Chemical synthesis method of b-type haemophilus influenzae capsular polysaccharide derivative

Abstract

The invention discloses a chemical synthesis method of a type b haemophilus influenzae capsular polysaccharide derivative, and belongs to the field of carbohydrate chemistry. The invention provides a modular synthesis strategy based on a phosphoramidite method. According to the strategy, a dimer receptor with a connecting arm at the O3 site of ribose at a non-reducing end is used as a main starting compound, and a phosphoramidite monomer donor and a dimer donor are respectively used as extension units of an odd-numbered polymer synthesis route and an even-numbered polymer synthesis route. Through optimization of a coupling catalysis strategy and optimization of a protecting group removal method, chemical synthesis of 1-12 repetitive units is efficiently completed. According to the synthesis method, the raw materials are cheap and easy to obtain, the preparation method is simple and easy to repeat, the yield of each step is relatively good, and the method has a very good application prospect in vaccine development of the haemophilus influenzae type b.

Description

Technical Field

[0001] The invention relates to a chemical synthesis method of a capsular polysaccharide derivative of Haemophilus influenzae type b, belonging to the technical field of sugar chemistry. Background Art

[0002] Haemophilus influenzae (Hi) is a Gram-negative bacterium that parasitizes the human nasopharynx and can be divided into two forms: non-capsulated (NiHi) and encapsulated. The encapsulated type includes six serotypes, and type b is the most important clinical subtype. Hib mainly infects infants and young children under the age of five and can cause serious invasive diseases such as meningitis, pneumonia, and sepsis (Ulanova, Clinical Infectious Diseases, 2021, 73(2): e380-e382). Since the introduction of Hib conjugate vaccines, the incidence of invasive Hib disease has dropped significantly worldwide. However, vaccine coverage is not balanced worldwide, and countries with low coverage still face a significant burden of Hib disease, and the disease burden of Hib is unevenly distributed geographically. Even in countries with high vaccination coverage, sporadic outbreaks of Hib infection occur from time to time. At the same time, the problem of Hib resistance is becoming increasingly serious. Multidrug-resistant or even extensively drug-resistant Hib strains have been reported many times around the world, limiting available treatment options (Slack et al., Clinical Microbiology Reviews, 2021, 34(3): e00028-21). In addition, the clinical manifestations of invasive Hib disease have also changed. In countries with high vaccination coverage, cases of Hib-related meningitis and epiglottitis have decreased dramatically, while bacteremia and pneumonia have become more common clinical manifestations (Gilsdorf, J Infect Dis, 2021, 224: S321-S330). This shows that it is crucial to continue to pay attention to the optimization of Hib disease and its related vaccines.

[0003] The core component of Hib vaccine is the capsular polysaccharide (CPS) on the surface of the bacteria, which is composed of polyribosyl-ribitol-phosphate (PRP) repeating units and is its most important virulence factor, enabling it to evade the host's immune system. PRP is an important antigenic epitope. In 1970, researchers first developed a polysaccharide vaccine based on PRP, but this vaccine had limited effect on children under 2 years old and was difficult to stimulate immune memory. In 1987, researchers combined PRP with diphtheria toxoid (DT) protein, thereby improving the immunogenicity of the vaccine, especially for infants and young children. This marked the birth of the first generation of Hib conjugate vaccines (Khatuntseva et al., Russ J Bioorg Chem, 2021, 47 (1): 26-52). With the advancement of technology, a variety of dosage forms and formulations of Hib vaccines have emerged, including monovalent vaccines and combined vaccines (such as diphtheria, tetanus, and pertussis triple vaccines, etc.). Currently, these vaccines are widely sold in the global market.

[0004] The production of traditional Haemophilus influenzae type b capsular polysaccharide-protein conjugate vaccine first involves culturing Haemophilus influenzae type b, extracting its capsular polysaccharide by crushing the bacteria after the culture matures, and then covalently coupling the capsular polysaccharide to the carrier protein by chemical methods to make a glycoprotein conjugate vaccine. This type of conjugate vaccine has several shortcomings: (1) The capsular polysaccharide obtained by crushing the bacteria is not a single substance, and its capsular polysaccharide chains are of different lengths; (2) In the process of extracting capsular polysaccharides, it is inevitable that broken bacterial fragments will be mixed in; (3) The effect of each batch of conjugate vaccine may be different, with microscopic heterogeneity, and the product quality is difficult to control. The preparation of bacterial surface sugar chains with clear structures and uniformity by chemical synthesis is crucial for conducting research on their biological activity.

[0005] Currently, several research teams have obtained PRP oligosaccharide fragments through chemical methods. The most noteworthy of these is that in 2004, the Verez-Bencomo and Roy research teams developed a scheme based on the polycondensation reaction of donor ribitol (containing a free hydroxyl group on O5) and acceptor ribose (containing H-phosphonate on O3) to synthesize PRP-related compounds. In this scheme, the non-reducing end of the donor is connected to the 2-(2-azidoethoxy)ethanol linker arm through a phosphodiester bond, and the donor and acceptor complete the polycondensation under the catalysis of pivaloyl chloride to form an oligomer mixture containing an average of 6-8 repeating units. The optimized method for synthesizing PRP oligomers from monomers in one pot has been expanded to commercial production, and the resulting oligomers have been applied to Vaccine (HeberBiotec SA), which also became the world's first chemically synthesized carbohydrate vaccine used in humans (Verez-Bencomo et al., Science, 2004, 305 (5683): ​​522-525). In 2018, Seeberger et al. proposed a new method for synthesizing PRP-related compounds, using 1,2,3,5-tetra-O-acetyl-β-D-ribofuranosyl as the starting material, first constructing the ribitol unit structure, using the structure containing free hydroxyl groups on the reducing end ribitol O5 as the receptor, connecting the donor containing H-phosphonate on the non-reducing end ribose O3 to extend the sugar chain, and finally connecting the five-carbon amino linker arm to the reducing end ribitol O5 through a phosphodiester bond to terminate the extension, and finally obtaining tetramers, hexamers, octamers and decamers of PRP-related derivatives (Baek et al., Chem. Sci., 2018, 9 (5): 1279-1288). However, this method has harsh conditions, such as some temperature conditions need to be controlled at a low temperature of -78°C, and the yield is average. Summary of the invention

[0006] [Technical issues]

[0007] The invention provides a chemical synthesis method of a capsular polysaccharide derivative of Haemophilus influenzae type b or an intermediate thereof with mild reaction conditions and equivalent or even better yield.

[0008] [Technical solution]

[0009] The present invention provides a chemical synthesis method of an intermediate A, the structure of which is shown below:

[0010]

[0011] The chemical synthesis method comprises the following steps:

[0012]

[0013] (a) using compound A-1 and compound A-2 as substrates, and carrying out glycosylation reaction under the action of ytterbium trifluoromethanesulfonate to obtain compound A-3;

[0014] (b) Compound A-3 is treated in an alkaline solution to obtain compound A-4;

[0015] (c) Compound A-4 and dibutyltin oxide are mixed and reacted in toluene for a period of time, and then a protecting agent is added to protect the 2- and 5-hydroxyl groups to obtain an intermediate compound A;

[0016] Among them, R 2 , R 3 , R 2 '、R3 'are independently selected from: acetyl (Ac), benzoyl (Bz), pivaloyl (Piv), chloroacetyl (ClAc), levulinyl (Lev), allylcarbonyl (Alloc), benzyl (Bn), p-methoxybenzyl (pMBn), allyl (All);

[0017] R 4 , R 5 , R 6 Each of the following is independently selected from: benzyl (Bn), p-methoxybenzyl (pMBn), allyl (All), acetyl (Ac), benzoyl (Bz), pivaloyl (Piv), chloroacetyl (ClAc), levulinyl (Lev), allylcarbonyl (Alloc);

[0018] R 7 Selected from: 4-monomethoxytrityl (MMTr), allyl (All), trityl (Tr), 4,4'-dimethoxytrityl (DMTr), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triethylsilyl (TES).

[0019] The present invention provides a chemical synthesis method of an intermediate B, the structure of which is shown below:

[0020]

[0021] The chemical synthesis method comprises the following steps:

[0022]

[0023] According to the above (a)-(c) to obtain intermediate compound A;

[0024] (d) Compound A is mixed with a protecting agent to protect the 3-hydroxyl group to obtain Compound B-1;

[0025] (e) Removal of R from Compound B-1 7 , to obtain compound B;

[0026] Among them, R 2 , R 3 , R 2 '、R 3 '、R 4 , R 5 , R 6 , R 7 Same definition as above.

[0027] The present invention provides a chemical synthesis method of an intermediate compound III, the structure of which is shown below:

[0028]

[0029] The chemical synthesis method comprises the following steps:

[0030]

[0031] According to the above (a)-(c) to obtain intermediate compound A;

[0032] (f) Compound A is mixed with a phosphitylation agent to react to obtain Compound III;

[0033] Among them, R 2 , R 3 , R 2 '、R 3 '、R 4 , R 5 , R 6 , R 7 The definition of is the same as above; R 1 Selected from acetonitrile (CE), allyl (All), benzyl (Bn); R a , R b are independently selected from C 1-4 alkyl.

[0034] In one embodiment of the present invention, the glycosidation reaction comprises:

[0035] The disaccharide fragment was synthesized by glycosylation reaction using 5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol and tetraacetylribose.

[0036] In one embodiment of the present invention, in the glycosylation reaction, the reaction is carried out in a solvent, and the solvent is dichloromethane or N,N-dimethylformamide (DMF). The reaction concentration in the glycosylation reaction is 0.01-0.1 mmol / mL.

[0037] In one embodiment of the present invention, in the glycosylation reaction, the reaction temperature may be between 0°C and 60°C, and the reaction time may be between 3 hours and 72 hours.

[0038] In one embodiment of the present invention, the glycosidation reaction further comprises using Molecular sieve or Molecular sieve. This molecular sieve is used to remove water.

[0039] In one embodiment of the present invention, if R 2 , R 3 , R 2 '、R 3' are all acetyl groups, then step (b) involves an acetylation deprotection process.

[0040] In one embodiment of the present invention, the alkaline solution is selected from: sodium methoxide / methanol, sodium hydroxide / water, potassium hydroxide / methanol, barium hydroxide / methanol, ammonia water, dimethylammonia / methanol, potassium cyanide / methanol, etc.

[0041] In one embodiment of the present invention, in step (c), the hydroxyl groups at positions 2 and 5 of the ribose group are selectively protected, and the corresponding process is:

[0042] The 2 and 5 hydroxyl groups are benzyl protected, while the 3 hydroxyl group is retained unchanged: the method used can be to add dibutyltin oxide (Bu 2 SnO), sodium hydride, additives, and protective reagents are reacted to obtain the corresponding product. Among them, the additive is tetrabutylammonium iodide (Bu 4 NI) and / or tetrabutylammonium bromide (Bu 4 NBr). The protecting agent is benzyl bromide.

[0043] In one embodiment of the present invention, in step (c), the reaction concentration is 0.01-0.1 mmol / mL, the reaction temperature is between room temperature and 110° C., preferably 100-110° C. The reaction time is between 1 hour and 8 hours.

[0044] In one embodiment of the present invention, in step (e), the removal reaction temperature can be between room temperature and 80° C., preferably 70-80° C. The reaction time is between 1 hour and 5 hours.

[0045] In one embodiment of the present invention, if R 7 is an allyl group, then step (e) is to deprotect the allyl group at position 5 of ribitol.

[0046] In one embodiment of the present invention, in step (e), R 7 The reaction system is selected from: tris(triphenylphosphine)rhodium chloride ((Ph 3 P) 3 RhCl) / 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) / ethanol / toluene / water / acetic acid, tetrakis(triphenylphosphine)palladium (Pd(Ph 3 ) 4 ) / 1,3-dimethylbarbituric acid / methanol, palladium chloride (PdCl 2 ) / sodium acetate / acetic acid / ethyl acetate / water, N-bromosuccinimide (NBS) / carbon tetrachloride / aqueous sodium hydroxide solution, palladium on carbon (Pd / C) / p-toluenesulfonic acid (TsOH) / methanol / water, pentamethylcyclopentadienyltri(acetonitrile)ruthenium(II) hexafluorophosphate ([CpRu(CH3 CN) 3 ]PF 6 ) / quinaldic acid / methanol, palladium chloride (PdCl 2 ) / copper chloride / N,N-dimethylformamide, palladium chloride (PdCl 2 ) / dichloromethane / water.

[0047] In one embodiment of the present invention, the 5-position of ribitol is protected by 4-monomethoxytrityl:

[0048] The method for protecting 4-monomethoxytrityl may be dichloromethane / pyridine / 4-monomethoxytrityl chloride, and the catalyst for promoting the reaction may be 4-dimethylaminopyridine (DMAP) or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0049] In one embodiment of the present invention, the ribose 3-position levulinyl group is protected:

[0050] The method for protecting the levulinic acid group can be dichloromethane / 4-dimethylaminopyridine / levulinic acid, and the condensing agent for promoting the reaction of the carboxyl and hydroxyl groups can be 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC).

[0051] In one embodiment of the present invention, step (f) is phosphoramidite protection of position 3 of the ribose group, and the phosphitylation agent used for phosphoramidite protection can be 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (CEP-CL), bis(diisopropylamino)(2-cyanoethoxy)phosphine.

[0052] In one embodiment of the present invention, the method used in step (f) can be N,N-diisopropylethylamine / 1-methylimidazole / dichloromethane / phosphitylation agent system, and the reaction temperature can be between 0°C and room temperature.

[0053] The present invention provides a chemical synthesis method of an intermediate compound IV, the structure of which is shown below:

[0054]

[0055] The method comprises:

[0056]

[0057] S1, using the above compound III and compound B as substrates, carrying out a phosphoramidite coupling reaction under the action of a catalyst, followed by oxidation to obtain an intermediate C;

[0058] S2, removing the 3-hydroxy protecting group from intermediate C to obtain compound D;

[0059] S3, compound D is mixed with a phosphitylation agent to react to obtain compound IV;

[0060] Among them, R 2 , R 3 , R 3 '、R 4 , R 5 , R 6 , R 7 , R 1 , R a , R b Same definition as above.

[0061] The present invention also provides a method for synthesizing a capsular polysaccharide derivative of Haemophilus influenzae type b assembled with a connecting arm, wherein the structure of the capsular polysaccharide derivative is shown in general formula I:

[0062]

[0063] Wherein, n is any integer from 0 to 11;

[0064] R is a hydrogen ion (H + ), sodium ion (Na + ), ammonium ion (NH 4 + );

[0065] Linker is -L-NH 2 , wherein L represents a connecting arm; the connecting arm L is diethylene glycol, or a chain structure with 2 to 40 carbon atoms containing 0 to 6 heteroatoms, a substituted or unsubstituted three- to six-membered ring structure, an amide bond, or a urea group;

[0066] The method comprises the following steps:

[0067] (1) The intermediate compound III and the linker reagent are mixed to react to obtain compound E;

[0068] (2) Compound E is reacted in a mixed system of boron trifluoride ether complex / methanol / organic solvent to obtain Compound II;

[0069] (3) Compound II undergoes a phosphoramidite coupling reaction with the intermediate compound III to obtain compound F;

[0070] (4) Compound F is reacted in a mixed system of boron trifluoride ether complex / methanol / organic solvent to obtain compound V;

[0071] (5) Using the obtained product as a substrate, repeating steps (3)-(4) to obtain compound VI;

[0072] (6) reacting the obtained compound VI in a mixed system of boron trifluoride ether complex / methanol / organic solvent to obtain compound VII;

[0073] (7) Compound VII and intermediate compound III or intermediate compound IV undergo phosphoramidite coupling reaction in the presence of a catalyst, followed by oxidation to obtain compound G or compound H, respectively;

[0074] (8) Removing the protecting agent from compound E, compound G or compound H, and then reducing the azide group to obtain the target compound of formula I.

[0075]

[0076] Among them, R 2 , R 3 , R 3 '、R 4 , R 5 , R 6 , R 7 , R 1 , R a , R b , n is defined as above, and n' is 0-10.

[0077] In one embodiment of the present invention, in the phosphoramidite coupling reaction, the catalyst can be 1-phenyl-1H-imidazol-3-ium trifluoromethanesulfonate (N-PhIMT), 1H-tetrazole (TetH), 5-benzylthiotetrazolyl (BTT), 5-ethylthiotetrazolyl (ETT), 4,5-dicyanoimidazole (DCI), 2-bromo-4,5-dicyanoimidazole (BrDCI), trifluoroacetic acid (TFA), 2,4-dinitrobenzoic acid (DNBA), trichloroacetic acid (TCA), pyridinium trifluoroacetate (PyTFA), imidazolium trifluoromethanesulfonate (IMT), saccharin 1-methylimidazole (SMI), imidazolium perchlorate (IMP), 1H-benzimidazol-3-ium trifluoromethanesulfonate (BIT), N-methylbenzimidazole trifluoroate (N-MeBIT) Any one or more. 1-Phenyl-1H-imidazol-3-ium trifluoromethanesulfonate (N-PhIMT) is preferred.

[0078] In one embodiment of the present invention, in the phosphoramidite coupling reaction, the compound having a hydroxyl structure is called an acceptor, and the compound having a phosphoramidite structure is called a donor.

[0079] In one embodiment of the present invention, in the phosphoramidite coupling reaction, the amount of catalyst relative to the acceptor can be between 0.1eq and 2eq. (eq refers to molar equivalent)

[0080] In one embodiment of the present invention, in the phosphoramidite coupling reaction, the amount of donor relative to acceptor can be between 1 eq and 5 eq. (eq refers to molar equivalent)

[0081] In one embodiment of the present invention, in the phosphoramidite coupling reaction, the reaction is carried out in an acetonitrile medium, the reaction concentration is 0.01-0.1 mmol / mL, the reaction temperature is 0° C. to 25° C., and the reaction time is 1 minute to 3 hours.

[0082] In one embodiment of the present invention, in the phosphoramidite coupling reaction, the reaction system further includes adding a molecular sieve for removing water, which can be Molecular sieve or Molecular sieve.

[0083] In one embodiment of the present invention, in the phosphoramidite coupling reaction, after the phosphoramidite coupling reaction is completed, the trivalent phosphorus in its phosphate group must be immediately oxidized to pentavalent phosphorus, and the oxidant used for oxidation can be any one or more of iodine (I2) / water / pyridine, (+)-8,8-dichlorocamphorsulfonazine (DCSO), tert-butyl hydroperoxide (TBHP) / toluene.

[0084] In one embodiment of the present invention, the oxidant is directly added into the reaction system, and the amount of the oxidant is 1 eq to 10 eq.

[0085] In one embodiment of the present invention, the oxidation time is 1 minute to 60 minutes and the temperature is 0°C to 25°C.

[0086] In one embodiment of the present invention, step (2), (4) or (6) is respectively R at the 5th position of the reducing end ribitol in compound E, compound F or formula VI. 7 The process of deprotection of protecting groups.

[0087] In one embodiment of the present invention, in step (2), (4) or (6), a reaction system of trifluoroacetic acid (TFA) / trifluoroacetic anhydride (TFAA) / organic solvent can be used instead of boron trifluoride ether complex (BF 3 ·Et 2 O) / methanol / organic solvent.

[0088] In one embodiment of the present invention, boron trifluoride etherate (BF 3 ·E t2O) / methanol / organic solvent, the organic solvent can be dichloromethane, chloroform, dichloroethane, dichloropropane, tetrahydrofuran, ethyl acetate.

[0089] In one embodiment of the present invention, boron trifluoride etherate (BF 3 ·Et 2 O) / methanol / organic solvent, relative to compound E, compound F or compound VI, the amount of boron trifluoride ether complex can be between 1eq and 30eq, and the amount of methanol can be between 2eq and 60eq. (eq refers to molar equivalent)

[0090] In one embodiment of the present invention, the reaction temperature may be between 0°C and room temperature, and the reaction time may be between 30 minutes and 12 hours;

[0091] In one embodiment of the present invention, in step (7), when n' in the compound of formula VII is an even number of 1, 3, 5, 7, 9, etc., a phosphoramidite coupling reaction is carried out with the compound of formula IV; when n' in the compound of formula VII is an odd number of 0, 2, 4, 6, 8, 10, etc., a phosphoramidite coupling reaction is carried out with the compound of formula III.

[0092] In one embodiment of the present invention, step (8) is a full deprotection process, specifically comprising:

[0093] The reaction is carried out by introducing hydrogen under catalytic conditions, the hydrogen pressure can be between 0.1 bar and 0.5 bar, the catalyst used for catalytic hydrogenation can be 10% palladium carbon or palladium hydroxide, etc., the solvent used for the reaction can be a water / methanol / dichloromethane / acetic acid mixture, a water / methanol / ethyl acetate / acetic acid mixture, a water / tert-butanol / dichloromethane mixture, a water / tert-butanol / ethyl acetate mixture, a water / tert-butanol / tetrahydrofuran mixture, etc., and the reaction temperature can be between 0°C and 40°C.

[0094] When the hydroxyl protecting group of the phosphate group is acetonitrile, it is necessary to first remove the acetonitrile by using a strong alkaline solvent such as ammonia water, sodium hydroxide aqueous solution, triethylamine aqueous solution, and then completely remove the protecting group by catalytic hydrogenation;

[0095] When the hydroxyl protecting group of the phosphate group is allyl or benzyl, direct catalytic hydrogenation is used to completely remove the protecting group;

[0096] After full deprotection, an ion exchange column is used for ion replacement. The resin filler can be DOWEX(R)50WX4 or DOWEX(R)50WX8, and the ion type can be sodium ion (Na + ) or hydrogen ion (H + ).

[0097] Although scientists around the world have partially studied the relationship between PRP fragments and immunogenicity through chemical synthesis methods, due to the limitations of traditional synthesis methods such as high cost, complex procedures, and difficulty in separation and purification, a comprehensive oligosaccharide library has not yet been constructed to deeply study the immune epitopes of Hib, nor is there a universal method for efficiently constructing a PRP fragment library. In addition, in addition to the length of the oligosaccharide component, the type of protein carrier and the type of adjuvant are also key factors affecting the immunogenicity and induced antibody protection characteristics of conjugate vaccines based on PRP-related oligosaccharide ligands. Therefore, further exploring the optimal immune epitopes of Hib capsular polysaccharides and developing methods that can efficiently prepare structurally uniform PRP oligosaccharide compounds are of vital significance for the development of semi-synthetic or fully synthetic Hib vaccines. This study not only helps to clarify the immune mechanism of Hib vaccines, but also has important guiding significance for guiding the rational design of modern and efficient sugar conjugate vaccines.

[0098] Based on the synthetic method, the present invention obtains a library of synthetic oligosaccharides from one repeating unit fragment (i.e., a disaccharide) to 12 repeating units, as shown below:

[0099]

[0100] The present invention also provides an oligosaccharide chip for preparing a vaccine of Haemophilus influenzae type b, wherein the capsular polysaccharide derivative of Haemophilus influenzae type b assembled with a connecting arm is fixed on the surface of the chip via an amino connecting arm. The relationship between the oligosaccharide fragments and antigenicity in the oligosaccharide chip is detected by screening antibodies in antiserum, and the oligosaccharide chip can be applied to the analysis of synthetic oligosaccharide antigen epitopes.

[0101] The present invention also provides a glycoprotein conjugate for preparing a vaccine for preventing and treating diseases caused by Haemophilus influenzae type b infection, and the structure of the glycoprotein conjugate is shown below:

[0102]

[0103] Wherein, the Hib oligosaccharide antigen is a capsular polysaccharide derivative of the above-mentioned Haemophilus influenzae type b assembled with a connecting arm, or is selected from one of the above-mentioned compounds 1* to 12*;

[0104] The linker is a structural part obtained by directly or indirectly connecting the oligosaccharide antigen and the carrier protein, and is not immunogenic;

[0105] The chemical group in the linker that reacts with the amino linker arm of the Hib oligosaccharide antigen and the amino group on the surface of the carrier protein can be isothiocyanate, isocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate, NHS ester, imidate, epoxide, fluorophenyl ester;

[0106] The chemical group in the linker that reacts with the thiol group assembled on the surface of the carrier protein can be maleimide, iodoacetyl group, bromoacetyl group, chloroacetyl group, pyridyldithiol, vinyl sulfone;

[0107] m is the number of Hib oligosaccharide antigens coupled to the carrier protein, which is any number between 1 and 30;

[0108] The carrier protein is selected from the group consisting of bovine serum albumin (BSA), human serum albumin (HSA), tetanus toxoid protein (TT), diphtheria toxoid (DT), diphtheria toxin avirulent variant (CRM 197 ), Haemophilus influenzae protein D (PD) and group B meningococcal outer membrane protein complex (OMPC), etc.

[0109] The diseases caused by infection with Haemophilus influenzae type b include one or more of meningitis, pneumonia, epiglottitis, sepsis, cellulitis, pericarditis and myelitis.

[0110] According to the present invention, preferably, the oligosaccharide fragments with prominent antigenicity are compounds 4, 6, 8, 11, and 12;

[0111] Preferably according to the present invention, the chemical group in the linker that reacts with the amino linker arm of the Hib oligosaccharide antigen is NHS ester, and the sulfhydryl reactive group assembled with the surface of the carrier protein is maleimide.

[0112] According to the present invention, the carrier protein is preferably selected from tetanus toxoid protein (TT) and diphtheria toxin non-toxic variant (CRM 197 ).

[0113] Optimally, the structure of the glycoprotein conjugate is as shown in Formula VI:

[0114]

[0115] Wherein, n is any one of 3, 5, 7, 11, and 12, and m is any number from 1 to 30.

[0116] In one embodiment of the present invention, the specific steps of the preparation method of the glycoprotein conjugate are as follows:

[0117] 1) Activation of Hib oligosaccharide antigen: Dissolve Hib oligosaccharide antigen (3 mg) in 0.3 mL dimethyl sulfoxide (DMSO) solution, add 3-maleimidopropionic acid N-hydroxysuccinimide ester (SMP) dissolved in 0.2 mL dimethyl sulfoxide under stirring, the molar equivalent ratio of SMP to Hib oligosaccharide antigen is between 3:1 and 8:1, the reaction temperature is 0°C to room temperature, the reaction time is 6 hours to 12 hours, purify with Sephadex LH-20 column chromatography (H2O), and obtain activated Hib oligosaccharide antigen after reduced pressure distillation;

[0118] 2) Thiolation of carrier protein: Dissolve the carrier protein (1 mg) in 1 mL PBS buffer (pH 8, containing 1.86 mg / mL EDTA), add dithiobis(succinimidyl propionate) (DSP) dissolved in 50 μL dimethyl sulfoxide under argon protection, the molar equivalent ratio of DSP to carrier protein is between 4:1 and 10:1, the reaction temperature is from 0°C to room temperature, and the reaction time is from 6 hours to 12 hours. After overnight reaction, place it in an ice bath, add dithiothreitol (DTT) at a concentration of 0.025 mmol / mL under argon protection, and react at 0°C for 3 hours. Then dilute the mixture with PBS buffer (pH 7.2, containing 1.86 mg / mL EDTA), and centrifuge it 3 times on a 50 kDa ultrafiltration membrane with PBS buffer (PH 7.2, containing 1.86 mg / mL EDTA). The solution is stored at 0°C;

[0119] 3) Coupling of Hib oligosaccharide antigen with carrier protein: The activated Hib oligosaccharide antigen was dissolved in 0.5 mL PBS buffer (PH 7.2), and the thiol carrier protein dissolved in 2 mL PBS buffer (PH 7.2, containing 1.86 mg / mL EDTA) was added under stirring. After reacting at 4-8°C for 6 hours, 0.1 mg N-ethylmaleimide was added to terminate the reaction. The mixture was then diluted with PBS buffer (PH 7.2), and centrifuged three times with PBS buffer (PH 7.2) on a 50 kDa ultrafiltration membrane to obtain a glycoprotein conjugate.

[0120] Preferably according to the present invention, the molar equivalent ratio of SMP to Hib oligosaccharide antigen in step (1) is 6:1, the reaction temperature is room temperature, the reaction time is 10 hours, and the stirring speed should not be too fast.

[0121] Preferably according to the present invention, the molar equivalent ratio of DSP to carrier protein in step (2) is 8:1, the reaction temperature is room temperature, and the reaction time is 8 hours.

[0122] Beneficial effects:

[0123] The present invention proposes a modular synthesis strategy based on the phosphoramidite method to make up for the deficiency that a comprehensive oligosaccharide library has not yet been constructed for the study of Hib immune epitopes. The strategy uses a dimer with a connecting arm at the O3 position of the non-reducing end ribose as the main starting compound, and uses a phosphoramidite monomer donor and a dimer donor as extension units for the odd-numbered polymer and even-numbered polymer synthesis routes, respectively. The present invention successfully solves two key problems: one is to achieve efficient catalysis during the sugar chain extension process, and the other is to ensure that the protecting group at the fifth position of the ribitol at the reducing end of the sugar chain can be completely detached. Through this modular synthesis strategy, the synthesis of 1-12PRP is efficiently completed. At the same time, the present invention successfully synthesizes the target object with mild and simple operation and good yield effect.

[0124] The present invention fixes 1-12 repeating fragments on the chip surface through amino connecting arms, and conducts in-depth analysis on the relationship between oligosaccharide fragments and antigenicity on the oligosaccharide chip through antibody screening in antiserum. The results reveal that oligomer fragments containing 4, 6, 8, 11 and 12 repeating units show significant antigenicity. In addition, the present invention also conjugates the oligosaccharide fragments with carrier proteins through amino connecting arms to prepare an oligosaccharide protein conjugate experimental vaccine. Through animal immunization experiments, we found that the fragment containing 4 repeating units is the most critical immune epitope. Further studies have shown that tetanus toxoid (TT) is the best carrier protein and can induce higher antibody titers.

[0125] The present invention proposes a chemical synthesis method with simple steps, time-saving, labor-saving and low cost, which is used to assemble a capsular polysaccharide derivative of Haemophilus influenzae type b (Hib) with a connecting arm. The method can prepare a compound monomer with high purity and consistent structure, so as to deeply explore its immune epitope and structure-activity relationship at the molecular level. In addition, by determining the minimum immune epitope, the complexity and cost of chemical synthesis are significantly reduced. At the same time, the present invention also screened the immune effects of different carrier proteins, clarified that the 4PRP-TT glycoprotein conjugate is an excellent vaccine candidate, and successfully developed a Hib semi-synthetic oligosaccharide conjugate vaccine with uniform structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 This is the synthetic route of phosphoramidite monomer donor A.

[0127] Figure 2 This is the synthetic route of phosphoramidite dimer donor B.

[0128] Figure 3 Synthetic route for assembled monomers and dimers with tethers.

[0129] Figure 4 A modular synthetic route for odd- and even-numbered sugar repeat units.

[0130] Figure 5 A roadmap for total de-insurance.

[0131] Figure 6 Comparison of results of evaluating the antigenicity of synthetic Hib CPS glycan fragments using glycan microarray. (A) Template for printing microarray slides. Synthetic oligosaccharides were spotted in duplicate at concentrations of 1 mM and 5 mM, TT protein at 0.01 mg / ml, and other samples at 0.05 mg / ml; (B) Representative array scans of immune rabbit antisera. Hib CPS antiserum (left, dilution 1:50) and CPS-TT antiserum (right, dilution 1:100); (C) Quantification of glycan mean fluorescence intensity (MFI) values ​​of oligosaccharide fragments bound to the two antisera. Error bars represent the SEM of two spots from two independent arrays.

[0132] Figure 7 The results of ELISA for the IgG antibody levels induced by five oligosaccharide protein conjugates after 35 days of immunization are compared. P / N is the ratio of the absorbance of the rabbit serum in the experimental group to that in the negative control group. The data are the mean ± SD of n = 4 rabbits in each group.

[0133] Figure 8 This is a comparison of the results of ELISA detection of IgG antibody levels induced by oligosaccharide protein conjugates 4PRP-TT and 4PRP-CRM197 35 days after immunization. DETAILED DESCRIPTION

[0134] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0135] All commercial reagents used in the experiment were used directly without treatment. The anhydrous solvents used in the reaction were prepared by MBraun MBSPS800 solvent drying system. The solvents used in silica gel column chromatography were all analytically pure and used after vacuum distillation. The silica gel plates used for thin layer chromatography (TLC) were glass-based or aluminum foil-based silica gel plates prepared from 60-F254 silica gel. The TLC colorimetric reagents were sugar colorimetric reagents (90.1% (v / v) 3-methoxyphenol, 2.5% (v / v) sulfuric acid ethanol solution), or ninhydrin colorimetric reagents (1.5% (w / v) ninhydrin and 3% (v / v) acetic acid n-butanol solution), or CAM colorimetric reagents (5% (w / v) ammonium molybdate, 1% (w / v) cerium (II) sulfate and 10% (v / v) sulfuric acid aqueous solution). The silica gel used for normal phase silica gel column chromatography was 200-300 mesh silica gel.

[0136] The yield of each reaction step was calculated separately, and the yield calculation method was: (amount of target product substance / amount of raw material substance)×100%. The structure of the product was identified by nuclear magnetic spectrum, infrared spectrum, optical rotation, and high-resolution mass spectrum, and the purity of the product was analyzed by nuclear magnetic spectrum. The hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum were measured by Bruker Ascend 600 mega nuclear magnetic resonance instrument, or Bruker Ultrashield Plus 400 mega nuclear magnetic resonance instrument at 25°C. The high-resolution mass spectrum was measured by Agilent 6220 electrospray ion source-time of flight mass spectrometer. The infrared spectrum was measured by ThermoFisher Scientific Nicolet iS5 infrared instrument. The optical rotation was measured by Schmidt & Haensch UniPol L1000 fully automatic polarimeter at 589nm, and the unit of concentration (c) was g / 100mL.

[0137] The above-mentioned Hib oligosaccharide antigen synthesis route:

[0138]

[0139] Reaction module A: phosphoramidite coupling reaction;

[0140] Reaction module B: R at position 5 of the reducing end ribitol 7 Deprotection of protecting groups;

[0141] Reaction module C: full deprotection strategy.

[0142] Embodiment 1:

[0143] Synthesis of 2,3,4-tri-O-acetyl-β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (15*);

[0144] The reaction equation is Figure 1 As shown;

[0145] Under nitrogen protection, the raw materials 5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol 14* (20 g, 43.24 mmol) and tetraacetyl ribose 13* (20.6 g, 64.86 mmol) were co-evaporated three times with toluene, and dry dichloromethane (800 mL) and activated Molecular sieves, the resulting reaction solution was stirred at room temperature for 30 minutes. At room temperature, ytterbium trifluoromethanesulfonate (32.2 g, 51.89 mmol) was added, and the reaction solution was then refluxed at 40 ° C for 48 hours. After the reaction was detected by TLC (petroleum ether / ethyl acetate, 2:1), the molecular sieves were removed by diatomaceous earth filtration. The filtrate was washed three times with deionized water, and the aqueous phase was extracted with dichloromethane. After further extraction and washing of the organic phase with saturated sodium bicarbonate solution, the organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 4:1, v / v) to obtain a colorless oily compound 15* (23.4 g, 32.43 mmol) with a yield of 75%. 1 H NMR (400 MHz, CDCl 3 )δ7.41–7.19(m,16H),5.98–5.78(m,1H),5.31(dd,J=6.7,4.8Hz,1H),5.27(q,J=1.7Hz ,1H),5.23–5.21(m,1H),5.15(dd,J=10.4,1.6Hz,1H),4.93(s,1H),4.76–4.54(m,6H), 4.32–4.22(m,2H),4.13–4.02(m,1H),3.96(dt,J=5.6,1.5Hz,2H),3.91(dd,J=10.3,6. 0Hz,1H),3.89–3.77(m,3H),3.72–3.58(m,3H),2.09(s,3H),2.05(s,3H),1.98(s,3H); 13 C NMR (151 MHz, CDCl 3 )δ170.6,169.64,169.58,138.5,138.44,138.41,134.9,128.3,127.99,127.97,127.9,127.6,127.5,116.9,105. 2,78.5,78.3,78.1,78.0,74.7,73.8,72.42,72.38,72.3,71.7,69.8,67.8,64.7,20.7,20.6,20.5; HRMS(ESI)m / z calcd forC40 H 48 NaO 12 :[M+Na] + :743.3043,found:743.3047.

[0146] Comparative Example 1:

[0147] Synthesis of 2,3,4-tri-O-acetyl-β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (15*);

[0148] Under nitrogen protection, the raw materials 5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol 14* (2 g, 4.32 mmol) and tetraacetyl ribose 13* (2.1 g, 6.49 mmol) were co-evaporated three times with toluene, and dry dichloromethane (120 mL) and activated Molecular sieves, the resulting reaction solution was stirred at room temperature for 30 minutes. The reaction system was cooled to 0°C in an ice bath, and then boron trifluoride ether complex (2.7mL, 21.6mmol) was slowly added, and the reactants were stirred at 0°C for 30min, then the ice bath was removed and slowly warmed to room temperature, and the reaction was continued for 10 hours. After the reaction was detected by TLC (petroleum ether / ethyl acetate, 2:1), the reaction was terminated with triethylamine (3mL). The molecular sieves were removed by diatomaceous earth filtration. The filtrate was washed three times with deionized water, and the aqueous phase was extracted with dichloromethane. After further extraction and washing of the organic phase with saturated sodium bicarbonate solution, the organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 4:1, v / v) to obtain a colorless oily compound 15* (1.96g, 2.68mmol) with a yield of 61%.

[0149] Embodiment 2:

[0150] Synthesis of β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (16*);

[0151] The reaction equation is Figure 1 As shown;

[0152] Compound 15* (23.4 g, 32.43 mmol) was dissolved in methanol (320 mL), and sodium methoxide (1.7 g, 32.42 mmol) was added and stirred at room temperature for 5 hours. After the reaction was completed by TLC (petroleum ether / ethyl acetate, 1:3), the reaction solution was neutralized to pH 7 with Dowex amberlite-120 hydrogen ion resin, and the mixed solution was filtered and concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:2, v / v) to obtain colorless oily compound 16* (17.9 g, 30.16 mmol) with a yield of 93%. 1 HNMR (400MHz, CDCl 3 )δ7.30(td,J=7.1,6.1,2.8Hz,15H),5.88(ddt,J=17.3,10.7,5.6Hz,1H),5.31–5.12(m,2 H),4.83(s,1H),4.74–4.54(m,6H),4.36(t,J=5.4Hz,1H),4.01(dt,J=6.1,3.2Hz,1H),3.9 5(q,J=4.7,4.0Hz,3H),3.90(d,J=3.6Hz,2H),3.84(dd,J=5.9,3.6Hz,1H),3.75(dt,J=9.7 ,3.0Hz,2H),3.67(dd,J=10.5,3.3Hz,1H),3.59(ddt,J=9.4,7.1,3.3Hz,3H),2.50(s,3H); 13 C NMR (101 MHz, CDCl 3 )δ138.3,138.0,134.7,128.4,128.1,128.0,127.7,117.0,107.4,84.5,77.3, 77.0,76.7,75.6,73.8,72.3,72.3,72.2,70.6,69.6,67.9,62.5; HRMS(ESI)m / z calcd for C 34 H 42 NaO 9 :[M+Na] + :617.2727,found:617.2728.

[0153] Embodiment 3:

[0154] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (17*);

[0155] The reaction equation is Figure 1 As shown;

[0156] Under nitrogen protection, compound 16* (17.9 g, 30.16 mmol) was co-evaporated three times with toluene, and dry toluene (1200 mL) and activated Molecular sieves, the resulting reaction solution was stirred at room temperature for 30 minutes, and dibutyltin oxide (11.3 g, 45.24 mmol) was added to the reaction solution. The reaction bottle was moved to an oil bath and refluxed at 110°C for 1 hour. After returning to room temperature, sodium hydride (1.8 g, 75.4 mmol), tetrabutylammonium iodide (27.8 g, 75.4 mmol) and benzyl bromide (28.7 mL, 241.3 mmol) were added, and then the temperature was raised to 110°C and refluxed for 2 hours. After the reaction was completed by TLC (petroleum ether / ethyl acetate, 2:1), the reaction solution was cooled to room temperature, the molecular sieve was filtered through diatomaceous earth, and the solvent was removed by concentrated vacuum distillation. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by distillation under reduced pressure. The resulting crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 15:1 to 7:1, v / v) to obtain a colorless oily compound 17* (16.4 g, 21.11 mmol) with a yield of 70%. 1 HNMR (400MHz, CDCl 3 )δ7.30–7.23(m,25H),5.88(dddd,J=15.9,10.7,6.3,4.7Hz,1H),5.25(dt,J=17.2,1.7Hz,1H),5.15(dt, J=10.4,1.6Hz,1H),5.01(s,1H),4.72(d,J=11.7Hz,1H),4.66(t,J=2.1Hz,2H),4.63(d,J=3.5Hz,1H),4. 61–4.55(m,3H),4.52(d,J=11.7Hz,1H),4.49–4.35(m,2H),4.17–4.00(m,2H),3.94(dt,J=5.6,1.6Hz,3H ),3.85(dtd,J=10.9,4.7,2.3Hz,4H),3.70(dq,J=10.5,2.4Hz,2H),3.66–3.56(m,2H),3.54–3.47(m,1H); 13 C NMR (101 MHz, CDCl 3)δ138.6,138.5,138.2,137.2,134.9,128.6,128.3,128.1,128.0,127.9,127.8,127.6,127.5,116.8,1 04.9,83.1,81.8,78.7,78.5,78.1,73.8,73.3,72.6,72.4,72.3,72.1,71.9,70.1,67.5; HRMS(ESI)m / z calcd for C 48 H 54 NaO 9 :[M+Na] + :797.3661,found:797.3674.

[0157] Comparative Example 2:

[0158] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (17*);

[0159] Under nitrogen protection, compound 16* (100 mg, 0.17 mmol) was dissolved in methanol (10 mL), and dibutyltin oxide (63.5 mg, 0.26 mmol) was added to the reaction solution. The reaction flask was moved to an oil bath and refluxed at 65 ° C for 1 hour. After the reaction, it was cooled to room temperature and the solvent was removed by vacuum distillation. Then, N, N-dimethylformamide (10 mL) was used to dissolve the crude product of the first step, and the activated Molecular sieves were added and stirred at room temperature for 30 minutes. Sodium hydride (8 mg, 0.34 mmol), tetrabutylammonium iodide (124.3 mg, 0.34 mmol) and benzyl bromide (81.3 μL, 0.67 mmol) were added in sequence and reacted at room temperature for 8 hours. After the reaction was detected by TLC (petroleum ether / ethyl acetate, 2:1), the reaction solution was cooled to room temperature, the molecular sieves were filtered through diatomaceous earth and concentrated by vacuum distillation to remove the solvent. The reaction solution was diluted with dichloromethane, the organic phase was extracted and washed with saturated sodium bicarbonate solution, the organic phase was treated with anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 15:1 to 7:1, v / v) to obtain a colorless oily compound 17* (56 mg, 0.073 mmol) with a yield of 43%.

[0160] Comparative Example 3:

[0161] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (17*);

[0162] Compound 16* (100 mg, 0.17 mmol) and dibutyltin oxide (63.5 mg, 0.26 mmol) were dissolved in anhydrous toluene (5 mL) and heated to reflux for 6 hours. After the mixture was cooled to room temperature, it was concentrated. The crude product was dissolved in DMF (7 mL), and then cesium fluoride (39.5 mg, 0.26 mmol), tetrabutyl sodium bromide (83.8 mg, 0.26 mmol), and benzyl bromide (81.3 μL, 0.67 mmol) were added, and the reaction solution was heated and stirred at 70 ° C for 12 hours. After the reaction was completed by TLC (petroleum ether / ethyl acetate, 2:1), the reaction solution was cooled to room temperature, the molecular sieve was filtered through diatomaceous earth, and the solvent was removed by concentrated vacuum distillation. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by distillation under reduced pressure. The resulting crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 15:1 to 7:1, v / v) to obtain a colorless oily compound 17* (68.5 mg, 0.088 mmol) with a yield of 52%.

[0163] Comparative Example 4:

[0164] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (17*)

[0165] Compound 16* (100 mg, 0.17 mmol) was co-evaporated three times with toluene, and dry toluene (10 mL) and activated Molecular sieves, the resulting reaction solution was stirred at room temperature for 30 minutes, and dibutyltin oxide (63.5 mg, 0.26 mmol) was added to the reaction solution. The reaction bottle was moved to an oil bath and refluxed at 80 ° C for 1 hour. After returning to room temperature, sodium hydride (6.1 mg, 0.26 mmol), tetrabutylammonium iodide (96 mg, 0.26 mmol) and benzyl bromide (81.3 μL, 0.68 mmol) were added, and then the temperature was raised to 80 ° C and refluxed for 2 hours. After the reaction was detected by TLC (petroleum ether / ethyl acetate, 2:1), the reaction solution was cooled to room temperature, the molecular sieve was filtered through diatomaceous earth, and the solvent was removed by concentrated vacuum distillation. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by distillation under reduced pressure. The resulting crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 15:1 to 7:1, v / v) to obtain a colorless oily compound 17* (80.3 mg, 0.1 mmol) with a yield of 61%.

[0166] Embodiment 4:

[0167] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-2,3,4-tri-O-benzyl-D-ribitol (18*);

[0168] The reaction equation is Figure 1 As shown;

[0169] Compound 17* (10 g, 13.43 mmol) was dissolved in a mixed solvent of dichloromethane / water (67 mL, 3:1, v / v), palladium chloride (238.1 mg, 1.34 mmol) was added, and the resulting reaction solution was heated to 50°C for reflux reaction for 6 hours. After TLC (petroleum ether / ethyl acetate, 2:1) detection showed that the reaction was complete, the reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane, palladium chloride was filtered through diatomaceous earth, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The resulting crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 18* (7.2 g, 9.81 mmol) with a yield of 75%. 2.5 g of raw material was recovered with a recovery rate of 93%. 1 HNMR (600MHz, CDCl 3 )δ7.36–7.21(m,25H),5.00(d,J=1.3Hz,1H),4.70–4.60(m,4H),4.58–4.53(m,4H),4.4 8(q,J=12.2Hz,2H),4.16–4.11(m,1H),4.09(td,J=6.3,3.8Hz,1H),3.94(dd,J=10.8,5. 5Hz,1H),3.88–3.83(m,2H),3.79(q,J=4.8Hz,1H),3.75(d,J=5.0Hz,2H),3.71(t,J=4. 7Hz,1H),3.70–3.66(m,1H),3.61(dd,J=10.4,3.9Hz,1H),3.51(dd,J=10.4,6.3Hz,1H); 13 C NMR (151 MHz, CDCl 3 )δ138.2,138.1,138.0,137.1,128.6,128.5,128.44,128.37,128.3,128.2,128.1,127.9,127.84,127.80,127.7, 127.5,104.9,83.1,81.8,79.1,78.9,77.9,74.0,73.3,72.6,72.3,71.98,71.94,71.7,67.2,61.4; HRMS(ESI)m / z calcd for C45 H 50 NaO 9 :[M+Na] + :758.3348,found:758.3381.

[0170] Comparative Example 5:

[0171] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-2,3,4-tri-O-benzyl-D-ribitol (18*);

[0172] Compound 17* (100 mg, 0.13 mmol) was dissolved in a mixed solution of ethanol / toluene / water (60 mL, 6:2:1, v / v / v), followed by the addition of tri(triphenylphosphine)rhodium chloride (I) (22 mg, 0.03 mmol), and then 1,8-diazabicycloundec-7-ene (6.7 μL, 0.04 mmol). The mixture was refluxed for 2 hours. After the reaction was completed as determined by TLC, the reaction mixture was concentrated and dissolved in a mixed solution of acetic acid / water (4.3 mL, 9:1, v / v), and heated to 80°C for reflux reaction for 1 hour. After TLC (petroleum ether / ethyl acetate, 2:1) showed that the reaction was complete, the reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by distillation under reduced pressure. The resulting crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 18* (59.2 mg, 0.081 mmol) with a yield of 62%. Unrecyclable raw materials.

[0173] Comparative Example 6:

[0174] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-2,3,4-tri-O-benzyl-D-ribitol (18*);

[0175] Compound 17* (100 mg, 0.13 mmol) and 1,3-dimethylbarbituric acid (61 mg, 0.39 mmol) were dissolved in methanol (3 mL), and then tetrakis(triphenylphosphine)palladium (15 mg, 0.013 mmol) was added. The resulting mixture was stirred at 40°C for 5 hours. After TLC (petroleum ether / ethyl acetate, 2:1) showed that the reaction was complete, the mixture was cooled to room temperature. The reaction solution was diluted with ethyl acetate, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 18* (59.2 mg, 0.91 mmol) with a yield of 70%. Unrecyclable raw materials.

[0176] Comparative Example 7:

[0177] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-2,3,4-tri-O-benzyl-D-ribitol (18*);

[0178] Compound 17* (100 mg, 0.13 mmol) was dissolved in a mixed solvent of 90% acetic acid aqueous solution and water (3 mL), palladium chloride (0.24 mg, 0.13 mmol) and sodium acetate were added, and the resulting reaction solution was reacted at room temperature for 3 hours. After TLC (petroleum ether / ethyl acetate, 2:1) detection showed that the reaction was complete, the reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The resulting crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 18* (55.7 mg, 0.072 mmol) with a yield of 55%. Unrecyclable raw materials.

[0179] Comparative Example 8:

[0180] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-2,3,4-tri-O-benzyl-D-ribitol (18*);

[0181] Compound 17* (100 mg, 0.13 mmol) was dissolved in a mixed solvent of dichloromethane / water (2.2 mL, 10:1, v / v), palladium chloride (0.24 mg, 0.13 mmol) was added, and the resulting reaction solution was heated to 80°C for reflux reaction for 16 hours. After TLC (petroleum ether / ethyl acetate, 2:1) detection showed that the reaction was complete, the reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane, palladium chloride was filtered through diatomaceous earth, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation. The resulting crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 18* (66.8 mg, 0.091 mmol) with a yield of 70%. 24 mg of raw material was recovered with a recovery rate of 84%.

[0182] Embodiment 5:

[0183] Synthesis of 2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-5-O-monomethoxytrityl-2,3,4-tri-O-benzyl-D-ribitol (19*);

[0184] The reaction equation is Figure 1 As shown;

[0185] Compound 18* (7.2 g, 9.81 mmol) was dissolved in dry dichloromethane (110 mL), and pyridine (15.8 mL, 196.2 mmol), N,N-dimethylpyridine (599.2 mg, 4.91 mmol) and 4-monomethoxytrityl chloride (12.2 g, 39.24 mmol) were added, and the resulting reaction solution was stirred at room temperature for 10 hours. After the reaction was completed as detected by TLC (petroleum ether / ethyl acetate, 2:1), the reaction solution was diluted with dichloromethane, the organic phase was extracted and washed with saturated sodium bicarbonate solution, the organic phase was treated with anhydrous sodium sulfate, and the solvent was removed by reduced pressure distillation. The resulting crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 3:1, v / v) to obtain yellow oily compound 19* (9.1 g, 9.03 mmol) with a yield of 92%. 1 H NMR (400 MHz, CDCl 3)δ7.52–7.29(m,20H),7.28–7.19(m,14H),7.13(dd,J=6.6,3.0Hz,2H),6.77(d,J=8.8Hz,2 H),5.01(s,1H),4.81(d,J=11.5Hz,1H),4.61(dd,J=20.6,10.3Hz,5H),4.54(d,J=3.8Hz,2H ),4.52–4.41(m,3H),4.17–4.07(m,2H),3.98–3.84(m,5H),3.78(s,3H),3.73(dd,J=3.2Hz ,1H),3.61(dd,J=3.9Hz,1H),3.53(dd,J=10.4,6.4Hz,1H),3.49–3.38(m,2H),2.59(s,1H); 13 C NMR (101 MHz, CDCl 3 )δ158.5,144.7,138.7,138.6,138.4,138.2,137.1,135.8,130.5,128.6,128.32,128.28,128.2,128.1,127.91,127.88,127.8,127.7,1 27.6,127.5,127.4,126.8,113.0,86.4,83.1,81.8,78.9,78.3,73.7,73.3,72.7,72.6,72.3,72.1,71.9,67.6,63.8,55.2; HRMS(ESI)m / z calcd for C 65 H 66 NaO 10 :[M+Na] + :1029.4549,found:1029.4553.

[0186] Embodiment 6:

[0187] Synthesis of 3-O-levulinyl-2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (21*);

[0188] The reaction equation is Figure 1 As shown;

[0189] Compound 17* (6.4 g, 8.27 mmol) was dissolved in dichloromethane (66 mL), and levulinic acid (2.8 g, 24.81 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.2 g, 16.54 mmol), and N,N-dimethylpyridine (2.0 g, 16.54 mmol) were added respectively, and the reaction solution was stirred at room temperature for 12 hours. After the reaction was completed by TLC (petroleum ether / ethyl acetate, 2:1), the reaction solution was diluted with dichloromethane, the organic phase was extracted and washed with saturated sodium bicarbonate solution, and the organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 3:1, v / v) to obtain colorless oily compound 21* (6.7 g, 7.69 mmol) with a yield of 93%. 1 H NMR (600 MHz, CDCl 3 )δ7.35–7.20(m,25H),5.88(ddq,J=16.5,10.8,5.5Hz,1H),5.25(dd,J=17.5,5.0Hz,1H),5.15(t,J=6.2Hz, 2H),5.02(s,1H),4.71(dd,J=11.9,7.4Hz,1H),4.64(d,J=24.3Hz,2H),4.60–4.51(m,3H),4.43(q,J=13.0, 12.5Hz,2H),4.32(d,J=5.5Hz,1H),4.07(d,J=18.6,5.5Hz,1H),3.98–3.90(m,3H),3.87–3.82(m,2H),3.73 –3.66(m,2H),3.62(dd,J=10.7,5.7Hz,1H),3.52(m,2H),2.68–2.64(m,2H),2.63–2.55(m,2H),2.13(s,3H); 13 C NMR (101 MHz, CDCl 3 )δ206.3,172.2,138.3,138.2,138.1,137.7,135.9,135.0,128.6,128.5,128.42,128.39,128.36,128.2,128.0,127.8,127.7,1 16.8,106.1,80.8,80.2,78.7,75.4,78.2,73.9,73.8,72.4,72.3,72.2,71.5,70.1,67.9,66.5,37.9,29.8,28.0; HRMS(ESI)m / z calcd for C 48 H 53 NaO8 :[M+Na] + :780.3633,found:780.3641.

[0190] Embodiment 7:

[0191] Synthesis of 3-O-levulinyl-2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-2,3,4-tri-O-benzyl-D-ribitol (22*);

[0192] The reaction equation is Figure 1 As shown;

[0193] Compound 21* (6.7 g, 7.69 mmol) was dissolved in dichloromethane / water (38 mL, 3:1, v / v), palladium chloride (136.3 mg, 0.77 mmol) was added, and the resulting reaction solution was heated to 50°C and refluxed for 5 hours. After TLC (petroleum ether / ethyl acetate, 2:1) showed that the reaction was complete, the reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane, palladium chloride was filtered through diatomaceous earth, and the organic phase was extracted and washed with saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oil compound 22* (4.8 g, 5.77 mmol) with a yield of 75%. 1 H NMR (400 MHz, CDCl 3 )δ7.36–7.26(m,25H),5.20(t,J=5.3Hz,1H),5.05(d,J=2.2Hz,1H),4.75–4.66(m,3H),4.60(d,J=3.5Hz ,3H),4.58–4.55(m,2H),4.48(d,J=3.7Hz,2H),4.36(q,J=5.2Hz,1H),4.13(dd,J=5.1,2.2Hz,1H),3.98( dd,J=10.6,5.4Hz,1H),3.91(t,J=4.9Hz,1H),3.87–3.82(m,1H),3.78(d,J=4.3Hz,2H),3.74(dd,J=7.7, 3.2Hz,2H),3.57(d,J=5.0Hz,2H),2.71(dd,J=7.0,5.3Hz,2H),2.63(dd,J=7.0,5.3Hz,2H),2.17(s,3H); 13 C NMR (101 MHz, CDCl 3)δ206.3,172.2,138.2,138.09,138071,137.7,128.5,128.45,128.40,128.39,128.36,128.1,127.9,127.83,127.80,127.73,127.69, 127.6,127.5,106.1,80.7,80.3,79.0,78.8,77.9,74.0,73.7,73.3,72.9,72.4,72.0,71.3,67.4,61.4,37.9,29.8,28.0; HRMS(ESI)m / z calcdforC 45 H 49 NaO 8 :[M+Na] + :740.3320,found:740.3354.

[0194] Embodiment 8:

[0195] Synthesis of 3-O-cyanoethyl N,N-diisopropylchlorophosphoramidite-2,5-di-O-benzyl-β-D-ribofuranose-(1→1)-5-O-monomethoxytrityl-2,3,4-tri-O-benzyl-D-ribitol (20*);

[0196] The reaction equation is Figure 1 As shown;

[0197] Under nitrogen protection, compound 19* (2.0 g, 1.99 mmol) was dissolved in dry dichloromethane (20 mL) and activated The molecular sieve was stirred at room temperature for 30 minutes. The reaction solution was cooled to 0°C in an ice bath, N, N-diisopropylethylamine (1.4 mL, 7.96 mmol) and 1-methylimidazole (79 μL, 1 mmol) were added, and then 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (1.1 mL, 4.98 mmol) was added. The reaction solution was stirred for 2 hours. After the reaction was detected by TLC (petroleum ether / ethyl acetate, 3:1), the molecular sieve was filtered using diatomaceous earth and concentrated to remove the solvent by vacuum distillation. The reaction solution was diluted with dichloromethane, the organic phase was extracted and washed with saturated sodium bicarbonate solution, and the organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by alkaline silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 15:1 to 6:1, v / v) to obtain a light yellow oily compound 20* (2.16 g, 1.79 mmol) with a yield of 90%. 1 HNMR (600MHz, CDCl 3)δ7.46–7.42(m,4H),7.37–7.06(m,34H),6.72(dd,J=9.0,3.1Hz,2H),4.96(s,1H),4. 77–4.64(m,2H),4.62–4.55(m,4H),4.54–4.41(m,4H),4.35–4.24(m,2H),3.98–3.91(m ,1H),3.88–3.81(m,3H),3.73(s,3H),3.72–3.64(m,3H),3.63–3.54(m,3H),3.54–3.4 6(m,1H),3.44–3.37(m,1H),3.37–3.30(m,1H),2.44–2.26(m,2H),1.17–1.03(m,12H); 13 C NMR (151 MHz, CDCl 3 )δ158.4,144.7,138.7,138.44,138.37,138.2,138.1,138.0,135.7,130.5,128.5,128.3,128.2,128.1 ,127.84,127.77,127.72,127.68,127.60,127.56,127.52,127.48,127.44,127.40,127.37,127.3,126. 7,117.7,117.6,113.0,105.5,105.4,86.4,81.7,81.4,79.0,78.9,78.3,73.6,73.2,72.64,72.60,72.4,72.31,72.27,71.3,71.2,67.8,67.7,63.8,58.7,58.5,58.3,58.1,55.1,43.3,43.2,43.1,24.6,20.1; 31 P NMR (243 MHz, CDCl 3 )δ13.18.;HRMS(ESI)m / z calcd for C 74 H 83 N 2 NaO 11 P:[M+Na] + :1229.5627,found:1229.5656.

[0198] Embodiment 9:

[0199] Synthesis of compound 23*;

[0200] The reaction equation is Figure 2 As shown;

[0201] Compound 22* (2.2 g, 2.64 mmol) and phosphoramidite monomer donor 20* (3.5 g, 2.91 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (26 mL) and added Molecular sieves were added and stirred at room temperature for 30 minutes. 1-phenyl-1H-imidazole-3-ium trifluoromethanesulfonate N-PhIMT (0.85 g, 2.91 mmol) was then added to the reaction system and stirred at room temperature for 10 minutes. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1) until the receptor was completely reacted. TBHP (1M toluene solution, 2 eq) was then added to the reaction mixture and stirred for 10 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (4.93 g, 2.52 mmol) with a yield of 97%. 1 HNMR (600MHz, CDCl 3 )δ7.43(ddt,J=8.3,4.3,2.3Hz,4H),7.33–7.18(m,56H),7.08(dq,J=7.1,3.6Hz,2H),6.72(dd,J=8.9,3.6Hz,2H),5.14(td,J=5.3, 1.3Hz,1H),5.03(d,J=3.1Hz,2H),4.93–4.84(m,1H),4.73(dd,J=11.6,3.6Hz,1H),4.65–4.59(m,4H),4.58–4.53(m,5H),4.53–4.43 (m,7H),4.42–4.33(m,3H),4.32–4.24(m,4H),4.23–4.17(m,1H),4.08–3.99(m,2H),3.95–3.87(m,3H),3.87–3.80(m,5H),3.78(m, 3H),3.75–3.71(m,4H),3.67–3.62(m,1H),3.54–3.48(m,3H),3.43–3.33(m,3H),2.68–2.64(m,2H),2.60–2.56(m,2H),2.12(s,3H); 13 C NMR (151 MHz, CDCl 3)δ206.2,172.1,158.5,144.7,138.69,138.67,138.6,138.5,138.4,138.3,138.13,13 8.08,138.00,137.97,137.95,137.86,137.7,137.6,137.5,135.8,130.5,128.6,128.4 4,128.39,128.37,128.31,128.26,128.2,128.1,127.98,127.95,127.91,127.89,127.85,127.82,127.76,127.71,127.68,127.65,127.57,127.5,127.4,126.8,116.4,113. 0,106.1,106.0,105.7,105.5,86.4,81.5,81.3,81.0,80.90,80.85,80.8,80.2,78.9,78.8,78.3,78.0,77.85,77.79,77.7,73.82,73.77,73.69,73.68,73.66,73.6,73.4,73 .33,73.28,72.9,72.7,72.64,72.63,72.4,72.34,72.33,720.3,71.3,70.9,70.7,68.2,68.1,67.7,67.6,67.4,67.3,63.83,63.75,61.9,61.8,55.2,37.9,29.8,28.0,18.9; 31 P NMR (243 MHz, CDCl 3 )δ-1.68,-2.06; MALDI-TOF m / z calcdfor C 118 H 124 NNaO 23 P:[M+Na] + :1977.8228,found:1977.8238.

[0202] Comparative Example 9:

[0203] Synthesis of compound 23*

[0204] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (723 mg, 0.6 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (2 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then 1H-tetrazole (42 mg, 0.6 mmol) was added to the reaction system, stirred at room temperature for 2 hours, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1: 1). Iodine solution (1M pyridine aqueous solution, 5 eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10: 1 to 2: 1, v / v) to obtain a colorless oily compound 23* (195 mg, 0.1 mmol) with a yield of 83%.

[0205] Comparative Example 10:

[0206] Synthesis of compound 23*

[0207] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (434 mg, 0.36 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then 4,5-dicyanoimidazole (42.5mg, 0.36mmol) was added to the reaction system, stirred at room temperature for 1 hour, and the reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1). Subsequently, (+)-8,8-dichlorocamphorsulfonazine (0.5M acetonitrile solution, 4eq) was added to the reaction mixture and stirred for 30 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (208mg, 0.106mmol) with a yield of 89%.

[0208] Comparative Example 11:

[0209] Synthesis of compound 23*

[0210] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (434 mg, 0.36 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Pyridinium trifluoroacetate (69.5 mg, 0.36 mmol) was then added to the reaction system, stirred at room temperature for 30 minutes, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1). Iodine solution (1M pyridine aqueous solution, 5 eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (215.6 mg, 0.11 mmol) with a yield of 92%.

[0211] Comparative Example 12:

[0212] Synthesis of compound 23*

[0213] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (434 mg, 0.48 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then 5-benzylthiotetrazolyl (92.3 mg, 0.48 mmol) was added to the reaction system, stirred at room temperature for 1 hour, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1). Iodine solution (1M pyridine aqueous solution, 5eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (201 mg, 0.103 mmol) with a yield of 86%.

[0214] Comparative Example 13:

[0215] Synthesis of compound 23*

[0216] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (434 mg, 0.36 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then 5-ethylthiotetrazolyl (46.7 mg, 0.36 mmol) was added to the reaction system, stirred at room temperature for 2 hours, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1). Iodine solution (1M pyridine aqueous solution, 5eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (216 mg, 0.108 mmol) with a yield of 90%.

[0217] Comparative Example 14:

[0218] Synthesis of compound 23*

[0219] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (434 mg, 0.6 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves were added and stirred at room temperature for 30 minutes. 2,4-dinitrobenzoic acid (75.4 μL, 0.6 mmol) was then added to the reaction system, stirred at room temperature for 2 hours, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1). (+)-8,8-dichlorocamphorsulfonazine (0.5 M acetonitrile solution, 4 eq) was then added to the reaction mixture and stirred for 30 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (189.5 mg, 0.097 mmol) with a yield of 81%.

[0220] Comparative Example 15:

[0221] Synthesis of compound 23*

[0222] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (434 mg, 0.36 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then saccharin 1-methylimidazole (95.5mg, 0.36mmol) was added to the reaction system, stirred at room temperature for 1 hour, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1). Iodine solution (1M pyridine aqueous solution, 5eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (199.2mg, 0.102mmol) with a yield of 85%.

[0223] Comparative Example 16:

[0224] Synthesis of compound 23*

[0225] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (289 mg, 0.36 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves were added and stirred at room temperature for 30 minutes. 1H-benzimidazol-3-ium trifluoromethanesulfonate (64.4 mg, 0.24 mmol) was then added to the reaction system, stirred at room temperature for 30 minutes, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1). TBHP (1M toluene solution, 2 eq) was then added to the reaction mixture and stirred for 20 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (216 mg, 0.11 mmol) with a yield of 92%.

[0226] Comparative Example 17:

[0227] Synthesis of compound 23*

[0228] Compound 22* (100 mg, 0.12 mmol) and phosphoramidite monomer donor 20* (434 mg, 0.36 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then N-methylbenzimidazole trifluorate (101.6 mg, 0.36 mmol) was added to the reaction system, stirred at room temperature for 2 hours, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1). Iodine solution (1M pyridine aqueous solution, 5 eq) was then added to the reaction mixture and stirred for 2 hours. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 23* (203.9 mg, 0.104 mmol) with a yield of 87%.

[0229] Embodiment 10:

[0230] Synthesis of compound 24*

[0231] The reaction equation is Figure 2 As shown;

[0232] Compound 23* (2 g, 1.02 mmol) was dissolved in dichloromethane / methanol (20 / 1, v / v, 20 mL) and N 2 H 4 HOAC (141 mg, 1.54 mmol), the reaction was stirred at room temperature for 3 hours. After the reaction was completed by TLC (petroleum ether / ethyl acetate, 1:1), the reaction solution was diluted with dichloromethane, the organic phase was extracted and washed with a saturated sodium bicarbonate solution, the organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation, and the obtained crude product was purified by silica gel chromatography column (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a light yellow oily compound 24* (1.77 g, 0.95 mmol), with a yield of 93%. 1 HNMR (400MHz, CDCl 3) δ 7.43 (dt, J = 7.4, 1.8 Hz, 4H), 7.35–7.13 (m, 56H), 7.09–7.06 (m, 2H), 6.72 (dd, J = 9.0, 2.5 Hz, 2H), 4.97 (s, 2H), 4.93–4.84 (m, 1H), 4.73 (dd, J = 11.6, 2.5 Hz, 1H), 4.67–4.60 (m, 4H), 4.59–4.52 (m, 7H), 4.52–4.46 (m, 4H), 4.46–4.42 (m, 2H), 4.41–4.34 (m, 1H), 4.33–4.18 (m, 4H), 4.13–4.00 (m, 3H), 3.96–3.81 (m, 8H), 3.81–3.75 (m, 3H), 3.73 (s, 4H), 3.66–3.55 (m, 2H), 3.54–3.42 (m, 2H), 3.42–3.31 (m, 3H), 2.55 (s, 1H), 2.16–2.03 (m, 2H); 13 C NMR (101 MHz, CDCl 3 ) δ 158.5, 144.7, 144.6, 138.68, 138.65, 138.6, 138.4, 138.34, 138.28, 138.2, 138.1, 138.0, 137.92, 137.86, 137.6, 137.5, 137.1, 135.8, 130.5, 128.61, 128.57, 128.4, 128.3, 128.2, 128.0, 127.9, 127.81, 127.76, 127.7, 127.64, 127.59, 127.4, 126.8, 116.4, 113.0, 105.7, 104.9, 86.4, 83.1, 81.8, 81.5, 81.2, 78.9, 78.3, 78.0, 77.9, 77.8, 73.8, 73.7, 73.33, 73.26, 72.6, 72.5, 72.3, 72.0, 71.7, 70.7, 68.2, 67.7, 67.1, 63.8, 61.9, 55.21, 55.15, 19.0, 18.9; 31 P NMR (162 MHz, CDCl 3 ) δ -1.70, -2.07; MALDI-TOF m / z calcd for C 113 H 118 NNaO 21 P: [M+Na] + : 1879.7860, found: 1879.7866.

[0233] Example 11:

[0234] Synthesis of phosphoramidite dimer donor 25*;

[0235] The reaction equation is Figure 2 As shown;

[0236] Under nitrogen protection, compound 24* (0.5 g, 0.27 mmol) was dissolved in dry dichloromethane (3 mL) and activated The molecular sieve was stirred at room temperature for 30 minutes. The reaction solution was cooled to 0°C in an ice bath, N, N-diisopropylethylamine (0.2mL, 1.08mmol) and 1-methylimidazole (10μL, 0.14mmol) were added, and then 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (0.15mL, 0.67mmol) was added. The reaction solution was stirred for 2 hours. After the reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1), the molecular sieve was filtered using diatomaceous earth and concentrated to remove the solvent by vacuum distillation. The reaction solution was diluted with dichloromethane, the organic phase was extracted and washed with saturated sodium bicarbonate solution, and the organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by basic silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a light yellow oily compound 25* (0.48g, 0.23mmol) with a yield of 85%. 1 HNMR (600MHz, CDCl 3)δ7.46–7.42(m,4H),7.36–7.25(m,25H),7.24–7.10(m,31H),7.09–7.06(m,2H),6.72(dd,J=8.9,3.6Hz,2H),5.03–4.93(m,2H),4.92–4.83(m,1H),4.75–4.64(m,3H),4.63–4.56(m,8H),4.55–4.49(m,5H),4.49–4.40(m,4H),4.39–4.34(m,2H),4.32–4.29(m,2H),4.28–4.23(m,2H),4.22–4.16(m,1H),4.02(ddd,J=23.6,4.9,2.6Hz,1H),3.96–3.92(m,2H),3.88–3.83(m,4H),3.82–3.76(m,4H),3.73(s,3H),3.70–3.65(m,2H),3.65–3.62(m,1H),3.60–3.54(m,3H),3.53–3.50(m,1H),3.49–3.45(m,1H),3.43–3.32(m,3H),2.44–2.27(m,2H),2.18–2.03(m,2H),1.14(dd,J=6.8,3.5Hz,6H),1.11(d,J=6.8Hz,3H),1.07(d,J=6.8Hz,3H); 13 C NMR(151MHz,CDCl 3)δ158.4,144.64,144.61,138.64,138.62,138.56,138.4,138.34,138.31,138.2,138.14,138.07,138. 04,137.98,137.96,137.90,137.86,137.8,137.6,137.4,135.7,130.4,128.5,128.4,128.33,128.28, 128.2,128.1,128.0,127.92,127.90,127.88,127.86,127.80,127.78,127.73,127.67,127.66,127.6,127.53,127.52,127.48,127.41,127.39,127.3,126.7,117.8,117.6,116.4,113.0,105.73,105.69,10 5.64,105.56,105.5,86.4,81.7,81.6,81.4,81.2,80.9,80.8,78.9,78.7,78.3,78.1,77.74,77.72,73.81,73.76,73.6,73.34,73.28,73.2,72.9,72.7,72.61,72.59,72.5,72.4,72.30,72.26,71.1,71.0,7 0.8,70.6,68.2,68.0,67.22,67.16,63.8,63.7,61.82,61.79,61.7,58.6,58.5,58.2,58.1,55.1,43.3,43.21,43.15,43.1,24.63,24.58,24.52,24.47,20.20,20.15,20.13,20.08,19.0,18.92,18.88,18.8; 31 P NMR (243 MHz, CDCl 3 )δ12.52,12.10; MALDI-TOF m / z calcd for C 122 H 135 N 3 NaO 22 P 2 :[M+Na] + :2079.8939,found:2079.8945.

[0237] Embodiment 12:

[0238] Synthesis of compound 27*;

[0239] The reaction equation is Figure 3 As shown;

[0240] 2-(2-Azidoethoxy)ethanol 26* (500 mg, 3.82 mmol) and phosphoramidite monomer donor 20* (3.5 g, 3.82 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (20 mL) and added Molecular sieves were added and stirred at room temperature for 30 minutes. N-PhIMT (1.12 g, 3.82 mmol) was then added to the reaction system, stirred at room temperature for 10 minutes, and the reaction was detected by TLC (petroleum ether / ethyl acetate, 3:2) until the receptor was completely reacted. TBHP (1M toluene solution, 2 eq) was then added to the reaction mixture and stirred for 10 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 27* (4.75 g, 3.78 mmol) with a yield of 99%. 1 H NMR (600 MHz, CDCl 3 )δ7.44(dt,J=6.4,1.6Hz,4H),7.37-7.15(m,31H),7.09(dd,J=3.9,2.1Hz,2H),6.75–6.70(m,2H),5.01(dd,J=17.2,2.4Hz,1H),4.89(dtd ,J=7.9,4.9,2.8Hz,1H),4.75(d,J=11.5Hz,1H),4.63–4.54(m,5H),4.50(t,J=11.9Hz,2H),4.45–4.35(m,2H),4.31(q,J=5.1Hz,1H),4.14 –4.08(m,1H),4.08–4.03(m,2H),4.03–3.97(m,2H),3.93(dd,J=10.8,6.0Hz,1H),3.90–3.88(m,1H),3.87–3.82(m,2H),3.74(s,3H),3.72 –3.67(m,1H),3.61–3.55(m,4H),3.54–3.49(m,2H),3.41(dd,J=10.3,2.7Hz,1H),3.38–3.33(m,1H),3.32–3.26(m,2H),2.46–2.29(m,2H); 13 C NMR (151 MHz, CDCl 3)δ158.4,144.63,144.60,138.63,138.59,138.4,137.9,137.6,137.5,135.7,130.5,128.5,128.42,128.37,128.36,128.30,128 .26,128.2,127.95,127.88,127.84,127.79,127.77,127.76,127.74,127.66,127.6,127.43,127.40,126.8,116.5,116.4,113.0, 105.4,105.3,86.4,81.2,81.1,81.0,78.85,78.81,78.76,78.3,77.52,77.49,73.6,73.44,73.40,72.7,72.64,72.62,72.3,71.0,70.8,70.0,69.9,69.73,69.68,69.6,68.1,67.2,67.15,67.11,63.7,62.0,61.93,61.86,55.2,50.6,19.3,19.23,19.15,19.1; 31 P NMR (243 MHz, CDCl 3 )δ-2.43; MALDI-TOF m / z calcd for C 72 H 77 N 4 NaO 14 P:[M+Na] + :1275.5067,found:1275.5077.

[0241] Embodiment 13:

[0242] Synthesis of compound 28*;

[0243] The reaction equation is Figure 3 As shown;

[0244] Under nitrogen protection, compound 27* (4.0 g, 3.2 mmol) was dissolved in a mixed solvent of boron trifluoride ether complex / methanol / dichloromethane (1:2:50, v:v:v, 320 mL), and the resulting reaction solution was stirred at room temperature for 3 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1) until the bright yellow 4-monomethoxytrityl group was completely detached. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The resulting crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oil compound 28* (0.77 g, 0.78 mmol) with a yield of 97%. 1 H NMR (400 MHz, CDCl 3 )δ7.33–7.14(m,25H),5.05(dd,J=11.5,2.6Hz,1H),4.93(ddt,J=7.3,5.0,2.6Hz,1H),4.69–4.54(m,8H), 4.45(d,J=3.5Hz,2H),4.34(q,J=4.9Hz,1H),4.17–4.07(m,3H),4.07–4.00(m,2H),3.96(ddd,J=10.6,7.3, 5.4Hz,1H),3.89(td,J=4.9,3.3Hz,1H),3.83(q,J=4.6Hz,1H),3.74(d,J=6.6Hz,4H),3.70(d,J=4.3Hz,4H ),3.64–3.50(m,2H),3.30(dt,J=9.9,5.0Hz,2H),2.46(td,J=6.3,2.2Hz,1H),2.39(td,J=6.2,3.3Hz,1H); 13 C NMR (101 MHz, CDCl 3 )δ138.23,138.21,138.1,137.9,137.6,137.5,116.50,116.46,105.63,105.56,105.5,81.1,80.8,78.9,77.9,74.0,73.5,73.4,72.7,72 .4,72.0,70.8,70.7,70.0,69.9,69.8,69.7,67.7,67.33,67.27,67. 21,67.15,62.05,62.99,61.9,61.4,50.6,19.4,19.3,19.24,19.17; 31 PNMR (162MHz, CDCl 3 )δ-2.36; 31P NMR (243 MHz, CDCl 3 )δ-2.43; MALDI-TOF m / z calcd for C 52 H 61 N 4 NaO 13 P:[M+Na] + :1003.3865,found:1003.3873.

[0245] Comparative Example 18:

[0246] Synthesis of compound 28*;

[0247] Compound 27* (100 mg, 0.08 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (23.7 μL, 0.32 mmol) was added, and the reaction solution was stirred at room temperature for 6 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1). The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 28* (69.8 mg, 0.062 mmol) with a yield of 78%.

[0248] Comparative Example 19:

[0249] Synthesis of compound 28*;

[0250] Compound 27* (100 mg, 0.08 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (23.7 μL, 0.32 mmol) and trifluoroacetic anhydride (45 μL, 0.32 mmol) were added respectively, and the reaction solution was stirred at room temperature for 4 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1). The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 28* (69.8 mg, 0.071 mmol) with a yield of 89%.

[0251] Comparative Example 20:

[0252] Synthesis of compound 28*;

[0253] Compound 27* (100 mg, 0.08 mmol) was dissolved in acetic acid (0.5 mL), and the reaction solution was stirred at room temperature for 12 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1). The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 28* (39.3 mg, 0.04 mmol) with a yield of 50%.

[0254] Comparative Example 21:

[0255] Synthesis of compound 28*;

[0256] Compound 27* (100 mg, 0.08 mmol) was dissolved in a mixed solvent of dichloromethane / methanol (2:1, v:v, 3 mL), and then sodium bisulfate (48 mg, 0.4 mmol) supported on silica gel was added, and the reaction solution was stirred at room temperature for 4 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1). The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 28* (51 mg, 0.052 mmol) with a yield of 65%.

[0257] Comparative Example 22:

[0258] Synthesis of compound 28*;

[0259] Compound 27* (100 mg, 0.08 mmol) was dissolved in a mixed solvent of dichloromethane / methanol (6:1, v:v, 3.5 mL), and then the reaction solution was cooled to 0°C, and zinc bromide (41 mg, 0.16 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:1). The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oily compound 28* (62.8 mg, 0.064 mmol) with a yield of 80%.

[0260] Embodiment 14:

[0261] Synthesis of compound 1*;

[0262] The reaction equation is Figure 5 As shown;

[0263] Under argon protection, compound 28* (30 mg, 0.031 mmol) was dissolved in a mixed solvent of ammonia / methanol (3:7, v:v, 10 mL) before deprotection. After stirring at room temperature for 18 hours, the reaction was detected to be complete by TLC. The solvent was removed under reduced pressure, diluted with ethyl acetate and washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain an intermediate. The intermediate was then dissolved in Et 2 Ac / MeOH / H 2 10% palladium on carbon (10 mg) was added to a mixed solvent of O / AcOH (1:1:0.5:0.2, v:v:v:v, 2.7 mL). Hydrogen was introduced and stirred at room temperature for 36 hours at a pressure of 0.45 bar. After completion, the mixture was filtered through diatomaceous earth and the solvent was removed under reduced pressure. The crude product was purified by size exclusion chromatography (SephadeXLH20). The pure compound was dissolved in deionized water and filtered through a column containing DOWEX(R) 50WX8 Na + The residue was eluted through a cation exchange resin column and then freeze-dried to obtain a white compound 1* (14 mg, 0.03 mmol) with a yield of 98%. 1 H NMR (600MHz, D 2 O)δ5.04(s,1H,1-H),4.59(td,J=7.5,4.6Hz,1H,3-H),4.23(d,J=4.7Hz,1H,2-H),4.17(td,J=6.5,2.9Hz,1H,4-H),4.13–4.01(m,2H,linker-CH 2 ),3.92(td,J=6.5,2.7Hz,1H,2'-H),3.89–3.84(m,2H,5-CH 2 ,1'-CH 2 ),3.82–3.77(m,4H,4'-H,5'-CH 2 ,linker-CH 2 ),3.76(t,J=4.4Hz,2H,linker-CH 2 ),3.73–3.71(m,1H,1'-CH 2 ),3.71–3.68(m,1H,3'-H),3.66(dd,J=11.3,5.1Hz,1H,5-CH 2 ),3.65–3.59(m,1H,5'-CH 2 ),3.22(t,J=5.0Hz,2H,linker-CH 2 ); 13C NMR (151MHz, D 2 O)δ106.7(1-C),81.9,81.8,74.3,74.2,73.8,73.7,72.2,71.9,70.21,70.16,70.1,68.7(1'-C),66.5,65.2,65.1,62.4,62.3,62.1,39.2; 31 P NMR (243MHz, D 2 O)δ0.09,-0.03; MALDI-TOF m / z calcd for C 14 H 30 NO 13 P:[M+H]:451.1455,found:451.1454.

[0264] Embodiment 15:

[0265] Synthesis of compound 29*;

[0266] The reaction equation is Figure 2 As shown;

[0267] Compound 28* (1 g, 0.5 mmol) and phosphoramidite monomer donor 20* (7720 mg, 0.6 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (5 mL) and added Molecular sieves were added and stirred at room temperature for 30 minutes. N-PhIMT (177 mg, 00.6 mmol) was then added to the reaction system, stirred at room temperature for 10 minutes, and the reaction was detected by TLC (petroleum ether / ethyl acetate, 2:3) until the receptor was completely reacted. TBHP (1M toluene solution, 2 eq) was then added to the reaction mixture and stirred for 10 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 29* (1.03 g, 0.49 mmol) with a yield of 98%. 1 HNMR (600MHz, CDCl 3)δ7.43(dt,J=6.3,2.0Hz,4H),7.33–7.15(m,56H),7.07(p,J=3.7,3.1Hz,2H),6.72(dd,J=8.8,3.6Hz,2H),5.07–4.98(m,2H),4.95–4.85(m,2H),4.73(dd,J=11.6,3.8Hz,1H),4.67–4.58(m,7H),4.58–4.52(m,6H),4.51–4.46(m,3H),4.45–4.40(m,3H),4.39–4.34(m,2H),4.33–4.28(m,2H),4.29–4.24(m,1H),4.23–4.18(m,1H),4.13–4.06(m,3H),4.05–4.00(m,3H),3.94–3.89(m,2H),3.88–3.81(m,5H),3.81–3.76(m,3H),3.73(s,4H),3.66(ddd,J=17.5,10.9,3.1Hz,2H),3.60–3.56(m,3H),3.55–3.50(m,3H),3.44–3.32(m,4H),3.32–3.27(m,2H),2.50–2.33(m,2H),2.19–2.03(m,2H); 13 C NMR(151MHz,CDCl 3)δ158.4,144.64,144.60,138.64,138.62,138.56,138.4,138.3,138.2,138.1,137.94,137.89,137.86,137.8.1 37.6,137.5,137.44,137.43,135.7,130.4,128.53,128.45,128.41,128.38,128.36,128.35,128.31,128.28,12 8.2,128.1,128.02,128.00,127.98,127,95127.93,127.91,127.88,127.86,127.83,127.81,127.78,127.73,12 7.68,127.65,127.6,127.53,127.48,127.42,127.40,127.37,127.35,126.7,116.5,116.4,116.42,116.38,113 .0,105.63,105.56,105.5,86.4,81.51,81.46,81.22,81.15,81.1,81.0,80.8,78.9,78.7,78.3,77.9,77.8,77. 7,73.81,73.76,73.6,73.43,73.36,73.4,73.3,72.69,72.67,72.6,72.4,72.30,72.25,70.8,70.7,70.6,70.0 69.9,69.72,69.68,69.6,68.2,68.1,67.6,67.5,67.3,67.24,67.16,67.1,63.8,63.7,61.99,61.96,61.92,61. 89,61.84,61.81,61.75,61.7,55.1,50.6,29.7,29.4,22.7,19.31,19.26,19.2,19.1,19.0,18.89,18.84,18.8; 31 P NMR(243MHz,CDCl 3 )δ-1.66(d,J610.6Hz),-2.11,-2.37,-2.40; 31 P NMR (243MHz, CDCl 3 )δ-2.43;MALDI-TOF m / z calcd for C 120 H 129 N 5 NaO 25 P.S 2 :[M+Na]+ :2125.8379,found:2125.8393.

[0268] Embodiment 16:

[0269] Synthesis of compound 30*;

[0270] The reaction equation is Figure 3 As shown;

[0271] Under nitrogen protection, compound 29* (1.03 g, 0.49 mmol) was dissolved in a mixed solvent of boron trifluoride ether complex / methanol / dichloromethane (1:2:50, v:v:v, 49 mL), and the resulting reaction solution was stirred at room temperature for 3 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 2:3) until the bright yellow 4-monomethoxytrityl group was completely detached. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation. The resulting crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 2:1, v / v) to obtain a colorless oil compound 30* (879 mg, 0.48 mmol) with a yield of 98%. 1 H NMR (600 MHz, CDCl 3 )δ7.26(dt,J=12.8,5.0Hz,56H),5.08–4.97(m,2H),4.95–4.88(m,2H),4.68–4.56(m,11H),4. 56–4.49(m,5H),4.48–4.36(m,4H),4.35–4.26(m,4H),4.25–4.20(m,1H),4.16–4.00(m,6H),3 .98–3.89(m,3H),3.87–3.82(m,3H),3.81–3.77(m,3H),3.76–3.65(m,4H),3.58(m,2H),3.53( m,4H),3.42(ddd,J=21.5,10.5,5.6Hz,1H),3.29(m,2H),2.49–2.33(m,2H),2.19–1.95(m,2H); 13 C NMR (101 MHz, CDCl 3)δ138.1,137.9,137.6,137.5,128.4,127.9,127.8,127.7,127.6,127.53,127.48,127 .42,127.40,127.5,116.42,116.38,113.0,105.63,105.56,105.581.6,81.1,78.9,77 .8,74.0,73.8,73.4,72.7,72.3,71.9,70.7,70.0,69.7,68.2,68.1,67.6,67.5,67.3,63.7,62.0,61.9,61.4,55.1,50.6,19.31,19.26,19.2,19.1,19.0,18.89,18.84,18.8; 31 P NMR (162 MHz, CDCl 3 )δ-1.63,-2.04,-2.37,-2.39; MALDI-TOF m / z calcd for C 100 H 113 N 5 NaO 24 P 2 :[M+Na] + :1853.7177,found:1853.7199.

[0272] Embodiment 17:

[0273] Synthesis of compound 2*;

[0274] The reaction equation is Figure 5 As shown;

[0275] Under argon protection, compound 30* (36 mg, 0.02 mmol) was dissolved in a mixed solvent of ammonia / methanol (3:7, v:v, 10 mL) before deprotection. After stirring at room temperature for 18 hours, the reaction was detected by TLC. The solvent was removed under reduced pressure, diluted with ethyl acetate and washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain an intermediate. The intermediate was then dissolved in Et 2 Ac / MeOH / H 2 10% palladium on carbon (10 mg) was added to a mixed solvent of O / AcOH (1:1:0.5:0.2, v:v:v:v, 2.7 mL). Hydrogen was introduced and stirred at room temperature for 36 hours at a pressure of 0.45 bar. After completion, the mixture was filtered through diatomaceous earth and the solvent was removed under reduced pressure. The crude product was purified by size exclusion chromatography (SephadeX LH20). The pure compound was dissolved in deionized water and filtered through a column containing DOWEX(R) 50WX8 Na+ The residue was eluted through a cation exchange resin column and then freeze-dried to obtain a white compound 2* (15.7 mg, 0.0187 mmol) with a yield of 95%. 1 HNMR (600MHz, D 2 O) δ5.07 (dd, J=3.8, 1.2Hz, 2H, 1-H), 4.62 (dtd, J=8.3, 6.5, 4.6Hz, 2H, 3-H), 4.26 (ddd, J=4. 5,3.1,1.2Hz,2H,2-H),4.19(tt,J=6.3,3.1Hz,2H,4-H),4.10–4.06(m,2H),3.99(ddd,J=9.5 ,8.2,4.7Hz,2H),3.96–3.92(m,2H),3.91–3.85(m,4H),3.83–3.80(m,3H),3.80–3.77(m,4H ),3.76–3.71(m,4H),3.71–3.68(m,2H),3.65(dd,J=11.7,7.0Hz,1H),3.24(t,J=5.0Hz,2H); 13 C NMR (151MHz, D 2 O) δ106.72,106.68,81.89,81.87,74.3,73.8,72.2,71.9,71.4,70.98,70.97,70.95,70.94,70.21,70.1 7,70.16,68.7,68.64,68.61,68.58,66.72,66.71,66.68,66.5,65.19,65.15,62.4,62.31,62.28,39.19; 31 PNMR(243MHz,D 2 O)δ0.24,-0.09; MALDI-TOF m / z calcd for C 24 H 48 NO 24 P 2 :[M+H] - :796.2047,found:796.2040.

[0276] Experimental operation and steps for sugar chain extension and complete deprotection:

[0277] General Procedure A: Phosphoramidite Coupling Reaction

[0278] The acceptor and donor (1-3 eq) were co-evaporated in toluene three times. Under argon protection, the donor and acceptor were dissolved in anhydrous acetonitrile (0.1 mmol / mL) and added Molecular sieves were added and stirred at room temperature for 30 minutes. N-PhIMT (1.2-3eq) was then added to the reaction system, stirred at room temperature for 5-30 minutes, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:1 to 1:4) until the receptor reacted completely. TBHP (1M toluene solution, 2eq) was then added to the reaction mixture and stirred for 5-30 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:3, v / v) to obtain a pure product.

[0279] General Procedure B: Deprotection of MMTr Group

[0280] Under nitrogen protection, the MMTr protected compound was dissolved in a mixed solvent of boron trifluoride ether complex / methanol / dichloromethane (1:2:50, v:v:v, 0.01 mmol / mL), and the resulting reaction solution was stirred at room temperature for 3 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 2:3 to 1:4) until the bright yellow 4-monomethoxytrityl group was completely detached. The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation. The resulting crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:3, v / v) to obtain a colorless oily compound.

[0281] General Procedure C: Full Deprotection

[0282] Under argon protection, the compound before deprotection was dissolved in a mixed solvent of ammonia water / methanol (3:7, v:v, 10 mL), stirred at room temperature for 18 to 24 hours, and the reaction was detected by TLC to be complete. The solvent was removed under reduced pressure, diluted with ethyl acetate and washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain an intermediate. The intermediate was then dissolved in Et 2 Ac / MeOH / H 2 10% palladium on carbon (10 mg) was added to a mixed solvent of O / AcOH (1:1:0.5:0.2, v:v:v:v, 2.7 mL). Hydrogen was introduced and stirred at room temperature for 36 to 72 hours at a pressure of 0.45 bar. After completion, the mixture was filtered through diatomaceous earth and the solvent was removed under reduced pressure. The crude product was purified by size exclusion chromatography (SephadeX LH20). The pure compound was dissolved in deionized water and filtered through a column containing DOWEX(R) 50WX8 Na + The product was eluted from a cation exchange resin column and then freeze-dried to obtain a white compound.

[0283] Embodiment 18:

[0284] Compound 3*:

[0285] The reaction equation is Figure 4 B and Figure 5 As shown;

[0286] The dimer acceptor 30* (50 mg, 0.027 mmol) was condensed with the phosphoramidite donor 20* (39.6 mg, 0.033 mmol) according to the above general procedure A to give the trimer 31* (80 mg, 0.033 mmol, 98%), and then the MMTr group was removed according to the general procedure B, and finally the general deprotection was carried out according to the general procedure C to obtain the pure trimer 3* (26.3 mg, 0.022 mmol) with an overall yield of 79%. 1 H NMR (600MHz, D 2 O)δ5.08–5.00(m,3H,1-H),4.58(tt,J=7.0,4.4Hz,3H,3-H),4.22(dd,J=4.6,1.4Hz,3H,2-H), 4.15(tt,J=5.9,2.6Hz,3H,4-H),4.09–4.01(m,4H),3.99–3.94(m,3H),3.94–3.89(m,4H),3.8 8–3.84(m,3H),3.84–3.81(m,3H),3.81–3.77(m,3H),3.76(s,1H),3.75(d,J=2.4Hz,2H),3.74 –3.72(m,2H),3.72–3.69(m,2H),3.69–3.66(m,3H),3.65–3.58(m,3H),3.19(t,J=5.0Hz,2H); 13 C NMR (151MHz, D 2 O)δ109.25,109.23,109.18,84.51,84.47,84.46,84.40,84.37,84.36,76.88, 76.86,76.84,76.80,76.78,76.75,76.33,76.26,76.24,76.21,74.7,74.4,73 .92,73.50,73.45,73.43,73.40,73.39,73.38,73.36,73.35,72.71,72.68,72.64,72.63,71.2,71.1,69.21,69.19,69.18,69.0,64.86,64.83,64.79,41.7; 31 P NMR (243MHz, D 2O)δ2.77,2.43; MALDI-TOF m / z calcd forC 34 H 67 NO 35 P 3 - :[M+2H]:1142.2712,found:1142.2704.

[0287] Embodiment 19:

[0288] Compound 4*:

[0289] The reaction equation is Figure 4 A and Figure 5 As shown;

[0290] According to the above general procedure A, the dimer acceptor 30* (800 mg, 0.437 mmol) was condensed with the phosphoramidite donor 25* (1.1 g, 0.524 mmol) to obtain the tetramer 33* (1.61 g, 0.424 mmol, 97%). Then, the MMTr group was removed according to the general procedure B to obtain the tetramer acceptor 34* (1.45 g, 0.411 mmol, 97%). Finally, compound 34* (50 mg, 0.014 mmol) was fully deprotected according to the general procedure C to obtain the pure tetramer 4* (19.8 mg, 0.013 mmol) with an overall yield of 86%. 1 HNMR (600MHz, D 2 O)δ5.01(s,4H,1-H),4.57(dt,J=12.0,5.9Hz,4H,3-H),4.24–4.18(m,4H,2-H),4.14(td,J=6.4,2.8Hz,4H,4-H) ,4.07–4.01(m,4H),3.97–3.94(m,2H),3.94–3.92(m,3H),3.92–3.89(m,3H),3.89–3.87(m,2H),3.86(s,2H),3. 85–3.84(m,1H),3.84–3.81(m,4H),3.81–3.78(m,2H),3.77(s,1H),3.76(s,1H),3.74(d,J=6.2Hz,4H),3.72–3. 71(m,2H),3.69(d,J=3.2Hz,2H),3.68–3.66(m,2H),3.66–3.64(m,2H),3.64–3.56(m,4H),3.18(t,J=5.0Hz,2H); 13 C NMR (151MHz, D 2O)δ109.23,109.22,109.21,109.18,84.51,84.47,84.39,84.36,76.89,76.86,76.84 ,76.81,76.80,76.78,76.75,76.28,76.26,76.24,74.7,74.4,73.92,73.90,73.87,7 3.85,72.67,73.51,73.48,73.45,73.43,73.41,73.40,73.38,73.36,73.35,72.71,72.68,72.63,72.62,71.2,71.1,69.22,69.20,69.18,69.0,64.7,64.83,64.79,41.7; 31 P NMR (243MHz, D 2 O)δ2.77,2.43; MALDI-TOF m / z calcdfor C 44 H 85 NNaO 46 P 4 :[M+Na+2H] - :1510.3196,found:1510.3169.

[0291] Comparative Example 23:

[0292] Synthesis of compound 33*;

[0293] Compound 30* (50 mg, 0.027 mmol) and phosphoramidite monomer donor 25* (172 mg, 0.082 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then 5-ethylthiotetrazolyl (10.7 mg, 0.082 mmol) was added to the reaction system, stirred at room temperature for 2 hours, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:2). Iodine solution (1M pyridine aqueous solution, 5eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 33* (89.2 mg, 0.0235 mmol) with a yield of 87%.

[0294] Comparative Example 24:

[0295] Synthesis of compound 33*;

[0296] Compound 30* (50 mg, 0.027 mmol) and phosphoramidite monomer donor 25* (172 mg, 0.082 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Pyridinium trifluoroacetate (15.8 mg, 0.082 mmol) was then added to the reaction system, stirred at room temperature for 30 minutes, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:2). Iodine solution (1M pyridine aqueous solution, 5 eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The resulting crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 33* (93.4 mg, 0.0246 mmol) with a yield of 91%.

[0297] Comparative Example 25:

[0298] Synthesis of compound 33*;

[0299] Compound 30* (50 mg, 0.027 mmol) and phosphoramidite monomer donor 25* (340 mg, 0.162 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves were stirred at room temperature for 30 minutes. 4,5-dicyanoimidazole (19.1 mg, 0.162 mmol) was then added to the reaction system, stirred at room temperature for 2 hours, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:2). (+)-8,8-dichlorocamphorsulfonazine (0.5 M acetonitrile solution, 4 eq) was then added to the reaction mixture and stirred for 1 hour. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 33* (81 mg, 0.021 mmol) with a yield of 79%.

[0300] Comparative Example 26:

[0301] Synthesis of compound 33*;

[0302] Compound 30* (50 mg, 0.027 mmol) and phosphoramidite monomer donor 25* (172 mg, 0.082 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves, stirred at room temperature for 30 minutes. Then 1H-benzimidazol-3-ium trifluoromethanesulfonate (21.7 mg, 0.082 mmol) was added to the reaction system, stirred at room temperature for 30 minutes, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:2). TBHP (1M toluene solution, 2 eq) was then added to the reaction mixture and stirred for 30 minutes. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated aqueous ammonium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:1, v / v) to obtain a colorless oily compound 33* (92.3 mg, 0.0243 mmol) with a yield of 90%.

[0303] Embodiment 20:

[0304] Compound 5*:

[0305] The reaction equation is Figure 4 B and Figure 5 As shown;

[0306] According to the above general procedure A, the tetramer acceptor 34* (50 mg, 0.014 mmol) was condensed with the phosphoramidite donor 20* (25.6 mg, 0.021 mmol) to give the pentamer 35* (64 mg, 0.014 mmol, 97%), and then the MMTr group was removed according to the general procedure B, and finally the general deprotection was carried out according to the general procedure C to obtain the pure pentamer 5* (23 mg, 0.012 mmol) with an overall yield of 84%. 1 HNMR (600MHz, D 2O)δ5.05(d,J=4.8Hz,5H,1-H),4.61(q,J=6.7Hz,H,3-H),4.28–4.23(m,5H,2-H),4.18(td,J=6.4,3.3Hz,5H, 4-H),4.11–4.08(m,4H),4.07–4.05(m,2H),4.02–3.99(m,2H),3.99–3.96(m,5H),3.95–3.92(m,5H),3.89(s ,2H),3.88–3.86(m,5H),3.86–3.84(m,2H),3.82–3.79(m,4H),3.78(dd,J=6.2,1.8Hz,4H),3.76(s,3H),3.7 4(s,2H),3.73–3.71(m,3H),3.71–3.70(m,2H),3.70–3.67(m,4H),3.66–3.62(m,2H),3.23(t,J=5.0Hz,2H); 13 C NMR (151MHz, D 2 O) δ109.25,109.24,109.23,109.19,109.18,84.51,84.48,84.46,84.40,84.38,84.36,76.89,76. 87,76.84,76.81,76.78,76.75,76.33,76.28,76.26,76.24,74.7,74.4,73.92,73.90,73.86,73.8 5,73.51,73.48,73.46,73.43,73.41,73.40,73.38,73.37,73.35,72.71,72.68,72.63,72.62,71.19,71.15,71.12,71.09,69.22,69.20,69.18,69.0,68.1,67.69,67.66,64.9,64.83,64.79,41.7; 31 P NMR (243MHz, D 2 O)δ2.77,2.43; MALDI-TOF m / z calcd for C 54 H 103 NNa 2 O 57 P 5 :[M+2Na+2H] - :1878.3681,found:1879.3662.

[0307] Embodiment 21:

[0308] Compound 6*:

[0309] The reaction equation is Figure 4 A and Figure 5 As shown;

[0310] According to the above general procedure A, the tetramer acceptor 34* (600 mg, 0.170 mmol) was condensed with the phosphoramidite donor 25* (535 mg, 0.255 mmol) to obtain the hexamer 37* (879 mg, 0.16 mmol, 94%). Then, the MMTr group was removed according to the general procedure B to obtain the hexamer acceptor 38* (510 mg, 0.155 mmol, 97%). Finally, compound 38* (50 mg, 0.0096 mmol) was fully deprotected according to the general procedure C to obtain the pure hexamer 6* (18.3 mg, 0.008 mmol) with an overall yield of 76%. 1 H NMR (600MHz, D 2 O)δ5.17(s,3H,1-H),5.03(s,3H,1-H),4.55(q,J=5.1,2.7Hz,3H,3-H),4.43( dd,J=8.2,4.6Hz,3H,2-H),4.31–4.27(m,3H,3-H),4.23(d,J=4.5Hz,3H,2-H), 4.13(td,J=6.4,2.9Hz,3H,4-H),4.09(d,J=4.5Hz,1H),4.05(td,J=6.6,3.0H z,3H,4-H),3.94(d,J=16.5Hz,2H),3.91(dq,J=6.3,3.1Hz,6H),3.88(d,J=3.1 Hz,2H),3.86(d,J=3.8Hz,2H),3.85–3.84(m,3H),3.84–3.83(m,3H),3.83–3. 82(m,3H),3.81(d,J=3.7Hz,3H),3.81–3.80(m,2H),3.79–3.78(m,4H),3.77(d ,J=3.0Hz,4H),3.74(d,J=2.8Hz,3H),3.72(d,J=3.9Hz,2H),3.71(d,J=3.1Hz, 4H),3.69(s,3H),3.69(d,J=1.2Hz,2H),3.68–3.65(m,5H),3.65–3.60(m,8H); 13 C NMR (151MHz, D 2O) δ106.61,106.60,106.59,105.83,105.81,105.78,82.71,82.69,82.68,82.02,81.98,77.35,77.32,73.89,73.87,73.83,73. 82,73.78,72.18,72.15,72.13,71.96,71.89,71.86,71.84,70.21,70.18,70.15,70.1,68.61,68.58,62.4,62.33,62.32,62.2; 31 P NMR (243MHz, D 2 O)δ0.95,0.77; MALDI-TOF m / z calcd for C 64 H 125 N 2 Na 2 O 68 P 6 :[M+NH 4 +2Na+2H] - :2241.4611,found:2241.4586.

[0311] Comparative Example 27:

[0312] Synthesis of compound 38*;

[0313] Compound 37* (30 mg, 0.0055 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (2.1 μL, 0.0273 mmol) and trifluoroacetic anhydride (3.8 μL, 0.0273 mmol) were added respectively, and the reaction solution was stirred at room temperature for 6 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:3). The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 1:2, v / v) to obtain a colorless oily compound 38* (23.5 mg, 0.0045 mmol) with a yield of 82%.

[0314] Embodiment 22:

[0315] Compound 7*:

[0316] The reaction equation is Figure 4 B and Figure 5 As shown;

[0317] According to the above general procedure A, the hexameric acceptor 38* (50 mg, 0.01 mmol) was condensed with the phosphoramidite donor 20* (23 mg, 0.019 mmol) to give the heptamer 39* (57 mg, 0.009 mmol, 94%), and then the MMTr group was removed according to the general procedure B, and finally the general deprotection was carried out according to the general procedure C to obtain the pure heptamer 7* (19.7 mg, 0.0074 mmol) with an overall yield of 77%. 1 H NMR (600MHz, D 2 O)δ5.18(s,3H,1-H),5.05(s,4H,1-H),4.54–4.48(m,4H,3-H),4.43(dd,J=7.8,4.9Hz,3H,2-H),4.28–4.22(m,7H,2-H,3-H),4.21– 4.17(m,1H),4.12(q,J=6.1Hz,4H,4-H),4.07(dt,J=8.4,3.8Hz,3H,4-H),4.00–3.95(m,2H),3.95–3.91(m,7H),3.89–3.87(m,2H),3 .87–3.86(m,4H),3.85(d,J=2.7Hz,7H),3.84–3.82(m,8H),3.82–3.81(m,4H),3.81–3.78(m,6H),3.78–3.76(m,3H),3.76–3.74(m, 3H),3.74–3.73(m,2H),3.73–3.72(m,3H),3.72–3.71(m,3H),3.71(s,3H),3.70–3.68(m,4H),3.67–3.66(m,3H),3.66–3.62(m,6H); 13 C NMR (151MHz, D 2 O) δ106.65,106.64,106.63,106.28,106.27,106.24,106.23,83.1,82.33,82.32,82.29,82.28,76.75,76.72,74.22,74.21,74.2 0,73.60,74.57,72.19,72.16,72.14,72.0,71.9,70.56,70.53,70.2,70.1,68.56,68.54,62.7,62.6,62.45,62.40,62.36,62.3; 31 P NMR (243MHz, D 2 O)δ3.51,3.20; MALDI-TOF m / zcalcdfor C 74 H 139NNa 4 O 79 P 7 :[M+4Na+2H] - :2614.4650,found:2614.4301.

[0318] Embodiment 23:

[0319] Compound 8*:

[0320] The reaction equation is Figure 4 A and Figure 5 As shown;

[0321] According to the above general procedure A, the hexameric acceptor 38* (300 mg, 0.057 mmol) was condensed with the phosphoramidite donor 25* (301 mg, 0.143 mmol) to obtain the octamer 41* (392 mg, 0.054 mmol, 95%). Then, the MMTr group was removed according to the general procedure B to obtain the octamer acceptor 42* (366 mg, 0.053 mmol, 97%). Finally, compound 42* (50 mg, 0.0072 mmol) was fully deprotected according to the general procedure C to obtain the pure octamer 8* (17.2 mg, 0.0056 mmol) with an overall yield of 72%. 1 H NMR (600MHz, D 2 O)δ5.07(d,J=5.4Hz,8H,1-H),4.63(s,8H,3-H),4.27(s,8H,2-H),4.19(d,J=6.3Hz,8H,4-H),4.1 1(d,J=9.7Hz,8H),4.08–4.06(m,2H),4.03–4.01(m,3H),4.00–3.98(m,8H),3.97–3.94(m,9H),3.9 2–3.90(m,3H),3.89(d,J=2.9Hz,6H),3.87(d,J=6.6Hz,6H),3.83–3.81(m,3H),3.80(d,J=5.7Hz, 4H),3.79(s,3H),3.78(s,4H),3.75(s,4H),3.73(d,J=5.5Hz,5H),3.71(s,5H),3.70–3.61(m,8H); 13 C NMR (151MHz, D 2O)δ=106.76,106.75,106.74,106.73,106.72,106.71,106.69,106.6 8,82.04,82.02,82.01,81.97,81.89,81.87,81.85,74.33,74.31,74. 2,73.79,73.77,73.74,72.2,72.1,71.9,71.7,71.4,70.94,70.90,70 .85,70.2,70.1,68.7,68.6,66.72,66.69,62.5,62.36,62.32,62.28; 31 P NMR (243MHz, D 2 O)δ0.27,-0.09; MALDI-TOF m / z calcd for C 84 H 167 N 4 Na 3 O 90 P 8 :[M+3NH 4 +3Na] 2- :1494.3109,found:1494.3026.

[0322] Comparative Example 28:

[0323] Synthesis of compound 41*

[0324] Compound 38* (50 mg, 0.0096 mmol) and phosphoramidite monomer donor 25* (100 mg, 0.0478 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves were stirred at room temperature for 30 minutes. Pyridinium trifluoroacetate (10 mg, 0.0478 mmol) was then added to the reaction system, stirred at room temperature for 1 hour, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:3). Iodine solution (1M pyridine aqueous solution, 5 eq) was then added to the reaction mixture and stirred for 2 hours. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:3, v / v) to obtain a colorless oily compound 41* (55 mg, 0.0079 mmol) with a yield of 83%.

[0325] Embodiment 24:

[0326] Compound 9*:

[0327] The reaction equation is Figure 4 B and Figure 5 As shown;

[0328] The octamer acceptor 42* (50 mg, 0.0072 mmol) was condensed with the phosphoramidite donor 20* (23 mg, 0.018 mmol) according to the above general procedure A to give the nonamer 43* (55 mg, 0.0068 mmol, 94%), and then the MMTr group was removed according to the general procedure B, and finally the general deprotection was carried out according to the general procedure C to give the pure nonamer 9* (13.3 mg, 0.0039 mmol) with an overall yield of 54%. 1 HNMR (600MHz, D 2 O)δ5.16(s,3H,1-H),5.03(s,6H,1-H),4.49(q,J=6.5,5.9Hz,6H,3-H),4.42(t,J=6.5Hz,3H,2-H),4.25–4.21(m,9H,2-H ,3-H),4.20–4.15(m,2H),4.12–4.08(m,6H,4-H),4.06(t,J=6.6Hz,3H,4-H),3.98–3.95(m,2H),3.95–3.90(m,10H),3.89 –3.86(m,4H),3.86–3.85(m,5H),3.85–3.83(m,9H),3.83–3.81(m,7H),3.81(s,5H),3.78(s,6H),3.76(d,J=2.1Hz,4H), 3.73(d,J=7.0Hz,7H),3.72–3.70(m,7H),3.69(d,J=3.4Hz,5H),3.69–3.67(m,3H),3.67–3.65(m,5H),3.65–3.58(m,9H); 13 C NMR (151MHz, D 2 O) δ106.71,106.70,106.69,106.68,106.67,106.66,106.39,106.37,106.35,83.2,82.5,82.4,76.6,74. 5,74.3,73.6,73.4,73.3,72.21,72.16,72.06,71.9,70.7,70.6,70.5,70.2,70.1,68.6,62.7,62.5,62.4; 31 P NMR (243MHz, D 2 O)δ4.07,3.74; MALDI-TOF m / zcalcd for C 94 H178 NNa 3 O 101 P 9 :[M+3Na+5H] - :3284.6160,found:3284.6089.

[0329] Comparative Example 29:

[0330] Synthesis of compound 43*

[0331] Compound 42* (50 mg, 0.0072 mmol) and phosphoramidite monomer donor 20* (43.3 mg, 0.0361 mmol) were co-evaporated in toluene for 3 times. Under argon protection, the donor and the acceptor were dissolved in anhydrous acetonitrile (1 mL) and added Molecular sieves were stirred at room temperature for 30 minutes. Pyridinium trifluoroacetate (7 mg, 0.0361 mmol) was then added to the reaction system, stirred at room temperature for 1 hour, and the reaction was detected using TLC (petroleum ether / ethyl acetate, 1:3). Iodine solution (1M pyridine aqueous solution, 5 eq) was then added to the reaction mixture and stirred for 2 hours. After the mixture was diluted with ethyl acetate to quench the reaction, the molecular sieves were removed by diatomaceous earth filtration, and the filtrate was washed twice with saturated ammonium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography column chromatography (petroleum ether / ethyl acetate, 10:1 to 1:3, v / v) to obtain a colorless oily compound 43* (49.3 mg, 0.0061 mmol) with a yield of 85%.

[0332] Embodiment 25:

[0333] Compound 10*:

[0334] The reaction equation is Figure 4 A and Figure 5 As shown;

[0335] According to the above general procedure A, the octamer acceptor 42* (200 mg, 0.029 mmol) was condensed with the phosphoramidite donor 25* (151 mg, 0.072 mmol) to obtain the decamer 45* (231 mg, 0.026 mmol, 90%). Then, the MMTr group was removed according to the general procedure B to obtain the decamer acceptor 46* (212 mg, 0.0246 mmol, 95%). Finally, compound 46* (50 mg, 0.0058 mmol) was fully deprotected according to the general procedure C to obtain the pure decamer 10* (15 mg, 0.0042 mmol) with an overall yield of 61%. 1 H NMR (600MHz, D 2O)δ5.16(s,4H,1-H),5.03(s,6H,1-H),4.49(td,J=7.4,4.5Hz,6H,3-H),4.42(dd,J=7.9,4.7Hz,4H,2-H),4.23(d,J=4.8Hz,10H,2 -H,3-H),4.19–4.15(m,2H),4.11(dd,J=6.5,4.1Hz,6H,4-H),4.06(td,J=6.6,3.1Hz,4H,4-H),3.98–3.95(m,2H),3.92(qd,J=6.4, 2.5Hz,11H),3.88–3.85(m,10H),3.85–3.83(m,11H),3.82(d,J=3.1Hz,6H),3.82–3.80(m,7H),3.80–3.77(m,6H),3.76(d,J=2.8Hz ,5H),3.74–3.72(m,7H),3.71(d,J=3.1Hz,8H),3.70–3.68(m,6H),3.68(d,J=2.9Hz,3H),3.66(d,J=6.2Hz,6H),3.65–3.60(m,8H); 13 C NMR (151MHz, D 2 O) δ106.72,106.70,106.68,106.66,106.64,106.62,106.42,106.38,106.35,106.3,83.2,83.1,83.5,82.41 ,83.36,76.65,76.63,74.5,74.3,73.6,72.21,72.15,72.06,71.9,70.6,70.2,70.1,68.6,62.7,62.5,62.4; 31 PNMR(243MHz,D 2 O)δ4.07,3.74; MALDI-TOF m / z calcd for C 104 H 196 NNa 4 O 112 P 10 :[M+4Na+5H] - :3652.6645,found:3652.6577.

[0336] Comparative Example 30:

[0337] Synthesis of compound 46*;

[0338] Compound 45* (30 mg, 0.0034 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1.3 μL, 0.0135 mmol) and trifluoroacetic anhydride (1.9 μL, 0.0135 mmol) were added respectively, and the reaction solution was stirred at room temperature for 6 hours. The reaction was detected by TLC (petroleum ether / ethyl acetate, 1:4). The reaction solution was diluted with dichloromethane, and the organic phase was extracted and washed with a saturated sodium bicarbonate solution. The organic phase was treated with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate, 10:1 to 1:4, v / v) to obtain a colorless oily compound 46* (22.1 mg, 0.0026 mmol) with a yield of 76%.

[0339] Embodiment 26:

[0340] Compound 11*:

[0341] The reaction equation is Figure 4 B and Figure 5 As shown;

[0342] According to the above general procedure A, the decamer acceptor 46* (50 mg, 0.0058 mmol) was condensed with the phosphoramidite donor 20* (21 mg, 0.017 mmol) to give the undecamer 47* (46 mg, 0.0053 mmol, 91%), and then the MMTr group was removed according to the general procedure B, and finally the general deprotection was carried out according to the general procedure C to obtain the pure undecamer 11* (14.4 mg, 0.0035 mmol) with an overall yield of 60%. 1 HNMR (600MHz, D 2O)δ5.22–5.16(m,4H,1-H),5.06–5.04(m,7H,1-H),4.65–4.47(m,7H,3-H),4.43(dd,J=7.9,4.7Hz,4H,2-H),4.26–4.22(m,11H,2-H,3- H),4.20–4.17(m,2H),4.11(td,J=6.5,3.9Hz,7H,4-H),4.07(td,J=6.5,3.0Hz,4H,4-H),3.98–3.96(m,2H),3.96–3.91(m,12H),3.89–3 .87(m,3H),3.86(t,J=2.6Hz,8H),3.85–3.84(m,11H),3.83(d,J=3.0Hz,5H),3.83–3.80(m,11H),3.80–3.78(m,8H),3.77(d,J=2.8Hz,6 H),3.75–3.74(m,4H),3.73(s,3H),3.73–3.72(m,9H),3.71–3.69(m,8H),3.69(d,J=2.9Hz,3H),3.68–3.66(m,5H),3.66–3.60(m,11H); 13 C NMR (151MHz, D 2 O) δ106.72,106.70,106.68,106.66,106.64,106.63,106.61,106.41,106.39,106.36,106.3,83.2,82.8,82.5,8 2.4,82.3,76.6,76.8,74.5,74.3,73.6,73.4,72.21,72.17,72.1,71.9,70.6,70.2,70.1,68.6,62.7,62.5,62.4; 31 P NMR (243MHz, D 2 O)δ4.07,3.74; MALDI-TOF m / zcalcdfor C 114 H 227 N 4 O 123 P 10 :[M+3NH 4 +6H] 2- :1980.9394,found:1980.9313.

[0343] Embodiment 27:

[0344] Compound 12*:

[0345] The reaction equation is Figure 4 A and Figure 5 As shown;

[0346] According to the above general procedure A, the decamer acceptor 46* (50 mg, 0.0058 mmol) was condensed with the phosphoramidite donor 25* (37 mg, 0.017 mmol) to give the dodecamer 49* (54 mg, 0.0052 mmol, 89%), and then the MMTr group was removed according to the general procedure B, and finally the general deprotection was carried out according to the general procedure C to obtain the pure dodecamer 12* (14.7 mg, 0.0032 mmol) with an overall yield of 56%. 1 HNMR (600MHz, D 2 O)δ5.18(s,5H,1-H),5.04(d,J=7.8Hz,7H,1-H),4.54–4.48(m,7H,3-H),4.43(dd,J=7.9,4.7Hz,5H,2-H),4.28–4.22(m,12H,2-H,3-H) ,4.19–4.17(m,2H),4.14–4.11(m,7H,4-H),4.08–4.05(m,5H,4-H),3.98–3.96(m,2H),3.95–3.92(m,12H),3.89–3.88(m,4H),3.87(d,J =3.6Hz,5H),3.86–3.84(m,10H),3.84–3.83(m,7H),3.83(s,4H),3.82–3.81(m,8H),3.80(d,J=3.1Hz,9H),3.78(s,2H),3.78–3.76(m, 4H),3.76–3.74(m,5H),3.74(s,2H),3.73–3.72(m,10H),3.71(d,J=6.1Hz,7H),3.69(s,4H),3.67(d,J=5.7Hz,7H),3.66–3.60(m,12H); 13 C NMR (151MHz, D 2 O)δ106.66,106.65,106.64,106.63,106.62,106.28,106.27,106.26,1 06.24,106.23,83.1,82.33,82.28,76.8,76.7,74.22,74.20,74.19,73. 60,73.59,73.57,72.19,72.15,72.14,72.13,72.0,71.9,70.6,70.5,70 .2,70.1,68.56,68.54,62.65,62.61,62.45,62.40,62.36,62.35,62.3;31 PNMR(243MHz,D 2 O)δ3.51,3.20; MALDI-TOF m / z calcd for C 124 H 235 NNa 2 O 134 P 12 :[M+2Na+8H] 2- :2150.4148,found:2150.4033.

[0347] Embodiment 28:

[0348] Construction and testing of oligosaccharide arrays;

[0349] The chemically synthesized 1-12PRP oligosaccharide fragments were fixed on the chip surface through amino linker arms, and the binding affinity between the antibodies and oligosaccharide fragments in the polysaccharide antiserum and polysaccharide protein antiserum was detected.

[0350] Specifically include the following steps:

[0351] 1) A solution of oligosaccharides and CPS in coupling buffer (50 mM sodium phosphate, pH 8.5) was printed onto a "CodeLink" glass slide (SurModics Inc.) using a biochip spotter (Rayme, China). After incubation overnight in a humidified chamber (25°C, 65% humidity), the slide was placed in a quenching buffer (50 nM Na 2 HPO 4 , 100 nM ethanolamine, pH 9) for 1 h.

[0352] 2) The slides were blocked with 3% BSA / PBS (w / v) at 4°C overnight. The slides were rinsed once with PBST and twice with PBS. The residual liquid was removed by centrifugation and the multiwell culture chamber (ProPlate) was placed on top of the slide. Rabbit serum diluted 1:50 with 1% BSA / PBS (w / v) was added to the incubation chamber on the microarray. There were at least two replicates for each sample. The microarray was incubated overnight in a dark and humid chamber at 4°C. The diluted serum was removed and each well was washed three times with PBST. Goat anti-rabbit IgG (Alexa Fluor 532, Thermo Fisher) or anti-rabbit IgM (DyLight 550, Abcam) was diluted 1:400 in 1% BSA / PBS (w / v) and added to the wells. The microarray was incubated in a dark and humid chamber at 37°C for 45 minutes. After rinsing with PBST, the culture chamber was removed and the slides were rinsed with distilled water for 10 minutes. After centrifugation to remove residual liquid, the slides were scanned with LuxScan 10K / B (CapitalBio 9Technology). Image analysis was performed using GenePix Pro 7 software (Molecular Devices).

[0353] The results are as follows Figure 4 As shown, almost all oligosaccharide fragments were recognized by IgG antibodies in both antisera, among which 2PRP, 4PRP, 6PRP, 11PRP and 12PRP showed strong binding, 8PRP, 5PRP and 7PRP showed moderate binding, and other fragments showed only weak binding.

[0354] Embodiment 29:

[0355] Preparation of glycoprotein conjugates of oligosaccharide compounds and TT;

[0356] To further validate the optimal antigenic epitope of Hib CPS, the synthesis of oligosaccharide protein conjugates was performed. Based on the glycan microarray results and reports of suboptimal antibody levels against the 2PRP antigen (Evenberg et al., J Infect Dis, 1992, 165: S152-S155.), we selected 4PRP, 6PRP, 8PRP, 11PRP and 12PRP as Hib vaccine candidate antigens for further evaluation. The results are shown in Figure 2. Figure 5 As shown in the figure, 4PRP-TT induced a higher level of antibodies. Therefore, 4PRP is a key immune epitope and can be used as an important antigenic substance for semi-synthetic sugar vaccines.

[0357] Specifically include the following steps:

[0358] 1) Activation of Hib oligosaccharide antigen: Dissolve Hib oligosaccharide antigen (3 mg) in 0.3 mL dimethyl sulfoxide (DMSO) solution, add 3-maleimidopropionic acid N-hydroxysuccinimide ester (SMP) dissolved in 0.2 mL dimethyl sulfoxide under stirring, the molar equivalent ratio of SMP to Hib oligosaccharide antigen is between 6:1, the reaction temperature is room temperature, the reaction time is 10 hours, purify by Sephadex LH-20 column chromatography (H2O), and obtain activated Hib oligosaccharide antigen after reduced pressure distillation;

[0359] 2) Thiolation of carrier protein: Dissolve the carrier protein (1 mg) in 1 mL PBS buffer (pH 8, containing 1.86 mg / mL EDTA), add dithiobis(succinimidyl propionate) (DSP) dissolved in 50 ul dimethyl sulfoxide under argon protection, the molar equivalent ratio of DSP to carrier protein is between 8:1, the reaction temperature is room temperature, and the reaction time is 8 hours. After overnight reaction, place it in an ice bath, add dithiothreitol (DTT) at a concentration of 0.025 mmol / mL under argon protection, and react at 0°C for 3 hours. Then dilute the mixture with PBS buffer (PH 7.2, containing 1.86 mg / mL EDTA), and centrifuge it 3 times on a 50 kDa ultrafiltration membrane with PBS buffer (PH 7.2, containing 1.86 mg / mL EDTA). The solution is stored at 0°C;

[0360] 3) Coupling of Hib oligosaccharide antigen with carrier protein: The activated Hib oligosaccharide antigen was dissolved in 0.5 mL PBS buffer (PH 7.2), and the thiol carrier protein dissolved in 2 mL PBS buffer (PH 7.2, containing 1.86 mg / mL EDTA) was added under stirring. After reacting at 4-8°C for 6 hours, 0.1 mg N-ethylmaleimide was added to terminate the reaction. The mixture was then diluted with PBS buffer (PH 7.2), and centrifuged three times with PBS buffer (PH 7.2) on a 50 kDa ultrafiltration membrane to obtain Hib oligosaccharide protein conjugate.

[0361] The structural formula of the synthetic conjugate is as follows:

[0362]

[0363] Where n is any one of 3, 5, 7, 11, 12, and m is any number from 1 to 30.

[0364] The calculated sugar loadings in the oligosaccharide conjugates were 18.9, 7, 5, 7.6, 8.2, and 8.1, respectively.

[0365] Comparative Example 31:

[0366] Oligosaccharide compounds 4PRP and CRM197 Preparation of glycoprotein conjugates;

[0367] According to the preparation method of the oligosaccharide compound and TT glycoprotein conjugate described in Example 5, the oligosaccharide compound 4PRP was conjugated with CRM 197 The conjugation was performed to obtain the sugar conjugate 4PRP-CRM197, in which the sugar loading amount was calculated to be 16.8, and its structure is shown below:

[0368]

[0369] Embodiment 30:

[0370] Oligosaccharide protein conjugates 4PRP-TT and 4PRP-CRM 197 Determination of immunogenic antibody titer;

[0371] Freund's adjuvant and oligosaccharide protein conjugates 4PRP-TT and 4PRP-CRM 197 New Zealand white rabbits were immunized subcutaneously with the mixture. At the same time, a commercial Hib vaccine (whose PRP was obtained by fermentation and then covalently coupled with TT) was used as a positive control and PBS (containing Freund's adjuvant) was used as a negative control. The injection volume was calculated based on the amount of oligosaccharide, 5 μg / rabbit, four rabbits per group, and three immunizations were performed with an interval of two weeks. Antiserum was collected 7 days after the last injection for immunological evaluation. CPS was used as a fixed antigen, and the titer of polysaccharide-specific antibodies was detected by enzyme-linked immunosorbent assay (ELISA).

[0372] ELISA analysis of sera after immunization Figure 6 As shown, 4PRP-CRM 197 Both oligosaccharide protein conjugates and 4PRP-TT can induce high levels of IgG antibodies that specifically recognize Hib CPS. This means that the carrier protein CRM 197 It has the ability to promote and help Hib oligosaccharides to convert into T cell-dependent antigens similar to TT. 197 The specific antibody titer induced by 4PRP-TT was lower than that induced by 4PRP-TT, which revealed the advantage of TT as a carrier protein in inducing immune response. This result shows that the oligosaccharide protein conjugate 4PRP-TT is a very promising candidate for anti-Haemophilus influenzae type b vaccine.

[0373] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A chemical synthesis method of an intermediate A, the structure of the intermediate A is as follows: The chemical synthesis method comprises the following steps: (a) using compound A-1 and compound A-2 as substrates, and carrying out glycosylation reaction under the action of ytterbium trifluoromethanesulfonate to obtain compound A-3; (b) Compound A-3 is treated in an alkaline solution to obtain compound A-4; (c) Compound A-4 and dibutyltin oxide are mixed and reacted in toluene for a period of time, and then a protecting agent is added to protect the 2- and 5-hydroxyl groups to obtain an intermediate compound A; in, R2, R3, R2', R3' are independently selected from: acetyl, benzoyl, pivaloyl, chloroacetyl, levulinyl, allylcarbonyl, benzyl, p-methoxybenzyl, allyl; R4, R5, and R6 are independently selected from the group consisting of benzyl, p-methoxybenzyl, allyl, acetyl, benzoyl, pivaloyl, chloroacetyl, levulinyl, and allylcarbonyl; R7 is selected from the group consisting of: 4-monomethoxytrityl, allyl, trityl, 4,4'-dimethoxytrityl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triethylsilyl.

2. The method according to claim 1, characterized in that The glycosidation reaction comprises: A disaccharide fragment is synthesized by using 5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol and tetraacetyl ribose for glycosylation reaction; the reaction is carried out in a solvent, which is dichloromethane or N,N-dimethylformamide; the reaction concentration in the glycosylation reaction is 0.01-0.1 mmol / mL; the reaction temperature is between 0°C and 60°C, and the reaction time is between 3 hours and 72 hours.

3. The method according to claim 1, characterized in that In step (c), the hydroxyl groups at positions 2 and 5 of the ribose group are selectively protected, and the corresponding process is: adding dibutyltin oxide, sodium hydride, an additive, and a protecting agent, and reacting to obtain a corresponding product; wherein the additive is tetrabutylammonium iodide and / or tetrabutylammonium bromide; and the protecting agent is benzyl bromide.

4. A chemical synthesis method of intermediate B, the structure of which is shown below: The chemical synthesis method comprises the following steps: Obtaining intermediate compound A according to steps (a) to (c) of claim 1; (d) Compound A is mixed with a protecting agent to protect the 3-hydroxyl group to obtain Compound B-1; (e) removing R7 from compound B-1 to obtain compound B; in, R2, R3, R2', R3', R4, R5, R6 and R7 have the same definitions as in claim 1.

5. A chemical synthesis method of an intermediate compound III, the structure of the intermediate compound III is shown below: The chemical synthesis method comprises the following steps: Obtaining intermediate compound A according to steps (a) to (c) of claim 1; (f) Compound A is mixed with a phosphitylation agent to react to obtain Compound III; in, R2, R3, R2', R3', R4, R5, R6, R7 are as defined in claim 1; R1 is selected from acetonitrile, allyl, benzyl; R a , R b are independently selected from C 1-4 alkyl.

6. The method according to claim 5, characterized in that Step (f) is a phosphoramidite protection process for the ribose 3-position, and the phosphitylation reagent used for phosphoramidite protection is selected from: 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite), bis(diisopropylamino)(2-cyanoethoxy)phosphine.

7. A chemical synthesis method of an intermediate compound IV, the structure of the intermediate compound IV is shown below: The method comprises: S1, using the above compound III and compound B as substrates, carrying out a phosphoramidite coupling reaction under the action of a catalyst, followed by oxidation to obtain an intermediate C; S2, removing the 3-hydroxy protecting group from intermediate C to obtain compound D; S3, compound D is mixed with a phosphitylation agent to react to obtain compound IV; Among them, R2, R3, R3', R4, R5, R6, R7, R1, R a , R b The definition is the same as that of claim 5.

8. A method for synthesizing a capsular polysaccharide derivative of Haemophilus influenzae type b assembled with a linker arm, wherein the structure of the capsular polysaccharide derivative is shown in general formula I: Wherein, n is any integer from 0 to 11; R is hydrogen ion, sodium ion, ammonium ion; Linker is -L-NH2, wherein L represents a linker arm; the linker arm L is diethylene glycol, or a chain structure with 2-40 carbon atoms containing 0-6 heteroatoms, a substituted or unsubstituted three- to six-membered ring structure, an amide bond, or a urea group; The method comprises the following steps: (1) The intermediate compound III and the linker reagent are mixed to react to obtain compound E; (2) Compound E is reacted in a mixed system of boron trifluoride ether complex / methanol / organic solvent to obtain Compound II; (3) Compound II undergoes a phosphoramidite coupling reaction with the intermediate compound III to obtain compound F; (4) Compound F is reacted in a mixed system of boron trifluoride ether complex / methanol / organic solvent to obtain compound V; (5) Using the obtained product as a substrate, repeating steps (3)-(4) to obtain compound VI; (6) reacting the obtained compound VI in a mixed system of boron trifluoride ether complex / methanol / organic solvent to obtain compound VII; (7) Compound VII and intermediate compound III or intermediate compound IV undergo phosphoramidite coupling reaction in the presence of a catalyst, followed by oxidation to obtain compound G or compound H, respectively; (8) removing the protecting agent from compound E, compound G or compound H, and then reducing the azide group to obtain the target compound of formula I; Among them, R2, R3, R3', R4, R5, R6, R7, R1, R a , R b The definition is the same as that of claim 5, and n' is 0-10.

9. The method according to claim 7 or 8, characterized in that: In the phosphoramidite coupling reaction, the catalyst is selected from any one or more of the following: 1-phenyl-1H-imidazol-3-ium trifluoromethanesulfonate, 1H-tetrazole, 5-benzylthiotetrazole, 5-ethylthiotetrazole, 4,5-dicyanoimidazole, 2-bromo-4,5-dicyanoimidazole, trifluoroacetic acid, 2,4-dinitrobenzoic acid, trichloroacetic acid, pyridinium trifluoroacetate, imidazolium trifluoromethanesulfonate, saccharin 1-methylimidazole, imidazolium perchlorate, 1H-benzimidazol-3-ium trifluoromethanesulfonate, and N-methylbenzimidazole trifluorate.

10. The method according to claim 7 or 8, characterized in that: In the phosphoramidite coupling reaction, when the phosphoramidite coupling reaction is completed, the trivalent phosphorus in the phosphoric acid group is immediately oxidized to pentavalent phosphorus; the oxidant used for oxidation is any one or more of iodine (I2) / water / pyridine, (+)-8,8-dichlorocamphorsulfonazine (DCSO), tert-butyl hydroperoxide (TBHP) / toluene.

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