dHh-5,4"-H),3.61–3.49(m,1H,Linker-OCH2),3.37(d,J＝33.8Hz,2H,Linker-OCH2,5"-H),3.25(d,J＝27.4Hz,2H,Linker-NCH2),2.61–2.48(m,2H,RHb-2),2.36(dd,J＝14.2,5.7Hz,1H,dHh-2),2.30–2.21(m,1H,dHh-2),1.77–1.69(m,3H,NHAc-CH3),1.64–1.44(m,6H,dHh-4,Linker-CH2),1.34(d,J＝6.1Hz,3H,RHb-4),1.33–1.21(m,2H,Linker-CH2),1.16(dd,J＝9.9,6.1Hz,3H,6-CH3),1.10(d,J＝6.1Hz,3H,6"-CH3),1.06(d,J＝6.1Hz,3H,dHh-6),0.95(d,J＝6.4Hz,3H,6'-CH3).13C NMR(151MHz,Methanol-d4)δ172.9(NH-C＝O),172.0(NH-C＝O),164.7(NH-C＝O),141.8–137.4(Ar),136.3(Ar),133.1(Ar),126.0–123.8(Ar),100.7(1"-C),98.2(1'-C),97.0(1-C),78.5(4-C,dHh-5),76.0(3-C,3"-C),74.9(4'-C),73.4(dHh-3),73.1(RHb-3,Ar-CH2),72.1(Ar-CH2),71.4(4"-H),70.9(Ar-CH2),70.5(5"-H),69.9(Ar-CH2),67.6(Ar-CH2),67.1(Ar-CH2)66.5(5'-C,5-C),56.9(2"-C),56.2(4"-C),53.6(4'-C),50.1(Ar-CH2,2-C),46.4(Linker-NCH2),43.1(RHb-2),42.9(dHh-4),41.4(dHh-2),28.8(Linker-CH2),23.2(Linker-CH2),21.8(NHAc-CH3),18.8(RHb-4,dHh-6),17.1(6"-C),15.7(6-CH3,6'-CH3,),HR-ESI-MS(m/z)：calcd for C98H115Cl2N5O18Na+(M+Na)+:1742.7506found:1742.7547。
化合物34*：在氮气的保护，将三糖31*(30mg,20.4μmol)溶于水(1mL)、Et3N(124.8μl，0.90mmol)和吡啶(4mL)的混合溶液中，随后加入1，3-丙二硫醇(122μl，1.22mmol)，室温下搅拌6小时。之后浓缩反应混合物，得到粗品氨基三糖，直接进行下一步反应。在室温下将3，5-二苄基己酸20*(10mg，30.6μmol)和氨基三糖溶解在在乙腈(5mL)溶液中，加入碳酸氢钠(5.1mg，61.2μmol)，10分钟后，依次加入1-羟基苯并三唑(0.55mg，4.08μmol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(6.25mg，32.6μmol)，在室温下搅拌6小时。TLC检测原料反应完全后，将粗产物溶解在乙酸乙酯中，并用饱和氯化钠溶液洗涤，分离的有机层用无水硫酸钠干燥，真空浓缩，并通过柱色谱法(DCM:MeOH＝20∶1)纯化，得到化合物34*(21.4mg,12.4μmol，61％).[α]25 D＝+8.5°(c＝0.3,CHCl3)；IR νmax(film)2956,2857,1739,1660,1543,1499,1464,1374,1240,1081,1046,819,757,698cm-1；1H NMR(600 MHz,Methanol-d4)δ7.80–7.65(m,4H,Ar),7.54–7.12(m,38H,Ar),5.95(s,1H,DCA-H),5.18–5.07(m,2H,Ar-CH2),4.97–4.84(m,4H,Ar-CH2,1'-H,1-H,1"-H),4.77–4.64(m,4H,Ar-CH2),4.61–4.55(m,2H,Ar-CH2),4.53–4.36(m,7H,Ar-CH2,4'-H,2-H),4.30(d,J＝11.3Hz,1H,Ar-CH2),4.16(dd,J＝11.4,6.2Hz,3H,Ar-CH2,3'-H,5'-H),4.05(ddd,J＝14.4,7.1,5.5Hz,2H,RHb-3,dHh-3,),4.00(d,J＝23.2Hz,1H,3-H),3.93–3.84(m,2H,4-H,5-H),3.82–3.71(m,3H,2"-H,4"-H,2'-H),3.70–3.50(m,3H,3"-H,dHh-5,Linker-OCH2),3.36(s,2H,Linker-OCH2,5"-H),3.23(s,2H,Linker-NCH2),2.62–2.46(m,2H,RHb-2),2.37–2.22(m,2H,dHh-2),1.72(d,J＝7.8Hz,3H,NHAc-CH3),1.64–1.46(m,6H,dHh-4,Linker-CH2),1.35(d,J＝6.2Hz,3H,RHb-4),1.28(s,2H,Linker-CH2),1.16(t,J＝7.9Hz,3H,6-CH3),1.13–1.02(m,3H,6"-CH3),0.97(dd,J＝25.3,6.3Hz,6H,dHh-CH3,6'-CH3).13C NMR(151MHz,Methanol-d4)δ172.9(NH-C＝O),172.2(NH-C＝O),171.9(NH-C＝O),164.7(NH-C＝O),138.8(Ar),138.5(Ar),138.2(Ar),135.8(Ar),133.3(Ar),133.0(Ar),128.3(Ar),128.2(Ar),127.9(Ar),127.3(Ar),127.1(Ar),125.8(Ar),125.6(Ar),125.4(Ar),100.6(1"-C),98.2(1'-C),97.1(1-C),78.5(3"-C),76.2(3-C,2'-C),74.9(3'-C),73.6(Ar-CH2),72.9(RHb-3,dHh-3),71.4(Ar-CH2),71.3(dHh-5,Ar-CH2),70.5(5"-C,Ar-CH2),69.8(Ar-CH2),67.6(Ar-CH2),66.6(5-C,Ar-CH2),66.4(5'-C)57.07(2"-C),55.9(4"-C),53.6(4'-C),50.1(Ar-CH2,2-C),46.6(Linker-NCH2),43.1(RHb-2),42.4(dHh-4),41.8(dHh-2),28.7(Linker-CH2),23.1(Linker-CH2),21.8(NHAc-CH3),18.8(RHb-4),18.7(dHh-6),17.0(6"-C),15.8(6-C),15.7(6"-C).HR-ESI-MS(m/z)：calcd for C98H115Cl2N5O18Na+(M+Na)+:1742.7506found:1742.7512。
化合物35*：在氮气的保护，将三糖31*(30mg,20.4μmol)溶于水(1mL)、Et3N(124.8μl，0.90mmol)和吡啶(4mL)的混合溶液中，随后加入1，3-丙二硫醇(122μl，1.22mmol)，室温下搅拌6小时。之后浓缩反应混合物，得到粗品氨基三糖，直接进行下一步反应。在室温下将3，5-二苄基己酸21*(10mg，30.6μmol)和氨基三糖溶解在在乙腈(5mL)溶液中，加入碳酸氢钠(5.1mg，61.2μmol)，10分钟后，依次加入1-羟基苯并三唑(0.55mg，4.08μmol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(6.25mg，32.6μmol)，在室温下搅拌6小时。TLC检测原料反应完全后，将粗产物溶解在乙酸乙酯中，并用饱和氯化钠溶液洗涤，分离的有机层用无水硫酸钠干燥，真空浓缩，并通过柱色谱法(DCM:MeOH＝20∶1)纯化，得到化合物35*(23.86mg,13.87μmol，68％)。[α]25 D＝-7.8°(c＝0.3,CHCl3)；IR νmax(film)2989,2851,1749,1660,1545,1499,1454,1384,1240,1081,1046,819,757,698cm-1；1H NMR(600MHz,Methanol-d4)δ7.77–7.66(m,4H,Ar),7.45–7.13(m,39H,Ar),5.95(s,1H,DCA-CH),5.17–5.07(m,2H,Ar-CH2),4.98–4.84(m,4H,1'-H,1-H,1"-H,Ar-CH2),4.75(d,J＝11.1Hz,1H,Ar-CH2),4.71–4.64(m,3H,Ar-CH2),4.59(d,J＝11.3Hz,2H,Ar-CH2),4.49(d,J＝12.0Hz,3H,Ar-CH2),4.45–4.35(m,5H,Ar-CH2,4'-H,2-H),4.29(d,J＝11.5Hz,1H,Ar-CH2),4.19–4.10(m,2H,3'-H,5'-H),4.06(h,J＝6.4Hz,1H,RHb-3),3.98(p,J＝6.3Hz,2H,dHh-3,3-H),3.88(dt,J＝21.2,8.7Hz,2H,4-H,5-H),3.77(dd,J＝10.3,3.9Hz,3H,2"-H,2'-H,4"-H),3.62(dd,J＝28.1,7.7Hz,2H,3"-H,Linker-OCH2),3.49(p,J＝6.2Hz,1H,dHh-5),3.35(m,2H,5"-H,Linker-OCH2),3.23(m,2H,Linker-NCH2),2.55(ddd,J＝47.7,14.3,6.3Hz,2H,RHb-2),2.26(pd,J＝15.3,14.4,8.6Hz,2H,dHh-2),1.88(dq,J＝13.4,6.2Hz,1H,dHh-4),1.78–1.66(m,3H,NHAc-CH3),1.65–1.42(m,5H,Linker-CH2,dHh-4),1.35(d,J＝6.1Hz,3H,RHb-4),1.28(m,2H,Linker-CH2),1.17(d,J＝6.1Hz,3H,6-CH3),1.04(dd,J＝13.5,6.1Hz,6H,6"-CH3,dHh-6),0.95(d,J＝6.4Hz,3H,6'-CH3).13C NMR(151MHz,Methanol-d4)δ172.9(NH-C＝O),172.3(NH-C＝O),164.8(NH-C＝O),139.6–137.7(Ar),135.9(Ar),133.1(Ar),126.2–125.1(Ar),100.6(1"-H),98.3(1'-H),97.1(1-H),78.6(3"-C),76.2(3-C),74.9(3'-C,2'-C),73.7(dHh-3),72.9(RHb-3),71.7(dHh-5),70.8(5"-H,Ar-CH2),70.5(Ar-CH2),69.9(Ar-CH2),67.6(Ar-CH2),66.8(5-C)66.5(5'-C),57.1(2"-C),55.8(4"-C),53.6(4'-C),50.1(Ar-CH2,2-C),46.2(Linker-NCH2),43.1(RHb-2),41.4(dHh-2),40.7(dHh-4),28.8(Linker-CH2),23.0(Linker-CH2)21.8(NHAc-CH3),18.8(RHb-4),18.6(dHh-6),17.0(6"-C),15.7(6-C),15.7(6'-C).HR-ESI-MS(m/z)：calcd for C98H115Cl2N5O18Na+(M+Na)+:1742.7506found:1742.7526。
实施例5
化合物1*和2*，3*，4*，5*的合成
如图6所示：将全保护的三糖32*和33*，34*，35*在钯碳加氢的条件下进行脱保护，合成4种不同构型的目标三糖1*和2*、3*、4*。将三糖31*在锌粉和醋酸、醋酸酐的条件下，将叠氮基团转换为NHAc，随后直接利用钯碳加氢脱保护得到三糖5*。
化合物1*和2*，3*，4*，5*的NMR二维HSQC图谱，如图10-14所示。
具体实验操作和步骤：
化合物1*：将三糖32*(12mg，6.0μmol)溶于二氯甲烷和叔丁醇、水(3∶6∶1，v/v/v，3mL)的混合溶液中。用氮气置换反应瓶中的空气，加入适量10％钯-碳，用氢气吹扫溶液5分钟，然后在氢气下搅拌24小时，使用硅藻土过滤并浓缩，残留物通过HPLC使用半制备型(Thermo Scientific Hypercarb)柱以1mL/min的流速纯化，用含有0.1％甲酸的超纯水(溶剂A)和乙腈(溶剂B)以10％至30％的溶剂B的线性梯度洗脱30分钟，得到化合物1*(4.67mg,5.58μmol,80％)。1H NMR(600MHz,Deuterium Oxide)δ5.00(d,J＝4.3Hz,1H,1'-H),4.83(d,1H,1-H),4.68(d,J＝8.4Hz,1H,1"-H),4.40(d,J＝4.8Hz,1H,4'-H),4.33–4.23(m,2H,2-H,5'-H),4.23–4.08(m,4H,RHb-3,dHh-3,3'-H,5-H),3.98(h,J＝6.5Hz,1H,dHh-5),3.91(dd,J＝11.0,3.1Hz,1H,3-H),3.82(d,J＝3.2Hz,1H,4-H),3.75(dd,J＝10.6,4.3Hz,1H,2'-H),3.68(td,J＝10.0,5.5Hz,2H,2"-H,Linker-OCH2),3.57(q,J＝7.3,6.2Hz,3H,3"-H,4"-H,5"-H),3.48(dt,J＝10.1,6.4Hz,1H,Linker-OCH2),3.02–2.96(m,2H,Linker-NCH2),2.54–2.41(m,4H,RHb-2,dHh-2),2.01(d,J＝4.1Hz,6H,NHAc-CH3),1.78–1.55(m,6H,dHh-4,Linker-CH2),1.45(dq,J＝14.9,7.4,7.0Hz,2H,Linker-CH2),1.26(d,J＝6.3Hz,3H,RHb-4),1.24–1.15(m,9H,6-CH3,6"-CH3,dHh-6),1.08(d,J＝6.5Hz,3H,6'-CH3).13C NMR(151MHz,Deuterium Oxide)δ174.7(NH-C＝O),174.2(NH-C＝O),171.0(NH-C＝O),101.7(1"-H),101.1(1'-H),97.1(1-H),76.7(3-C),76.6(3'-C),71.7(5"-C),71.4(4-C),70.9(3"-C),67.8(2'-C,Linker-CH2),66.6(dHh-3),66.5(5-C),66.0(5'-C),65.5(dHh-5),65.2(RHb-3),57.0(4"-C),56.4(2"-C),53.0(4'-C),48.5(2-C),44.8(RHb-2),44.5(dHh-4),43.8(dHh-2),39.4(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2),22.3(NHAc-CH3),22.2(Linker-CH2),22.0(RHb-4),21.7(6"-CH3),17.0(dHh-6),15.5(6'-CH3),15.3(6-CH3).HR-ESI-MS(m/z)：calcd for C37H67N5O16Na+(M+Na)+:860.4475found:860.4478
化合物2*：将三糖33*(17mg,9.89μmol)溶于二氯甲烷和叔丁醇、水(3∶6∶1，v/v/v，3mL)的混合溶液中。用氮气置换反应瓶中的空气，加入适量10％钯-碳，用氢气吹扫溶液5分钟，然后在氢气下搅拌24小时，使用硅藻土过滤并浓缩，残留物通过HPLC使用半制备型(Thermo Scientific Hypercarb)柱以1mL/min的流速纯化，用含有0.1％甲酸的超纯水(溶剂A)和乙腈(溶剂B)以10％至30％的溶剂B的线性梯度洗脱30分钟，得到化合物2*(7mg,8.40μmol,85％)。1HNMR(600MHz,Deuterium Oxide)δ5.03(d,J＝4.3Hz,1H,1'-H),4.85(d,J＝3.8Hz,1H,1-H),4.70(d,J＝8.4Hz,1H,1"-H),4.44–4.40(m,1H,4'-H),4.32(dd,J＝11.1,3.8Hz,1H,2-H),4.30–4.26(m,1H,5'-H),4.22(p,J＝6.8Hz,2H,RHb-3,dHh-3),4.14(m,J＝13.5,12.0,5.6Hz,2H,3'-H,5-H),4.02(q,J＝6.3Hz,1H,dHh-5),3.93(dd,J＝11.0,3.1Hz,1H,3-H),3.85(d,J＝3.2Hz,1H,4-H),3.77(dd,J＝10.6,4.2Hz,1H,2'-H),3.70(m,J＝8.1Hz,2H,2"-H,Linker-OCH2),3.59(d,J＝6.0Hz,3H,3"-H,4"-H,5"-H),3.51(m,J＝10.3,6.2Hz,1H,Linker-OCH2),3.02(t,J＝7.7Hz,2H,Linker-NCH2),2.54–2.43(m,4H,RHb-2,dHh-2),2.03(d,J＝4.4Hz,6H,NHAc-CH3),1.70(td,J＝15.4,7.7Hz,4H,Linker-CH2),1.63(dt,J＝7.9,3.9Hz,2H,dHh-4),1.47(q,J＝7.1Hz,2H,Linker-CH2),1.28(d,J＝6.3Hz,3H,RHb-4),1.25(d,J＝6.5Hz,3H,6-CH3),1.22(d,J＝6.0Hz,6H,dHh-6,6"-CH3),1.10(d,J＝6.5Hz,3H,6'-CH3).13C NMR(151MHz,Deuterium Oxide)δ174.7(NH-C＝O),174.6(NH-C＝O),174.5(NH-C＝O),174.2(NH-C＝O),101.7(1"-C),101.1(1'-C),97.1(1-C),76.7(3-C),76.6(3'-C),71.7(5"-C),71.4(4-C),70.9(3"-C),67.8(2'-C,Linker-OCH2),66.5(5-C),66.0(5'-C),65.5(RHb-3),65.2(dHh-3),64.3 (dHh-5),57.0(4"-C),56.4(2"-C),53.0(4'-C),48.5(2-C),44.8-44.8(RHb-2,dHh-4),44.1(dHh-2),39.4(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2),22.7(dHh-6-CH3),22.3(Linker-CH2),22.2(NHAc-CH3),22.0(NHAc-CH3),22.02(RHb-4-CH3),17.0(6"-CH3),15.5(6'-CH3),15.3(6-CH3).HR-ESI-MS(m/z)：calcd for C37H67N5O16Na+(M+Na)+:860.4475found:860.4490
化合物3*：将三糖34*(9mg,3.1μmol)溶于二氯甲烷和叔丁醇、水(3∶6∶1，v/v/v，3mL)的混合溶液中。用氮气置换反应瓶中的空气，加入适量10％钯-碳，用氢气吹扫溶液5分钟，然后在氢气氛下搅拌24小时，使用硅藻土过滤并浓缩，残留物通过HPLC使用半制备型(Thermo Scientific Hypercarb)柱以1mL/min的流速纯化，用含有0.1％甲酸的超纯水(溶剂A)和乙腈(溶剂B)以10％至30％的溶剂B的线性梯度洗脱30分钟，得到化合物3*(2.2mg,2.64μmol,85％)。1H NMR(600MHz,Deuterium Oxide)δ5.01(d,J＝4.2Hz,1H,1'-H),4.84(d,1H,1-H),4.69(d,J＝8.6Hz,1H,1"-H),4.41(m,1H,4'-H),4.31(dd,J＝11.1,3.6Hz,1H,2-H),4.27(t,J＝6.8Hz,1H,5'-H),4.20(m,J＝6.6Hz,2H,RHb-3,dHh-3),4.13(dq,J＝14.2,7.1,5.8Hz,2H,3'-H,5-H),4.01(q,J＝6.4Hz,1H,dHh-5),3.92(d,J＝11.3Hz,1H,3-H),3.84(m,1H,4-H),3.76(dd,J＝10.7,4.1Hz,1H,2'-H),3.70(t,J＝9.5Hz,2H,2"-H,Linker-OCH2),3.62(t,J＝9.9Hz,1H,4"-H),3.56(t,J＝9.8Hz,2H,3"-H,5"-H),3.50(m,1H,Linker-OCH2),3.01(t,J＝7.9Hz,2H,Linker-NCH2),2.53–2.38(m,4H,RHb-2,dHh-2),2.02(d,J＝3.3Hz,6H,NHAc),1.74–1.59(m,6H,Linker-CH2,dHh-4),1.46(dt,J＝15.0,7.5Hz,2H,Linker-CH2),1.27(m,J＝6.3Hz,3H,RHb-4),1.22(m,J＝15.0,6.3Hz,9H,6-CH3,6"-CH3,dHh-6),1.09(d,J＝6.5Hz,3H,6'-CH3).13C NMR(151MHz,Deuterium Oxide)δ174.6(NH-C＝O),174.6(NH-C＝O),174.4(NH-C＝O),101.7(1"-H),101.1(1'-H),97.1(1-H),76.7(3-C),76.6(3'-C),71.8(5"-C),71.4(4-C),70.1(3"-C),67.82(2'-C,Linker-OCH2),66.5(5-C),66.0(5'-C),65.6-65.2(RHb-3,dHh-3),64.3(dHh-5),56.9(4"-C),56.4(2"-C),53.0(4'-C),48.5(2-C),45.0(dHh-4),44.8(RHb-2),44.3(dHh-2),39.3(Linker-NCH2),28.0(2-C),26.5(2-C),22.7(dHh-6),22.3(Linker-CH2),22.2(NHAc-CH3),22.0(RHb-4),17.0(6"-CH3),15.5(6'-CH3),15.3(6-CH3).HR-ESI-MS(m/z)：calcd for C37H67N5O16Na+(M+Na)+:860.4475found:860.4476
化合物4*：将三糖35*(7.8mg,4.536μmol)溶于二氯甲烷和叔丁醇、水(3∶6∶1，v/v/v，3mL)的混合溶液中。用氮气置换反应瓶中的空气，加入适量10％钯-碳，用氢气吹扫溶液5分钟，然后在氢气下搅拌24小时，使用硅藻土过滤并浓缩，残留物通过HPLC使用半制备型(Thermo Scientific Hypercarb)柱以1mL/min的流速纯化，用含有0.1％甲酸的超纯水(溶剂A)和乙腈(溶剂B)以10％至30％的溶剂B的线性梯度洗脱30分钟，得到化合物4*(3.15mg,3.765μmol,83％)。1H NMR(600MHz,Deuterium Oxide)δ5.03(d,J＝4.3Hz,1H,1'-H),4.86(s,1H,1-H),4.71(d,J＝8.4Hz,1H,1"-H),4.43(d,J＝4.7Hz,1H,4'-H),4.33(dd,J＝11.2,3.8Hz,1H,2-H),4.29(d,J＝6.6Hz,1H,5'-H),4.22(q,J＝6.4Hz,1H,RHb-3),4.18–4.11(m,3H,dHh-3,3'-H,5-H),4.01(q,J＝6.4Hz,1H,dHh-5),3.94(dd,J＝11.2,3.2Hz,1H,3-H),3.85(d,J＝3.2Hz,1H,4-H),3.78(dd,J＝10.6,4.2Hz,1H,2'-H),3.71(q,J＝8.7,8.0Hz,2H,2"-H,Linker-OCH2),3.65–3.61(m,1H,4"-H),3.60–3.54(m,2H,3"-H,5"-H),3.52(m,J＝10.4,6.4,5.2Hz,1H,Linker-OCH2),3.07–2.97(m,2H,Linker-NCH2),2.55–2.49(m,3H,RHb-2,dHh-2),2.43(dd,J＝14.2,8.5Hz,1H,dHh-2),2.04(d,J＝3.7Hz,6H,NHAc-CH3),1.78–1.63(m,6H,dHh-4,Linker-CH2),1.47(m,J＝7.2Hz,2H,Linker-CH2),1.29(d,J＝6.3Hz,3H,RHb-4),1.25(d,J＝6.6Hz,3H,6-CH3),1.23(d,J＝6.1Hz,6H,6"-CH3,dHh-6),1.11(d,J＝6.5Hz,3H,6'-CH3).13C NMR(151MHz,Deuterium Oxide)δ174.7(NH-C＝O),174.6(NH-C＝O),174.6(NH-C＝O),174.3(NH-C＝O),101.7(1"-C),101.2(1'-C),97.1(1-C),76.7(3-C),76.6(3'-C),71.8(5"-C),71.4(4-C),71.0(3"-C),67.9(2'-C,Linker-OCH2),66.7(dHh-3),66.5(C-5),66.1(5'-C),65.5(dHh-5),65.2(RHb-3),56.9(4"-C),56.5(2"-C),53.0(4'-C),48.5(2-C),44.9(dHh-4),44.7(RHb-2),44.0(dHh-2),39.5(Linker-NCH2),28.1(Linker-CH2),26.8(Linker-CH2),22.3(Linker-CH2),22.2(NHAc-CH3),22.1(RHb-4),21.8(dHh-6),17.0(6"-CH3),15.5(6'-CH3),15.3 (6-CH3).HR-ESI-MS(m/z)：calcd for C37H67N5O16Na+(M+H)+:838.4656found:838.4647
化合物5*：将三糖31*(30mg；20.41μmol)溶于四氢呋喃和醋酸酐、醋酸(3/2/1，v/v/v，3mL)的混合溶液中，加入新活化的Zn(1g)，并在室温下搅拌过夜，TLC检测原料反应完全后，用二氯甲烷稀释反应液并过滤。滤液用饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤。随后将合并的有机层用无水硫酸钠干燥，过滤，真空蒸发，并在油泵上抽真空干燥。将粗产物溶解在二氯甲烷和叔丁醇和水(3/6/1，v/v/v，2mL)中，并向溶液中加入适量10％钯-碳。在氢气的氛围下(4atm)搅拌36小时，随后用硅藻土过滤混合物并用水洗涤，重复洗涤三次，并在真空中蒸发溶剂。残留物通过HPLC使用半制备型(Thermo Scientific Hypercarb)柱以1mL/min的流速纯化，用含有0.1％甲酸的超纯水(溶剂A)和乙腈(溶剂B)以10％至30％的溶剂B的线性梯度洗脱30分钟，得到化合物5*(9.9mg,13.27μmol,两步的总产率为65％)。1HNMR(600MHz,Deuterium Oxide)δ4.93(t,J＝3.3Hz,1H,1'-H),4.76(d,J＝3.3Hz,1H,1-H),4.60(dd,J＝7.8,2.0Hz,1H,1"-H),4.33(d,J＝4.7Hz,1H,4'-H),4.23(d,J＝11.3,3.1Hz,1H,2-H),4.18(m,J＝6.8Hz,1H,5-H),4.12(q,J＝6.1Hz,1H,RHb-3),4.08–4.00(m,2H,3'-H,5'-H),3.83(dt,J＝11.1,2.9Hz,1H,3-H),3.75(d,J＝3.0Hz,1H,4-H),3.68(dt,J＝10.6,3.2Hz,1H,2'-H),3.60(m,J＝7.9,7.4Hz,2H,2"-H,Linker-OCH2),3.44(m,J＝28.2,9.7,4.4Hz,4H,3"-H,4"-H,5"-H,Linker-OCH2),2.98–2.89(m,2H,Linker-NCH2),2.45–2.36(m,2H,RHb-2),1.94(dt,J＝6.4,2.2Hz,9H,NHAc-CH3),1.66–1.54(m,4H,Linker-CH2),1.37(q,J＝7.4Hz,2H,Linker-CH2),1.18(m,3H,RHb-4),1.15(m,3H,6'-CH3),1.11(m,3H,6"-CH3)1.00(m,3H,6-CH3).13C NMR(151MHz,Deuterium Oxide)δ174.6(NH-C＝O),171.0(NH-C＝O),101.7(1"-H),101.1(1'-H),97.1(1'-H),76.7(3'-C,3-C),71.8(4-C),71.4(3"-C),70.9(5"-C),67.8(2'-H,Linker-OCH2),66.5(5'-C),66.0(5-C),65.2(RHb-3)57.0(4"-C),56.3(2"-H),44.8(RHb-2),39.3(Linker-NCH2),28.0(Linker-CH2),26.5(Linker-CH2),22.3(Linker-CH2),22.2(NHAc-CH3),22.1(NHAc-CH3),22.0(NHAc-CH3),16.9(6"-H),15.5(6-H),15.3(6'-CH3).HR-ESI-MS(m/z)：calcd for C33H59N5O14Na+(M+Na)+:772.3951found:772.3968。
实施例6
应用合成的四种寡糖通过NMR分析，阐明dHh的绝对构型。如图7-8所示
具体分析过程：为了尽可能的减少误差，合成的寡糖(1*～4*)所使用的核磁测试仪器均为600M，并且在相同的温度(25℃)下测试。利用合成的四种寡糖与天然OPS进行NMR谱图比对，尝试确定dHh侧链的可能的绝对构型。值得注意的是，四种合成的三糖(1*-4*)中没有一种显示出与天然提取的O-抗原(OPS)三糖重复单元相同的NMR-1H和NMR-13C数据。我们推测，这种现象主要是由于合成低聚糖的长度与天然的OPS的差异，以及还原端氨基连接臂(Linker)和非还原端D-奎诺糖3号位羟基(D-Qui-C3-OH)的存在也增加了误差。由于位于三糖结构中间的RHb侧链推测会较少受到Linker和D-Qui-C3-OH的影响，我们在比较dHh差异的同时，策略性的选择了RHb侧链作为对照。NMR-13C分析显示，四种合成三糖的RHb与天然三糖比较相关，而dHh的NMR-13C化学位移与天然三糖明显差异。与合成糖2*和3*相比，合成糖1*和4*的dHh-NMR-13C化学位移与天然三糖的差异较小，如图7所示，初步分析便可以确定合成糖1和4*是最可能的天然构型。通过整合和处理核磁图谱进一步分析发现，如图8所示，四种异构体在dHh-2号位和4号位NMR-13C的明显不同，合成糖4*与天然构型展现出了较好的匹配度。因此霍乱弧菌O100血清型OPS中dHh修饰基团的绝对构型被指认为3S,5S。
实施例7
利用合成得到的五种寡糖进行免疫学评估，如图9所示。
具体步骤和方法如下：
糖芯片制作：将合成得到的五种寡糖(1*～5*，图9A)和不相关的合成糖(6*，D-FucNAc) 溶于50mM磷酸盐溶液(pH 8.5)，分别配置0.1mM、0.5mM的糖溶液；并且相应配置1mg/mL、5mg/ml的霍乱弧菌O100血清型脂多糖(LPS1)和奇异变形杆菌脂多糖(LPS2)溶液；然后使用芯片点样仪按照图9B所示的分布格打印于芯片(9mm长×9mm宽)的区域上，在室温，65％湿度条件下过夜孵育，使糖片段与芯片共价结合。孵育完成后，使用100nM乙醇胺和50nM的磷酸钠混合溶液(pH＝9)在50℃条件下处理1小时，随后用超纯水清洗。
使用含3％BSA的PBS溶液封闭糖芯片，在室温条件下封闭1h。使用0.1％Tween 20的PBS溶液(PBST溶液)洗1次，PBS溶液洗2次，离心甩干，糖芯片装入16孔孵育器中(ProPlate)。每孔加入1：200稀释于含1％BSA的PBS溶液的兔血清样品120微升，在4℃湿盒中避光过夜孵育，使识别合成糖片段的兔血清IgG抗体与糖芯片上的合成寡糖结合，移除样品，使用200微升含有PBST溶液洗3次，除去未结合的血清IgG抗体。随后，使用cy3荧光标记的山羊抗兔血清IgG抗体作为二抗，每孔加入1：400稀释于含1％BSA的PBS溶液的二抗120微升，在湿盒中室温避光孵育45min，移除二抗溶液；使用200微升PBST溶液洗3次，洗去未结合的二抗，拆开16孔孵育器，使用超纯水清洗1次，再使用超纯水清洗15分钟，得到最终的检测芯片。
在微阵列扫描仪上进行扫描发出荧光信号，结果显示(图9C-D)，IgG抗体对所有合成糖1*-5*都表现出优异的结合能力。合成糖6*和LPS2没有抗体结合，与预期相符合。与三糖4*相比，合成糖1*-3*和5*没有表现出结合能力的消失或显著降低。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的原则和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种霍乱弧O100血清型O抗原寡糖的化学合成方法，其特征在于，所述方法是利用三个单糖砌块和五种羧酸衍生物作为原料；
    所述霍乱弧O100血清型O抗原寡糖的结构如下式(1)～(5)所示：
    其中，Linker*为-(CH2)nNH2或者-(CH)nSH，其中n＝1～25；
    所述的三个单糖砌块的结构分别如式(6)～(8)所示，五种羧酸衍生物的结构分别如式(9)～(13)所示，
    PG2，PG3，PG4，PG6，PG7为羟基临时保护基，分别独立地选自苄基、2-萘甲基、叔丁基二甲基硅烷基、叔丁基二苯基硅烷基、三乙基硅烷基；
    PG8，PG9，PG11，PG12，PG14，PG15，PG17，PG18，PG20为羟基临时保护基，分别独立地选自苄基、2-萘甲基、乙酰基、苯甲酰基、新戊酰基、9-戊甲氧羰基、2-对甲氧基苄基；
    PG10，PG13，PG16，PG19，PG21分别独立地选自羟基、氯、溴、氟、C1-4烷氧基中的一种；
    PG1为氨基临时保护基，选自三氯乙酰基、二氯乙酰基、氯乙酰基中；
    PG5为氨基临时保护基，选自乙酰基、三氯乙酰基、二氯乙酰基、氯乙酰基、三氯乙氧羰基、邻苯二甲酰基、9-芴甲氧羰基、叔丁基氧羰基；
    Linker为-(CH2)nN-Y1Y2或者-(CH)nS-Y1，其中n＝1～25，Y1和Y2为氢、酰基、苄基、2-萘甲基、苄甲氧羰基中的一种；
    离去基团LG1为N-苯基三氟乙酰亚胺酯；
    离去基团LG2选自三氯乙酰亚胺酯、N-苯基三氟乙酰亚胺酯、甲硫基、硒苯基、乙硫基、苯硫基、对甲苯硫基、二丁基膦酸基中的一种；
    所述合成方法包括如下步骤：
    (1)构建二糖受体：单糖砌块8脱除3号位的羟基保护基团PG6，得到受体14；受体14与单糖砌块7在无水二氯甲烷和乙醚的混合体系中进行糖基化反应，得到二糖15；利用还原剂将二糖15中的叠氮基团还原为氨基，并加入化合物13进行酰胺化，得到化合物16；脱除化合物16中单糖砌块7上3号位的羟基保护基PG4，得到二糖受体17；
    (2)构建目标三糖：
    二糖受体17和单糖砌块6在活化剂的作用进行糖基化反应，并控制反应的温度为0℃逐步升温至室温，反应得到三糖18；再利用还原剂还原三糖18的叠氮基团，并加入式(9)～(12)中任意一种羧酸衍生物进行酰胺化，相应得到化合物19～22；将化合物19～22进行催化氢化，脱保护得到目标化合物1～4；
    或者，三糖18经还原酰化、将叠氮基团转化为乙酰氨基，随后催化氢化，脱保护得到目标化合物5；
    PGa、PGb为羟基临时保护基，分别独立地选自苄基、2-萘甲基、乙酰基、苯甲酰基、新戊酰基、9-戊甲氧羰基、2-对甲氧基苄基。
  2. 根据权利要求1所述的化学合成方法，其特征在于，步骤(1)中，糖基化反应的浓度为0.01～0.1M；使用活化试剂进行糖基化反应，所述活化试剂为TMSOTf，NIS/TMSOTf，NIS/TfOH中的一种；单糖砌块7和受体14的摩尔比为(1～3):1或者1:(1～3)。
  3. 根据权利要求1所述的化学合成方法，其特征在于，步骤(1)中，糖基化反应的条件具体为：将单糖砌块7和受体14溶于二氯甲烷、乙醚的混合溶剂中，在氩气的保护下进行搅拌，加入分子筛，反应温度为-20℃～0℃，加入相对单糖砌块7摩尔量0.1～0.3当量的活化试剂，反应时间为2～8h。
  4. 根据权利要求1所述的化学合成方法，其特征在于，步骤(1)中，所述二糖15中还原叠氮基团使用的还原剂为锌粉、三苯基磷、1，3-丙二硫醇、氢化铝锂、三甲基磷、二水氯化亚锡、硼氢化钠、氰基硼氢化钠中的一种。
  5. 根据权利要求1所述的化学合成方法，其特征在于，步骤(2)中，所述的糖基化反应浓度为0.01～0.1M；所述活化剂为TMSOTf，NIS/TMSOTf，NIS/TfOH中的一种。
  6. 根据权利要求1所述的化学合成方法，其特征在于，步骤(2)中，糖基化反应是在溶剂中进行的，所述溶剂为无水二氯甲烷、乙醚、甲苯、甲醇、四氢呋喃、乙腈、N,N-二甲基甲酰胺或水其中的一种或多种；所述单糖砌块6和二糖受体17的摩尔比为(1～3):1或者1:(1～3)。
  7. 根据权利要求1所述的化学合成方法，其特征在于，步骤(2)中，所述的糖基化反应的条件包括：将二糖受体17和单糖砌块6溶于二氯甲烷溶剂中，加入分子筛，加入相对二糖受体17摩尔量0.2～1当量的活化试剂，控制反应的温度为0℃逐步升温至室温，反应时间为2～8h。
  8. 根据权利要求1所述的化学合成方法，其特征在于，步骤(2)中，所述还原剂为1，3-丙二硫醇；所述还原的过程中还包括加入缩合剂，所述缩合剂选自：HATU、EDC。
  9. 根据权利要求1所述的化学合成方法，其特征在于，步骤(2)中，所述催化氢化所用的催化剂可以是10％钯碳催化剂或氢氧化钯。
  10. 权利要求1-9任一项所述方法在糖芯片或者霍乱弧菌糖蛋白缀合物的制备中的应用，所述应用包括如下过程：
    S1：利用权利要求1-9任一项所述方法制备具有连接臂的霍乱弧O100血清型O抗原寡糖片段；
    S2：随后利用所得寡糖片段的连接臂与芯片或者载体蛋白质结合，得到相应的糖芯片或者霍乱弧菌糖蛋白缀合物。
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