Process for the synthesis of reactive carbohydrate containing antigen precursors for conjugation with carrier materials

Abstract

Carbohydrates of formula (A) [Formula 1] JPEG2024504658000078.jpg20170 (In the formula, R 1 is H and n is an integer from 1 to 5) is a compound (A 0 ) [chemical 2] JPEG2024504658000079.jpg21170 HO-(CH 2 ) n -CH=CH 2 and (B) in the presence of an acid capable of liberating a proton, while heating, and cooling the reaction mixture to obtain a cooled mixture containing compound (A). Formula (AA PG ) carbohydrates [C3] JPEG2024504658000080.jpg27170 (In the formula, R 2 is a monovalent protecting group, PG is a divalent protecting group, and n is an integer of 1 to 5), is reacted with a compound represented by the formula (A PG ) Compound [C4] JPEG2024504658000081.jpg28170 The formula (A 1 ) Compound [C5] JPEG2024504658000082.jpg20170 (wherein X is -I, -Br, -Cl, -CN, -OTf (triflate), -OMs (mesylate), -OTs (tosylate), methyl sulfate, or trichloroacetimidate). PG ) is deprotected to obtain compound (AA) [C6] JPEG2024504658000083.jpg24170 is formed. The carbohydrates (A) and (AA) can be coupled with a thiolated linker to form a glycoconjugate precursor, which can then be conjugated to a carrier material such as a protein, peptide, or polypeptide.

Description

[Technical field]

[0001] The present specification relates to processes for the synthesis of reactive carbohydrates, such as antigen precursors, useful for conjugation to carrier materials (e.g., proteins), as well as processes for the preparation of glycoconjugates from reactive carbohydrates, useful as immunogens and as therapeutic / diagnostic tools.

[0002] This specification refers to a number of documents, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0003] The ultimate goal of immunotherapy is to treat diseases such as infections or cancer by modulating the innate and adaptive responses of the immune system to improve its ability to fight foreign agents such as bacteria, viruses, and cancer cells. Innate immunity is considered the first line of immune defense, triggered at the early stage of exposure to pathogens. The responsible cells include natural killer (NK) cells, dendritic cells (DCs), macrophages, monocytes, γδT cells, and natural killer T (NKT) cells. Unlike the innate immune system, adaptive immunity is slower to develop upon initial exposure to foreign antigens, but develops highly specific responses and creates immune memory for long-lasting protection. This involves the clonal expansion of T and B cells and their humoral and cellular mediators, cytokines, and antibodies. The main interface between the innate and adaptive immune responses are professional antigen-presenting cells (pAPCs); macrophages, B cells, and especially dendritic cells (DCs). pAPCs can process and present antigens from endogenous and exogenous sources to T cells. They recognize microorganisms through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). In recognition of microbial surface determinants or abnormal and non-natural antigens, microorganisms or tumors and their associated antigenic markers can be engulfed by pAPCs via endocytic pathways, where they are typically degraded into peptide fragments and the released antigens are bound on intracellular MHC class I or class II molecules (pMHC). pAPCs undergo maturation and activation, resulting in redistribution of pMHC complexes from intracellular compartments to the cell surface and secretion of cytokines and chemokines. In addition to pAPCs, all nucleated cell types present only endogenous peptides on the cell membrane. In contrast to pAPCs, peptides originating from within the cell itself, including viruses and intracellular pathogens, are presented by MHC class I molecules coupled to b2-microglobulin.APCs typically do not express MHC class II.

[0004] Peptides presented on MHC class II molecules are typically recognized by T cell antigen receptors (TCRs) on CD4+ T helper cells, which then undergo functional maturation into different subsets, such as Th1 or Th2 cells, when costimulatory signals are received from pAPCs. Th1 cells cause proinflammatory responses, mainly with the secretion of IFN-γ and TNF-α, while Th2 cells secrete typical cytokines. Although Th1 cells are primarily associated with cell-mediated responses, both types of Th cells support the production of antibodies by B cells, which in turn influences antibody isotype and function. For example, IL-12 and TNF-α are associated with the differentiation of Th1 cells and the production of type 1 IgG subclass, while IL-6 and other Th2 cytokines contribute to type 2 IgG subclass (IgG1) production.

[0005] APCs presenting peptides on MHC class I molecules are recognized by the TCR of CD8+ cytotoxic T cells. Several further interactions between costimulatory molecules expressed by the two cell types trigger activation of cytotoxic T cells into effector cells, but when dendritic cells interact with both activated T helper cells and T cytotoxic cells, potent and long-lasting memory T cells are generated. Once activated, T cells undergo clonal selection and proliferation with the help of cytokines. This increases the number of cells specific for the dysfunctional target antigen, which can then migrate throughout the body in search of dysfunctional antigen-positive somatic cells. Upon docking to a target cell, activated cytotoxic T cells release a payload of cytotoxins such as perforin and ganzymes. The action of perforin allows granzymes to penetrate the target cell, where their proteases cause cell death. Cytotoxic T cells can also induce target cell death by the FAS signaling pathway.

[0006] It is therefore desirable to be able to tailor vaccine-induced immunity to an appropriate response to address a pathogen or tumor antigen of interest.

[0007] Carbohydrates, in contrast to proteins and peptides, are T-cell-independent antigens that are not adequately equipped to trigger Th cell engagement and therefore are unable to induce immune cell proliferation, antibody class switching, and affinity / specificity maturation. The first major early advances made with carbohydrate-based vaccines were supported by the discovery that bacterial capsular polysaccharides, when appropriately conjugated to carrier proteins that serve as T-cell-dependent epitopes, were now able to acquire the immunochemical capabilities required to generate opsonophagocytic antibodies.

[0008] Traditionally, strategies for conjugating carbohydrate antigens to carrier proteins rely on either reductive amination of aldehyde-derived sugars onto the ε-amino groups of lysine residues or simply amide coupling reactions. In both cases, partial and random carbohydrate antigen conjugation generally occurs. Furthermore, if all amide partners (amines from lysine or acids from glutamic / aspartic acid) are used for carbohydrate conjugation, too many carbohydrate antigens are attached to the carrier protein, resulting in masking of potentially essential T cell peptide epitopes with an inherent reduction / elimination of immunogenicity. For this reason, current strategies for preparing glycoconjugate vaccines are insufficient and face significant regulatory and / or commercial obstacles because the preparations lack the necessary uniformity in terms of their carbohydrate distribution and reproducibility (i.e., the attachment points of sugars to proteins are randomly distributed and of varying density from batch to batch). For this reason, glycoconjugate vaccines with higher carbohydrate antigen homogeneity, more precisely characterizable structures, and batch-to-batch reproducibility are highly desirable.

[0009] In addition, the synthesis of reactive carbohydrates useful as antigen precursors for conjugation with proteins can present a variety of challenges: current synthetic routes can be challenging with respect to stereoselectivity control, leading to the acquisition of intermediate stereoisomers that can be difficult to separate and / or low yields of the desired stereoisomer.

[0010] SARS-CoV-2, the causative pathogen of the COVID-19 pandemic that began in late 2019, is an ongoing threat to global human health that has also crippled the global economy. Early vaccine development efforts were primarily focused on protein antigens and epitopes present on the spike (S) glycoprotein that mediates cell entry and membrane fusion of SARS-CoV-2 into host cells. However, health experts around the world have strongly encouraged scientists to explore different strategies in parallel to develop therapeutic interventions against SARS-CoV-2 to mitigate the possibility of failure or complications that may arise for a single strategy, including new mutations in the virus that may evade current vaccine strategies directed at protein epitopes. For this reason, there remains a need to develop vaccines and other therapeutic tools against SARS-CoV-2 in parallel with those focused on protein antigens present on the S protein of SARS-CoV-2. Summary of the Invention

[0011] In a first aspect, a reactive carbohydrate of formula (A)

[0012] [ka] (In the formula, R 1 is H and n is an integer from 1 to 5, comprising the steps of:

[0013] [ka] HO-(CH2) nDescribed herein is a process that includes reacting with heating, in the presence of an acid capable of liberating a proton, a -CH=CH2 to produce a reaction mixture comprising a compound of formula (A), and cooling the reaction mixture to obtain a cooled mixture comprising a compound of formula (A).

[0014] In some embodiments, reacting the compound of Formula (A0) comprises reacting the compound of Formula (A0) with HO—(CH) n A compound of formula (A0), HO-(CH2) n The method includes forming a low temperature intermediate reaction mixture containing -CH=CH2, and HCl.

[0015] In a further embodiment, the compound of formula (AA PG ) reactive carbohydrates

[0016] [ka] (In the formula, R 2 is a monovalent protecting group, PG is a divalent protecting group, and n is an integer from 1 to 5, comprising reacting a compound of formula (A PG ) Compound

[0017] [ka] with a compound of formula (A1)

[0018] [ka] where X is -I, -Br, -Cl, -CN, -OTf (triflate), -OMs (mesylate), -OTs (tosylate), methylsulfate, or trichloroacetimidate.

[0019] In some embodiments, the formula (A PG The reaction between the compound of formula (A1) and the compound of formula (A2) is carried out in the presence of molecular sieves.

[0020] In some embodiments, the formula (A PG ) is a compound of formula (A)

[0021] [ka] (In the formula, R 1 is H and has a protecting group PG.

[0022] In some embodiments, the compound of formula (A) is obtained by a process defined herein.

[0023] In a further embodiment, the reactive carbohydrate of formula (AA)

[0024] [ka] wherein n is an integer from 1 to 5, PG Described herein is a process that includes deprotecting a compound of formula (I).

[0025] In some embodiments, the deprotection comprises a compound of formula (AA PG ) with a basic solution to form an intermediate product, and then treating the intermediate product with an acidic solution.

[0026] In a further embodiment, the reactive carbohydrate of formula (AA)

[0027] [ka] A process for synthesizing a compound of formula (AA PG )

[0028] [ka] (In the formula, n is an integer of 1 to 5, and R 2 is a monovalent protecting group and PG is a divalent protecting group, PG Described herein is a process that includes reacting a compound of formula (I) with a basic solution to form an intermediate crude product, and then treating the intermediate crude product with an acidic solution.

[0029] In a further embodiment, the reactive carbohydrate of formula (B) or formula (BB)

[0030] [ka] wherein n is an integer from 1 to 5, m is an integer from 1 to 5, and LG is a leaving group that allows for amide bond formation when a compound of formula (B) or (BB) reacts with an amino group, comprising:

[0031] [ka] with a compound of formula (A)

[0032] [ka] (In the formula, R 1 is H) to form a compound of formula (B) or a compound of formula (AA)

[0033] [ka] to form a compound of formula (BB).

[0034] In some embodiments, the compound of formula (A) is obtained by a process defined herein.

[0035] In some embodiments, the compound of formula (AA) is obtained by a process defined herein.

[0036] In some embodiments, reacting the compound of Formula (C) with the compound of Formula (A) or (AA) is carried out in the presence of a photoinitiator.

[0037] In some embodiments, reacting the compound of Formula (C) with the compound of Formula (A) or (AA) is carried out under UV irradiation.

[0038] In some embodiments, reacting the compound of Formula (C) with the compound of Formula (A) or (AA) is carried out under UV irradiation in the presence of a photoinitiator.

[0039] In some embodiments, reacting the compound of Formula (C) with the compound of Formula (A) or (AA) is carried out under visible light in the presence of a visible light absorbing transition metal photocatalyst.

[0040] In some embodiments, m in formula (B), (BB), and / or (C) is an integer from 1 to 3. In some embodiments, m is 2.

[0041] In a further embodiment, the glycoconjugate of formula (I) or (II)

[0042] [ka] (In the formula, n is an integer of 1 to 5, m is an integer of 1 to 5, and p is an integer of 1 to 50,

[0043] [ka] is a carrier material comprising at least one amino group available for conjugation, which comprises conjugating at least one free amino group of the carrier material with a compound of formula (B) to form a glycoconjugate of formula (I) or with a compound of formula (BB) to form a glycoconjugate of formula (I), wherein the compounds of formula (B) and (BB) are obtained by the process defined herein.

[0044] In some embodiments, the carrier material comprises a protein, polypeptide, or peptide. In some embodiments, in the processes defined herein, n is an integer from 1 to 3. In some embodiments, n is 1 or 2. In some embodiments, n is 1.

[0045] In a further embodiment, the compound of formula (AA PG ) Compound

[0046] [ka] wherein n is an integer from 1 to 5; and R 2 is Ac and PG is a divalent protecting group selected from PhCH, Me2C, O=C, Me2Si, and 2,6-bis(trifluoromethyl)phenyl-B.

[0047] In some embodiments, the compound (AA PG ) PG is PhCH.

[0048] In some embodiments, the compound (AA PG ) n is an integer from 1 to 3. In some embodiments, n is 1 or 2. In some embodiments, n is 1.

[0049] In some embodiments, the compound (AA PG ) is expressed as follows:

[0050] [ka] has.

[0051] In a further aspect, the compound of formula

[0052] [ka] The compounds of the formula are described herein.

[0053] general definition Headings and other identifiers, e.g., (a), (b), (i), (ii), etc., are provided merely to facilitate the readability of the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0054] The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0055] The term "about" is used to indicate that a value includes the standard deviation of error of the device or method used to determine the value. In general, the term "about" is meant to indicate a possible variation of up to 10%. Thus, variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of a value are included within the term "about". Unless otherwise indicated, the use of the term "about" before a range applies to both ends of the range.

[0056] As used in this specification and the claims, the terms "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "includes" and "include"), or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0057] The phrase "leaving group" or "LG," as used herein, refers to a leaving group that provides improved reaction efficiency and / or specificity (i.e., improved conjugation to free amine groups of a polypeptide or protein) compared to the corresponding functional group prior to replacement by the leaving group. In some embodiments, the leaving group can be an O-fluorophenyl group, such as OPhF5 or OPhF4(para SO3Na), or an O-(N-succinimidyl) group.

[0058] As used herein, the term "support material" refers to a material that can be conjugated to a carbohydrate antigen (e.g., an antigenic monosaccharide, disaccharide, oligosaccharide, or polysaccharide, e.g., a natural or synthetic antigen). In some aspects, the support material may be conjugable to a carbohydrate antigen via a thiol linker that is terminated with a group that can form an amide bonded with at least one amino present on the support material. In some embodiments, the support material may be a carrier protein. In other embodiments, the support material may include a sensor chip, a microassay, or a bead, to name a few, that carries an amino group.

[0059] As used herein, the term "protein" (e.g., in the expression "carrier protein") means any peptide-linked chain of amino acids that may or may not include any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfation, sumoylation, prenylation, ubiquitination, etc.), as long as the modification does not destroy the immunogenicity of the glycoconjugate immunogens and glycoconjugate vaccines described herein. For further clarity, the terms "protein" and "carrier protein" as used herein encompass both peptides and polypeptides, although both embodiments may be listed together, such as in the expression "carrier protein or peptide".

[0060] As used herein, the term "glycoconjugate" refers to a carbohydrate antigen (e.g., an antigenic mono-, di-, oligo-, or polysaccharide, e.g., a natural or synthetic antigen) coupled to a carrier material. When the carrier material is a carrier protein or peptide, the coupling may enhance the immunogenicity of the carbohydrate antigen in the subject of interest. The expressions "carbohydrate antigen" and "saccharide antigen" have the same meaning as used herein. The term "immunogen" refers to an agent that can be specifically bound by components of the immune system (e.g., by antibodies and / or lymphocytes) and generate a humoral and / or cellular immune response in the subject of interest. As used herein, the term "immunogen" in expressions such as "glycoconjugate immunogen" refers to the capacity (i.e., physical characteristics or properties) of a glycoconjugate without limiting the glycoconjugate itself to a particular use (e.g., as an immunogen for generating an immune response in a subject). For example, in some embodiments, the glycoconjugate immunogens described herein may be used in diagnostic assays or methods (e.g., in vitro methods) to detect the presence or absence of antibodies that bind to the glycoconjugate immunogen in a biological sample (e.g., from a subject). In some embodiments, the glycoconjugate immunogens described herein may be used to screen, identify, or evaluate antibodies (e.g., monoclonal antibodies with diagnostic or therapeutic applications) that specifically bind to the glycoconjugate immunogen.

[0061] As used herein, the term "synthetic" refers to a compound that is not a natural product, produced through human intervention.

[0062] As used herein, the term "conjugable" refers to the ability or potential for at least two molecules (e.g., a carbohydrate antigen and a thiolinker, or a carbohydrate antigen and a carrier material such as a protein or peptide) to be covalently bonded to one another via a chemical reaction, regardless of whether the molecules are actually covalently bonded to one another. In contrast, the term "conjugated" refers to at least two molecules (e.g., a carbohydrate antigen and a thiolinker, or a carbohydrate antigen and a carrier material such as a protein or peptide) that are covalently bonded to one another.

[0063] As used herein, the term "administration" may include routes of administration such as parenteral (e.g., subcutaneous, intradermal, intramuscular, or intravenous), oral, transdermal, intranasal, etc., so long as the route of administration results in the production of an immune response in the subject.

[0064] As used herein, the term "subject" generally refers to an organism (e.g., an animal or a human) that is capable of mounting an immune response against the glycoconjugates described herein, and preferably resulting in the production of antibodies and / or lymphocytes that specifically bind to the glycoconjugates and / or cells presenting the glycoconjugates. In some embodiments, the subjects described herein may be patients to be treated therapeutically (e.g., via vaccination with the glycoconjugate immunogens described herein) or may be used as a means to generate tools (e.g., antibodies) for research, diagnostic, and / or therapeutic purposes.

[0065] The meanings of various abbreviations used in this disclosure are provided in Table 1 below.

[0066] [Table 1]

[0067] Other objects, advantages and features of the present description will become more apparent from a reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. [Brief description of the drawings]

[0068] The accompanying diagram is as follows: [Figure 1] FIG. 1 shows ELISA results demonstrating the reactivity of the TF-specific lectin peanut agglutinin (PNA) and the anti-TF monoclonal antibody JAA-F11 to 0.01 μg of unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios of NHS-TF (compound 11b) to total lysines in the protein (0.2, 0.6, 3). [Diagram 2] FIG. 1 shows a Coomassie stained SDS-PAGE gel of 1 μg of unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios of NHS-TF (compound (11b)) to total lysines in the protein. [Diagram 3] FIG. 1 shows a Western blot of 1 μg of unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios of NHS-TF (compound (11b)) to total protein lysines detected using the TF-specific lectin peanut agglutinin (PNA) and anti-TF monoclonal antibody JAA-F11. [Figure 4] FIG. 1 shows an overlay of MALDI-TOF spectra of unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios of NHS-TF (compound (11b)) to the total number of lysines in the protein.

[0069] Sequence Listing This application contains a sequence listing in computer readable form, approximately 15 Kb in size, created on Jan. 11, 2022. The computer readable form is incorporated herein by reference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] The present specification relates to reactive carbohydrates, including antigen precursors, as well as processes for synthesizing glycoconjugates suitable for uses such as immunogens in vaccines, in diagnostics, or for generating analytical or therapeutic tools (e.g., generating novel anti-glycoconjugate antibodies).

[0071] Conjugation of carbohydrate antigens terminating in terminal acid functional groups to the amine groups of carrier proteins is traditionally performed via random activation with succinimide or carbodiimide reagents. One of the major drawbacks of such carbohydrate antigen-carrier protein conjugation methods is the uncontrolled / unwanted autocrosslinking that occurs within the carrier protein itself, in which the side chains of the carrier protein's own aspartic acid / glutamic acid residues become coupled to the ε-amine groups of the carrier protein's own lysine residues. This approach leads to disruption or destruction of the native structure of the carrier protein, often resulting in substantial loss of important peptide sequences that are otherwise highly immunogenic, as well as the possibility of undesired crosslinking of the carrier protein. In addition, carbohydrate antigen-carrier protein conjugation methods described in the art often use linkers, such as squaric acid, which can induce an immune response against the linker itself, and not just against the carbohydrate antigen to which the linker is coupled. Furthermore, carbohydrate antigen-carrier protein conjugation methods described in the art do not allow adequate control over the degree to which the carrier protein is glycosylated, often resulting in heterogeneous glycoconjugate species, which is a major barrier to production for human therapeutics. In addition, the synthesis of reactive carbohydrate intermediates useful as antigen precursors for conjugation with proteins can present various challenges. Current synthetic routes can be problematic in controlling stereoselectivity, which can make the intermediate stereoisomers obtained difficult to separate and / or result in low yields of the desired stereoisomer.

[0072] In contrast, the present specification provides a process that allows the synthesis of intermediate reactive carbohydrates in fewer steps and with better stereoselective control than currently known processes, resulting in synthetic antigens suitable for conjugation to carrier materials such as carrier proteins. The present specification also provides a process for synthesizing glycoconjugates that uses simple and limited synthetic steps, while still allowing adequate control over the degree to which the carrier material is glycosylated.

[0073] In a first aspect, the present disclosure relates to an improved process for producing reactive carbohydrates that can be conjugated to a carrier material (e.g., a carrier protein or peptide) having one or more free amine groups to produce glycoconjugates. The reactive carbohydrates can be purified and then used in a coupling reaction with a carrier material having one or more free amine groups. The coupling reaction conjugates one or more of the purified reactive carbohydrates to a carrier material with one or more free amine groups via an amide bond, thereby producing a glycoconjugate.

[0074] In a further aspect, described herein are processes for producing complex glycoconjugates from synthetic reactive carbohydrates prepared according to the processes described herein.

[0075] In another aspect, described herein is a process for producing intermediate reactive carbohydrates that can be reacted to yield conjugable reactive carbohydrates.

[0076] Scheme 1 below illustrates the synthetic steps that may be involved in these various processes.

[0077] [ka]

[0078] Process Reaction 1 In some embodiments, the reactive carbohydrate of formula (A)

[0079] [ka] The synthesis of 1 is H, and n is an integer from 1 to 5, for example 1 to 3, preferably 1 or 2, most preferably 1. The process comprises the step of preparing a compound of formula (A0)

[0080] [ka] and the formula HO-(CH2) n The reaction may involve a reaction between an alcohol of -CH=CH2 and an acid. The reaction may be carried out with heating to obtain a reaction mixture containing the compound of formula (A), which may then be cooled. Thus, the synthesis of the compound of formula (A) may involve the reaction of an alcohol HO-(CH2) n It involves the etherification of the carbohydrate (A0) at the C1 carbon under acidic conditions with -CH=CH2. To catalyze the reaction that can lead to the formation of the compound of formula (A), several acids can be used to carry out the reaction, which can include the addition of protons H in the reaction mixture. + In some embodiments, the acid can be a strong acid or a Lewis acid. In some embodiments, the Lewis acid can be different from BF3.Et2O.

[0081] In some embodiments, compound (A0) and alcohol HO-(CH2) n The acid used in the reaction to form the compound of formula (A) from -CH=CH2 can be acetyl chloride, acetic acid, an acidic cation exchange resin, camphorsulfonic acid, p-toluenesulfonic acid monohydrate, HCl, or any mixture thereof.

[0082] In some embodiments, the compound of formula (A0) is reacted with the alcohol HO-(CH) in the presence of acetyl chloride as an acid. n -React with CH=CH2. HO-(CH2) n A solution containing -CH=CH2 and acetyl chloride can be first prepared at a low temperature, such as a temperature of about 0° C. to about 25° C. In some embodiments, HO-(CH2) n -CH=CH2 and acetyl chloride can be prepared at a temperature of about 0° C. In another embodiment, HO-(CH2) n The temperature at which the solution of -CH=CH2 and acetyl chloride is prepared may be about 0°C to about 10°C, or about 0°C to about 20°C, or about 10°C to about 20°C, or about 10°C to about 25°C, or any temperature within these ranges.n When preparing a solution of -CH=CH2 and acetyl chloride, an excess of alcohol can be used. Thus, the alcohol can be used as a reactant and simultaneously function as a solvent. When the alcohol and acetyl chloride are mixed, some AcO-(CH2) n Some HCl is formed along with -CH=CH2. The solution can preferably be prepared at low temperatures to keep some HCl in solution in the alcohol. In some embodiments, HO-(CH2) n -CH=CH2 and acetyl chloride can be prepared under an inert atmosphere such as argon or N2. HO-(CH2) n After preparation of the solution of -CH=CH2 and acetyl chloride is complete, the compound of formula (A0) is added to the solution, which may still be at a low temperature, for example, from about 0° C. to about 25° C., to obtain at least the compound of formula (A0), HO-(CH2) n -CH=CH2, HCl, and some AcO-(CH2) n An intermediate reaction mixture containing -CH=CH2 is formed at a low temperature. The intermediate reaction mixture is then heated to promote the formation of the compound of formula (A). Upon heating, the compound of formula (A0) and HO-(CH2) n The reaction between -CH=CH2 and proton H +In the presence of, the reaction can occur rapidly, thereby forming an ether of formula (A). In addition to activating the oxygen atom at the C1 position of compound (A0), the proton can also activate the acetamide group on the carbon at the 2 position of the sugar, increasing the anomeric effect that favors the alpha conformation. Thus, reaction in the presence of an acid such as acetyl chloride can promote the formation of a compound of formula (A) having an alpha conformation using a single reaction, as opposed to the previous synthetic process that required a protection-deprotection step to obtain the same compound. In some embodiments, heating of the intermediate mixture can be carried out at a temperature of about 40°C to about 80°C. In some embodiments, the reaction temperature can be about 60°C to about 80°C, for example, about 70°C. Any temperature within these temperature ranges can be used as the heating temperature. The reaction time can be comprised between about 30 minutes and 5 hours. In some embodiments, the reaction time can be between about 30 minutes and about 1 hour. When acetyl chloride is used as the acid, a short reaction time, such as between about 30 minutes and about 45 minutes, can be sufficient. Even about 30 minutes can be sufficient. In some embodiments, the progress of the reaction can be monitored and the reaction time adjusted accordingly. In further embodiments, at least the compound of formula (A0), HO-(CH2) n The intermediate mixture, which includes -CH=CH2, and HCl at a low temperature, is contacted with a heat source at a temperature high enough to heat the mixture rapidly and instantly. In other words, in some embodiments, the low-temperature intermediate mixture may be contacted with the heat source immediately, as opposed to stepwise heating from a low temperature to a heating temperature. On a laboratory scale, this can be done by placing the round-bottom flask containing the low-temperature intermediate mixture in an oil bath that is already at a heating temperature, as opposed to placing the round-bottom flask in a low-temperature oil bath and then heating the oil bath to increase the temperature to reach the desired heating temperature. On an industrial scale, any conventional heating means that allows the intermediate mixture to be "contacted" with the heat source can be used. For example, these can include heat exchangers. In some embodiments, the heat source can be at a temperature of about 40°C to about 80°C.

[0083] In further embodiments, the acid source capable of liberating a proton to catalyze the reaction to obtain the compound of formula (A) may include an acid selected from acetic acid, an acidic cation exchange resin, camphorsulfonic acid, p-toluenesulfonic acid monohydrate, HCl, or any mixture thereof. In some embodiments, the reaction with these other types of acids may result in the formation of the compound of formula (A0), HO-(CH2) n This can be done by preparing a solution containing -CH=CH2 and an acid, and then heating the solution. The solution can also include an organic solvent. In some embodiments, the solvent can be selected from dichloromethane, chloroform, 1,3-dioxolane, diethoxymethane, dimethoxymethane, 2,5,7,10-tetraoxaundecane, dipropoxymethane, and any combination thereof, to name a few. In some embodiments, the compound of formula (A0), HO-(CH2) n The reaction mixture containing -CH=CH2 and an acid can be heated to a temperature of about 30°C to about 70°C. Any temperature within this range can be used. The reaction time can be varied and can be adjusted by monitoring the progress of the reaction. In some embodiments, the reaction can be carried out for a reaction time of about 0.5 hours to about 15 hours, or about 0.5 hours to about 10 hours, or about 0.5 hours to about 5 hours, or about 0.5 hours to about 2 hours.

[0084] In some embodiments, the reaction temperature and reaction time for preparing a compound of formula (A) from compound (A0) can be further adjusted by monitoring the formation of the equivalent beta conformer of compound of formula (A). Although the reaction conditions described above (i.e., selection of acid, temperature, reaction time and / or preparation of intermediate mixture) should favor the formation of alpha conformer compound (A), some beta conformer may be formed during the reaction. The formation of the beta conformer can be assessed by analyzing the reaction mixture during heating, and if any beta conformer is observed, heating can be stopped immediately and the mixture cooled.

[0085] Once the heating step is complete and the reaction mixture is cooled, the cooled mixture can be neutralized by any conventional method (e.g., adding a base and checking the pH until a pH of about 7 is reached). The compound of formula (A) can then be isolated and purified by any conventional method. For example, the isolated crude product can be lyophilized, reprecipitated, or purified by chromatography. In some embodiments, the purification step may allow the compound of formula (A) to be separated from the equivalent beta conformer, some of which was formed during the reaction.

[0086] Process Reaction 2 In some embodiments, the formula (A PG ) Compound

[0087] [ka] Also provided is the synthesis of: wherein PG is a protecting group and n is an integer from 1 to 5. The reaction is carried out by reacting a compound of formula (A)

[0088] [ka] wherein R 1 is H and has a divalent protecting group PG.

[0089] In some embodiments, n is an integer from 1 to 3, preferably 1 or 2, and most preferably 1.

[0090] In some embodiments, the compound of formula (A) can be prepared by process reaction 1 above. In further embodiments, protection of the compound of formula (A) is carried out by reaction with a compound selected from the group consisting of PhCH(OMe)2, triphosgene, Me2SiCl2, Me2C(OMe)2, 2-methoxypropene, and 2,6-bis(trifluoromethyl)phenylboronic acid ester. Thus, in some embodiments, this reaction provides the protected compound of formula (A PG) where PG is selected from the group consisting of PhCH, Me2C, O=C, Me2Si, and 2,6-bis(trifluoromethyl)phenyl-B. In a preferred embodiment, the protecting group is PhCH.

[0091] In some embodiments, the protection reaction is carried out in an organic solvent selected from the group consisting of DMF, dimethyl acetamide (DMAC), DMSO, THF, dioxane, 1-3 dioxolane, acetonitrile, 2,5-dimethyl tetrahydrofuran (DMTHF), gamma-vaterolactone (GVL), dihydrolevoglucoseron (Cyrene), methyl levulinate (ML), ethyl levulinate (EL), ethyl levulinate propyleneglycol ketal (ELPK), dimethyl glutarate (DMG), dimethylpropylene urea (dimethylpropylene urea), and the like. The reaction is carried out in an organic solvent selected from the group consisting of urea, DMPU), poly(propylene glycol) (PPG), glycofurol (THFP), 1-ethyl-3-methylimidazolium acetate ([emim][OAc]), and any combination thereof.

[0092] In some embodiments, the formula (A PG A compound of formula (A) can be prepared by reacting a compound of formula (A) with PhCH(OMe)2 in the presence of a catalytic amount of p-toluenesulfonic acid monohydrate in DMF.

[0093] In some embodiments, the reaction can be carried out at room temperature and is represented by the formula (A PG The formation of the compound of formula (I) can be monitored and the reaction stopped when the reaction is substantially complete. In some embodiments, the reaction time can be about 5 hours. The compound can then be isolated by conventional methods.

[0094] Process Reaction 3 In some embodiments, the formula (AA PG ) reactive carbohydrate compounds

[0095] [ka] Also provided is the synthesis of 2 is a monovalent protecting group, PG is a divalent protecting group, and n is an integer of 1 to 5.

[0096] In some embodiments, the synthesis includes a method of preparing a zeolite having a metal-containing zeolite having the formula (A PG ) Compound

[0097] [ka] and a compound of formula (A1)

[0098] [ka] and wherein X is -I, -Br, -Cl, -CN, -OTf (triflate), -OMs (mesylate), -OTs (tosylate), methylsulfate, or trichloroacetimidate.

[0099] In some embodiments, the formula (A PG ) can be prepared by process reaction 2 above.

[0100] In some embodiments, the formula (AA PGThe reaction for preparing the compound of formula (I) can be carried out in an organic solvent. For example, the organic solvent can be selected from the group consisting of toluene, dichloromethane-toluene mixture, DMSO-toluene mixture, and N-methylpyrrolidone-toluene mixture. In some embodiments, the solvent can be toluene. The reaction is preferably carried out under an inert atmosphere (e.g., under argon or N2).

[0101] In some embodiments, the monovalent protecting group R 2 may be selected from the group consisting of Ac, Bz, allyl, benzoate, methoxymethyl (MOM), tetrahydropropyranyl (THP), t-butyl, pivalate, t-butyldimethylsilyl (TBDMS), and t-butyldiphenylsilyl (TBDPS). Any other possible hydroxyl protecting group may also be used. In a preferred embodiment, R 2 can be Ac or Bz.

[0102] As mentioned above, in some embodiments, the divalent protecting group PG can be selected from PhCH, Me2C, O=C, Me2Si, and 2,6-bis(trifluoromethyl)phenyl-B, preferably PhCH. The leaving group X in the compound of formula (A1) can be preferably a halogen selected from I, Br, or Cl, most preferably Br.

[0103] In some embodiments, n is an integer from 1 to 3, preferably 1 or 2, and most preferably 1.

[0104] In some embodiments, the metal-containing zeolite may be a zeolite having at least one metal that is part of the zeolite framework itself or that has at least one metal present within the zeolite pores and / or on the zeolite surface. Thus, in some embodiments, the zeolite may be a metal-impregnated zeolite. In further embodiments, the metal in the metal-containing zeolite may include Ag, Al, Cd, Co, Cu, Fe, Ga, In, Mo, Pd, Pt, Sn, Sb, V, Zr, or any mixture thereof. In some embodiments, the metal-containing zeolite can be a zeolite selected from the group consisting of Sn-Beta, Zr-Beta, Al-Beta(OH), Al-Beta(F), Pt@MCM-22, K-PtSn / MFI, 0.3Pt / 0.5Sn-Si-Beta, Pt / Sn 2.0-Beta, 0.5CoSi-Beta, V-Beta, H-[Fe]ZSM-5, Fe-BEA, Ga-Beta, Ga-Beta-200, Mo / HZSM-5, or any mixture thereof, to name a few. However, the metal-containing zeolite may be selected from the group consisting of Sn-Beta, Zr-Beta, Al-Beta(OH), Al-Beta(F), Pt@MCM-22, K-PtSn / MFI, 0.3Pt / 0.5Sn-Si-Beta, Pt / Sn 2.0-Beta, 0.5CoSi-Beta, V-Beta, H-[Fe]ZSM-5, Fe-BEA, Ga-Beta, Ga-Beta-200, Mo / HZSM-5, or any mixture thereof, to name a few. PG Any type of metal-containing zeolite capable of catalyzing the ether linkage reaction between a compound of formula (AA) and a compound of formula (A1) can be used. PG In a preferred embodiment, the metal-containing zeolite may comprise a silver-containing zeolite, such as a silver-exchanged zeolite. Such zeolites are generally commercially available.

[0105] In some embodiments, the glycosylation reaction carried out in the presence of a metal-containing zeolite can be carried out at room temperature or with heating. In some embodiments, the glycosylation temperature can be from about 20° C. to about 80° C. In other embodiments, the reaction is carried out at a temperature of from about 20° C. to about 70° C., from about 20° C. to about 60° C., from about 20° C. to about 50° C., from about 20° C. to about 40° C., or from about 20° C. to about 30° C. Thus, the glycosylation reaction can be carried out at any temperature within these ranges. In further embodiments, the glycosylation can be carried out for a period of from about 5 hours to about 50 hours. In other embodiments, the glycosylation time may last for about 5 hours to about 30 hours, or about 5 hours to about 25 hours, or about 5 hours to about 20 hours, or about 10 hours to about 30 hours, or about 10 hours to about 25 hours, or about 10 hours to about 20 hours, or about 15 hours to about 30 hours, or about 15 hours to about 25 hours, or about 15 hours to about 20 hours, or about 20 hours to about 25 hours, or about 20 hours to about 30 hours. In some embodiments, the glycosylation reaction may be carried out at about 60 to about 80° C. for about 15 hours to about 20 hours. In other embodiments, the glycosylation may be carried out at about 20° C. to about 25° C. for about 20 hours to about 30 hours.

[0106] Formula (A PG By using metal-containing zeolites as catalysts for the reaction between the two carbohydrates (A1) and (A2), it was surprisingly possible to obtain a glycosylated product (AA PG It has become possible to obtain essentially the same product (AA) as that obtained in the process described herein. This is an important advantage compared to conventional glycosylation reactions (e.g., using trichloroacetimidate and BF3.Et2O or TMSOTf), which generate a mixture of alpha and beta conformers. PGPurification of the zeolite can be easily achieved by filtration at the end of the reaction to remove the zeolite. Furthermore, glycosylation in the presence of zeolites can be more easily controlled than conventional reactions. For example, glycosylation in the presence of trichloroacetimidate and BF3.Et2O or TMSOTf may require the reaction temperature to be kept very low, such as about -30°C, which in turn may slow down the reaction. Furthermore, the use of metal-containing zeolites is preferred over the use of other catalysts such as Hg(CN)2, which are also known to catalyze glycosylation but contain toxic heavy metals and are therefore poisonous. Overall, the use of metal-containing zeolites allows glycosylation to be performed more smoothly with high stereoselectivity control.

[0107] In some embodiments, the formula (AA PG Preparation of a compound of formula (A PG The coupling between compounds (A1) and (A2) can further be carried out in the presence of molecular sieves. Examples of molecular sieves that can be used include molecular sieves of type 3 Å, type 4 Å, type 5 Å, type 13X. Any mixtures of these can also be used. Carrying out the reaction in the presence of molecular sieves allows for the scavenging of moisture / trace amounts of water, which in turn can facilitate the reaction.

[0108] As explained above, the final crude product (AA PG ) can be readily isolated by filtering the reaction mixture followed by removal of the solvent. In some embodiments, the crude product can then be purified by any conventional method.

[0109] Process Reaction 4 In some embodiments, a reactive carbohydrate compound of formula (AA)

[0110] [ka] Also provided is the synthesis of:

[0111] This reaction is carried out by reacting the compound of formula (AA PG The method may include deprotecting the compound of formula (I).

[0112] In some embodiments, the formula (AA PG ) can be obtained by the processes described herein.

[0113] In some embodiments, n is an integer from 1 to 3, preferably 1 or 2, and most preferably 1.

[0114] In some embodiments, the formula (AA PG The deprotection of the compound of formula (AA PG ) with a basic solution to form intermediate product (AAi), and then treating intermediate product (AAi) with an acidic solution.

[0115] In some embodiments, the basic solution required to obtain intermediate product (AAi) may comprise a base selected from the group consisting of methoxide, ethoxide, and 2-methylpropan-2-olate. In preferred embodiments, the base may be sodium methoxide. In some embodiments, the basic solution may comprise a solvent selected from the group consisting of methanol, ethanol, and isopropanol. In some embodiments, the solvent may be preferably methanol. In some embodiments, the (AAi) to form intermediate product (AAi) may comprise a base selected from the group consisting of methoxide, ethoxide, and 2-methylpropan-2-olate. In preferred embodiments, the base may be sodium methoxide. In some embodiments, the basic solution may comprise a solvent selected from the group consisting of methanol, ethanol, and isopropanol. In some embodiments, the solvent may be preferably methanol. PG The reaction of (AAi) with the basic solution can be carried out at a temperature of about 0° C. to about 50° C., preferably at room temperature. The reaction with the basic solution to form the intermediate product (AAi) can be carried out for a period of about 0.5 hours to about 5 hours, preferably about 1 hour to about 2 hours.

[0116] In some embodiments, the intermediate product (AAi) in crude form can be directly treated with an acidic solution without the need for any purification steps. This is advantageous as it can reduce the number of steps in the protection process. Thus, in some embodiments, both the base treatment and the subsequent acid treatment can be carried out in the same reaction vessel. In some embodiments, once the base treatment is complete, the solution can be neutralized and the liquid can be evaporated to obtain the intermediate product (AAi) in crude form. The intermediate product (AAi) in crude form can then be directly treated with an acidic solution as described above without the need for any purification steps.

[0117] In some embodiments, the subsequent acid treatment can be carried out by reacting intermediate product (AAi) with an acidic solution comprising an acetic acid-water mixture or an ethanolic acid-water mixture. In a preferred embodiment, the acidic solution can be an aqueous solution of acetic acid. In a further embodiment, intermediate product (AAi) can be treated with the acidic solution at a temperature of about 50° C. to about 70° C., preferably about 60° C. Treatment with the acidic solution can be carried out for about 1 hour to about 5 hours, preferably about 1 hour to about 2 hours, to obtain a solution comprising the compound of formula (AA).

[0118] In some embodiments, the compound of formula (AA) can then be isolated by conventional methods including, for example, extraction, precipitation, crystallization, lyophilization, chromatography on silica gel under normal or reverse phase on C-18, simulated moving bed (SMB) chromatography, and / or purification by membrane techniques such as membrane nanofiltration.

[0119] Process Reactions 5a and 5b In some embodiments, a process for synthesizing a reactive carbohydrate of formula (B) or formula (BB) is also provided,

[0120] [ka] wherein n is an integer from 1 to 5, m is an integer from 1 to 5, and LG is a leaving group that allows for amide bond formation when the compound of formula (B) or (BB) reacts with an amino group. The compound of formula (B) or (BB) is also referred to herein as a carbohydrate antigen precursor. The compound of formula (B) or (BB) comprises a thiol-linker to which a carbohydrate antigen is linked.

[0121] In some embodiments, the process for obtaining a compound of formula (B) comprises the step of:

[0122] [ka] wherein m and LG are as defined above, Compound of formula (A)

[0123] [ka] (In the formula, R 1 is H and n is as defined above.

[0124] In some embodiments, the compound of formula (A) may be obtained by the process defined herein above.

[0125] In some embodiments, the process for obtaining a compound of formula (BB) comprises reacting a compound of formula (C) with a compound of formula (AA)

[0126] [ka] where n is as defined above.

[0127] In some embodiments, the compound of formula (AA) may be obtained by the process defined herein above.

[0128] In some embodiments, in compounds of formula (A), (AA), (B), and (BB), n is an integer from 1 to 3, preferably 1 or 2, and most preferably 1.

[0129] In some embodiments, in the compound of Formula (C), m is an integer from 1 to 3. In a preferred embodiment, m is 2.

[0130] In some embodiments, in the compound of formula (C), LG can be an O-fluorophenyl group, such as OPhF5 or OPhF4(para SO3Na), or an O-(N-succinimidyl) group. In a preferred embodiment, LG in the compound of formula (C) is an O-(N-succinimidyl) group.

[0131] In some embodiments, the reaction of a compound of formula (C) with a compound of formula (A) to obtain a compound of formula (B) or with a compound of formula (AA) to obtain a compound of formula (BB) is carried out in the presence of a photoinitiator. In some embodiments, the photoinitiator can be selected from the group consisting of free radical generating azo compounds, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), metal or metal ion based photoinitiators, peroxides, ammonium persulfate, 2,2-dimethoxy-2-phenylacetophenone (DMPA), and any combination thereof.

[0132] In some embodiments, when the photoinitiator is a free radical generating azo compound, it can be azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropionitrile), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 1,1'-azobis(cyanocyclohexane) (ACHN), diazenedicarboxylic acid bis(N,N-dimethylamide) (TMAD), azodicarboxylic acid dipiperidine, etc. dihydrochloride (ADD), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), 2,2'-azobis(2-methylpropionitrile), 4,4'-(diazene-1,2-diyl)bis(4-cyanopentanoic acid), 2,2'-azodi(2-methylbutyronitrile), or any combination thereof.

[0133] In some embodiments, when the photoinitiator is a peroxide, the peroxide can include tert-butyl peroxyisobutyrate, tert-butyl hydroperoxide, benzoyl peroxide, and any combination thereof.

[0134] In some embodiments, the reaction of the compound of formula (C) with the compound of formula (A) or (AA) can be carried out in water or in an organic solvent. In some embodiments, the solvent can be selected depending on the solubility of the photoinitiator in this solvent. In some embodiments, a mixture of different solvents can be used if necessary to increase the solubility of the photoinitiator. In some embodiments, the solvent used to carry out the reaction of the compound of formula (C) with the compound of formula (A) or (AA) can be selected from water, dioxane, acetonitrile, tetrahydrofuran (THF), diisopropyl ether, isopropanol, chlorobenzene, methyl-tert-butyl ether, methanol, ethanol, tert-butanol, chloroform, and any combination thereof.

[0135] In some embodiments, the reaction of a compound of formula (C) with a compound of formula (A) to obtain a compound of formula (B) or with a compound of formula (AA) to obtain a compound of formula (BB) can be carried out at a temperature ranging from about 40° C. to about 110° C. In some embodiments, the reaction of a compound of formula (C) with a compound of formula (A) or (AA) can be carried out for a period of about 1 hour to about 10 hours.

[0136] In some embodiments, the reaction of the compound of formula (C) with the compound of formula (A) to obtain the compound of formula (B) or with the compound of formula (AA) to obtain the compound of formula (BB) can be carried out under UV irradiation. In some embodiments, the UV irradiation can be short-wavelength, medium-wavelength, or long-wavelength UV irradiation. In some embodiments, the reaction can further be carried out under UV irradiation in the presence of a photoinitiator. In such embodiments, the photoinitiator can be preferably selected from 2,2'-azidobis[2-imidazolin-2-yl)propane]dihydrochloride, 2,2-dimethoxy-2-phenylacetophenone (DMPA), and combinations thereof. In some embodiments, when the reaction to obtain the compound of formula (B) or (BB) is carried out under UV irradiation, optionally in the presence of a photoinitiator, the reaction can be carried out in an alcohol solvent, such as methanol, ethanol, isopropanol, or any combination thereof. In some embodiments, the synthesis of the compound of formula (B) or (BB) under UV irradiation can be carried out at a temperature ranging from about 20°C to about 30°C. In some embodiments, irradiation can occur for a period of about 1 hour to about 24 hours.

[0137] In some embodiments, the reaction of a compound of formula (C) with a compound of formula (A) to obtain a compound of formula (B) or with a compound of formula (AA) to obtain a compound of formula (BB) can be carried out under visible light in the presence of a visible light absorbing transition metal photocatalyst. In some embodiments, the visible light absorbing transition metal photocatalyst can be a ruthenium polypyridyl complex, such as Ru(bpz)3(PF6)2, and the light source can include a blue LED light. In some embodiments, the reaction carried out in the presence of a visible light absorbing transition metal photocatalyst can be carried out in a solvent, such as acetonitrile, at a temperature ranging from about 20°C to about 30°C.

[0138] In some embodiments, the compound of formula (B) or (BB) can be isolated using conventional methods. In some embodiments, the reaction mixture containing the compound of formula (B) or (BB) can be treated to remove the solvent and any unreacted compound of formula (C) to recover the compound of formula (B) or (BB). The compound of formula (B) or (BB) can then be used directly in crude form, i.e., without further purification, for conjugation with a carrier material carrying at least one amino group, as described in more detail below.

[0139] Process Reactions 6a and 6b In some embodiments, a glycoconjugate of formula (I) or (II)

[0140] [ka]

[0141] Also provided is a process for preparing the compound represented by the formula:

[0142] [ka] is a support material carrying at least one free amino group. The process comprises conjugating the support material with a compound of formula (B) prepared by the process defined herein to form a glycoconjugate of formula (I) or with a compound of formula (BB) prepared by the process defined herein to form a glycoconjugate of formula (I).

[0143] In some embodiments, the support material has at least one amino group available for conjugation, i.e.,

[0144] [ka] The carrier material comprises at least one amino group capable of reacting with an activated ester group (CO)LG of a compound of formula (B) or (BB) to form a bond in a glycoconjugate of formula (I) or (II). In some embodiments, the carrier material may comprise 1 to 50 amino groups, and may comprise 1 to 50

[0145] [ka] This allows a bond to be formed in the glycoconjugate of formula (I) or (II).

[0146] In some embodiments, in compounds of Formula (I) and (II), n is an integer from 1 to 3, preferably 1 or 2, and most preferably 1.

[0147] In some embodiments, in the compounds of Formula (I) and (II), m is an integer from 1 to 3. In a preferred embodiment, m is 2.

[0148] In some embodiments, conjugation between a compound of formula (B) or (BB) and a carrier material may involve any conventional conjugation method.

[0149] In some embodiments, the support material may comprise a protein, polypeptide, or peptide. In other embodiments, the support material may comprise a sensor chip, a microassay, or a bead, to name a few, bearing free amino groups that can be conjugated with a compound of formula (B) or (BB).

[0150] In some embodiments, the conjugation reaction between a protein, peptide, or polypeptide and a compound of formula (B) or (BB) may advantageously minimize or avoid self-crosslinking of the carrier protein or peptide between the side chains of aspartic acid / glutamic acid residues and the ε-lysine amines present in the carrier protein or peptide itself.

[0151] In some embodiments, the conjugation reactions described herein allow the number of carbohydrate antigens conjugated to a carrier protein or peptide to be controlled by reactant availability and / or stoichiometry (e.g., the molar ratio of carrier protein or peptide to compound of formula (B) or (BB)). In some embodiments, the conjugation reactions described herein may comprise reacting 1-500, 1-400, 1-300, 1-200, 5-500, 5-400, 5-300, or 5-200 molar equivalents of a compound of formula (B) or (BB) per carrier protein or peptide.

[0152] In some embodiments, the carrier protein or peptide described herein comprises one or more free amine groups. As used herein, "free amine" or "free amine group" refers to a carrier protein or peptide that has one or more amino groups available for chemical modification and / or conjugation (e.g., to carbohydrate antigens as described herein, such as solvent-accessible lysine residues that tend to be exposed at the periphery of the carrier protein). In some embodiments, it may be advantageous to avoid conjugating too many multiple carbohydrate antigens to adjacent positions on the carrier protein. In some embodiments, the carrier protein or peptide may preferably lack a lysine-rich domain (e.g., a segment of at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids that contain at least 50% lysine residues).

[0153] In some embodiments, the carrier protein comprises at least a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 102, 104, 105, 106, 107, 108, 109, 2, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 lysine residues. In some embodiments, the carrier protein may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 free amino acid residues.

[0154] In some embodiments, the carrier protein or peptide comprises one or more lysine residues with one or more free amine groups, or is optionally engineered to add one or more additional lysine residues, for example, at the amino terminus, carboxy terminus, or solvent accessible positions of the carrier protein or peptide. In some embodiments, the carrier protein comprises a T cell epitope and / or induces a cellular immune response in a subject. In some embodiments, the carrier protein or peptide comprises a B cell epitope and / or induces a humoral immune response in a subject. In some embodiments, the carrier protein comprises both a B cell epitope and a T cell epitope, and / or induces both a humoral immune response and a cellular immune response in a subject.

[0155] Preferably, the carrier proteins described herein may be proteins that have already been approved by a regulatory agency (eg, the FDA) for administration to human subjects (eg, in an approved vaccine). In some embodiments, the carrier protein is, is derived from, or comprises Tetanus Toxoid (TT), Diphtheria Toxoid (DT), cross-reacting material 197 (CRM197), Meningococcal Outer Membrane Protein Complex (OMPC), H. Influenzae Protein D (HID), a virus-like particle (VLP), a cytokine, an immunogenic peptide, such as Tetanus Toxoid 831-844 (SEQ ID NO: 1 or 2), Tetanus Toxoid 830-843 (SEQ ID NO: 5), albumin (such as bovine serum albumin or human serum albumin), keyhole limpet hemocyanin (KLH), or an immunogenic fragment thereof.

[0156] In some embodiments, the carrier protein or peptide is exogenous to the subject to which it is administered, and preferably has no (close) orthologue in the subject. In the context of human vaccine production, the carrier protein described herein refers to a "carrier protein suitable for human use" or simply a "suitable carrier protein", which means a carrier protein that is antigenically different from a human protein such that the carrier protein is not considered a "self-antigen" in humans. Using a carrier protein that is too antigenically similar to the corresponding human protein may cause the carrier protein to be considered a "self-antigen", which may not be ideal in a human vaccine. For example, glycoconjugate immunogens consisting of TF antigens randomly conjugated to the ε-amino groups of lysine residues of bovine serum albumin (BSA) have been previously described and characterized (e.g., Demian et al., 2014; Rittenhouse-Diakun et al., 1998; Heimburg et al., 2006; Tati et al., 2017). However, not only was the level of carbohydrate on the 59 lysine residues of BSA random and inefficient (no more than 4-6 TF antigens were conjugated per BSA molecule), but BSA was too antigenically similar to human albumin to be suitable as a carrier protein in a human vaccine. In some embodiments, the carrier protein is not albumin (e.g., bovine serum albumin).

[0157] In some embodiments, the glycoconjugates of formula (I) or (II) described herein may be glycoconjugate immunogens, wherein a carrier protein or peptide is immunogenic when administered to a subject, and wherein the carbohydrate antigen is conjugated to the carrier protein or peptide via a thiolinker, thereby increasing the immunogenicity of the carbohydrate antigen when administered to a subject, compared to the corresponding administration of the unconjugated carbohydrate antigen.

[0158] In some embodiments, the carbohydrate antigen or carbohydrate antigen is not cleavable from the carrier protein or peptide by endogenous enzymes of the subject after coupling to the carrier protein or peptide.

[0159] In some embodiments, a carrier protein or peptide described herein may comprise a T cell epitope and / or may induce a cellular immune response in a subject upon administration.

[0160] In some embodiments, the synthetic glycoconjugates of formula (I) or (II) described herein may induce a cellular immune response against a carbohydrate antigen upon administration to a subject.

[0161] In a further aspect, a method for producing a glycoconjugate vaccine or immune response eliciting composition is described herein. The method may include formulating a glycoconjugate of formula (I) or (II) prepared by the method described herein with a pharma- ceutically acceptable excipient and / or adjuvant. In some embodiments, the adjuvant is or comprises an inorganic compound, a mineral oil, a microbial derivative, a botanical derivative, a cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate, hydroxyapatite, a toll-like receptor agonist, an immunostimulatory polynucleotide (e.g., CPG), an immunostimulatory lipid, Freund's adjuvant, RIBI adjuvant, QS-21, muramyl dipeptide, TiterMax®, Steviune®, Stimune®, or any combination thereof.

[0162] The vaccine composition may be administered in doses and with techniques well known to those skilled in the medical or veterinary arts, taking into account factors such as the age, sex, weight, species, and condition of the recipient animal, as well as the route of administration. The route of administration may be transdermal, via mucosal administration (e.g., oral, nasal, intraocular), or via parenteral routes (e.g., intradermal, intramuscular, subcutaneous). The vaccine composition may be administered alone or co-administered or sequentially administered with other treatments or therapies. Administration forms may include suspensions and preparations for parenteral, subcutaneous, intradermal, or intramuscular administration (e.g., injection administration), such as sterile suspensions or emulsions. The vaccine may be administered as a spray, mixed into food and / or water, or delivered mixed with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, and the like. Depending on the route of administration and the desired preparation, the compositions may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc. Suitable preparations can be prepared without undue experimentation with reference to standard pharmaceutical texts, such as "Remington's Pharmaceutical Sciences," 1990.

[0163] In a further aspect, described herein is a glycoconjugate vaccine or adaptive immune response eliciting composition comprising a glycoconjugate of formula (I) or (II) prepared by the methods described herein and a pharma- ceutically acceptable excipient and / or adjuvant as described herein. In embodiments, the glycoconjugate vaccine may be a prophylactic or therapeutic vaccine. In embodiments, the vaccine composition described herein may comprise one or more TACAs, and the vaccine composition may be an anti-cancer vaccine against a cancer expressing a TACA. In embodiments, the cancer may be B-cell lymphoma, breast cancer, colon cancer, non-small cell lung cancer, melanoma, neuroblastoma, ovarian, prostate, sarcoma, small cell lung cancer, or gastric cancer.

[0164] In some aspects, described herein are methods of immunizing, vaccinating, or treating a subject comprising administering to the subject a glycoconjugate of formula (I) or (II) produced by a method described herein, a synthetic glycoconjugate of formula (I) or (II) described herein, a glycoconjugate vaccine or an adaptive immune response eliciting composition produced by a method described herein, or a glycoconjugate vaccine described herein.

[0165] In some embodiments, described herein are glycoconjugates of formula (I) or (II) produced by the methods described herein, synthetic glycoconjugates of formula (I) or (II) described herein, glycoconjugate vaccines or adaptive immune response eliciting compositions produced by the methods described herein, or glycoconjugate vaccines described herein for use in immunizing, vaccinating, or treating a subject with a disease, or for detecting the presence of antibodies that specifically bind to a glycoconjugate of formula (I) or (II), or for detecting said immunization, vaccination, or treatment (e.g., in a biological sample from a subject).

[0166] In some embodiments, described herein are glycoconjugates of formula (I) or (II) produced by the methods described herein, synthetic glycoconjugates described herein, or adaptive immune response eliciting compositions produced by the methods described herein for producing a vaccine for immunizing or treating a subject with a disease, or for detecting the presence of antibodies that specifically bind to a glycoconjugate of formula (I) or (II), or for detecting said immunization or treatment (e.g., in a biological sample from a subject).

[0167] Described herein, in some embodiments, is a glycoconjugate of formula (I) or (II) produced by the methods described herein, a synthetic glycoconjugate of formula (I) or (II) described herein, a glycoconjugate vaccine or an adaptive immune response eliciting composition produced by the methods described herein, or a glycoconjugate vaccine described herein, for use in treating a subject having a disease associated with increased expression of said carbohydrate antigen.

[0168] In some embodiments, described herein are glycoconjugates of formula (I) or (II) produced by the methods described herein, synthetic glycoconjugates of formula (I) or (II) described herein, glycoconjugate vaccines or adaptive immune response eliciting compositions produced by the methods described herein, or glycoconjugate vaccines described herein for detecting or screening for the presence of antibodies that specifically bind to a carbohydrate antigen or tumor circulating cells containing the carbohydrate antigen, or for detecting the presence of antibodies resulting from immunization or vaccination with the carbohydrate antigen. In some embodiments, detection or screening may be performed via any suitable detection method, such as immunosorbent assay, ELISA, microarray, or immunoblot analysis.

[0169] In a further aspect, described herein is a method of treating a subject comprising administering a glycoconjugate of formula (I) or (II) produced by the methods described herein to generate an immune response in the subject against a carbohydrate antigen, and optionally screening a biological sample from the subject for the presence of an antibody that specifically binds to the carbohydrate antigen.

[0170] In a further aspect, glycoconjugates of formula (I) or (II) are described herein for use as therapeutic and / or diagnostic tools related to SARS-CoV-2. More particularly, glycoconjugates of formula (I) or (II) are described herein for use in immunizing a subject against SARS-CoV-2, for use in eliciting the production of anti-SARS-CoV-2 antibodies in a subject, for use in inducing a cellular immune response against SARS-CoV-2 in a subject, or any combination thereof. Also described herein are glycoconjugates of formula (I) or (II) for use in detection / diagnostic tools related to SARS-CoV-2. For example, glycoconjugates of formula (I) or (II) are described herein for use in detecting the presence of anti-SARS-CoV-2 antibodies in a sample from a subject. In this regard, results showing the presence of carbohydrate antigens (e.g., TF, Tn, and their sialylated variants) on the S protein of SARS-CoV-2 and their availability for binding by anti-carbohydrate ligands are presented in PCT / CA2020 / 051253.

[0171] As used herein, the expression "anti-SARS-CoV-2 antibodies" refers to antibodies capable of binding to antigens (e.g., carbohydrate antigens) in their native conformation as expressed on native recombinant proteins and / or present on assembled virion particles. In contrast, antibodies that only bind to denatured antigens (e.g., under denaturing conditions, such as after SDS-PAGE) but not to the same antigens in their native conformation are excluded from the expression "anti-SARS-CoV-2 antibodies". In some embodiments, the glycoconjugates of formula (I) or (II) or vaccines described herein may induce the production of antibodies with neutralizing activity. As used herein, the expression "neutralizing activity" refers to ligands (e.g., antibodies) that bind to SARS-CoV-2 virion particles and inhibit their ability to infect susceptible host cells.

[0172] In some embodiments, the glycoconjugates of formula (I) or (II) described herein may comprise carbohydrate antigens conjugated to a suitable carrier material (e.g., a carrier protein or peptide, or a non-proteinaceous polymeric material), which carbohydrate antigens comprise or consist of a TF antigen, a Tn antigen, or any combination thereof.

[0173] In some embodiments, the carbohydrate antigens described herein may be conjugated to a carrier material comprising a B cell epitope or a T cell epitope, depending, for example, on whether it is desired to elicit a humoral or cellular immune response. In some aspects, the carbohydrate antigen may be covalently conjugated to a SARS-CoV-2 S protein fragment of SEQ ID NO: 3 or 4, for example, at positions 4 and / or 6 of SEQ ID NO: 3, or at positions 323, 325, and / or 678 of SEQ ID NO: 4. In particular, position 678 of SEQ ID NO: 4 (close to the furin cleavage site of the spike protein at R682) has been reported to be O-glycosylated by core-1 and core-2 structures.

[0174] In some embodiments, the carrier protein or peptide may comprise an immunogenic fragment of the SARS-CoV-2 S protein sequence of SEQ ID NO: 4, which comprises one or more carbohydrate antigens conjugated to positions 323, 325, and / or 678 of SEQ ID NO: 4. In some embodiments, the carbohydrate antigen may be covalently conjugated to a variant of the SARS-CoV-2 S protein fragment of SEQ ID NO: 3, e.g., a variant in which residues at positions 4 and / or 6 may be replaced with lysine and / or cysteine ​​residues that may facilitate chemical conjugation to the carbohydrate antigen. In some embodiments, the carrier protein or peptide may comprise an immunogenic fragment of a variant of the SARS-CoV-2 S protein sequence of SEQ ID NO: 4 having a lysine or cysteine ​​at positions 323, 325, and / or 678, which comprises one or more carbohydrate antigens conjugated to the lysine or cysteine ​​residues at positions 323, 325, and / or 678 of SEQ ID NO: 4. In the case of lysine residues, the carbohydrate antigen may be conjugated to the carrier protein via the conjugation methods described herein. In the case of cysteine ​​residues, the carbohydrate antigen may be conjugated to the carrier protein via the conjugation methods described herein. Thus, in some embodiments, the carrier material described herein may comprise or consist of a peptide of SEQ ID NO: 3, or a variant of the peptide of SEQ ID NO: 3 that includes a cysteine ​​or lysine at positions 4 and / or 6. In some embodiments, the peptide of SEQ ID NO: 3 or a peptide variant may be included in the carrier material (e.g., recombinantly engineered into the amino acid sequence) or fused to the carrier material (e.g., as a fusion protein).

[0175] In some embodiments, the carrier material is, is derived from, or comprises Tetanus Toxoid (TT), Diphtheria Toxoid (DT), cross-reacting material 197 (CRM197), Meningococcal Outer Membrane Protein Complex (OMPC), H. Influenzae Protein D (HID), a virus-like particle (VLP), a cytokine, an immunogenic peptide, such as Tetanus Toxoid 831-844 (SEQ ID NO: 1 or 2), Tetanus Toxoid 830-843 (SEQ ID NO: 5), albumin (such as bovine serum albumin or human serum albumin), keyhole limpet hemocyanin (KLH), or an immunogenic fragment thereof.

[0176] In some aspects, described herein is a SARS-CoV-2 or COVID-19 vaccine or adaptive immune response-inducing composition comprising one or more glycoconjugates of formula (I) or (II) as defined herein and a pharma- ceutically acceptable excipient and / or adjuvant. The glycoconjugates generally comprise one or more carbohydrate antigens expressed on the SARS-CoV-2 virion, such as, for example, carbohydrate antigens expressed on the S (or S1) protein of SARS-CoV-2. Carbohydrate antigens suitable for the SARS-CoV-2 vaccines described herein are carbohydrate antigens with an aberrant glycosylation pattern, i.e., carbohydrate antigens that are not expressed on normal or healthy cells and tissues of the subject, in order to reduce the risk of eliciting an autoimmune response in the subject to which the vaccine is administered.

[0177] In some embodiments, the glycoconjugates of formula (I) or (II) described herein or the SARS-CoV-2 vaccines described herein induce the production of antibodies that bind to SARS-CoV-2 virion particles and preferably have neutralizing activity (e.g., inhibit the ability of SARS-CoV-2 virion particles to infect susceptible host cells).

[0178] In another aspect, the present disclosure also provides compounds of formula (AA PG ) Compound

[0179] [ka] (In the formula, n is an integer of 1 to 5, and R 2 is Ac and PG is a divalent protecting group selected from PhCH, Me2C, O=C, Me2Si, and 2,6-bis(trifluoromethyl)phenyl-B.

[0180] In some embodiments, the divalent PG protecting group is PhCH.

[0181] In some embodiments, the formula (AA PG ), n is an integer of 1 to 3. In a preferred embodiment, n is 1 or 2. In another embodiment, n is 1.

[0182] In some embodiments, the formula (AA PG Compounds of formula (AA) can be used as intermediates in the production of glycoconjugates of formula (I) or (II) described herein. In some embodiments, compounds of formula (AA) can be used as intermediates in the production of glycoconjugates of formula (I) or (II) described herein. PG ) can itself be prepared by the processes described herein.

[0183] In some embodiments, the compound (AA PG ) is expressed as follows:

[0184] [ka] has.

[0185] In a further aspect, the present disclosure also provides a compound of formula

[0186] [ka] The present invention relates to a compound of the formula:

[0187] item One or more of the following items are described herein.

[0188] 1. Reactive carbohydrates of formula (A)

[0189] [ka] (In the formula, R 1 is H, and n is an integer from 1 to 5, Under heating, a compound of formula (A0)

[0190] [ka] HO-(CH2) n -CH=CH2 in the presence of an acid capable of liberating a proton to produce a reaction mixture comprising a compound of formula (A); and cooling the reaction mixture to obtain a cooled mixture comprising the compound of formula (A).

[0191] 2. The process of claim 1, wherein the acid comprises acetyl chloride, acetic acid, an acidic cation exchange resin, camphorsulfonic acid, p-toluenesulfonic acid monohydrate, HCl, or any mixture thereof.

[0192] 3. The process of clause 1 or 2, wherein the acid comprises acetyl chloride.

[0193] 4. Reacting the compound of formula (A0) with HO-(CH2) n A compound of formula (A0), HO-(CH2) n 4. The process of any one of clauses 1 to 3, comprising forming a low temperature intermediate reaction mixture comprising -CH=CH2, and HCl.

[0194] 5. The process according to clause 4, wherein the mixing temperature is about 0°C, or from about 0°C to about 10°C, or from about 0°C to about 20°C, or from about 10°C to about 20°C, or from about 10°C to about 25°C.

[0195] 6.HO-(CH2) n The process of any one of clauses 4 to 5, wherein a solution containing -CH=CH2 and acetyl chloride is prepared under an inert atmosphere at a temperature of about 0°C.

[0196] 7. The process of any one of clauses 4-6, wherein reacting the compound of formula (A0) comprises contacting the intermediate reaction mixture with a heat source at a temperature of from about 40° C. to about 80° C.

[0197] 8. The process of any one of clauses 1-7, wherein the heating is carried out at a reaction temperature of about 40° C. to about 80° C. for a reaction time of about 30 minutes to 5 hours.

[0198] 9. The process according to clause 8, wherein the reaction temperature is from about 60° C. to about 80° C.

[0199] 10. The process of clause 8 or 9, wherein the reaction temperature is about 70° C.

[0200] 11. The process according to any one of clauses 8 to 10, wherein the reaction time is from about 30 minutes to about 1 hour.

[0201] 12. The process according to any one of clauses 8 to 11, wherein the reaction time is from about 30 minutes to about 45 minutes.

[0202] 13. The process of any one of clauses 8 to 12, wherein the reaction time is about 30 minutes.

[0203] 14. The process of any one of clauses 1 to 2, wherein the acid comprises acetic acid, an acidic cation exchange resin, camphorsulfonic acid, p-toluenesulfonic acid monohydrate, HCl, or any mixture thereof.

[0204] 15. The compound of formula (A0) is reacted with HO-(CH2) n -CH=CH2 to give a compound of formula (A0), HO-(CH2) n 15. The process of claim 14, comprising heating a solution comprising -CH=CH2 and an acid.

[0205] 16. The process of clause 15, wherein the solution further comprises a solvent selected from the group consisting of dichloromethane, chloroform, 1,3-dioxolane, diethoxymethane, dimethoxymethane, 2,5,7,10-tetraoxaundecane, dipropoxymethane, and any combination thereof.

[0206] 17. The process of clause 15 or 16, wherein the reaction is carried out at a temperature of about 30° C. to about 70° C.

[0207] 18. The process according to any one of clauses 15 to 17, wherein the reaction is carried out for a reaction time of about 0.5 hours to about 15 hours, or about 0.5 hours to about 10 hours, or about 0.5 hours to about 5 hours, or about 0.5 hours to about 2 hours.

[0208] 19. The process of any one of clauses 1-18, further comprising neutralizing the cooled mixture.

[0209] 20. The process according to any one of clauses 1 to 19, further comprising isolating the compound of formula (A).

[0210] 21. Expression (AA PG ) reactive carbohydrates

[0211] [ka] (In the formula, R 2 is a monovalent protecting group, PG is a divalent protecting group, and n is an integer from 1 to 5, In the presence of a metal-containing zeolite, PG ) Compound

[0212] [ka] with a compound of formula (A1)

[0213] [ka] wherein X is -I, -Br, -Cl, -CN, -OTf (triflate), -OMs (mesylate), -OTs (tosylate), methylsulfate, or trichloroacetimidate.

[0214] 22. The process of claim 21, wherein the metal in the metal-containing zeolite comprises Ag, Al, Cd, Co, Cu, Fe, Ga, In, Mo, Pd, Pt, Sn, Sb, V, Zr, or any mixture thereof.

[0215] 23. The process of claim 21, wherein the metal-containing zeolite comprises a zeolite selected from the group consisting of Sn-Beta, Zr-Beta, Al-Beta(OH), Al-Beta(F), Pt@MCM-22, K-PtSn / MFI, 0.3Pt / 0.5 Sn-Si-Beta, Pt / Sn 2.0-Beta, 0.5 CoSi-Beta, V-Beta, H-[Fe]ZSM-5, Fe-BEA, Ga-Beta, Ga-Beta-200, Mo / HZSM-5, or any mixture thereof.

[0216] 24. The process of claim 21, wherein the metal-containing zeolite comprises a silver-containing zeolite.

[0217] 25. The process according to any one of clauses 21 to 24, wherein the reaction is carried out in an organic solvent selected from the group consisting of toluene, dichloromethane-toluene mixtures, DMSO-toluene mixtures, and N-methylpyrrolidone-toluene mixtures.

[0218] 26. The process of any one of clauses 21-25, wherein the reaction is carried out in toluene.

[0219] 27. The process of any one of clauses 21 to 26, wherein the reaction is carried out in the presence of molecular sieves.

[0220] 28. The process of any one of clauses 21 to 27, wherein the reaction is carried out in the presence of molecular sieves of type 3 Å, type 4 Å, type 5 Å, type 13X, or any mixture thereof.

[0221] 29. The process of any one of clauses 21-28, wherein the reaction is carried out at a temperature of about 20°C to about 80°C.

[0222] 30. The process of any one of clauses 21-29, wherein the reaction is carried out at a temperature of about 20° C. to about 50° C.

[0223] 31. The process of any one of clauses 21 to 30, wherein the reaction is carried out at a temperature of about 20° C. to about 30° C.

[0224] 32. The process according to any one of clauses 21 to 31, wherein the reaction is carried out for about 5 hours to about 50 hours.

[0225] 33. The process of any one of clauses 21-32, wherein the reaction is carried out for about 5 hours to about 30 hours.

[0226] 34. The process of any one of clauses 21 to 31, wherein the reaction is carried out at about 60 to about 80° C. for about 15 hours to about 20 hours.

[0227] 35. The process according to any one of clauses 21 to 31, wherein the reaction is carried out at about 20° C. to about 25° C. for about 20 hours to about 30 hours.

[0228] 36.R 2 36. The process of any one of clauses 21-35, wherein is selected from the group consisting of Ac, Bz, allyl, benzoate, methoxymethyl (MOM), tetrahydropropanyl (THP), t-butyl, pivalate, t-butyldimethylsilyl (TBDMS), and t-butyldiphenylsilyl (TBDPS).

[0229] 37.R 2 37. The process according to any one of clauses 21 to 36, wherein is Ac or Bz.

[0230] 38. The process according to any one of clauses 21 to 37, wherein PG is selected from the group consisting of PhCH, Me2C, O=C, Me2Si, and 2,6-bis(trifluoromethyl)phenyl-B.

[0231] 39. The process of any one of clauses 21 to 38, wherein the PG is PhCH.

[0232] 40. The process of any one of clauses 21 to 39, wherein X is Br.

[0233] 41. The process of any one of clauses 21 to 40, wherein the reaction is carried out under an inert atmosphere.

[0234] 42. Shiki (AA PG 42. The process of any one of clauses 21 to 41, further comprising isolating the compound of formula (1).

[0235] 43.(A PG ) is a compound of formula (A)

[0236] [ka] (In the formula, R 1is H and has a protecting group PG.

[0237] 44. The process according to clause 43, wherein protecting the compound of formula (A) is carried out by reaction with a compound selected from the group consisting of PhCH(OMe)2, triphosgene, Me2SiCl2, Me2C(OMe)2, 2-methoxypropene, and 2,6-bis(trifluoromethyl)phenylboronic acid ester.

[0238] 45. The process according to clause 43 or 44, wherein protecting the compound of formula (A) is carried out in an organic solvent selected from the group consisting of DMF, dimethylacetamide (DMAC), DMSO, THF, dioxane, 1-3 dioxolane, acetonitrile, 2,5-dimethyltetrahydrofuran (DMTHF), gamma-baterolactone (GVL), dihydrolevoglucoseron (Cyrene), methyl levulinate (ML), ethyl levulinate (EL), ethyl levulinate propylene glycol ketal (ELPK), dimethyl glutarate (DMG), dimethylpropylene urea (DMPU), poly(propylene glycol) (PPG), glycofurol (THFP), 1-ethyl-3-methylimidazolium acetate ([emim][OAc]), and any combination thereof.

[0239] 46. ​​The process according to any one of clauses 43 to 45, wherein the compound of formula (A) is obtained by a process according to any one of clauses 1 to 20.

[0240] 47. Reactive carbohydrates of formula (AA)

[0241] [ka] wherein n is an integer from 1 to 5, 47. The process according to claim 21, wherein the compound of formula (AA PG) deprotecting the compound of formula (I).

[0242] 48. Deprotection provides a compound of formula (AA PG 48. The process of claim 47, comprising reacting a compound of formula (I) with a basic solution to form an intermediate product and then treating the intermediate product with an acidic solution.

[0243] 49. The process of claim 48, wherein the basic solution comprises a base selected from the group consisting of methoxide, ethoxide, and 2-methylpropan-2-olate, preferably the base is sodium methoxide.

[0244] 50. The process of clause 48 or 49, wherein the basic solution comprises a solvent selected from the group consisting of methanol, ethanol, and isopropanol, preferably the solvent is methanol.

[0245] 51. The process according to any one of clauses 48 to 50, wherein the acidic solution comprises an acetic acid-water mixture or an ethanolic acid-water mixture, preferably the acidic solution is an aqueous solution of acetic acid.

[0246] 52. The process according to any one of clauses 48-51, wherein the reacting with the basic solution is carried out at a temperature of about 0° C. to about 50° C., preferably at room temperature.

[0247] 53. The process according to any one of clauses 48-52, wherein the reacting with the basic solution is carried out for a period of about 0.5 hours to about 5 hours, preferably about 1 hour to about 2 hours.

[0248] 54. The process according to any one of clauses 48 to 53, wherein the treating with an acidic solution is carried out at a temperature of about 50°C to about 70°C, preferably about 60°C.

[0249] 55. The process according to any one of clauses 48 to 54, wherein the treatment with the acidic solution is carried out for a period of about 1 hour to about 5 hours, preferably about 1 hour to about 2 hours.

[0250] 56. Shiki (AA PG 56. The process of any one of clauses 48-55, further comprising neutralizing the basic solution after reacting with the compound of formula (I) and evaporating any liquid to obtain the intermediate product in crude form.

[0251] 57. The process of any one of clauses 48-56, wherein the intermediate product is crude when treated with an acidic solution.

[0252] 58. A process for synthesizing reactive carbohydrates of formula (AA), comprising:

[0253] [ka] The process is represented by the formula (AA PG ) Compound

[0254] [ka] (In the formula, n is an integer of 1 to 5, and R 2 is a monovalent protecting group and PG is a divalent protecting group, The deprotection is carried out to give a compound of formula (AA PG ) with a basic solution to form an intermediate crude product, and then treating the intermediate crude product with an acidic solution.

[0255] 59. The process of clause 58, wherein the basic solution comprises a base selected from the group consisting of methoxide, ethoxide, and 2-methylpropan-2-olate, preferably the base is sodium methoxide.

[0256] 60. The process of clause 58 or 59, wherein the basic solution comprises a solvent selected from the group consisting of methanol, ethanol, and isopropanol, preferably the solvent is methanol.

[0257] 61. The process of any one of clauses 58-60, wherein the acidic solution comprises an acetic acid-water mixture or an ethanolic acid-water mixture, preferably the acidic solution is an aqueous solution of acetic acid.

[0258] 62. The process according to any one of clauses 58-61, wherein the reacting with the basic solution is carried out at a temperature of about 0° C. to about 50° C., preferably at room temperature.

[0259] 63. The process according to any one of clauses 58-62, wherein the reacting with the basic solution is carried out for a period of about 0.5 hours to about 5 hours, preferably about 1 hour to about 2 hours.

[0260] 64. The process according to any one of clauses 58 to 63, wherein the treating with an acidic solution is carried out at a temperature of about 50°C to about 70°C, preferably about 60°C.

[0261] 65. The process according to any one of clauses 58 to 64, wherein the treatment with the acidic solution is carried out for a period of about 1 hour to about 5 hours, preferably about 1 hour to about 2 hours.

[0262] 66. Shiki (AA PG 66. The process of any one of clauses 58-65, further comprising neutralizing the basic solution after reacting with the compound of formula (I) and evaporating any liquid to obtain a crude intermediate product.

[0263] 67.R 2 67. The process according to any one of clauses 58 to 66, wherein is Ac or Bz, preferably Bz.

[0264] 68. The process according to any one of clauses 58 to 67, wherein PG is selected from the group consisting of PhCH, Me2C, O=C, Me2Si, and 2,6-bis(trifluoromethyl)phenyl-B.

[0265] 69. The process according to any one of clauses 58 to 68, wherein the PG is PhCH.

[0266] 70. Reactive carbohydrates of formula (B) or formula (BB)

[0267] [ka] wherein n is an integer from 1 to 5, m is an integer from 1 to 5, and LG is a leaving group that enables amide bond formation when a compound of formula (B) or (BB) reacts with an amino group, comprising: Compound of formula (C)

[0268] [ka] with a compound of formula (A)

[0269] [ka] (In the formula, R 1 is H) to form a compound of formula (B); or Compound of formula (AA)

[0270] [ka] to form a compound of formula (BB).

[0271] 71. The process according to clause 70, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out in the presence of a photoinitiator.

[0272] 72. The process of claim 71, wherein the photoinitiator is selected from the group consisting of free radical generating azo compounds, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), metal or metal ion based photoinitiators, peroxides, ammonium persulfate, 2,2-dimethoxy-2-phenylacetophenone (DMPA), and any combination thereof.

[0273] 73. Free radical generating azo compounds include azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropionitrile), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 1,1'-azobis(cyanocyclohexane) (ACHN), diazenedicarboxylic acid bis(N,N-dimethylamide) (TMAD), azodicarboxylic acid dipiperidide (ADD), 2,2'-azobis[2-(2 73. The process of claim 72, wherein the compound is selected from the group consisting of 2,2'-azobis(2-amidinopropane)dihydrochloride, 2,2'-azobis(2-methylpropionitrile), 4,4'-(diazene-1,2-diyl)bis(4-cyanopentanoic acid), 2,2'-azodi(2-methylbutyronitrile), and any combination thereof.

[0274] 74. The process of clause 72 or 73, wherein the peroxide is selected from the group consisting of tert-butyl peroxyisobutyrate, tert-butyl hydroperoxide, benzoyl peroxide, and any combination thereof.

[0275] 75. The process according to any one of clauses 70-74, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out in water or in an organic solvent.

[0276] 76. The process according to any one of clauses 70-75, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out in a solvent selected from the group consisting of water, dioxane, acetonitrile, tetrahydrofuran (THF), diisopropyl ether, isopropanol, chlorobenzene, methyl-tert-butyl ether, methanol, ethanol, tert-butanol, chloroform, and any combination thereof.

[0277] 77. The process of any one of clauses 70-76, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out at a temperature in the range of about 40°C to about 110°C.

[0278] 78. The process according to any one of clauses 70-77, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out for a period of about 1 hour to about 10 hours.

[0279] 79. The process according to clause 70, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out under visible light in the presence of a visible light absorbing transition metal photocatalyst.

[0280] 80. The process according to clause 70, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out under ultraviolet irradiation.

[0281] 81. The process according to clause 70, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out in the presence of a photoinitiator and under ultraviolet irradiation.

[0282] 82. The process of claim 81, wherein the photoinitiator is selected from the group consisting of 2,2'-azidobis[2-imidazolin-2-yl)propane]dihydrochloride, 2,2-dimethoxy-2-phenylacetophenone (DMPA), and combinations thereof.

[0283] 83. The process according to any one of clauses 80-82, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out in an alcoholic solvent such as methanol, ethanol, isopropanol, or any combination thereof.

[0284] 84. The process according to any one of clauses 80-83, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out at a temperature in the range of about 20°C to about 30°C.

[0285] 85. The process according to any one of clauses 80-84, wherein reacting the compound of formula (C) with the compound of formula (A) or (AA) is carried out for a period of from about 1 hour to about 24 hours.

[0286] 86. The process of any one of clauses 80 to 85, wherein the ultraviolet radiation is short-wavelength, medium-wavelength, or long-wavelength ultraviolet radiation.

[0287] 87. The process of any one of clauses 70 to 86, wherein m is an integer from 1 to 3.

[0288] 88. The process of any one of clauses 70 to 87, wherein m is 2.

[0289] 89. The process of any one of clauses 70-88, wherein LG is an O-fluorophenyl group, such as OPhF-5 or OPhF4(para SO3Na), or an O-(N-succinimidyl) group.

[0290] 90. The process of any one of clauses 70-89, wherein LG is an O-(N-succinimidyl) group.

[0291] 91. The process according to any one of clauses 70 to 90, wherein the compound of formula (A) is obtained by a process according to any one of clauses 1 to 20.

[0292] 92. The process according to any one of clauses 70 to 90, wherein the compound of formula (A) is obtained by a process according to any one of clauses 47 to 69.

[0293] 93. Glycoconjugates of formula (I) or (II)

[0294] [ka] (In the formula, n is an integer of 1 to 5, m is an integer of 1 to 5, and p is an integer of 1 to 50,

[0295] [ka] is a carrier material comprising at least one amino group available for conjugation, A process comprising conjugating at least one free amino group of a carrier material with a compound of formula (B) to form a glycoconjugate of formula (I) or with a compound of formula (BB) to form a glycoconjugate of formula (I), wherein the compounds of formula (B) and (BB) are obtained by the process according to any one of clauses 70 to 92.

[0296] 94. The process according to clause 93, wherein the carrier material comprises a protein, polypeptide, or peptide.

[0297] 95. The process of any one of clauses 1 to 94, wherein n is an integer from 1 to 3.

[0298] 96. The process of any one of clauses 1 to 95, wherein n is 1 or 2.

[0299] 97. The process of any one of clauses 1 to 96, wherein n is 1.

[0300] 98. Shiki (AA PG )

[0301] [ka] In the formula, n is an integer of 1 to 5, and R 2 is Ac and PG is a divalent protecting group selected from PhCH, Me2C, O=C, Me2Si, and 2,6-bis(trifluoromethyl)phenyl-B.

[0302] 99. The compound according to clause 98, wherein PG is PhCH.

[0303] 100. The compound according to clause 98 or 99, wherein n is an integer from 1 to 3.

[0304] 101. The compound according to any one of clauses 98 to 100, wherein n is 1 or 2.

[0305] 102. The compound according to any one of clauses 98 to 101, wherein n is 1.

[0306] 103.Formula

[0307] [ka] 99. The compound according to claim 98, having the formula:

[0308] 104. Expression

[0309] [ka] Compound. EXAMPLES

[0310] Various reactive carbohydrates were synthesized as further detailed below according to Reaction Scheme 2. In addition, protein conjugation was performed to form glycoconjugates (I) and (II), and the conjugation was evaluated by ELISA, Western blot, MALDI-TOF, and Bradford assay.

[0311] [ka]

[0312] The reactions were carried out under an argon atmosphere using commercially available HPLC grade. Commercially available reagents (Sigma Aldrich and Fisher Scientific, Canada) were used without further purification. N-acetyl-D-galactosamine was provided by Rose Scientific Ltd., Alberta, Canada. Reaction progress was monitored using silica gel 60 F. 254 The reactions were monitored by thin layer chromatography using coated plates (E. Merck). Flash chromatography was performed using ZEOprep™ silica gel 60 (40-63 μm) from Canadian Life Science or a FlasuPure™ system from Buchi. Detection was performed under UV light or by spraying with 20% ethanolic sulfuric acid or molybdate or KMnO4 solutions followed by heating. NMR spectra were recorded on a Bruker ULTRASHIELD™ 300 MHz and a Bruker Avance™ III HD 400 and 600 MHz spectrometer. Proton and carbon chemical shifts (δ) were 7.27 ppm ( 1 H) and 77.00 ppm ( 13 Coupling constants (J) are reported in Hertz (Hz) and the following abbreviations are used for peak multiplicities: singlet (s), doublet (d), doublet of doublets (dd), doublet of doublets with equal coupling constants (t ap ), triplet (t), multiplet (m). Analysis and assignments were performed using COSY (correlation spectroscopy) and HSQC (heteronuclear single quantum coherence spectroscopy) experiments. High-resolution mass spectra (HRMS) were measured using a Thermo Scientific LC-MS-TOF (liquid chromatography mass spectrometry time-of-flight) instrument in positive and / or negative electrospray mode. Protonated ions (M+H) + or sodium adduct (M+Na) +Either of the following was used for experimental confirmation. LC method: Samples were injected (2 μL) onto a PrePure™ C18 150×4.6 mm column with 5 μm particles using a Dionex Ultimate™ 3000 system (Thermo Scientific) with water (A) and acetonitrile (B), both containing 0.1% acetic acid, at room temperature with a flow rate of 800 μL / min. The gradient started at 5% B and was held for 0.5 min. This was increased to 15% B in 1 min, then to 27% B in 14.5 min, then to 95% B in 4 min. After the gradient was held at 95% B for 2 min, it was decreased to 5% B in 1 min. Finally, the gradient was held at 5% B for 1 min. Total time was 24 min. MS Methods: MS spectra were collected on a TSQ Quantum Access Max™ (Thermo Scientific) equipped with a HESI ion source in positive ion mode set at a source voltage of 4.5 kV and a source temperature of 320° C. MS acquisition was from m / z 200 to 1000 in SCAN mode. Data were analyzed using Thermo XCalibur Qual Browser™.

[0313] Example 1: Synthesis of allyl 2-acetamido-2-deoxy-α-D-galactopyranoside (2) Acetyl chloride (4.85 mL, 68.0 mmol, 3.4 equiv) was added to allyl alcohol (80 mL) at 0° C. under N2. After the solution was stirred at this temperature for 1 h, N-acetylgalactosamine (GalNAc, (1), 4.42 g, 20.0 mmol, 1.0 equiv) was added to the solution at room temperature. The mixture was stirred at 70° C. for 1 h. The solution was cooled to room temperature and then kept at 0° C. The solution was diluted with MeOH (40 mL) and neutralized with solid NaHCO3 (8.0 g) until a pH of 7.0 was reached. The mixture was filtered under a pad of Celite™ and washed with MeOH. The solvent was then removed under reduced pressure. The dry crude product was 1) dissolved in water (50 mL) and washed with DCM (4 x 200 mL) followed by EtOAc (200 mL), the aqueous layer was separated and then kept at -80 °C followed by lyophilization, or 2) precipitated in MeOH / hexane or EtOH / diisopropyl ether, or 3) purified by flash chromatography on silica gel using a gradient (EtOAc 100% to EtOAc / MeOH 4:1) to give the desired product arylGalNAc (2) as a white solid (4.18 mg, 1.60 mmol, 80%). Rf = 0.30; ACN / H2O 95:5; 1 H NMR(CD3OD,600MHz):δ 5.99-5.88(m,1H,OCH2CH=CH2),5.31(dd,1H,J trans =17.3,J gem =1.3Hz,OCH2CH=CH2),5.17(dd,1H,J cis =10.5Hz,OCH2CH=CH2),4.86(d,1H,J 1,2 =3.8Hz,H-1),4.27(dd,1H,J 2,3 =11.0Hz,H-2),4.20(m,1H,OCH2),4.00(m,1H,OCH2),3.89(dd,J 3,4 =J 4,5 = 2.6Hz, H-4), 3.85-3.77 (m, 2H, H-3 and H-5), 3.72 (m, 2H, H-6a and H-6b) and 1.99ppm (s, 3H, CH3); 13C NMR(CD3OD,150MHz):δ 172.5(NHCO),134.2(OCH2CH=CH2),116.1(OCH2CH=CH2),96.6(C-1),71.2(C-3), 69.0(C-4),68.3(C-5).67.8(OCH2),61.4(C-6),50.2(C-2) and 21.2ppm(CH3).ESI + -HRMS:C 11 H 20 About O6N [M+H] + Calculated, 262.1285; Found, 262.1294. LC-MS: rt=4.94 min.

[0314] Example 2: Synthesis of allyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (3) To a solution of allylGalNAc (2) (2.35 g, 9.0 mmol, 1.0 equiv.) and benzaldehyde dimethyl acetal (6.75 mL, 45.0 mmol, 5.0 equiv.) in dry DMF (20 mL) was added a catalytic amount of p-toluenesulfonic acid monohydrate. The mixture was stirred at room temperature. After 5 h, the mixture was diluted with CHCl3 and washed with saturated aqueous NaHCO3. The organic layer was separated, washed with water, dried over Na2SO4, and concentrated to give a white solid. The benzylidene acetal (compound (3)) was isolated as a white solid by precipitation in EtOAc / hexanes (2.64 g, 7.56, 84%). Rf=0.21; DCM / MeOH 9.0:0.5; 1 H NMR(CDCl3,300MHz):δ 7.59-7.46(m,2H,H-ar),7.43-7.31(m,3H,H-ar),5.91(m,1H,OCH2CH=CH2),5.75(d,1H,J NH,H2 =9.0Hz,NH),5.58(s,1H,PhCH),5.34-5.17(m,2H,OCH2CH=CH2),5.01(d,1H,J 1,2 =3.5Hz,H-1),4.56-4.42(ddd,1H,J 2,3 =10.9Hz,J 2,OH =9.1Hz,H-2),4.34(dd,1H,J 5,6a =1.5Hz,J6a,6b = 12.5Hz, H-6a), 4.19 (m, 2H, H-4 and OCH2), 4.04 (m, 1H, dd, 1H, J 5,6b =1.6Hz,J 6a,6b =12.5Hz,H-6b),4.01(m,OCH2),3.86(dd,1H,J 3,4 =10.9Hz,H-3),3.71(sb,1H,H-5),2.80(d,1H,J 3,OH = 10.7Hz, OH-3) and 2.05ppm (s, 3H, CH3).ESI + -HRMS:C 18 H 24 About O6N [M+H] + Calculated, 350.1598; Found, 350.1608. LC-MS: rt=19.78 min.

[0315] Example 3: Synthesis of allyl(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→3)-2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (5a) Compound 3 (657 mg, 1.88 mmol, 1.0 equiv.) and 2,3,4,6-tetra-O-benzoyl-α-D-galactopyranosyl bromide (compound (4b), 3.48 g, 8.46 mmol, 4.5 equiv.) were stirred with silver-exchanged zeolite (2.56 g) in anhydrous toluene (60 mL) containing 4 Å molecular sieves (800 mg) at 70° C. under argon atmosphere for 18 h. The mixture was then filtered off under a pad of Celite™. The solvent was removed under reduced pressure. The crude product was purified by chromatography on silica gel using a gradient from 100% hexane to hexane / EtOAc 1:4 to give the desired disaccharide compound (5a) as a white solid (817 mg, 1.20 mmol, 64%). Rf=0.24; hexane / EtOAc 1:4; 1 H NMR(CDCl3,400MHz):δ 7.57-7.50(m,2H,H-ar),7.42-7.30(m,3H,H-ar),5.97-5.80(m,1H,OCH2CH=CH2),5.63-5.545(m,3H),5.38(dd,1H,J 3,4’ =3.3Hz,J4,5’ =1.0Hz,H-4 II ),5.33-5.14(m,4H),5.05(d,1H,J=3.5Hz,H-1),5.11-5.00(m,1H,H-2),4.76(d,1H,J=7.9Hz,H-1'),4. 68(m,1H),4.30-3.80(m,7H),3.65(m,1H),2.15(s,3H),2.04(s,6H),1.99(s,3H) and 1.97ppm(s,3H).ESI + -HRMS:C 32 H 42 O 15 [M+H] for N + Calculated, 680.2549; Found, 680.2549 (0.0 ppm). LC-MS: rt = 13.11 min.

[0316] Example 4: Synthesis of allyl(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (5b) Compound 3 (101 mg, 0.29 mmol, 1.0 equiv.) and 2,3,4,6-tetra-O-benzoyl-α-D-galactopyranosyl bromide (compound (4b), 239 mg, 0.58 mmol, 2.0 equiv.) were stirred with silver-exchanged zeolite (430 mg) in anhydrous toluene (10 mL) containing 4 Å molecular sieves at 70° C. for 18 h under argon atmosphere. The mixture was then filtered off under a pad of Celite™. The solvent was removed under reduced pressure. The crude product was purified by chromatography on silica gel using a gradient from 100% hexane to hexane / EtOAc 1:2 to give the desired disaccharide compound (5b) as a white solid (250 mg, 0.27 mmol, 94%). mp: 110-111° C.; Rf=0.20; hexane / EtOAc 1:2; 1 H NMR(CDCl3,600MHz):δ 8.06-7.19(m,25H,H-ar),5.98(dd,1H,J 3,4’ =3.3Hz,J 4,5’ =1.0Hz,H-4 II ),5.85-5.78(m,2H,OCH2CH=CH2and H-2II ),5.60(dd,1H,J 2,3’ =10.2Hz,J 3,4’ =3.4Hz,H-3 II ),5.48(sb,1H,NH),5.23(m,3H,OCH2CH=CH2and H-1),4.68(dd,1H,J 5’,6a’ =6.9Hz,J 6a’,6b’ =11.4Hz, H-6a I ),4.63-4.58(m,1H,H-2),4.46-4.36(m,3H,H-4.H-5 and H-6b II ), 4.14-4.07(m,3H,H-6a,OCH2and H-3), 3.96(m,1H,OCH2), 3.75(m,1H,H-6b), 3.51(m,1H,H-5) and 1.40ppm(s,3H,CH3); 13 C NMR (CDC 13 ,150MHz):δ 170.0(NHCO), 166.0, 165.5, 165.4, 165.2(CO), 137.6-126.2(multiple, 30 C-arom), 133.2(OCH2CH=CH2), 117.8(OCH2CH=CH2), 102.0(C-1 II ), 100.9(CPhCH), 97.3(C-1 I ),76.1(C-3),75.4(C-4),71.7(C-3 II and C-5 II ), 70.2(C-2 II ),69.1(C-6),68.6(OCH2),68.1(C-4 II ), 62.9(C-5), 62.6(C-6 I ), 48.2(C-2) and 22.5ppm(CH3).ESI + -HRMS:C 52 H 50 O 15 [M+H] for N + Calculated value: 928.3175; Measured value: 928.3133.

[0317] Example 5: Synthesis of allyl(β-D-galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-α-D-galactopyranoside (6) A solution of compound (5b) (1.12 g, 1.20 mmol, 1.0 equiv) in 1 M sodium methoxide in methanol (12 mL, pH 8-9) was stirred at room temperature until the starting material was consumed. After 1.5 h, the solution was chromatographed on an ion exchange resin (Amberlite™ IR 120, H + ), filtered, washed with MeOH, and the solvent was removed under reduced pressure to give the intermediate as a white solid. The white solid intermediate was then dissolved in 10 mL of 60% aqueous acetic acid, and the resulting solution was stirred at 60 °C for 1.5 h. The solvent was removed under reduced pressure, and the residue was dissolved in water and washed several times with dichloromethane and twice with EtOAc. The aqueous layer was then lyophilized to give the deprotected allyl compound (6) as a white solid (400 mg, 0.94 mmol, 79%). mp=230-232 °C; Rf=0.53; CHCl3 / MeOH / H2O 11:6:1; 1 H NMR(D2O,600MHz):δ 5.80(m,1H,OCH2CH=CH2),5.19(dd,1H,J trans =17.3Hz,OCH2CH=CH2),5.09(dd,1H,J cis =10.4Hz,OCH2CH=CH2),4.77(d,1H,J 1,2 =3.7Hz,H-1),4.29(d,1H,J 1,2 =3.7Hz,H-1),4.29(d,1H,J 1,2 =7.8Hz,H-1 II ), 4.16(dd,1H,J 2,3 =11.2Hz,J 1,2 =3.7Hz,H-2),4.08-4.01(m,2H,H-4 and OCH2),3.92-3.82(m,3H,H-3,H-5 and OCH2),3.73(dd,1H,H-4 II ),3.63-3.52(m,4H,H-6a,b and H-6'a,b),3.47(m,2H,H-3 II and H-5 II ),3.39(dd,1H,J 2’,3’ =10.0Hz,J 1’,2’ =7.7Hz,H-2 II ) and 1.85 ppm (s,3H,CH3); 13C NMR(D2O,150MHz):δ 174.6(NHCO),133.7(OCH2CH=CH2),117.9(OCH2CH=CH2),104.7(C-1 II ),96.4(C-1),77.2(C-3),75.0(C-5 II ), 72.5(C-3 II ), 70.7(C-5), 70.6(C-2 II ), 68.8(C-4), 68.6(C-4 II ), 68.4(OCH2), 61.2(C-6 II ), 61.0(C-6), 48.6(C-2) and 22.0ppm(CH3).ESI + -HRMS:C 17 H 29 O 11 [M+Na] for NNa + Calculated, 446.1633; Found, 446.1613. LC-MS: rt=4.77 min.

[0318] Example 6: Formation of glycoconjugate (II) by conjugation of N-succinimidyl-3-{[3-(β-D-galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-α-D-galactopyranosyl)oxypropyl]thio}propanoate (11b) with protein CRM197 Compound (6) (138 mg, 0.53 mmol, 1.0 equiv.), compound (7) (538 mg, 2.65 mmol, 5 equiv. - commercially available from Sigma-Aldrich), and 4,4'-azobis(4-cyanovaleric acid) (ACVA, 1.5 mg, 0.05 mmol, 0.1 equiv.) were stirred in degassed acetonitrile (5 mL) at reflux (70 °C) for 2 h. After returning to room temperature, the solvent was removed under reduced pressure. Compounds (6) and (7) can also be reactivated with DMPA (0.1 equiv.) in THF / water (1:1; v / v) under UV irradiation (280-320 nm) at 25 °C for 20 min. The crude product was suspended in water and extracted by washing several times with dichloromethane and twice with EtOAc to remove the succinimide reagent. The final aqueous layer containing crude compound (11b; NHS-TF) was cooled to -80°C and lyophilized. Crude compound (11b) was then used directly in the conjugation step with protein CRM197 without any further purification to give glycoconjugate (II; CRM-TF). The crude white solid was also purified by reverse preparative HPLC on C-18 to give white solid 11b. Pure compound (11b) was used in the conjugation step with protein CRM197 to give glycoconjugate II; CRM-TF). 1 H NMR(D2O,400MHz):δ 4.83(d,1H,J=3.7Hz,H-1),4.40(d,1H,J=7.7Hz,H-1 II ),4.25(dd,1H,J=17.0,6.9Hz),4.18(d,1H,J=2.6Hz),4.02-3.90(m,2H),3.84(d,1H,J=3.1Hz),3.80-3.62(m,5H),3.6 2-3.40(m,4H),3.02(t,2H,J=6.7Hz),2.96-2.83(m,5H),2.79-2.55(m,3H),1.96(s,3H) and 1.87(dd,2H,J=12.9,6.4Hz); 13C NMR(D2O,100MHz,):δ 176.6,174.6,173.3,169.2,104.8,97.2,77.3,75.0,72.6,70.6,68.8,68.6,66.4,66.3 ,61.2,61.0,48.7,34.3,31.4,28.4,28.2,26.3,25.7,25.6,25.2,and 22.0ppm(CH3).ESI + -HRMS:C 24 H 38 O 15 [M+Na] for N2SNa + Calculated value, 649.18851; Found value, 649.18838, 0.2 ppm. HPLC: Tr=10.747.

[0319] For the conjugation step, the carrier protein was buffer exchanged into PBS at pH 8 by centrifugal filtration (Amicon™ MWCO; 10 or 30K) and its concentration was adjusted to 2 mg / ml. Protein concentration was measured using Bradford assay with BSA as standard. Conjugation to protein was initiated by adding a fresh 20 mM solution of compound 11b in water to the protein to reach a final protein concentration of 1 mg / ml. The amount of compound 11b added to the protein can be adjusted depending on the number of surface accessible lysines of the protein and the final ratio of intended conjugation. The solution was then vortexed for 90 minutes and then washed by centrifugal filtration (Amicon™ MWCO; 10 or 30K) using PBS at pH 7.4, or by gel filtration.

[0320] The conjugation of protein to compound (11b), i.e. obtaining glycoconjugate (II), was demonstrated by ELISA, Western blot, MALDI-TOF, and Bradford assay.

[0321] The reactivity of the TF-specific lectin peanut agglutinin (PNA) and the anti-TF monoclonal antibody JAAF11 against 0.01 μg of unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios (0.2, 0.6, 3) of NHS-TF (compound (11b)) to the total number of lysines in the protein was determined by ELISA. As shown in Figure 1, the ELISA shows a correlation between the conjugation conditions and the reactivity of the resulting glycoconjugates.

[0322] Figure 2 shows a Coomassie stained SDS-PAGE gel of 1 μg of unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios of NHS-TF (compound (11b)) to total lysines in the protein. A decrease in the mobility of the glycoconjugate band as a function of the conjugation conditions is observed compared to unconjugated CRM, indicating that the CRM is conjugated.

[0323] Western blots of 1 μg of unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios of detected NHS-TF (compound (11b)) to total protein lysines were recorded using the TF-specific lectin peanut agglutinin (PNA) and the anti-TF monoclonal antibody JAAF11. As shown in Figure 3, the western blot revealed reactive bands for both PNA and JAAF11 of increasing molecular weight relative to the conjugation conditions, indicating that the CRM was conjugated and the absence of reactivity for unconjugated CRM.

[0324] MALDI-TOF spectra were recorded for unconjugated CRM197 and CRM197 conjugated to TF (CRM-TF:glycoconjugate (II)) at three different ratios of NHS-TF (compound (11b)) to the total number of lysines in the protein. The spectra show the mass increase as a function of the conjugation conditions, as shown in Figure 4. The table in the inset shows the mass increase and the average molar ratio of conjugation to TF.

[0325] Example 7: Formation of glycoconjugate (I) by conjugation of N-succinimidyl-3-[3-(2-acetamido-2-deoxy-α-D-galactopyranosyloxypropyl)thio]-propanoate (11a) with protein CRM197 Compound (2) (200 mg, 0.765 mmol, 1.0 equiv.), compound (7) (933 mg, 4.59 mmol, 6.0 equiv.), and 4,4'-azobis(4-cyanovaleric acid) (ACVA, 2 mg, 0.08 mmol, 0.01 equiv.) were stirred in degassed acetonitrile and isopropyl alcohol (4.0 mL, 1:1) at reflux (70° C.) for 2 h. After returning to room temperature, the solvent was removed under reduced pressure. The crude product was suspended in water and purified by A) chromatography on C18 silica gel or C18 HPLC, or B) extraction by washing several times with dichloromethane and twice with EtOAc to remove the succinimide reagent. The final aqueous layer containing crude compound (11a; NHS-TN) was cooled to −80° C. and lyophilized. The crude compound (11a) was then used directly in the conjugation step with protein CRM197 without any further purification to give glycoconjugate (I; CRM-TN).

[0326] For the conjugation step, the carrier protein was buffer exchanged into PBS at pH 8 by centrifugal filtration (Amicon™ MWCO; 10 or 30K) and its concentration was adjusted to 2 mg / ml. Protein concentration was measured using Bradford assay with BSA as standard. Conjugation to protein was initiated by adding a fresh 20 mM solution of compound 11a in water to the protein to reach a final protein concentration of 1 mg / ml. The amount of compound 11a added to the protein can be adjusted depending on the number of surface accessible lysines of the protein and the final ratio of intended conjugation. The solution was then vortexed for 90 minutes and then washed by centrifugal filtration (Amicon™ MWCO; 10 or 30K) using PBS at pH 7.4, or by gel filtration.

Claims

1. Reactive carbohydrates of formula (A) 【Chemistry 1】 (In the formula, R １ is H and n is an integer from 1 to 5, Formula (A ０ ) Compound 【Chemistry 2】 HO-(CH ２ ) ｎ -CH=CH ２ and acetyl chloride at a mixing temperature of about 0° C. to about 25° C. to produce a cold intermediate reaction mixture containing the compound of formula (A 0 ), HO—(CH 2 ) n —CH═CH 2 , and HCl; heating the intermediate reaction mixture with a heat source to a temperature of about 60° C. to about 80° C.; and cooling the reaction mixture to obtain a cooled mixture comprising the compound of formula (A).

2. 2. The process of claim 1, wherein the mixing temperature is from about 10° C. to about 25° C.

3. The process of claim 1, wherein the mixing temperature is from about 10°C to about 20°C.

4. HO-(CH ２ ) ｎ -CH=CH ２ 4. The process according to any one of claims 1 to 3, wherein the solution comprising acetyl chloride and acetyl chloride is prepared under an inert atmosphere at a temperature of about 0°C.

5. 5. The process of any one of claims 1 to 4, wherein the heating is carried out at a reaction temperature of about 60°C to about 80°C for a reaction time of about 30 minutes to 5 hours.

6. 6. The process of claim 5, wherein the reaction temperature is about 70°C.

7. The process of claim 5 or 6, wherein the reaction time is from about 30 minutes to about 1 hour.

8. The process of any one of claims 5 to 7, wherein the reaction time is from about 30 minutes to about 45 minutes.

9. The process according to any one of claims 5 to 8, wherein the reaction time is about 30 minutes.

10. The process of any one of claims 1 to 9, further comprising neutralizing the cooled mixture.

11. The process of any one of claims 1 to 10, further comprising isolating the compound of formula (A).

12. Formula (AA ＰＧ ) reactive carbohydrates 【Chemistry 3】 (In the formula, R ２ is a monovalent protecting group, PG is a divalent protecting group, and n is an integer from 1 to 5, In the presence of a metal-containing zeolite, ＰＧ ) Compound 【Chemistry 4】 Formula (A １ ) Compound 【Chemistry 5】 wherein X is -I, -Br, -Cl, -CN, -OTf (triflate), -OMs (mesylate), -OTs (tosylate), methylsulfate, or trichloroacetimidate.

13. Reactive carbohydrates of formula (AA) 【Chemistry 6】 wherein n is an integer from 1 to 5, comprising the steps of: The compound of formula (AA) obtained by the process according to claim 12 ＰＧ ) deprotecting the compound of formula (I).

14. Reactive carbohydrates of formula (AA) 【Chemistry 7】 The process for synthesizing a compound of formula (AA ＰＧ ) Compound 【Chemistry 8】 (wherein n is an integer from 1 to 5; R ２ is a monovalent protecting group and PG is a divalent protecting group, The deprotection is carried out by reacting a compound of the formula (AA ＰＧ ) with a basic solution to form an intermediate crude product, and then treating said intermediate crude product with an acidic solution.

15. Reactive carbohydrates of formula (B) or (BB) 【Chemistry 9】 wherein n is an integer from 1 to 5, m is an integer from 1 to 5, and LG is a leaving group that allows for amide bond formation when the compound of formula (B) or (BB) reacts with an amino group, Compound of formula (C) 【Chemistry 10】 with a compound of formula (A) 【Chemistry 11】 (In the formula, R １ is H) to form a compound of formula (B) or Compound of formula (AA) 【Chemistry 12】 to form the compound of formula (BB).

16. The process according to claim 15, wherein the compound of formula (A) is obtained by the process according to any one of claims 1 to 11 and the compound of formula (AA) is obtained by the process according to claim 12.

17. Glycoconjugates of formula (I) or (II) 【Chemistry 13】 (In the formula, n is an integer from 1 to 5, m is an integer from 1 to 5, and p is an integer from 1 to 50, 【Chemistry 14】 is a carrier material comprising at least one amino group available for conjugation, 17. A process comprising conjugating at least one free amino group of the carrier material with a compound of formula (B) to form the glycoconjugate of formula (I) or with a compound of formula (BB) to form the glycoconjugate of formula (I), wherein the compounds of formula (B) and (BB) are obtainable by the process according to claim 15 or 16.

18. 20. The process of claim 17, wherein the support material comprises a protein, polypeptide, or peptide.

19. The process of any one of claims 1 to 18, wherein n is an integer from 1 to 3.

20. The process of any one of claims 1 to 19, wherein n is 1.

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