WO2021169100A1 - Application of trisaccharide repeating unit oligosaccharide chain in preparation of staphylococcus aureus vaccines - Google Patents

Abstract

An application of a trisaccharide repeating unit oligosaccharide chain in the preparation of staphylococcus aureus vaccines, which relates to the field of medicine. A trisaccharide repeating unit oligosaccharide chain is obtained by means of structure optimization on the basis of a chemical synthesis means; the oligosaccharide chain is coupled with a carrier protein and is immobilized on the surface of a matrix to prepare a glycochip; the glycochip is coupled with a protein to prepare a glycoprotein conjugate which has outstanding immunocompetence, and a further prepared monoclonal antibody can specifically recognize staphylococcus aureus. The raw materials are cheap and easily obtained, the preparation method is simple and easy to repeat, and the trisaccharide repeating unit oligosaccharide chain has very good application prospects in the development of staphylococcus aureus vaccines.

Description

Application of Trisaccharide Repeating Unit Oligosaccharide Chain in Preparation of Staphylococcus Aureus Vaccine Technical field

The invention relates to the application of a trisaccharide repeating unit oligosaccharide chain in preparing a Staphylococcus aureus vaccine, and belongs to the field of medicine.

Background technique

The carbohydrate antigens on the surface of pathogens such as bacteria, viruses, and parasites have high structural specificity and play an important role in the process of infecting the host. They are important targets for vaccine development. Glyco-conjugated protein vaccines based on the extraction of polysaccharides from bacterial surfaces, such as Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae vaccines, protect the lives and health of millions of people every year (C. Anish et al., Chem Biol ,2014,21:38). However, with the continuous improvement of vaccine quality, efficacy and safety requirements, the preparation of relatively uniform polysaccharide products through extraction methods often requires extremely cumbersome purification steps. In addition, the inevitable mixing of impurities and non-protective epitopes will cause side reactions and low reactivity, which has severely restricted the development and production of polysaccharide vaccines. The synthetic method to prepare oligosaccharides with a clear structure and a uniform composition provides an extremely valuable alternative technology, especially for the development of vaccines against pathogenic bacteria that cannot be cultured on a large scale (C.Anish et al., Chem Biol, 2014, 21:38 ). As Haemophilus influenzae type b synthesizes oligosaccharide protein conjugated vaccine QuimiHi

(CIGB, La Habana), researchers have done a lot of research work on the development of synthetic oligosaccharide vaccines for pathogenic bacteria (P. Kaplonek et al., PNAS, 2018, 115: 13353). Synthetic oligosaccharide antigens have a huge advantage in that their structure is clear. Oligosaccharides with a uniform composition will facilitate the study of epitopes and will greatly promote the optimal design of carbohydrate antigen structures. Not only that, for the surface sugar chains of pathogenic bacteria that usually contain rare monosaccharide building blocks and rare modification groups, the identification of the minimum epitope will help reduce the workload of oligosaccharide synthesis in order to produce cost-effective vaccines (C. Anish et al., Chem Biol, 2014, 21:38).

Since most of the surface sugar chains of pathogens are repetitive polysaccharides, their repeating unit oligosaccharide chains have become the preferred choice for antigen discovery and immunogen design. For polysaccharides composed of long-chain repeating units (usually containing more than six sugar units) and non-repetitive polysaccharides, it is usually necessary to prepare corresponding oligosaccharide fragment libraries based on factors such as chain length, terminal sugar groups, linking sequences and modification groups. , Will facilitate the use of patient antiserum to carry out antigenic analysis of oligosaccharides (C. Anish et al., Chem Biol, 2014, 21:38). For oligosaccharides with good antigenicity, the immunogenicity of oligosaccharides can be explored by protein conjugation and animal immune experiments. Its hallmark effect is to stimulate the immune system to produce antibodies that can recognize intact pathogens (G. Liao et al. ,ACS Cent Sci,2016, 2:210; F. Broecker et al., Nat Commun, 2016, 7: 11224; M. Emmadi et al., J Am Chem Soc, 2017, 139: 14783). Through the preparation of monoclonal antibodies, studies on the interaction between oligosaccharide antigens and antibodies can be carried out, and epitope information of carbohydrate antigens can be provided, which will significantly improve the development efficiency of oligosaccharide vaccines (B. Schumann et al., Sci Transl Med, 2017,9:eaaf5347).

Staphylococcus aureus is one of the most common human opportunistic pathogens. Its infection can cause a series of fatal diseases, such as endocarditis, abscess, bacteremia, sepsis and osteomyelitis. The highly prevalent antibiotic-resistant strains of Staphylococcus aureus, such as methicillin and vancomycin-resistant strains, make it a high-risk pathogen of hospital infections and increase the difficulty of dealing with their infections. In particular, antibiotic-resistant Staphylococcus aureus blood infections are considered to be the main cause of disability and death in hemodialysis patients (E.C.O’Brien et al., Trends Mol Med, 2019, 25: 171). Therefore, the development of a Staphylococcus aureus vaccine, especially for the clinically most important type 5 and type 8 capsular polysaccharide strains, is imminent (D. Gerlach et al., Nature, 563:705). In the past two decades, although researchers have carried out a lot of work on the development of Staphylococcus aureus vaccines, no vaccine has been successfully marketed (S. Ansari et al., Infect Drug Resist, 2019, 12:1243). Among them, the experimental vaccine StaphVAX (Nabi Biopharmaceuticals, Rockville, MD) based on type 5 and type 8 capsular polysaccharides and the experimental vaccine V710 (Merck, Kenilworth, NJ) based on the iron surface determinant IsdB are the only two that have entered clinical phase 3 The experimental vaccines against Staphylococcus aureus in China failed to provide effective protection. It should be pointed out that the experimental vaccine V710 has the safety risk of increasing the mortality of the test group, while the experimental vaccine StaphVAX has shown good safety in the study, and its low immune effect is considered to be inconsistent with the quality of the type 8 capsular polysaccharide used. Stability is related to a single antigen type (S. Ansari et al., Infect Drug Resist, 2019, 12:1243). Therefore, the researchers further developed an experimental vaccine containing a variety of Staphylococcus aureus antigens, which contained type 5 and type 8 capsular polysaccharide protein conjugates, recombinant bacterial surface protein aggregation factor A (ClfA) and recombinant manganese transporter C ( The quadrivalent vaccine (SA4Ag) of rMntC) produces a significant protective immune response in healthy adults. The clinical 2B study is currently underway (S. Ansari et al., Infect Drug Resist, 2019, 12:1243; E. Begier et al., Infect Drug Resist, 2019, 12:1243; E. Begier et al.) al., Vaccine, 2017, 35:1132).

It can be seen that the current preparation of Staphylococcus aureus vaccines involves the combined use of complex capsular polysaccharide compounds. On the one hand, such methods are difficult to control the stability of the vaccine, and the process is cumbersome and the cost is high. therefore. There is an urgent need to provide a simple and effective method for preparing a Staphylococcus aureus vaccine.

Summary of the invention

The present invention is based on a chemical synthesis method to obtain specific trisaccharide repeat fragments assembled with orthogonal connecting arms→3)-4-O-Ac-β-D-ManpNAcA-(1→3)-α-L-FucpNAc-(1 →3)-α-D-FucpNAc-(1→, the oligosaccharide protein conjugate experimental vaccine is prepared by conjugating the amino linking arm with the carrier protein, and its immune activity is verified by animal immune experiments. The present invention uses amino linking The arm fixes the synthetic oligosaccharide on the surface of the chip, and uses the oligosaccharide chip to detect antibodies in the antiserum in immunogenicity research. The synthetic oligosaccharide monoclonal antibody prepared further can specifically recognize Staphylococcus aureus. The structure-activity relationship of the trisaccharide repeat fragment has outstanding immune activity, and can be used to prepare or develop a Staphylococcus aureus vaccine to obtain a glycoprotein conjugate vaccine of the trisaccharide repeat unit.

The technical problem to be solved by the present invention is to overcome the structural performance diversity and unpredictability of the carbohydrate structure, based on chemical synthesis, to explore its immune epitope and structure-activity relationship at the molecular level, and to explore the trisaccharide repeat fragment [ 3) The immune activity of -4-O-Ac-β-D-ManpNAcA-(1→3)-α-L-FucpNAc-(1→3)-α-D-FucpNAc-(1→]; and through reduction The terminal amino linker is conjugated with the carrier protein to prepare an experimental vaccine of oligosaccharide protein conjugate, and the synthetic oligosaccharide is immobilized on the surface of the chip through the amino link arm to prepare a synthetic oligosaccharide chip. The triglycoprotein conjugate needs to be able to stimulate the production of experimental animals Specific antibodies, which can be analyzed by synthetic oligosaccharide chip. The triglycoprotein conjugate immunize animals to further prepare monoclonal antibodies, and the prepared monoclonal antibodies should be able to specifically recognize Staphylococcus aureus .

The present invention relates to a glycoprotein conjugate vaccine based on chemically synthesized trisaccharide repeating units (Figure 1). The present invention completes the chemical synthesis of trisaccharide repeat fragments assembled with orthogonal link arms for the first time. The oligosaccharide protein conjugate experimental vaccine is prepared by conjugating the amino link arms with the carrier protein, and its immunological activity is verified by animal immunization experiments. . The synthetic oligosaccharide is immobilized on the surface of the chip through the amino linking arm, and the oligosaccharide chip is used for the detection of antibodies in antiserum in immunogenicity research. Further prepare synthetic oligosaccharide monoclonal antibody and verify its ability to specifically recognize Staphylococcus aureus, in order to prove that the synthetic trisaccharide repeat fragment has outstanding immunological activity.

An object of the present invention is to provide the application of trisaccharide repeating unit oligosaccharide chain in the preparation of Staphylococcus aureus vaccine; the polysaccharide compound; the trisaccharide repeating unit oligosaccharide chain is assembled with amino linking arms, and the chemical structure can be Expressed as: U ₁ -U ₂ -U ₃ -OL-NH ₂ , where L represents the connecting arm, U ₁ , U ₂ and U ₃ are as follows:

The present invention also provides a protein conjugate for preparing Staphylococcus aureus vaccine; the chemical structural formula of the protein conjugate can be expressed as: [U ₁ -U ₂ -U ₃ -OL-NH-(C= O)-S-(C=O)-NH] _n -CP, where U ₁ , U ₂ and U _{3 are} as described above; n is 1-20; L represents the connecting arm; S represents the extension arm; CP represents the carrier protein .

In the present invention, the compound assembled at the reducing end of the oligosaccharide chain as a linker raw material can be represented by Formula 1.

Wherein PG ^a and PG ^b are benzyl and benzyloxycarbonyl.

In the present invention, the linking arm L may have a chain structure with 2-40 carbon atoms (including the carbon atoms of the side chain).

When the main chain length of the linking arm L in the present invention is 4-8 atoms, the chain may contain 1, 2 or 3 heteroatoms (O, N and S). When the main chain length of the linking arm is 9-14 atoms, the chain may contain 1, 2, 3, 4, 5 or 6 heteroatoms (O, N and S).

In the present invention, the linking arm -L- can be fully or partially substituted with fluorine. The linking arm -L- can contain a three-, four-, five- or six-membered saturated carbocyclic ring; it can also contain a five-membered unsaturated carbocyclic ring (non-aromatic ring); it can also contain a four-, five- or six-membered saturated oxygen heterocyclic ring; It can also contain a four-, five- or six-membered saturated nitrogen heterocycle; it can also contain a six-membered aromatic carbocyclic ring.

In the present invention, the linking arm -L- may also include an amide bond and/or a urea group.

In the present invention, the linking arm -L- may contain one or more substituent groups, and these substituents may include: -F, -Cl, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -C ₅ H ₉ , -C ₆ H ₁₃ , -OC ₂ H ₅ , -OCH ₃ , -CH ₂ F, -CF ₃ , -NHC(O)CH ₃ , -CHF ₂ , -C(O)-NH ₂ , -SCH ₃ , -N(CH ₃ ) ₂ , -SC ₂ H ₅ and -N(C ₂ H ₅ ) ₂ .

The extension arm used to covalently link the synthetic oligosaccharide and the carrier protein CP in the present invention can be expressed as general formula 2,

In the present invention, -S- in the extension arm can be a chain structure with 2-40 carbon atoms (including the number of carbon atoms in the side chain).

When the main chain length of -S- in the extension arm of the present invention is 4-8 atoms, the chain may contain 1, 2 or 3 heteroatoms (O, N and S). When the main chain length of the linking arm is 9-14 atoms, the chain may contain 1, 2, 3, 4, 5 or 6 heteroatoms (O, N and S).

In the present invention, -S- in the extension arm can be fully or partially substituted with fluorine. The linking arm -S- can contain a three-, four-, five-, or six-membered saturated carbocyclic ring; it can also contain a five-membered unsaturated carbocyclic ring (non-aromatic ring); it can also contain a four-, five-, or six-membered saturated oxygen heterocyclic ring; It can also contain a four-, five- or six-membered saturated nitrogen heterocycle; it can also contain a six-membered aromatic carbocyclic ring.

In the present invention, -S- in the extension arm may also contain an amide bond and/or a urea group.

In the present invention, -S- in the extension arm may contain one or more substituents, and these substituents may include: -F, -Cl, -CH ₃ , -C ₂ H ₅ , -C ₅ H ₉ , -C ₃ H ₇ , -C ₆ H ₁₃ , -OC ₂ H ₅ , -OCH ₃ , -NHC(O)CH ₃ , -CH ₂ F, -CF ₃ , -CHF ₂ , -C(O)-NH ₂ ,- SCH ₃ , -SC ₂ H ₅ , -N(CH ₃ ) ₂ and -N(C ₂ H ₅ ) ₂ .

The sugar chain structure synthesized in the present invention contains basic (amino) and acidic (carboxy) groups, which can form corresponding salts with organic or inorganic acids or bases. Acids that can be used for salt formation include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, gluconic acid, lactic acid, malic acid, fumaric acid, butane Diacid, ascorbic acid, nitric acid, formic acid, phosphonic acid, perchloric acid, o-toluene tartaric acid, propionic acid, tartaric acid, tartaric acid, naphthalenesulfonic acid, p-aminobenzenesulfonic acid, hydroxymaleic acid, pyruvic acid, camphor sulfonic acid Acid, mandelic acid, phenylacetic acid, maleic acid, sulfonic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, vinylsulfonic acid, p-toluene Sulfonic acid, o-methylmandelic acid, hydroxybenzene sulfonic acid, picric acid, adipic acid, amino naphthalene sulfonic acid, and other mineral acids or carboxylic acids. The inorganic or organic bases that can be used for salt formation include potassium hydroxide, sodium hydroxide, tetraalkylammonium hydroxide, ammonia, lysine, arginine and the like.

Since the sugar chain structure synthesized in the present invention contains both basic (amino) and acidic (carboxy) groups, it can also migrate through intramolecular protons, that is, the protons of acidic groups are transferred to basic groups, and the general formula I It can be an amphiphilic molecule ^{containing -O-} and -NH ₃ ^+.

In the present invention, _{the connection mode between the monosaccharide building blocks (U 1} , U ₂ , U ₃ ) is that one monosaccharide building block passes through the terminal carbon (carbon at position 1) and the corresponding hydroxyl group of the other monosaccharide building block. Glycosidic bond formed by oxygen.

When the glycoprotein conjugate is prepared in the present invention, the connection mode between the connecting arm L and the extending arm S is that the amino group on the connecting arm L and the carboxyl group on the extending arm S form an amide bond.

When the glycoprotein conjugate is prepared in the present invention, the connection mode between the extension arm S and the carrier protein is that the carboxyl group on the extension arm S forms an amide bond with the side chain amino groups of lysine and arginine in the protein structure.

When preparing the oligosaccharide chip in the present invention, the connecting arm L and the chip surface are connected in a manner that the amino group on the connecting arm L and the carboxyl group modified on the surface of the chip form an amide bond.

When the glycoprotein conjugate is prepared in the present invention, the carrier protein CP used may include: keyhole blue protein, detoxified diphtheria toxin, detoxified diphtheria toxin mutant, chemically or enzymatically modified detoxified diphtheria toxin, and chemically modified diphtheria toxin. Or enzymatically modified diphtheria toxin detoxified mutants, detoxified tetanus toxoid, tetanus toxoid detoxified mutants, chemically or enzymatically modified detoxified tetanus toxoid, chemically or enzymatically modified Cold toxoid detoxified mutant, outer membrane protein, bovine serum albumin, detoxified cholera toxoid, cholera toxoid detoxified mutant, chemically or enzymatically modified detoxified cholera toxoid, chemically or enzymatically modified Detoxification mutant of cholera toxoid. The above chemical or enzymatic modification may include the modification of the side chain carboxyl group of aspartic acid or glutamic acid to form an amide bond, and may include the modification of the side chain sulfhydryl group of cysteine to form a disulfide bond, but does not include Modification to form an amide bond with the side chain amino group of lysine or arginine.

The present invention provides a method for synthesizing D-fucosamine in an oligosaccharide chain using D-glucose as a raw material, which is characterized in that deoxygenation at position 6 and translocation at position 4 are carried out in sequence, including the following steps:

1) 6-P-toluenesulfonyl-D-glucosamine 3 ^{is transiodized to produce 6-iodo-D-glucosamine 4, R 1} I can be KI, NaI, TBAI, etc., and the reaction temperature can be 40- Between 80°C;

2) 6-iodo-D-glucosamine 4 is treated with sodium cyanoborohydride to remove the iodine at the 6th position to obtain D-quinosamine 5;

3) D-quinosamine 5 is trifluoromethanesulfonated at position 4 to prepare 4-trifluoromethanesulfonyl-D-quinosamine 6, and the reaction temperature is between -40°C and room temperature;

4) 4-Trifluoromethanesulfonyl-D-quinolosamine 6 undergoes the Lattrell-Dax reaction to achieve the configuration reversal at position 4 to obtain D-fucosamine 7, R ² NO ₂ can be potassium nitrite ( KNO ₂ ), sodium nitrite (NaNO ₂ ), tetrabutylammonium nitrite (TBANO ₂ ), etc. The reaction temperature can be between room temperature and 80°C.

Among them, PG ¹ is a hydroxyl protecting group, which can be selected from the following groups: acetyl (Ac), levulinyl (Lev), benzoyl (Bz), chloroacetyl (ClAc), dichloroacetyl (DCA) ), trichloroacetyl (TCA), pivaloyl (Piv), allyloxycarbonyl (Alloc), 2-naphthylmethyl (Nap), p-methoxybenzyl (PMB), tert-butyldimethyl Silyl group (TBDMS), tert-butyldiphenylsilyl group (TBDPS), triethylsilyl group (TES), etc. PG ² is a terminal protecting group, which can be selected from the following groups: selenophenyl (SePh), ethylthio (SEt), phenylthio (SPh), p-tolylthio (STol), allyl (OAll ), enpentyl (OPent), tert-butyldimethylsiloxy (OTBDMS) and so on.

Beneficial effects:

The present invention completes the assembly of trisaccharide repeat fragments [3)-4-O-Ac-β-D-ManpNAcA-(1→3)-α-L-FucpNAc-(1→3) with orthogonal connecting arms based on chemical methods )-α-D-FucpNAc-(1→]; the oligosaccharide protein conjugate experimental vaccine is prepared by conjugating the reducing end amino linker with the carrier protein, and the synthetic oligosaccharide is immobilized on the surface of the chip by the amino linker Obtain a synthetic oligosaccharide chip. Combining animal immune experiments and synthetic oligosaccharide chip analysis, carry out the detection of antibodies in antiserum, and explore the good immunogenicity of synthetic oligosaccharide protein conjugates. Further prepared synthetic oligosaccharide monoclonal antibodies can be The specific recognition of Staphylococcus aureus proves that the trisaccharide repeat fragment can generate an immune response against intact cells and can be used to prepare and develop Staphylococcus aureus vaccines.

In the present invention, oligosaccharide fragments are prepared by chemical synthesis, and compound monomers with high purity and consistent structure determination can be obtained, and the immune epitope and structure-activity relationship can be explored from the molecular level. At the same time, determining the minimum immune epitope can greatly reduce the difficulty and cost of chemical synthesis, and optimize the structure of the minimum immune epitope to achieve the successful development of a Staphylococcus aureus vaccine.

Description of the drawings

Figure 1: Glycoprotein conjugate vaccine based on chemical synthesis of repetitive units of the capsular polysaccharide trisaccharide of Staphylococcus aureus type 8.

Figure 2: Compounds represented by U ₁ , U ₂ , and U ₃ in the general formula I.

Figure 3: The linker assembled at the reducing end of the oligosaccharide chain.

Figure 4: Extension arms covalently linking synthetic oligosaccharides to carrier proteins.

Figure 5: Synthesis of D-Fucosamine 7 from D-Glucose 3.

Figure 6: Chemical synthesis reaction formula of compound 10*.

Figure 7: Chemical synthesis reaction formula of compound 14*.

Figure 8: Chemical synthesis reaction formula of compound 23*.

Figure 9: Characterization of glycoprotein conjugates (A) SDS-PAGE detection of glycoprotein conjugates; 1: Protein molecular weight standard (Marker), 2: CRM ₁₉₇ , 3: glycoconjugates in aqueous solution, 4: PBS Glycoconjugate in solution; (B) MALDI-TOF/TOF-MS matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer to analyze the average molecular mass of _{CRM197 and glycoprotein conjugate.}

Figure 10: Glycochip detection of mouse serum; (A) using anti-mouse IgG-labeled Alexa Fluor 488 secondary antibody detection; the sugar chip spotting pattern is shown in the figure; among them, 1: trisaccharide 23, spotting concentrations are respectively 0.1, 0.5, 1M; 2: connecting arm, spotting concentration is 0.1, _{0.5, 1mM; 3: CRM 197} , spotting concentration is 0.1, 0.05μM; 4: E. coli O55: B5LPS, spotting concentration is 0.2mg /mL; 5: Spotting buffer, 50mM sodium phosphate solution, pH 8.5; 6: Synthetic P. shiga O51 serotype O antigen 3 sugar, spotting concentration 0.5mM; 7: Synthetic α-1 -6-glucotriose, spot concentration 0.5mM; mice 1-3 are PBS-immunized control group, mice 4-6 are glycoconjugate-immunized experimental group; (B) quantitative detection of PBS and glycoprotein conjugate The average fluorescence intensity of mice in the compound immunized group; the error bar is the standard deviation of 3 different points from two different detection areas.

Figure 11: SDS-PAGE detection of ascites purification; 1: unpurified ascites, 2: purified ascites, 3: marker. The heavy chain is the upper band of the middle lane, and the light chain is the lower band.

Figure 12: Laser confocal detection of the binding of mouse serum and bacteria (a) Staphylococcus aureus type 8 (ATCC 49525) and 1:50 dilution of pre-immune mouse serum, (b) Staphylococcus aureus type 8 (ATCC 49525) ) And 1:50 dilution of immunized mouse serum, (c) E. coli (BL21) and 1:50 dilution of immunized mouse serum. The scale bar is 5μm.

Figure 13: Laser confocal detection of mouse serum and monoclonal antibody binding, (a) Staphylococcus aureus type 8 (ATCC49525); (b) Escherichia coli (BL21) and 1:50 dilution of mouse serum after immunization, use The monoclonal antibody concentration is 186μg/mL, and the scale is 5μm.

Detailed ways

Illustrate based on the content contained in the claims.

All commercial reagents used in the experiment were used directly without treatment. The anhydrous solvent used in the reaction is prepared by MBraun MB-SPS 800 solvent drying system. The silica gel plate used for thin layer chromatography (TLC) is a glass-based or aluminum-foil-based silica gel plate made of 60-F254 silica gel. TLC color reagent is sugar developer (0.1% (v/v) 3-methoxyphenol, 2.5% (v/v) sulfuric acid ethanol solution), or CAM developer (5% (w/ v) Ammonium molybdate, 1% (w/v) cerium (II) sulfate and 10% (v/v) sulfuric acid aqueous solution), or ninhydrin developer (1.5% (w/v) ninhydrin and 3 %(V/v) acetate n-butanol solution). The silica gel used for normal phase silica gel column chromatography is 200-300 mesh silica gel.

The yield of each reaction step is calculated separately, and the calculation method of the yield is: (amount of target product substance/amount of raw material)×100%. Use nuclear magnetic spectrum, infrared spectrum, optical rotation, high resolution mass spectrometry to identify the structure of the product, and use nuclear magnetic spectrum and high performance liquid chromatography to analyze the purity of the product. The hydrogen spectrum, carbon spectrum and two-dimensional NMR spectrum were measured by Bruker Ultrashield Plus 400M NMR spectrometer at 25°C. The high resolution mass spectrum was measured by Agilent 6220 electrospray ion source-time-of-flight mass spectrometer. The optical rotation is measured by Schmidt&Haensch UniPol L 1000 automatic polarimeter at 589nm, and the unit of concentration (c) is g/100mL. The infrared spectrum is measured by Thermo Fisher Scientific Nicolet iS5 infrared instrument.

Example 1:

Synthesis of selenophenyl 3-O-acetyl-2-azido-2-deoxy-α-D-glucopyranose (1*)

The reaction equation is shown in Figure 6;

The selenophenyl 4,6-O-benzylidene-3-O-acetyl-2-azido-2-deoxy-α-D-glucopyranose (C.Qin et al., J Am Chem Soc ,2018,140:3120) (6g, 12.6mmol) was dissolved in 80% acetic acid solution (94mL), heated to 55°C, stirred for 5 hours, TLC monitored the raw material reaction to be complete. Cool to room temperature, spin off the solvent directly, and purify by silica gel column chromatography (petroleum ether: ethyl acetate=8:1→5:1→4:1→3:1→2:1) to obtain 1*(4.9g , 12.6mmol, equivalent reaction). ¹ HNMR (400MHz, CDCl ₃ ) δ = 7.26 (s, 5H, ArH), 5.89 (d, J = 5.4 Hz, 1H, H-1), 5.11 (dd, J = 10.2, 9.2 Hz, 1H, H- 3), 4.16 (dt, J = 9.8, 3.8 Hz, 1H, H-5), 3.97 (dd, J = 10.2, 5.4 Hz, 1H, H-2), 3.80 (ddd, J = 5.5, 3.8, 1.8 Hz, 2H, H-6a, H-6b), 3.71 (td, J = 9.6, 5.5 Hz, 1H, H-4), 3.01 (d, J = 5.7 Hz, 1H, 4-OH), 2.21 (s ,3H,-CH3),1.76(t,J=6.4Hz,1H,6-OH); ¹³ CNMR(100MHz,CDCl ₃ )δ=172.1,135.0,129.3,128.3,83.7,77.2,76.6,74.3,69.8 , 62.2, 61.7, 21.0.

Example 2:

Synthesis of selenophenyl 6-O-p-methylbenzenesulfonyl-3-O-acetyl-2-azido-2-deoxy-α-D-glucopyranose (2*)

The reaction equation is shown in Figure 6;

Dissolve 1* (4.9g, 12.6mmol) in anhydrous pyridine (90mL), add p-toluenesulfonyl chloride (4.1g, 21.4mmol) under argon protection, and stir at room temperature for 12 hours. TLC monitors the complete reaction of the raw materials. Dilute with ethyl acetate, wash the organic phase with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The combined organic phase is dried over anhydrous sodium sulfate and concentrated to obtain a crude product purified by silica gel column chromatography (petroleum ether: ethyl acetate =10:1→8:1→7:1→6:1→4:1) to obtain 2*(5.9g, 10.9mmol, 87%). ¹ HNMR (400MHz, CDCl ₃ ) δ = 7.26 (s, 10H, ArH), 5.81 (d, J = 5.4 Hz, 1H, H-1), 5.06 (t, J = 9.7 Hz, 1H, H-3) , 4.35 (dd, J = 11.2, 4.1 Hz, 1H, H-6a), 4.28 (ddd, J = 9.9, 4.1, 1.8 Hz, 1H, H-5), 4.10 (dd, J = 11.0, 1.9 Hz, 1H, H-6b), 3.93 (dd, J = 10.2, 5.4 Hz, 1H, H-2), 3.69 (td, J = 9.5, 5.5 Hz, 1H, H-4), 3.01 (d, J = 5.5 Hz, 1H, 4-OH), 2.44 (s, 3H, -CH3), 2.20 (s, 3H, -CH3).

Example 3:

Synthesis of selenophenyl 6-iodo-3-O-acetyl-2-azido-2,6-dideoxy-α-D-glucopyranose (3*)

The reaction equation is shown in Figure 6;

Under the protection of argon, dissolve 2*(5.9g, 10.9mmol) in methyl ethyl ketone solution (134mL), add acetic acid solution (0.8mL), add sodium iodide (8.1g, 53.7mmol), stir and heat to 80℃ , Stirred for 3 hours, TLC monitored the raw material reaction to be complete. Return to normal temperature, add DCM solution (100mL), wash with 1M sodium thiosulfate solution, water, combine the organic phases, dry with anhydrous sodium sulfate, spin off the solvent to obtain the crude product and purify by silica gel column chromatography (petroleum ether: ethyl acetate Ester=15:1→10:1→8:1→7:1) to obtain 3* (4.9g, 9.8mmol, 91%). ¹ HNMR(400MHz,CDCl ₃ )δ=7.26(s,5H),5.90(d,J=5.3Hz,1H,H-1),5.09(dd,J=10.2,9.1Hz,1H,H-3) ,3.99(dd,J＝10.2,5.3Hz,1H,H-2), 3.84(ddd,J＝9.3,4.8,2.9Hz,1H,H-5),3.57(t,J＝9.2Hz,1H, H-4), 3.48(dd,J=11.0,4.8Hz,1H,H-6a),3.41(dd,J=11.0,2.9Hz,1H,H-6b),2.22(s,3H,-CH3) ; ¹³ C NMR (100MHz, CDCl ₃ ) δ = 172.3, 134.7, 129.3, 128.2, 127.8, 84.0, 77.2, 76.3, 73.6, 72.5, 62.2, 29.7, 20.9, 6.4.

Example 4:

Synthesis of selenophenyl 3-O-acetyl-2-azido-2-deoxy-α-D-quinopyranoose (4*)

The reaction equation is shown in Figure 6;

Under the protection of argon, dissolve 3* (4.8g, 9.7mmol) in DMF (108mL), add sodium cyanoborohydride (4.8mg, 77.9mmol), heat to 95°C, continue to stir and react for 3 hours, monitored by TLC The reaction of the raw materials was complete, the temperature was reduced to room temperature, water (80 mL) was added to quench the reaction, the system was extracted with DCM, the organic phase was collected, dried with anhydrous sodium sulfate, and spin-dried to obtain the crude product. Purification by silica gel column chromatography (petroleum ether: ethyl acetate Ester=10:1→8:1→7:1) to obtain the product 4* (1.4g, 3.9mmol, 40%). ¹ HNMR(400MHz,CDCl ₃ )δ=7.86-6.91(m,5H,ArH), 5.82(d,J=5.3Hz,1H,H-1), 5.04(dd,J=10.2,9.2Hz,1H, H-3), 4.15 (dq, J = 9.6, 6.2 Hz, 1H, H-4), 3.97 (dd, J = 10.2, 5.4 Hz, 1H, H-2), 3.33 (td, J = 9.4, 5.9 Hz,1H,H-5), 2.66(d,J=6.0Hz,1H,4-OH),2.21(s,3H,-CH3), 1.28(d,J=6.2Hz,3H,-CH3); ¹³ CNMR(100MHz, CDCl ₃ )δ=172.3, 134.7, 129.2, 128.1, 84.0, 78.5, 75.3, 75.0, 71.0, 62.5, 21.0, 17.2.

Example 5:

Synthesis of selenophenyl 4-O-trifluoromethanesulfonyl-3-O-acetyl-2-azido-2-deoxy-α-D-quinopyranos (5*)

The reaction equation is shown in Figure 6;

Under the protection of argon, dissolve 4*(1.1g, 2.8mmol) in anhydrous DCM (20mL), add anhydrous pyridine (2mL), cool the system to -20°C, stir for 20 minutes, add trifluoride dropwise Methanesulfonic anhydride (1mL), stir the reaction while the temperature slowly rises to -10°C, after 2 hours TLC detects that the raw material has reacted completely, add DCM (20mL) to dilute, use 1M hydrochloric acid solution, saturated sodium bicarbonate solution, water extraction and wash, collect The organic phase was subjected to anhydrous sodium sulfate to remove water, and the solvent was spin-dried to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate=80:1→70:1→50:1→40:1→30:1→ 20:1) to obtain 5* (1.1 g, 2.1 mmol, 74%). ¹ HNMR(400MHz, CDCl ₃ )δ=7.90-6.90(m,6H,ArH), 5.86(d,J=5.5Hz,1H,H-1), 5.45(t,J=9.7Hz,1H,H- 3),4.59(t,J=9.5Hz,1H,H-4),4.55-4.43(m,1H,H-2),4.00(dd,J=10.2,5.5Hz,1H,H-5), 2.19(s,3H,-CH3),1.31(d,J=6.1Hz,3H,-CH3); ¹³ CNMR(100MHz,CDCl ₃ )δ=169.3,134.8,129.4,128.4,127.3,84.6,83.2,78.9 , 75.0, 70.9, 67.7, 63.3, 20.6, 17.0.

Example 6:

Synthesis of selenophenyl 3-O-acetyl-2-azido-2-deoxy-α-D-fucose(6*)

The reaction equation is shown in Figure 6;

Dissolve 5* (1g, 2mmol) in anhydrous DMF (20mL) under the protection of argon, add potassium nitrite (0.9g, 10mmol), heat up to 50°C and stir the reaction. TLC detects that the raw material reaction is complete after 1 hour. Add DCM (10mL) to dilute, extract and wash with saturated brine, collect the organic phase and dry with anhydrous sodium sulfate to remove water, spin off the solvent to obtain the crude product and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1→ 8:1→6:1→5:1) to obtain 6* (514 mg, 1.4 mmol, 69%). ¹ HNMR (400MHz, CDCl ₃ ) δ = 7.26 (s, 6H, ArH), 5.93 (d, J = 5.5 Hz, 1H, H-1), 5.06 (dd, J = 10.8, 3.0 Hz, 1H, H- 3), 4.43 (q, J = 6.6 Hz, 1H, H-5), 4.31 (dd, J = 10.7, 5.4 Hz, 1H, H-2), 3.99 (s, 1H, H-4), 2.19 ( s,3H,-CH3),1.22(d,J=6.6Hz,3H,-CH3).

Example 7:

Synthesis of selenophenyl 4-O-benzyl-3-O-acetyl-2-azido-2-deoxy-α-D-fucose(7*)

The reaction equation is shown in Figure 6;

Under the protection of argon, dissolve 6* (450mg, 1.2mmol) in anhydrous DCM (12mL), place the system in an ice bath, add benzyl bromide (1.4mL, 12.2mmol) at 0°C, and stir for 20 Minutes, add silver oxide (855mg, 3.6mmol), keep stirring at 0℃ for 24 hours, TLC test the raw material reaction is complete, filter to remove silver oxide, spin dry the organic phase to obtain the crude product purified by silica gel column chromatography (petroleum ether: acetic acid Ethyl ester=70:1→50:1→30:1→20:1) to obtain 7* (249 mg, 0.54 mmol, 45%). ¹ HNMR (400MHz, CDCl ₃ ) δ = 7.68-7.21 (m, 9H, ArH), 5.91 (d, J = 5.3 Hz, 1H, H-1), 5.46 (dd, J = 3.3, 1.2 Hz, 1H, H-4), 4.75(d,J=10.7Hz,1H,PhCH ₂ -), 4.53(d,J=10.7Hz,1H,PhCH ₂ -), 4.48-4.37(m,1H,H-5), 4.12 (dd, J = 10.3, 5.4 Hz, 1H, H-2), 3.78 (dd, J = 10.4, 3.3 Hz, 1H, H-3), 2.15 (s, 3H, -CH ₃ ), 1.12 (d , J = 6.5 Hz, 3H, -CH ₃ ); ¹³ C NMR (100MHz, CDCl ₃ ) δ = 136.9, 134.5, 129.1, 128.5, 128.3, 128.1, 127.9, 85.1, 77.3, 71.7, 68.9, 67.8, 60.4, 20.8, 16.1.

Example 8:

Synthesis of 4-O-benzyl-3-O-acetyl-2-azido-2-deoxy-D-pyranofucose trichloroacetimide ester (8*)

The reaction equation is shown in Figure 6;

Compound 7* (160mg, 0.348mmol) was dissolved in 1:1 (v/v) tetrahydrofuran/water mixture (1mL), N-bromosuccinimide (150mg, 0.843mmol) was added, and the reaction solution was at room temperature React for 3 hours. After TLC detection showed that the reaction was complete, the reaction solution was diluted with dichloromethane (5 mL), and extracted and washed with 10% sodium thiosulfate solution/1M sodium bicarbonate solution (1:1, v/v). The organic phase was subjected to anhydrous sodium sulfate to remove water, and the crude product obtained by distillation under reduced pressure was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1, v/v) to obtain 1-hydroxy compound (108 mg, 0.336 mmol, 97%) ).

Under the protection of argon, the 1-hydroxy compound (108mg, 0.336mmol) was dissolved in anhydrous dichloromethane (4mL), and trichloroacetonitrile (0.3mL, 2.992mmol) and 1,8-dinitrogen were added at 0°C Heterobicycloundec-7-ene (DBU) (5 μL, 0.033 mmol), the reaction solution was stirred at room temperature for 4 hours. After TLC detection showed that the reaction was complete, the solvent was distilled off under reduced pressure at 30°C, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 10:1, v/v, containing 0.5% triethylamine) to obtain three Chloroacetimide ester 8* (149 mg, 0.320 mmol, 95%).

Example 9:

N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 4-O-benzyl-3-O-acetyl-2-azido-2-deoxy-D-fucose (9* )Synthesis

The reaction equation is shown in Figure 6;

Under the protection of argon, 8*(149mg, 0.320mmol), N-benzyl-N-benzyloxycarbonyl-3-aminopropan-1-ol (115mg, 0.384mmol) and thiophene (0.3mL, 3.747mmol) were dissolved To 3:1 (v/v) anhydrous ether/anhydrous dichloromethane mixture (8mL), add activated molecular sieve (Aw-300) and stir the reaction solution at room temperature for 30 minutes. Trimethylsilyl trifluoromethanesulfonate (70μL, 0.387mmol) was added at -30°C. After stirring the reaction solution for 2 hours, TLC showed that the reaction was complete. Triethylamine (0.1mL) was added to quench the reaction, and the filtrate was filtered. Extract and wash with saturated sodium bicarbonate solution. The organic phase was subjected to anhydrous sodium sulfate to remove water and then the solvent was distilled off under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 6:1, v/v) to obtain α and β products (80%, α: β=3.5:1), where the α configuration is the target product 9* (120mg, 0.199mmol, 62%). ¹ HNMR(400MHz,CDCl ₃ )δ=7.41-7.12(m,15H,3Ph), 5.26(s,1H,3-H), 5.22-5.13(m,2H,Bn-CH ₂ ), 4.84(s, 1H, 1-H), 4.69 (d, J = 11.3 Hz, 1H, Bn-CH ₂ ), 4.63-4.39 (m, 3H, Bn-CH ₂ ), 3.90 (m, 1H, 5-H), 3.76 (m,3H,2-H,4-H,linker-1H), 3.36(m,3H,linker-3H), 2.09(s,3H,CH ₃ CO), 1.87(m,2H,linker-2H) ,1.14(s,3H,6-CH ₃ ); ¹³ CNMR(100MHz,CDCl ₃ )δ=170.3,137.7,128.6,128.5,128.4,128.2,127.99,127.96,127.90,127.4,98.2,77.2,75.7,71.5 , 67.3, 66.3, 65.9, 57.7, 51.0, 20.9, 16.4.

Example 10:

Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 4-O-benzyl-2-azido-2-deoxy-α-D-pyranofucose (10*)

The reaction equation is shown in Figure 6;

Compound 9* (61 mg, 0.10 mmol) was dissolved in methanol (2 mL), sodium methoxide (3 mg, 0.06 mmol) was added, and the reaction solution was stirred at room temperature for 2 h. After the TLC test showed that the reaction was over, the ^{reaction solution was neutralized with Amberlite IR 120H +} cation exchange resin, and the filtrate was filtered to obtain the filtrate. The solvent was distilled off under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 6:1, v /v) The target product 10* (56 mg, 0.1 mmol, equivalent reaction) is obtained. ¹ HNMR(400MHz,CDCl ₃ )δ=7.46-7.11(m,15H,3Ph), 5.18(m,2H,Bn-2H), 4.78(m,2H,Bn-1H,1-H), 4.65(d ,J=11.5Hz,1H,Bn-1H),4.60-4.39(m,2H,Bn-2H),3.91(m,2H,3-H,5-H),3.59(m,2H,linker-2H ), 3.35(m,4H,2-H,4-H,linker-2H),2.05(d,J=9.0Hz,1H,3-OH),1.82(m,2H,linker-2H),1.23( d,J=6.4Hz,3H,6-CH ₃ ).

Example 11:

Synthesis of allyl 2-azido-2-deoxy-β-L-fucose(11*)

The reaction equation is shown in Figure 7;

Allyl 3,4-di-O-acetyl-2-azido-2-deoxy-β-L-fucose (C.Qin et al., J Am Chem Soc, 2018, 140: 3120) (447 mg, 1.43 mmol) was dissolved in methanol (15 mL), sodium methoxide (39 mg, 0.72 mmol) was added, and the reaction solution was stirred at room temperature for 4 hours. After TLC detection showed that the reaction was completed, the reaction solution was neutralized with Amberlite IR 120H+ cation exchange resin, and the filtrate was filtered to obtain the filtrate by distillation under reduced pressure to remove the solvent to obtain the white solid target product 11* (327 mg, 1.43 mmol, equivalent reaction). [α] _D ²⁰ =-27.6° (c = 1.00, CHCl ₃ ); IRν _max (film) 3312, 2865, 2111, 1348, 1279, 1162, 1071, 999, 925, 755 cm ^-1 ; ¹ H NMR (400MHz, CDCl ₃ )δ=6.04-5.85(m,1H,CH=C), 5.34(dq,J=17.3,1.6Hz,1H,C=CH _a ), 5.23(dd,J=10.5,1.6Hz,1H,C= CH _b ), 4.41 (ddt, J = 12.9, 5.3, 1.5 Hz, 1H, OCH _a ), 4.30 (d, J = 7.9 Hz, 1H, 1-H), 4.14 (ddt, J = 12.8, 6.1, 1.4 Hz, 1H, OCH _b ), 3.70 (d, J = 3.2 Hz, 1H, 4-H), 3.63-3.53 (m, 1H, 5-H), 3.53 (dd, J = 10.1, 7.8 Hz, 1H, 2-H), 3.45 (dd, J = 10.1, 3.3 Hz, 1H, 3-H), 3.04 (s, 1H, OH), 2.68 (s, 1H, OH), 1.35 (d, J = 6.5 Hz, 3H,6-Me); ¹³ C NMR(100MHz,CDCl ₃ )δ=133.5,117.8,101.1,72.6,70.9,70.6,70.3,64.0,16.3; HR-ESI-MS(m/z):calcd for C ₉ H ₁₅ N ₃ O ₄ Na ⁺ (M+Na ⁺ ): 252.0960, found: 252.0954.

Example 12:

Synthesis of allyl 3-O-p-methoxybenzyl-2-azido-2-deoxy-β-L-fucose(12*)

The reaction equation is shown in Figure 7;

Dissolve 11* (4.2g, 18.4mmol) in anhydrous toluene (184mL) under the protection of argon, add dibutyltin oxide (14.1g, 27.6mmol), heat to reflux at 120°C, stir and react for 4 hours, Rotate to dry to the remaining half of the solvent, add p-methoxybenzyl chloride (5.5mL, 22.1mmol), then add tetrabutylammonium bromide (6.5g, 20.2mmol), reheat to 120°C and stir for 3 hours. TLC detects that the raw material reaction is complete. The system was cooled to room temperature, and the solvent was directly spin-dried. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15:1→10:1→8:1→5:1) to obtain 12*(3.8 g, 10.9 mmol, 59%). ¹ HNMR(400MHz,CDCl ₃ )δ=7.26(s,5H,ArH), 6.05-5.86(m,1H,-OAll), 5.38-5.28(m,1H,-OAll), 5.21(dd,J=10.4 ,1.5Hz,1H,-OAll),4.64(s,2H,PhCH2-),4.43-4.34(m,1H,-OAll),4.22(d,J=8.1Hz,1H,H-1),4.16- 4.05(m,1H,-OAll),3.81(s,3H,-CH ₃ ),3.73-3.66(m,1H,H-4),3.59(dd,J=10.0,8.1Hz,1H,H-2 ), 3.47(q,J=6.5Hz,1H,H-5), 3.30(dd,J=10.0,3.3Hz,1H,H-3),2.34-2.25(m,1H,4-OH).

Example 13:

Synthesis of allyl 4-O-benzyl-3-O-p-methoxybenzyl-2-azido-2-deoxy-β-L-fucose(13*)

The reaction equation is shown in Figure 7;

Dissolve 12* (3.6g, 10.3mmol) in DMF (60mL), place in an ice bath for 10 minutes to cool to 0°C, slowly add sodium hydride (530mg, 20.7mmol) under the protection of argon, and stir in an ice bath For 0.5 hour, benzyl bromide (2mL, 15.5mmol) was added dropwise at 0°C, and after stirring for 0.5 hour under an ice bath, the ice bath was removed, and the mixture was returned to normal temperature and stirred for 2 hours. TLC monitored the complete reaction of the raw materials. Add DCM (30mL) solution to dilute the reaction solution, slowly add water (100mL), extract with DCM, combine the organic phases with saturated brine and anhydrous sodium sulfate to remove water, the filtrate is spin-dried and the crude product obtained is purified by silica gel column chromatography ( Petroleum ether: ethyl acetate=50:1→20:1→15:1→10:1) to obtain 13* (3.2g, 7.3mmol, 71%). ¹ HNMR (400MHz, CDCl ₃ )δ = 7.59-6.74 (m, 10H, ArH), 5.93 (dddd, J = 16.9, 10.9, 6.1, 5.0 Hz, 1H, -OAll), 5.31 (dq, J = 17.2, 1.7Hz,1H,-OAll),5.22-5.17(m,1H,-OAll), 4.93(d,J=11.7Hz,1H,PhCH2-),4.70-4.52(m,3H,PhCH2-), 4.38( ddt,J=12.9,5.1,1.6Hz,1H,-OAll),4.21(d,J=8.0Hz,1H,H-1),4.14-4.04(m,1H,-OAll),3.88–3.76(m ,4H,H-2,-CH3), 3.50(dd,J=2.9,1.0Hz,1H,H-4),3.45-3.36(m,1H,H-5), 3.29(dd,J=10.4, 2.8Hz,1H,H-3),1.19(d,J=6.4Hz,3H,-CH3); ¹³ CNMR(101MHz,CDCl ₃ )δ=138.3,133.8,129.8,129.5,128.4,128.2,127.7,117.4 ,113.9,100.9,80.8,74.9,74.6,72.3,70.6,69.9,63.0,55.3,16.9.

Example 14:

Synthesis of allyl 4-O-benzyl-2-azido-2-deoxy-β-L-fucose (14*)

The reaction equation is shown in Figure 7;

Dissolve 13* (3.2g, 7.3mmol) in DCM (300mL), add DDQ (2.5g, 11mmol), add water (18mL), stir at room temperature for 7 hours, TLC check the raw material reaction is complete, add DCM (100mL) Dilute, extract and wash with 5% sodium thiosulfate solution, collect the organic phase, remove water with anhydrous sodium sulfate, spin dry the solvent to obtain the crude product and purify it by silica gel column chromatography (petroleum ether: ethyl acetate = 40:1→30 :1→20:1→10:1) to obtain 14* (2.1g, 6.6mmol, 90%). ¹ H NMR(400MHz, CDCl ₃ )δ=7.46-7.18(m,5H), 5.94(dddd,J=16.9,10.9,6.1,5.1Hz,1H, H-OAll), 5.33(dd,J=17.2, 1.7Hz,1H,H-OAll),5.21(dd,J=10.4,1.5Hz,1H,H-OAll), 4.82(d,J=11.6Hz,1H,PhCH2-),4.72(d,J=11.6 Hz, 1H, PhCH2-), 4.41 (ddt, J = 12.9, 5.0, 1.5 Hz, 1H, H-OAll), 4.26 (d, J = 7.9 Hz, 1H, H-1), 4.18–4.06 (m, 1H, H-OAll), 3.64–3.48 (m, 3H, H-3, H-4, H-5), 3.45 (ddd, J = 10.2, 7.7, 3.4 Hz, 1H, H-2), 2.22 ( d,J=7.7Hz,1H,3-OH),1.32(d,J=6.5Hz,3H,-CH3); ¹³ CNMR(100MHz,CDCl ₃ )δ=137.9,133.6,128.6,128.2,128.1,117.5 ,101.0,78.5,76.0,73.0,70.9,70.1,64.6,16.9.

Example 15:

Allyl 4-O-benzyl-3-O-(4,6-O-benzylidene-3-O-benzyl-2-O-levulinyl-β-D-glucopyranose)-2 Synthesis of -azido-2-deoxy-β-L-fucose (15*)

The reaction equation is shown in Figure 8;

Under the protection of argon, p-methylphenylthio 4,6-O-benzylidene-3-O-benzyl-2-O-levulinyl-β-D-glucopyranose (S. David et al .,Carbohyd Res,1989,188:193; YHWang et al.,Carbohyd Res,2013,375:118; T.Li et al.,ChemMedChem,2014,9:1071) (180mg, 0.32mmol) and 14*( 51mg, 0.16mmol) was dissolved in anhydrous dichloromethane (7mL), added activated molecular sieve (AW-300) and stirred at room temperature for 30 minutes, at 0℃, added iodosuccinimide (86mg, 0.38mmol) ) And trimethylsilyl trifluoromethanesulfonate (29 μL, 0.16 mmol), and the reaction solution was stirred for 4 hours. After TLC detection showed that the reaction was complete, the reaction was quenched with triethylamine (0.5 mL), and the filtrate obtained by filtration was extracted with 5% sodium thiosulfate solution and saturated sodium bicarbonate solution. The organic phase was dewatered by anhydrous sodium sulfate and then the solvent was distilled off under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1, v/v) to obtain 15*(107mg, 0.14mmol, 88 %, single β configuration). ¹ H NMR (400MHz, CDCl ₃ ) δ=7.55-7.25 (m, 15H, 3Ph), 5.94 (m, 1H, allyl-1H), 5.60 (s, 1H, PhCH), 5.32 (m, 1H, allyl- 1H),5.21-5.16(m,1H,allyl-1H),5.10(dd,J=8.5,7.4Hz,1H,2'-H),4.93-4.83(m,2H,Bn-2H), 4.80( d,J=7.7Hz,1H,1'-H),4.70(d,J=11.7Hz,1H,Bn-1H),4.52(d,J=11.3Hz,1H,Bn-1H), 4.46(dd ,J=10.4,5.2Hz,1H,6'-CH _a ), 4.39(m,1H,allyl-1H),4.22(d,J=8.0Hz,1H,1-H),4.11(dd,J= 13.0,6.3Hz,1H,allyl-1H),3.91(m,2H,6'-CH _b ,4'-H),3.83-3.68(m,2H,3'-H,2-H),3.63( dd,J=10.3,2.7Hz,1H,3-H),3.54(m,2H,4-H,5'-H), 3.48(q,J=6.4,5.7Hz,1H,5-H), 2.69-2.25 (m, 4H, Lev-CH ₂ ), 2.06 (s, 3H, lev-CH ₃ ), 1.18 (d, J = 6.5 Hz, 3H, 6-CH ₃ ).

Example 16:

Allyl 4-O-benzyl-3-O-(4,6-O-benzylidene-3-O-benzyl-β-D-glucopyranose)-2-azido-2-deoxy Synthesis of -β-L-fucose pyranose (16*)

The reaction equation is shown in Figure 8;

Compound 15* (0.34g, 0.450mmol) was dissolved in a 20:1 (v/v) dichloromethane/methanol mixture (5mL), hydrazine acetate (51mg, 0.55mmol) was added, and the reaction solution was stirred at room temperature for 5 hours. After TLC detection showed that the reaction was complete, the solvent was distilled off under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 8:1, v/v) to obtain 16* (222 mg, 0.337 mmol, 75%). ¹ HNMR(400MHz, CDCl ₃ )δ=7.55-7.27(m,15H,3Ph), 5.94(m,1H,allyl-1H), 5.60(s,1H,PhCH), 5.33(d,J=16.9Hz, 1H, allyl-1H), 5.21 (d, J = 10.2Hz, 1H, allyl-1H), 5.00 (m, 2H, Bn-2H), 4.78 (d, J = 11.5Hz, 1H, Bn-1H), 4.75(d,J=11.4Hz,1H,Bn-1H), 4.64(d,J=5.3Hz,1H,1'-H), 4.40(m,2H,allyl-1H,6'-CH _a ), 4.26(d,J=7.8Hz,1H,1-H),4.09(dd,J=12.9,6.1Hz,1H,allyl-1H), 3.83(m,2H,3'-H,6'-CH _b ), 3.79-3.63(m,4H,4'-H,2-H,3-H,5'-H), 3.60(d,J=2.7Hz,1H,4-H),3.56-3.38(m ,2H,2'-H,H-5),2.37-2.27(m,1H,2'-OH),1.20(d,J=6.5Hz,3H,6-CH ₃ ); ¹³ CNMR(100MHz,CDCl ₃ )δ=138.31,138.29,137.2,133.7,129.7,129.1,128.47,128.45,128.3,128.24,128.20,128.0,127.9,127.7,126.0,117.7,101.7,101.3,100.8,81.2,80.4,78.6,77.2, 75.5, 74.72, 74.66, 73.2, 70.5, 70.0, 68.7, 66.9, 61.9, 16.8.

Example 17:

Allyl 4-O-benzyl-3-O-(4,6-O-benzylidene-3-O-benzyl-2-azido-2-deoxy-β-D-mannanose )-2-azido-2-deoxy-β-L-fucose (17*) synthesis

The reaction equation is shown in Figure 8;

Under argon protection, compound 16* (139mg, 0.211mmol) was dissolved in anhydrous dichloromethane (1.5mL), pyridine (0.3mL) was added, and trifluoromethanesulfonic anhydride (0.1mL, 0.585mmol), slowly increase the temperature to 10°C and continue stirring for 4h. After TLC detection showed that the reaction was over, add 5 mL of dichloromethane to dilute the reaction solution, extract and wash with 1M hydrochloric acid solution and saturated sodium bicarbonate solution, remove the water from the organic phase with anhydrous sodium sulfate, concentrate to obtain the crude product and purify it by silica gel column chromatography (petroleum Ether: ethyl acetate, 5:1, v/v) to obtain the trifluoromethanesulfonyl compound (107 mg, 0.134 mmol, 64%).

Under the protection of argon, the trifluoromethanesulfonyl compound (107 mg, 0.134 mmol) was dissolved in anhydrous DMF (2 mL), sodium azide (57 mg, 0.88 mmol) was added, and the reaction solution was heated to 60° C. for 3 hours. After TLC detection showed that the reaction was complete, 6 mL of dichloromethane was added to dilute the reaction solution, and after water extraction and washing, the organic phase was dehydrated with anhydrous sodium sulfate. The crude product was purified by distillation under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate, 5:1, v/v) to obtain 17* (81 mg, 0.120 mmol, 90%). ¹ H NMR (400MHz, CDCl ₃ )δ=7.51-7.27 (m, 15H, 3Ph), 5.95 (m, 1H, allyl-1H), 5.58 (s, 1H, PhCH), 5.34 (dd, J = 17.2, 1.9Hz, 1H, allyl-1H), 5.22 (dd, J = 10.7, 1.7 Hz, 1H, allyl-1H), 4.85 (d, J = 12.6 Hz, 1H, Bn-1H), 4.80 (d, J = 11.9Hz,1H,Bn-1H),4.74(d,J=12.5Hz,1H,Bn-1H), 4.64(d,J=11.8Hz,1H,Bn-1H),4.45-4.39(m,1H, allyl-1H), 4.38(d,J=1.2Hz,1H,1'-H),4.31(dd,J=10.4,4.8Hz,1H,6'-CH _a ), 4.26(d,J=7.5Hz ,1H,1-H),4.17-4.06(m,1H,allyl-1H),4.04 (t,J=9.3Hz,1H,4'-H),3.91(t,J=10.1Hz,1H,6 '-CH _b ), 3.77(dd,J=10.6,7.8Hz,1H,2-H), 3.69(dd,J=10.2,3.1Hz,1H,3-H), 3.60-3.51(m,2H, 4-H,3'-H),3.51-3.39(m,2H,2'-H,5-H),3.21(td,J=9.5,4.6Hz,1H,5'-H),1.35(d , J = 6.2 Hz, 3H, 6-CH ₃ ); ¹³ C NMR (100MHz, CDCl ₃ ) δ = 138.1, 137.8, 137.2, 133.7, 129.0, 128.53, 128.51, 128.47, 128.45, 128.41, 128.37, 128.3, 128.2 ,128.1,127.98,127.95,127.8,127.7,126.0,117.5,101.5,101.0,100.9,97.0,78.3,77.6,76.3,75.4,75.0,72.9,70.4,70.1,68.3,67.68,67.65,63.3,62.1,17.1 .

Example 18:

4-O-benzyl-3-O-(4,6-O-benzylidene-3-O-benzyl-2-azido-2-deoxy-β-D-mannanose)-2 Synthesis of -azido-2-deoxy-1-(N-phenyl)-2,2,2-trifluoroacetimide ester-L-fucose(18*)

The reaction equation is shown in Figure 8;

Compound 17* (26mg, 0.038mmol) was dissolved in 20:1 (v/v) acetic acid/water mixture (3mL), sodium acetate (183mg, 2.244mmol) and palladium chloride (11.4mg, 0.065mmol) were added to react The solution was stirred at room temperature for 3h. After TLC detection showed that the reaction was complete, 6 mL of ethyl acetate was added to dilute the reaction solution, and the filtrate was filtered through Celite and washed with saturated sodium bicarbonate solution. The organic phase was evaporated under reduced pressure with anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 2:1, v/v) to obtain 1-hydroxy compound (20mg, 0.031mmol, 82 %).

Under the protection of argon, the 1-hydroxy compound (20mg, 0.031mmol) was dissolved in anhydrous dichloromethane (2mL), and N-phenyltrifluoroacetimide chloride (10μL, 0.067mmol) and 1 , 8-diazabicycloundec-7-ene (DBU) (11 μL, 0.074 mmol), and the reaction solution was stirred at room temperature for 10 h. After TLC detection showed that the reaction was complete, the solvent was distilled off under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 5:1, v/v) to obtain 18* (15 mg, 0.018 mmol, 58%).

Example 19:

N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 4-O-benzyl-3-O-(4-O-benzyl-3-O-[4,6-O-benzylidene- 3-O-benzyl-2-azido-2-deoxy-β-D-mannanose)-2-azido-2-deoxy-α-L-fucose)-2- Synthesis of azido-2-deoxy-α-D-fucose(19*)

The reaction equation is shown in Figure 8;

Under the protection of argon, the donor 18* (60mg, 0.074mmol) and the acceptor 10* (30mg, 0.054mmol) were dissolved in a 3:1 (v/v) ether/dichloromethane mixture (2.5mL), and added After activated molecular sieve (AW-300) and thiophene (70 μL, 0.874 mmol), the reaction solution was stirred at room temperature for 30 minutes. Trimethylsilyl trifluoromethanesulfonate (1.5 μL, 0.008 mmol) was added at -10°C, and the reaction solution was stirred at -10°C for 5 hours. After TLC detection showed that the reaction was complete, triethylamine (0.5 mL) was added to quench the reaction, and the filtrate obtained by celite filtration was extracted and washed with saturated sodium bicarbonate solution. After the organic phase was dewatered by anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 3:1, v/v) to obtain 19* (52 mg, 0.044 mmol, 82 %, single α configuration). ¹ HNMR(400MHz, CDCl ₃ )δ=7.63-7.15(m,30H,6Ph), 5.58(s,1H,PhCH), 5.21(d,J=4.2Hz,1H,1'-H), 5.19-5.12 (m,2H,Bn-2H), 4.88(m,2H,1-H,Bn-1H), 4.79(d,J=11.2Hz,1H,Bn-1H),4.76-4.60(m,4H,Bn -4H),4.56(m,3H,1″-H,NBn-2H), 4.17(m,2H,3'-H,6″-CH _a ),4.06(m,2H,4″-H,3 -H), 3.91(m,2H,5-H,5'-H), 3.83(m,2H,2-H,6″-CH _b ), 3.73(dd,J=10.5,3.7Hz,1H, 2'-H),3.63(m,3H,linker-1H,3″-H,2″-H),3.56(d,J=2.6,1H,4'-H),3.53(s,1H,4 -H),3.42(m,1H,linker-1H),3.32(m,2H,linker-2H), 3.23(td,J=9.7,4.8Hz,1H,5″-H),1.83(m,2H ,linker-2H),1.21(m,6H,6-CH ₃ ,6'-CH ₃ ); ¹³ CNMR(100MHz,CDCl ₃ )δ=138.4,137.9,137.3,136.8,129.0,128.6,128.52,128.49, 128.4,128.3,128.2,128.1,128.0,127.94,127.9,127.82,127.78,127.7,127.4,126.1,101.6,99.9,98.2,98.1,79.9,78.4,77.22,77.17,76.9,76.5,76.4,75.7,75.3, 75.2, 73.0, 68.4, 67.8, 67.5, 67.3, 66.9, 66.0, 63.6, 60.3, 58.5, 16.8.

Example 20:

N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 4-O-benzyl-3-O-(4-O-benzyl-3-O-[4,6-O-benzylidene- 3-O-benzyl-2-acetylamino-2-deoxy-β-D-mannanose)-2-acetylamino-2-deoxy-α-L-fucose)-2-acetylamino Synthesis of -2-deoxy-α-D-fucose(20*)

The reaction equation is shown in Figure 8;

Compound 19* (12.2mg, 10μmol) was dissolved in pyridine (1mL), water (289μL, 16.05mmol), triethylamine (62μL, 0.45mmol) and 1,3-propanedithiol (60μL, 0.60mmol) were added, The resulting reaction solution was stirred at room temperature for 4 hours. After TLC detection showed that the reaction was complete, the solvent was distilled off under reduced pressure, and the obtained crude product was directly used in the next reaction.

Under argon protection, the crude amino product of the previous step was dissolved in methanol (0.5 mL), acetic anhydride (12 μL, 0.12 mmol) was added, and the reaction solution was stirred at room temperature for 18 hours. After TLC detection showed that the reaction was over, the solvent was distilled off under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol, 40:1, v/v) to obtain a yellow syrupy product 20* (8.3mg, 6.7μmol, two Step yield 67%). IRνmax(film)3326,2872,1665,1530,1453,1370,1087,1047,734,697,576cm ^-1 ; ¹ HNMR(400MHz,CDCl ₃ )δ=7.60-7.12(m,30H,6Ph),7.02(d, J=9.3Hz,1H,2-NH), 6.25(d,J=9.6Hz,1H,2'-NH), 6.16(d,J=8.4Hz,1H,2″-NH), 5.56(s, 1H,PhCH),5.24-5.05(m,2H,Bn-2H),4.94-4.81(m,3H,Bn-2H,1'-H),4.77(m,4H,Bn-2H,2-H, 2″-H),4.70-4.55(m,4H,Bn-2H,2'-H,1-H),4.51(d,J=2.1Hz,1H,1″-H), 4.46(d,J = 11.3 Hz, 1H, Bn-1H), 4.37 (d, J = 15.9 Hz, 1H, Bn-1H), 4.22 (dd, J = 10.8, 4.7 Hz, 1H, 6″-CHa), 4.00-3.89 ( m,2H,5-H,3-H),3.84(d,J=6.7Hz,1H,5'-H),3.79-3.57(m,6H,linker-2H,6″-CH _b ,4″ -H,3″-H, 3-H), 3.46(dd,J=5.8,2.6Hz,2H,4-H,4'-H), 3.37-3.10(m,3H,linker-2H,5″ -H), 2.12(s,3H,CH ₃ CO),2.01(s,6H,2CH ₃ CO),1.73(m,2H,linker-2H),1.26(d,J=6.5Hz,3H,6- CH ₃ ),1.22(d,J=6.4Hz,3H,6'-CH ₃ ); ¹³ CNMR(100MHz,CDCl ₃ )δ=172.0,171.4,156.5,138.6,138.5,138.2,137.4,137.3,136.4, 128.9,128.7,128.50,128.47,128.3,128.24,128.19,127.9,127.8,127.7,127.5,127.2,127.1,126.1,101.5,100.4,98.8,97.9,80.6,79.5,78.4,76.1,75.4,75.1,71.6, 68.6, 67.5, 67.0, 51.0, 49.7, 48.8, 47.7, 24.1,23.2, 16.9, 16.8.

Example 21:

N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 4-O-benzyl-3-O-(4-O-benzyl-3-O-[3-O-benzyl-2-acetyl Amino-2-deoxy-β-D-mannopyranoic acid benzyl ester]-2-acetylamino-2-deoxy-α-L-fucose)-2-acetylamino-2-deoxy-α -D- Fucose Pyranose (21*) Synthesis

The reaction equation is shown in Figure 8;

Compound 20* (8.3 mg, 6.7 μmol) was dissolved in dichloromethane (0.7 mL), water (2 μL) and trifluoroacetic acid (70 μL, 0.94 mmol) were added, and the reaction solution was stirred at room temperature for 18 hours. After TLC detection showed that the reaction was complete, triethylamine (0.1 mL) was added to quench the reaction. After the solvent was distilled off under reduced pressure, the crude product was purified by silica gel column chromatography (dichloromethane: methanol, 20:1, v/v) to obtain 4 , 6-Dihydroxy sugar (6mg, 5.2μmol, 78%).

Dihydroxytriose (12.7mg, 11μmol) was dissolved in dichloromethane (0.6mL), water (0.28mL), TEMPO (0.9mg, 5.5μmol) and diacetoxy iodobenzene (9mg, 27.7μmol) were added to obtain The reaction solution was stirred at room temperature for 4 hours. After TLC detection showed that the reaction was complete, the reaction solution was filtered through silica gel (dichloromethane: methanol, 5:1, v/v), and the crude product obtained by concentrating the filtrate was drained by an oil pump. Under argon protection, the crude product was dissolved in anhydrous DMF (1.1 mL), benzyl bromide (10 μL, 82.5 μmol) and sodium bicarbonate (5 mg, 50 μmol) were added, and the reaction solution was stirred at room temperature for 12 hours. After TLC detection showed that the reaction was complete, 2 mL of water was added to the reaction solution to quench the reaction, and after extraction with ethyl acetate (3×5 mL), the organic phase was extracted and washed with saturated brine (5 mL). The organic phase was treated with anhydrous sodium sulfate and the solvent was distilled off under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane: methanol, 50:1, v/v) to obtain a colorless syrupy compound 21* (12.0mg, 9.6 μmol, two-step yield 87%). IRνmax(film)2942,2865,1722,1366,1294,1242,1190,1104,1046,732cm ^-1 ; ¹ HNMR(400MHz,CDCl ₃ )δ=7.51-7.09(m,30H,5Ph),6.90(d ,J=9.3Hz,1H,NH),6.32(m,2H,N″-H,NH),5.43-5.24(m,2H,Bn-2H),5.13(s,2H,Bn-2H),4.99 -4.82(m,3H,Bn-2H,1'-H),4.83-4.69(m,4H,Bn-2H,2-H,2'-H),4.68-4.56(m,3H,1-H ,NBn-1H,2″-H),4.54(d,J=2.0Hz,1H,1″-H), 4.47(dd,J=11.3,3.4Hz,2H,Bn-2H), 4.38(d, J=15.8Hz,1H,NBn-1H),3.99-3.85(m,3H,5'-H,3-H,4″-H), 3.79(d,J=9.3Hz,2H,5″-H ,5-H), 3.71(dd,J=10.7,2.6Hz,2H,3'-H,linker-1H), 3.68-3.57(m,1H,linker-1H), 3.50-3.37(m,3H, 4-H,4'-H,3″-H), 3.28(s,1H, linker-1H), 3.20(d,J=13.9Hz,1H,linker-1H), 2.74(d,J=2.5Hz ,1H,4″-OH),2.05(s,3H,CH ₃ CO),2.00(s,6H,2CH ₃ CO),1.71(m,2H,linker-2H),1.26(d,J=7.0Hz ,3H,6'-CH ₃ ), 1.20(d,J=6.5Hz,3H,6-CH ₃ ).

Example 22:

N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 4-O-benzyl-3-O-(4-O-benzyl-3-O-[4-O-acetyl-3-O -Benzyl-2-acetamido-2-deoxy-β-D-mannopyranoic acid benzyl ester]-2-acetylamino-2-deoxy-α-L-fucose)-2-acetyl Synthesis of amino-2-deoxy-α-D-fucose(22*)

The reaction equation is shown in Figure 8;

Under argon protection, compound 21* (7 mg, 5.6 μmol) was dissolved in pyridine (0.2 mL), acetic anhydride (6 μL, 63.5 μmol) was added, and the reaction solution was stirred at room temperature for 3 hours. After TLC detection showed that the reaction was over, methanol (50μL) was added to quench the reaction, and the solvent was distilled off under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane: methanol, 70:1, v/v) to obtain a colorless syrupy product 22* (5.8 mg, 4.5 μmol, 80%). [α] _D ²⁰ =-13.47° (c = 0.50, CHCl ₃ ); IRνmax(film) 3334, 2923, 1754, 1671, 1526, 1454, 1368, 1232, 1095, 1051, 736, 698 cm ^-1 ; ¹ HNMR( 400MHz, CDCl ₃ )δ = 7.51-7.10 (m, 30H, 6Ph), 6.84 (d, J = 9.5 Hz, 1H, NH), 6.57 (d, J = 9.1 Hz, 1H, N″-H), 6.36 (d,J=9.6Hz,1H,NH), 5.38(t,J=6.5Hz,1H,4″-H), 5.27-5.00(m,5H,Bn-5H), 4.98(d,J=3.8 Hz,1H,1'-H),4.91-4.67(m,5H,1-H,2-H,2'-H,Bn-2H), 4.67-4.47(m,5H,1″-H,2 "-H,Bn-3H),4.46-4.34(m,2H,Bn-2H),4.29-4.18(m,1H,3'-H),4.04(d,J=6.1Hz,1H,5"- H), 3.96(m,3H,5-H,5'-H,3-H), 3.67(m,3H,4-H,linker-2H),3.51(m,2H,4'-H,3 "-H),3.38-3.15(m,2H,linker-2H),2.04(s,3H,CH ₃ CO),2.00(s,3H,CH ₃ CO),1.92(s,3H,CH ₃ CO) ,1.82(s,3H,CH ₃ CO),1.76(m,2H,linker-2H),1.25(d,J=6.4Hz,6H,6-CH ₃ ,6'-CH ₃ ); ¹³ CNMR(100MHz ,CDCl ₃ )δ=171.9,169.7,169.6,167.4,156.4,139.0,138.3,137.9,136.5,135.0,128.8,128.7,128.6,128.5,128.4,128.3,128.2,128.1,127.8,127.5,127.3,100.3( anomeric),98.0(anomeric),94.5(anomeric),74.7,74.4,72.1,67.9,67.7,67.5,67.2,63.5,49.7,48.8,47.7,47.0,29.7,23.6,22.9,20.8,17.3,17.0; HR -ESI-MS(m/z):calcd for C ₇₂ H ₈₄ N ₄ O ₁₈ Na ⁺ (M+Na ⁺ ):1315.5678,found:1315.5697.

Example 23:

3-Aminopropyl 3-O-(3-O-[4-O-Acetyl-2-Acetylamino-2-deoxy-β-D-Mannopyranoic acid]-2-Acetylamino-2- Synthesis of deoxy-α-L-fucose)-2-acetylamino-2-deoxy-α-D-fucose(23*)

The reaction equation is shown in Figure 8;

Trisaccharide 22* (4.7mg, 3.63μmol) is dissolved in tert-butanol/water/dichloromethane mixture (5mL, 5:2:1, v/v/v), after nitrogen replacement of the reaction system, add 10% Palladium-carbon hydrogenation catalyst, continue nitrogen replacement for 5 minutes. After further replacing the reaction system with hydrogen for 5 minutes, the reaction solution was stirred for 24 hours in a hydrogen environment, filtered through diatomaceous earth, and the crude product obtained by concentration was applied to a C ₁₈ cartridge (Macherey-Nagel, Düren, Germany) (the eluent was water and Methanol) was purified to obtain the target trisaccharide 23* (2.5 mg, 3.53 μmol, 97%) as a white solid. [α] _D ²⁰ = -45.25° (c = 0.20, H ₂ O); ¹ HNMR (400MHz, D ₂ O) δ = 5.13-5.05 (m, 2H, 4″-H, 1'-H), 5.02 (s,1H,1″-H), 4.83(d,J=3.8Hz,1H,1-H),4.59(d,J=4.4Hz,1H,2″-H),4.33(dd,J= 11.1,3.8Hz,1H,2-H),4.27-4.20(m,2H,2'-H,4'-H), 4.13(dd,J=9.6,4.5Hz,3H,3″-H,5 -H,5'-H),4.08(s,1H,3'-H),4.04-3.94(m,2H,5″-H,3-H), 3.85(d,J=3.2Hz,1H, 4-H), 3.81 (dd, J = 10.9, 5.6 Hz, 1H, linker-CH ₂ ), 3.57 (dt, J = 11.2, 6.0 Hz, 1H, linker-CH ₂ ), 3.16 (t, J = 7.6 Hz, 2H, linker-CH ₂ ), 2.18 (s, 3H, CH ₃ CO), 2.11 (s, 6H, 2CH ₃ CO), 2.03 (s, 5H, linker-CH ₂ , CH ₃ CO), 1.28 ( d, J = 2.3 Hz, 3H, 6'-CH ₃ ), 1.27 (d, J = 2.7 Hz, 3H, 6-CH ₃ ); ¹³ CNMR (100MHz, D ₂ O) δ = 175.6, 173.9, 173.8, 173.1, 98.9(1'-C), 97.2(1-C), 95.1(1″-C), 74.1, 73.3, 73.1, 71.1, 70.1, 69.5, 67.6, 66.9, 66.5, 65.0, 53.0, 48.5, 47.6 ,37.2,26.8,22.3,21.94,21.92,20.3,15.5,15.3; HR-ESI-MS(m/z):calcd for C ₂₉ H ₄₈ N ₄ O ₁₆ Na ⁺ (M+Na ⁺ ):731.2963,found :731.2962.

Example 24:

Preparation of protein conjugate of trisaccharide 23* and CRM-197

Add triethylamine (12μL, 86μmol) to a DMSO/pyridine solution (1:1, 25mL: 0.25mL) of bis(p-nitrophenyl adipate) (PNP, 26.33mg, 67.8μmol) (PNP, 26.33mg, 67.8μmol) and stir at room temperature for 5 minutes . Trisaccharide 23 (1.6 mg, 2.26 μmol) dissolved in DMSO/pyridine (1:1, 0.1 mL: 0.1 mL) was added dropwise, and the reaction was stirred at room temperature for 7 hours. TLC detection showed that the raw material had reacted completely, and sugar stain showed it. The reaction mixture was lyophilized. The lyophilized solid was washed 6 times with chloroform (1 mL) to obtain triose-PNP ester. CRM ₁₉₇ protein (1mg, 0.017μmol) was washed 3 times with sterile water (400μL) in the ultrafiltration tube, and then washed once with phosphate solution (pH 8.0, 400μL). The cleaned _CRM197 protein was added to the trisaccharide-PNP ester and stirred at room temperature for 24 hours. After the reaction, the mixture was washed with sterilized water and a phosphate solution to obtain a glycoprotein conjugate. MALDI-TOF-MS and SDS-PAGE were used to identify the obtained glycoprotein conjugates. According to the SDS-PAGE result of Fig. 9, it can be seen that _{after a certain amount of trisaccharide residues are connected to the CRM197} protein, glycoconjugates are formed. The molecular weight of the bands increases after glycosylation, and the aggregation is poor and widened. According to MALDI-TOF/TOF-MS (matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer) analysis, the average molecular weight of glycoprotein conjugate is about 63.5kDa, and it can be calculated that there are 6 trisaccharide residues attached to each protein. Calculate the amount of glycoprotein conjugate required for each mouse in the immunization experiment.

Example 25: Immunoassay and chip detection of glycoprotein conjugate

Glycoconjugate immunoassay:

Use anti-mouse IgG labeled Alexa Fluor 488 secondary antibody for detection. And quantitatively detect the average fluorescence intensity of mice in the PBS-immunized control group and the glycoconjugate-immunized experimental group. The error bar is the standard deviation of 3 different points from two different detection areas.

Twelve six-week-old Balb/c mice were randomly divided into two groups, six control groups and six experimental groups. The 6 control groups were PBS-immunized control groups. The results are shown in Figure 10(A), mice 1-3, and the 6 experimental groups were glycoconjugate immunized experimental groups. The results are shown in Figure 10(A). Rat 4-6.

On day 0, mice in the experimental group were subcutaneously immunized with 100 μL of glycoconjugate and Freund's complete adjuvant 1:1 mixed emulsion; control group mice were injected with 100 μL of PBS and Freud's complete adjuvant 1:1 mixed emulsion. On the 14th day, boost with Freund's incomplete adjuvant. The amount of antigen injected per mouse in the experimental group was equivalent to 4 μg of carbohydrate antigen. The mouse sera on days 0, 14, and 21 were tested on a chip to detect the immune response.

Sugar chip experiment:

The spot pattern is shown in Figure 10(B).

Chip inspection:

Spot 1: Trisaccharide 23*, spot concentration is 0.1, 0.5, 1mM;

Spotting 2: Connecting arm (HOCH ₂ CH ₂ CH ₂ NH ₂ ), spotting concentration is 0.1, 0.5, 1mM;

Spot 3: CRM ₁₉₇ , spot concentration is 0.1, 0.05μM;

Spot 4: E. coli O55: B5LPS, spot concentration 0.2mg/mL;

Spot 5: Spot buffer, 50mM sodium phosphate solution, pH 8.5;

Spot 6: Pseudomonas shiga-like O51 serotype O antigen 3 sugar (see CN108558961A), spot concentration 0.5mM;

Spot 7: α-1-6-glucotriose, spot concentration 0.5mM;

Immerse the APTES slide (Electron Microscopy Science, Cat. #63734-01) in solution A (1.58g tetraethylene glycol succinyl disuccinic acid, 257mL DMF, 3.6mL diisopropylamine), 40℃, 60-70 Incubate with shaking overnight. Ultrasound for 15 minutes and wash 3 times with absolute ethanol. The slides were centrifuged and dried under vacuum at 37°C for 3 hours.

The solid to be spotted was dissolved in 50 mM sodium phosphate solution, pH 8.5. Arrayjet sprint (Arrayjet) is used for spotting on modified glass slides. After spotting, incubate overnight at 26°C and 55% humidity. Then the slide was immersed in solution B (50 nM Na ₂ HPO ₄ , 100 nM ethanolamine in water) at 50° C. for 1 hour. The slides were washed 3 times with ultrapure water, and centrifuged to remove residual water. Use 3% BSA (w/v) in PBS and block overnight at 4°C. Wash with PBST (PBS containing 0.1% tween) once, wash twice with PBS, and centrifuge to dry. The slides are loaded into a 64-well incubator (ProPlate). Add 30 μL of mouse serum sample diluted 1:50 in 1% BSA (w/v) PBS solution to each well, and incubate for 1 hour in a humidified box at room temperature in the dark. Remove the sample, wash 3 times with 50 μL PBST, add a secondary antibody diluted 1:400 in 1% BSA (w/v) PBS solution, and incubate in a humid box at room temperature for 45 minutes in the dark. Remove the secondary antibody solution and wash 3 times with 50μL PBST. Remove the 64-well incubator, clean it with ultrapure water, and then rinse with ultrapure water for 15 minutes. Centrifuge to remove residual water.

Use the chip scanner to scan. According to the results shown in Figure 10, compared to the control group and the pre-immunization level, after immunization and one boost, the experimental group mice had a significant immune response and gradually produced IgG antibodies in the serum. According to the different degrees of binding to the CRM ₁₉₇ protein, spotting buffer and different sugar samples on the chip, the IgG antibody has the highest specificity for the synthesized trisaccharide 23*. Based on the results, the mice with the best immune response were selected for a booster immunization on the 28th day, and their spleen cells were harvested for hybridoma cell hybridization after 7 days.

Example 26:

Preparation of monoclonal antibodies

The hybridoma cells were prepared according to the general method (reference Broecker, F., Anish, C. & Seeberger, PH Generation of monoclonal antibodies against defined oligosaccharide antigens. Methods Mol. Biol. 1331, 57-80 (2015)). Six-week-old female Balb/c mice were used to prepare ascites. The mice were intraperitoneally injected with sterile 0.5 mL liquid paraffin, and one week later, 0.5 mL PBS ^{containing 0.5×10 6} to 1×10 ^{6 hybridoma cells was intraperitoneally injected.} About 9 days later, the mice were sacrificed and the ascites was collected. Centrifuge at 3000 rpm for 15 minutes to remove fat, and freeze the supernatant at -20°C. Use caprylic acid-ammonium sulfate precipitation to obtain purified ascites. As shown in Figure 11, the purified ascites IgG antibody has a high purity. The band at about 70 kDa can correspond to the antibody heavy chain, and the band at about 29 kDa can correspond to the antibody light chain.

Example 27:

Detection of Serum and Monoclonal Antibodies Recognizing Inactivated Staphylococcus aureus

Staphylococcus aureus (ATCC 49525) was cultured in Soybean-Casein Digest medium at 37°C overnight. Escherichia coli (BL21) was cultured in LB medium at 37°C overnight. Use 0.4% paraformaldehyde to inactivate for 48 hours. Bacteria are labeled with 0.1mg/mL FITC, diluted serum (serum collected 7 days after the second immunization of mice) or monoclonal antibody combined with FITC-labeled bacteria, use diluted goat anti-mouse IgG-Alexa Fluor The 635 secondary antibody was incubated. Laser confocal detection combination.

As shown in Figures 12 and 13, the cells of Staphylococcus aureus and Escherichia coli are both FITC-labeled and show green fluorescence. The binding of antibodies and cells mainly detects the red fluorescence displayed by IgG-Alexa Fluor 635. Both serum and monoclonal antibodies are It shows good recognition and binding ability against Staphylococcus aureus, but basically no binding to Escherichia coli, showing that the glycoprotein conjugate has a good effect of stimulating immune response and the specificity of related antibodies.

Claims (11)

1. The application of the trisaccharide repeating unit oligosaccharide chain in the preparation of Staphylococcus aureus vaccine is characterized in that the trisaccharide repeating unit oligosaccharide chain is assembled with amino linking arms, and the chemical structural formula can be expressed as: U ₁ -U ₂ -U ₃ -OL-NH ₂ , where L represents a linking arm, and is a chain structure of 2-40 carbon atoms without or containing heteroatoms, the heteroatoms being selected from O, N and S; U ₁ , U ₂ and U _{3 is} as follows:

   
2. The application according to claim 1, wherein when the chain length of the connecting arm L is 4-8 carbon atoms, the chain contains 1-3 heteroatoms; when the chain length of the connecting arm is 9-14 For the number of carbon atoms, the chain contains 1-6 heteroatoms.
3. The application according to claim 1, wherein the chain structure of the connecting arm L contains a three-, four-, five- or six-membered saturated carbocyclic ring; or a five-membered unsaturated carbocyclic ring (non-aromatic ring); Or it contains a four-, five- or six-membered saturated oxygen heterocycle; or it contains a four-, five- or six-membered saturated nitrogen heterocycle; or it contains a six-membered aromatic carbocyclic ring.
4. The application according to claim 1, wherein the chain structure of the connecting arm L contains an amide bond and/or a urea group.
5. The application according to claim 1, wherein the linking arm L contains one or more substituent groups selected from: -F, -Cl, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -C ₅ H ₉ , -C ₆ H ₁₃ , -OC ₂ H ₅ , -OCH ₃ , -CH ₂ F, -CF ₃ , -NHC(O)CH ₃ , -CHF ₂ , -C( O) -NH ₂ , -SCH ₃ , -N(CH ₃ ) ₂ , -SC ₂ H ₅ and -N(C ₂ H ₅ ) ₂ .
6. A glycoprotein conjugate for preparing a Staphylococcus aureus vaccine, characterized in that the chemical structural formula of the glycoprotein conjugate is: [U ₁ -U ₂ -U ₃ -OL-NH-(C= O)-S-(C=O)-NH] _n -CP; where n is 1-20; L represents a connecting arm; S represents an extended arm, which is 2-40 carbon atoms without or containing heteroatoms Chain structure; CP stands for carrier protein; U ₁ , U ₂ and U ₃ are as follows:

   
7. The glycoprotein conjugate according to claim 6, wherein the glycoprotein conjugate uses the linking arm L and the extension arm S in the trisaccharide repeating unit oligosaccharide chain of claim 1. It is connected by an amide bond, and the extension arm S is connected with the carrier protein by an amide bond.
8. An oligosaccharide chip for preparing Staphylococcus aureus vaccine, characterized in that the structural formula of the oligosaccharide chip is: [U ₁ -U ₂ -U ₃ -OL-NH-(C=O)-S- (C=O)-NH] _n -chip; where n is 1-20; L represents a connecting arm; S represents an extended arm, which is a chain structure of 2-40 carbon atoms without or containing heteroatoms; U ₁ , U ₂ and U ₃ are as follows:

   
9. The oligosaccharide chip according to claim 8, wherein the oligosaccharide chip uses the linking arm L and the extension arm S in the trisaccharide repeating unit oligosaccharide chain of claim 1 to be connected by an amide bond , The extension arm S and the chip are connected by an amide bond.
10. The use of the glycoprotein conjugate of any one of claims 6-7 in the development and preparation of a Staphylococcus aureus vaccine.
11. The application of the oligosaccharide chip according to any one of claims 8-9 in the development and preparation of a Staphylococcus aureus vaccine.

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