Synthesis of 6-azido-6-deoxy-2-N-acetyl-hexosaminyl-nucleoside diphosphates

Abstract

The present invention relates to a method for the synthesis of 6-azido-6-deoxy-2-N-acetyl-monosaccharide-nucleotide diphosphates, in particular 6-azido-6-deoxy-2-N-acetyl-D-galactosamine-nucleotide diphosphate or 6-azido-6-deoxy-2-N-acetyl-D-glucosamine-nucleotide diphosphate. The synthesis method according to the present invention is characterized by high efficiency and high yield. Another part of the present invention is a key intermediate of the method.

Description

Technical Field

[0001] This invention belongs to the field of functionalized nucleoside sugars and relates to the improved preparation of glyconucleotide diphosphates. More specifically, this invention relates to a method for chemically converting GalNAc or GlcNAc into their respective 6-azido derivatives via a cyclic sulfate ester, followed by an anomeric phosphorylation step and a UMP coupling step to convert them into their respective UDP derivatives. This invention also relates to various specific intermediates and purification steps. Background Technology

[0002] Carbohydrates play a crucial role in cell biology through their functions in energy metabolism and storage, as well as as key components of genetic material and other structural elements. Furthermore, carbohydrates linked to proteins or lipids (also known as glycans) are important for cell communication during cell differentiation and development. Glycans are defined as the oligosaccharide portion of glycoconjugates (such as glycoproteins) that can be linked to proteins via glycosidic ether bonds (as in O-glycoproteins) or amide bonds (as in N-glycoproteins). In both O- and N-glycoproteins, N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) are frequently encountered structural units that can be directly linked (GalNAc) to serine or threonine, as in O-glycoproteins, or directly linked (GlcNAc) to asparagine, as in N-glycoproteins. GalNAc or GlcNAc can also be part of a larger oligosaccharide chain in a glycoprotein, either internally or as the furthest monosaccharide, primarily linked to another sugar via β-glycosidic bonds. To incorporate the GalNAc moiety into proteins or oligosaccharide chains, a series of N-acetylgalactosamine transferases (GalNAc-transferases) have evolved in nature, capable of transferring the monosaccharide GalNAc from UDP-GalNAc (donor) to the alcohol moiety of serine / threonine or another sugar (acceptor). Similarly, N-acetyl-glucosamine transferases (GlcNAc-transferases) can link GlcNAc to an alcohol acceptor. For this purpose, the UDP-GalNAc donor substrate is produced from glucose-6-phosphate and glutamate in the so-called hexosamine pathway. Five subsequent enzymatic conversions result in the formation of UDP-GlcNAc, which is then converted back to UDP-GalNAc by UDP-galactose-4-epimerase, as disclosed, for example, in Yamamoto et al., Applied Environ. Microbiol. 1981, 41, 392, which is incorporated herein by reference.

[0003] To investigate in depth the mechanisms and functions of incorporating GalNAc into naturally occurring glycans, or for the identification of novel galactosamine transferases, large quantities of (labeled) UDP-GalNAc are required, such as those disclosed in Maley et al., Biochem. Biophys. Res. Commun. 1970, 39, 371, which is incorporated herein by reference. Therefore, various methods for the preparation of UDP-GalNAc (and its analogues) have been devised over the years, generally following chemical processes, enzymatic conversions, or combinations thereof.

[0004] The chemical synthesis of glyconucleotides typically follows one of two pathways, as summarized by Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which is incorporated herein by reference: (a) pyrophosphorylation using sugar-1-phosphate and activated nucleoside monophosphate (NMP), or (b) direct glycosylation of the glycosyl donor using nucleoside diphosphate (NDP), with the pyrophosphorylation pathway being more prevalent. Therefore, pyrophosphorylation first requires the synthesis of a sugar-1-phosphate derivative, for which numerous methods have been published, as summarized by Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which is incorporated herein by reference. One of the difficulties in these methods is obtaining the required sugar-1-phosphate, which exhibits unique α-selectivity, i.e., the anomeric conformation found in the vast majority of glyconucleotides. The second step in the pyrophosphorylation method involves coupling the sugar-1-phosphate with activated NMP. Besides the 1-H-tetrazole modification reported by Wittmann et al., J. Org. Chem. 1997, 62, 2144, the pioneering work by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756 (incorporated herein by reference), on coupling with phosphomorpholidate remains widely used. However, advances beyond this classic phosphomorpholidate strategy have provided a range of other methods, summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95 (incorporated herein by reference). One method particularly useful for glyconucleotides involves coupling sugar-1-phosphate with imidazolide-activated NMP, as reviewed in Wagner et al., Nat. Prod. Rep. 2009, 26, 1172 (incorporated herein by reference). The required imidazoline can be readily generated from nucleoside monophosphates and activated with ZnCl2 or MgCl2 via the method reported by Dabrowski-Tumanski et al., Eur. J. Org. Chem. 2013, 2147 (which is incorporated herein by reference). However, a universally accepted chemical pathway for glyconucleotides has not yet emerged. In particular, most methods revolve around common hexoses, with only a subset applicable to N-acetylated hexosamines, for example, due to the use of strongly basic conditions and / or azido-modified sugars, for example, due to the use of phosphate ester deprotection conditions incompatible with azides.

[0005] Enzymatic synthesis of glyconucleotides avoids the protecting and deprotecting steps required during chemical synthesis. Furthermore, enzymatic formation of pyrophosphate bonds generally proceeds with better efficiency and stereoselectivity compared to chemical bond formation. Different enzymatic methods have been reported, summarized in Bülter et al., Glycoconj. J. 1999, 16, 147, which is incorporated herein by reference. These methods vary in the number of enzymes used and the type of raw materials used, as reported by Cai et al., J. Carbohydr. Chem. 2012, 31, 535, which is also incorporated herein by reference. For example, Piller et al., Anal. Biochem. 1982, 127, 171 (which is incorporated herein by reference) reported that UDP derivatives of N-acetylglucosamine (GlcNAc) can be converted to UDP-GalNAc by mammalian Gal-4 epimerase. The main drawback of this method is the low yield (30% equilibrium GalNAc:GlcNAc) and the difficulty in isolating UDP-GalNAc from excess UDP-GlcNAc. The enzymatic pathway reported by Carlson et al., Biochemistry 1964, 3, 402 (incorporated herein by reference), begins with D-galactosamine and utilizes yeast ecdysonekinase to form galactosamine-1-phosphate (GalNH2-1-P). In the next step, the purified GalNH2-1-P is chemically or enzymatically coupled to UMP using yeast UDP-glucuronidyltransferase, as reported by Heidlas et al., J. Org. Chem. 1992, 57, 152 (incorporated herein by reference). In both cases, UDP-GalNH2 is chemically N-acetylated in the final step, resulting in a very low overall yield after purification (typically not exceeding 20%). Another enzymatic synthesis, as described by Bülter et al., Carbohydr. Res. 1997, 305, 469 (incorporated herein by reference), facilitates the production of UDP-GalNAc using a seven-enzyme coupled system that converts UMP, sucrose, and GalNH2-1-P into UDP-GalNH2, which is ultimately chemically converted to an N-acetylated product with a total yield of 34%. Zou et al., Carb. Res. 2013, 373, 76 (incorporated herein by reference), devised a concise one-pot three-enzyme scheme for the preparation of UDP-GalNAc and its derivatives, utilizing enzymes derived from Streptococcus pneumoniae: UTP-glucose-1-phosphate uridine transferase (SpGalU), galactokinase (SpGalK), and inorganic phosphatase (PPase).In the presence of ATP, SpGalK converts GalNAc to GalNAc-1-P, which binds to uridine triphosphate (UTP) as a substrate for SpGalK, producing UDP-GalNAc in a reasonable yield (32%). The third enzyme in this reaction is yeast inorganic pyrophosphatase (PPase), which drives the production of UDP-GalNAc by preventing the reverse reaction—the separation of PPi into two monophosphate (Pi) molecules. Following earlier work, Liu et al., Bioorg. Med. Chem. Lett. 2013, 23, 3764 (incorporated herein by reference), applied the same one-pot three-enzyme approach, using UDP-glucose pyrophosphatase (AtUSP) from Arabidopsis thaliana instead of SpGalU. Bourgeaux et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459 (incorporated herein by reference), applied a similar three-enzyme approach to produce UDP-GalNAc. Thus, starting with GalNAc, UTP, and ATP, UDP-GalNAc was synthesized in high yield (68%) using recombinant human GalNAc kinase (GK2) and UDP-GalNAc pyrophosphorylase (AGX1). Here, mammalian GK2 uses ATP as a phosphate donor to catalyze the phosphorylation of GalNAc. Then, mammalian AGX1 uses UTP to convert GalNAc-1-P to UDP-GalNAc, thereby using PPase to increase product formation and obtain large quantities of UDP-GalNAc. Subsequently, Pouilly et al., ACS Chem. Biol. 2012, 7, 753 (incorporated herein by reference), demonstrated the versatility of this method by generating several UDP-GalNAc analogues as substrates for the polypeptide GalNAc transferase T1 (ppGalNAcT1). In addition to the reported enzymatic protocols for the preparation of UDP-GalNAc, several chemoenzymatic methods, employing combinations of enzymatic and chemical steps, have been reported. For example, Lai et al., Bioorg. Med. Chem. Lett., 2009, 19, 18, 5433, and Guan et al., Chem. Commun., 2009, 6976 and Chem. Eur. J. 2010, 16, 13343 (incorporated herein by reference), described two strategies for synthesizing UDP-GalNAc and several analogues starting from N-acetylgalactosamine.

[0006] Although the (chemically)enzymatic synthesis of UDP-sugars is concise, particularly by eliminating lengthy synthesis involving excessive (de)protection steps, it is clear that the scalability of enzymatic UDP-sugar synthesis is challenging. Furthermore, recombinant expression of various enzymes is required, thus protocols involving (multiple) enzymes would be expensive. Obviously, the cost would be further increased if such UDP-sugars were produced according to GMP standards for the manufacture of clinical-grade materials, as disclosed by Warneck et al., Biotechnol. Bioengin. 2005, 92, 831 (which is incorporated herein by reference). Finally, it is likely that most of the required enzymes prevent the use of alternative N-substituted galactosamine variants, making the enzymatic synthesis of non-natural UDP-GalNAc analogs a challenging, if not impossible, task. In this regard, there has been considerable interest in the use of azido-modified sugars in, for example, metabolic reporter strategies, as reported by Hang et al., PNAS 2003, 100, 14846 (incorporated herein by reference), or through controlled labeling of glycoproteins, as reported by Zeglis et al., Bioconj. Chem. 2013, 24, 1057 and Li et al., Angew. Chem. Int. Ed. 2014, 53, 7179 (incorporated herein by reference). In the latter area, van Geel et al., Bioconj. Chem. 2015, 26, 2233 (incorporated herein by reference) demonstrated that UDP-GalNAz can be cleanly mounted on monoclonal antibodies, providing stable and homogeneous antibody-drug conjugates following metal-free click conjugation of the toxic payload. Recently, Verkade et al., Antibodies, 7, 12 (incorporated hereby by reference), showed that shifting an azide to the 6-position of GalNAc provides antibody-drug conjugates with a reduced tendency to aggregate compared to GalNAz-containing analogs. However, it is clear that the preparation of any ADC based on 6-azido-GalNAc incorporation requires several grams to kilograms of UDP-6-azidosaccharide, which is not obtainable by known means.

[0007] Several complete synthetic strategies for 6-azido-GalNAc have been reported and are disclosed herein. Without exception, the introduction of the azido group is achieved through the S-oxidation of N-protected D-galactosamine 6-O-sulfonated derivatives. N2. Nucleophilic substitution is achieved. However, the efficiency of this latter substitution is highly dependent on the specific protecting groups at the O-3 and O-4 positions, exhibiting a strong rate-dependent relationship in the order of diacetyl < isopropylidene < unprotected. This strong structure-reactivity relationship results in nucleophilic substitution being either slow, requiring prolonged treatment with azide anions at high temperatures (e.g., 5 days at 100°C) and yielding low yields (<50%), or requiring lengthy synthetic routes to obtain properly protected galactosamine derivatives (up to 10 synthetic steps). Furthermore, the chosen synthetic routes may require expensive, odorous, and / or hazardous reagents (e.g., thiophenol, trifluoromethanesulfonic anhydride, 15-crown-5, cerium ammonium nitrate), and ultimately necessitate cumbersome amine protecting group exchange and anomeric deprotection schemes (e.g., removal of phthalimide with hydrazine or removal of allyl group with palladium reagents). Finally, a method reported by Hang et al., PNAS 2003, 100, 14846 (incorporated herein by reference) describes a rapid three-step synthetic route from GalNAc to 6-azido-GalNAc. However, due to the lack of selectivity in the toluenesulfonation step, the desired product is obtained as a mixture of components that is almost difficult to process, thus requiring extensive and cumbersome silica gel purification. From a preparative standpoint, these characteristics are unacceptable, and therefore a short and high-yield route is needed to obtain a properly protected 6-azido-GalNAc derivative (formally 6-azido-6-deoxy-N-acetyl-D-galactosamine).

[0008] The second challenge in obtaining UDP 6-azido-GalNAc at a suitable scale lies in the cumbersome subsequent steps of converting the 6-azido-GalNAc monosaccharide into a uridine diphosphate derivative (UDP). While various pathways can be considered, this typically involves first phosphorylation at the anomeric position, followed by a coupling step with UMP, either of which can be carried out chemically or enzymatically. Regarding anomeric chemophosphorylation, for example, MacDonald et al., J. Org. Chem. 1966, 31, 513 and Masuko et al., J. Org. Chem. 2012, 77, 1449 (which are incorporated herein by reference), pure phosphoric acid has been used; however, the products are obtained as a mixture of anomeric α / β molecules in low yields (<50%), thus requiring cumbersome purification and significant loss of valuable later-stage materials. Alternatively, the anodic, selectively deprotected 6-N3-GalNAc derivative can be reacted with a phosphorylation agent (e.g., chlorophosphonite or phosphoramide), which can be activated to react with the anodic hydroxyl group in the presence of a proton scavenger or a weak acid, respectively. Any of these phosphorylation agents will carry a protecting group to be removed in the phosphorylation step and subsequently after oxidation of the intermediate phosphite to a phosphate triester with mCPBA, H2O2, iodine, or other oxidants. For example, Hang et al., J. Am. Chem. Soc. 2004, 126, 6 (which is incorporated herein by reference), demonstrated the use of a phosphoramide agent for anodic phosphorylation. The disadvantages of this approach are the high cost and sensitivity of the phosphoramide agent, and the fact that phosphorylation at O-1 typically yields a mixture of α-anodic and β-anodic forms.

[0009] Regarding the coupling step of sugar-monophosphate with UMP, known methods involve activating UMP via nucleoside 5'-aminophosphate, as described by Moffatt et al., J. Am. Chem. Soc. 1961, 83, 649 (which is incorporated herein by reference), and were later improved by Wittmann et al., J. Org. Chem. 1997, 62, 2144 (which is incorporated herein by reference), based on 1-H-tetrazole activation. Another common strategy for coupling sugar-1-phosphate and UMP involves the use of carbonylating reagents, such as those reported by Illarionov et al., Russ. Chem. Bull. 2001, 50, 1303 and Loureiro Morais, Can. J. Chem. 2006, 84, 587, which are incorporated herein by reference. Alternatively, sugar-1-phosphate can be coupled with morpholidate derivatives of UMP, as reported by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756 (incorporated herein by reference), with coupling optionally carried out in the presence of 1-H-tetrazole, as reported by Wittmann et al., J. Org. Chem. 1997, 62, 2144 (incorporated herein by reference). Coupling of sugar-1-phosphate with imidazoline-activated NMP (as described by Wagner et al., Nat. Prod. Rep. 2009, 26, 1172, incorporated herein by reference) may be particularly efficient. However, despite the availability of various methods, there is no universally accepted, high-yield, and scalable route to obtain monosaccharide UDP derivatives, particularly N-acetylated hexosamines. Furthermore, the presence of the 6-azido group in UDP 6-azido-N-acetyl-hexosamine further restricts the choice of conditions due to its electron-withdrawing properties and incompatibility with a range of (reducing) conditions. Therefore, there is a strong need for an improved method for preparing UDP 6-azido-6-deoxy-N-acetyl-hexosamine (e.g., UDP 6-azido-6-deoxy-GalNAc and UDP 6-azido-6-deoxy-GlcNAc). Summary of the Invention

[0010] The inventors have developed a method for synthesizing 6-azido-6-deoxy-2-N-acetyl-monosaccharide-nucleoside diphosphate, particularly 6-azido-6-deoxy-2-N-acetyl-D-galactosamine-nucleoside diphosphate or 6-azido-6-deoxy-2-N-acetyl-D-glucosamine-nucleoside diphosphate and their various salt forms. The target compound of this invention is represented herein by structure (IX):

[0011]

[0012] In this article, B is a nucleobase.

[0013] This invention relates to (partial) synthetic methods for target compounds according to the present invention, and key intermediates in these methods. The invention also relates to total synthetic methods for compounds having structure (IX).

[0014] The synthesis method according to the present invention is characterized by high efficiency and high yield. In particular, it eliminates the disadvantages of the aforementioned prior art methods. Through the present invention, 6-azido-6-deoxy-2-N-acetyl-D-galactosamine-nucleoside diphosphate and 6-azido-6-deoxy-2-N-acetyl-D-glucosamine-nucleoside diphosphate are readily available to those skilled in the art. Detailed Implementation

[0015] definition

[0016] The verb “comprising” and its inflections, as used in this specification and claims, are used in their non-limiting sense to indicate inclusion of the item following the word, but do not exclude items not specifically mentioned. Furthermore, the indefinite article “a / an” refers to an element and does not exclude the possibility of more than one element, unless the context explicitly requires the presence of one and only one element. Therefore, the indefinite article “a / an” generally means “at least one / a kind”.

[0017] The compounds disclosed in this specification and claims may contain one or more asymmetric centers, and the compounds may exist in different diastereomers and / or enantiomers. Unless otherwise stated, the description of any compound in this specification and claims is intended to include all diastereomers and mixtures thereof. Furthermore, unless otherwise stated, the description of any compound in this specification and claims is intended to include individual enantiomers, as well as any mixtures of enantiomers—racemic mixtures or other mixtures. When the structure of a compound is described as a particular enantiomer, it should be understood that the invention of this application is not limited to that particular enantiomer.

[0018] Compounds can exist in different tautomer forms. Unless otherwise stated, compounds according to the present invention are intended to include all tautomer forms. When the structure of a compound is described as a specific tautomer, it should be understood that the invention of this application is not limited to that specific tautomer.

[0019] Compounds according to the invention can exist in the form of salts, which are also included in this invention. Salts are generally pharmaceutically acceptable salts containing pharmaceutically acceptable anions. The term "its salt" means a compound formed when an acid proton (typically an acid proton) is replaced by a cation (e.g., a metal cation or an organic cation). Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts not intended for administration to patients. For example, in a salt of a compound, the compound may be protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid is the anionic component of the salt. Pharmaceutically acceptable salts are acceptable for administration to patients such as mammals (salts with counterions have acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound, which is derived from a variety of organic and inorganic counterions known in the art and includes, for example, sodium, potassium, calcium, magnesium, ammonium, alkylammonium, dialkylammonium, trialkylammonium, tetraalkylammonium, etc., and when the molecule contains a basic functional group, it includes salts of organic or inorganic acids, such as hydrochlorides, hydrobromides, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, etc. In a preferred embodiment, the counterion of the salt according to the invention is selected from trialkylammonium, ammonium, and sodium, more preferably ammonium and sodium, and most preferably sodium.

[0020] The term "monosaccharide" is used in its usual scientific sense in this article, referring to an oxygen-containing heterocycle formed by the intramolecular hemiacetalization of 5-9 (hydroxylated) carbon atoms, most commonly containing five carbon atoms (pentoses) or six carbon atoms (hexoses). Typical monosaccharides are glucose (Glu), galactose (Gal), and mannose (Man).

[0021] The term "hexosamine" is used herein to refer to a monosaccharide having an amino group at the 2-position of its carbon chain. Typical hexosamines are D-galactosamine (GalNH2) and D-glucosamine (GlcNH2). Hexosamines can be acetylated. Typical acetylated hexosamines are N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc).

[0022] The terms “large amount” or “substantially” are defined herein as the majority (i.e., >50% of the total) of a mixture or sample, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total.

[0023] The alkyl group can be substituted or unsubstituted, can be straight-chain or branched, and may optionally contain a cyclic moiety. Optionally, the alkyl group is substituted with one or more substituents. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, tert-butyl, etc. In the context of this invention, particularly in R... 1 and R 2 In the definition, the preferred alkyl group is C10. 1-6 Alkyl, more preferably C 1-2 Alkyl group, with methyl group being the most preferred.

[0024] Aryl groups can include monocyclic, bicyclic, and polycyclic structures. Optionally, the aryl group can be substituted. Examples of aryl groups include, for example, phenyl, naphthyl, anthracene, and other groups. In the context of this invention, particularly in R... 1 and R 2 In the definition, the preferred aryl group is C 5-6 Aryl, with phenyl being the most preferred.

[0025] An arylalkyl group comprises an alkyl (or alkylene) moiety and an aryl (or arylene) moiety and may be considered as a substituted alkyl moiety or a substituted aryl moiety. The aryl (or arylene) moiety may comprise monocyclic and bicyclic structures. Optionally, the arylalkyl group may be substituted with one or more substituents. Examples of arylalkyl groups are benzyl, naphthylmethyl, 4-tert-butylphenyl, etc. In the context of this invention, particularly in R... 1 and R 2 In the definition, the preferred aryl group is C10. 6-12 arylalkyl, more preferably C 6-8 Arylalkyl, with benzyl being the most preferred.

[0026] This invention

[0027] The inventors have developed an improved method for the synthesis of 6-azido-2-N-acetyl-hexosamine-nucleoside diphosphate or its salts in high yield, particularly wherein the hexosamine is galactosamine or glucosamine and the corresponding acetylated hexosamine is N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc). The target compound of this invention is represented herein by structure (IX):

[0028]

[0029] In this document, B is a nucleobase. Although any nucleobase can be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0030] In this document, the wavy bond at the carbon atom at position 4 of the monosaccharide moiety can be axial (galactose configuration) or planar (glucose configuration). Both products can be readily obtained by the method of the present invention. In a preferred embodiment, the product having the galactose configuration is prepared because the compound (GalNAc) can be applied to the terminal GlcNAc moiety of its transfer to the glycoproteoglycan, a reaction readily carried out in the presence of a mutant galactosyltransferase (GalT) or N-acetylgalactosamine transferase (GalNAcT). This application of the compound of the present invention is known in the art, for example, according to Ramakrishnan et al., J. Biol. Chem. 2002, 277, 20833 and WO 2016170186, the entire contents of which are incorporated herein by reference.

[0031] [A] Reaction steps

[0032] The method according to various aspects of the invention includes one or more of steps (a), (b), (c), (d), (e), (f), (g), (i), (j), (i1), (j1), (x1), (x2), (x3), (x4), (y1), (y2), and (z). These steps are defined below.

[0033] [A.1] Step (a)

[0034] In the method of this invention, step (a) involves converting N-acetylglucosamine or N-acetylglucosamine into a 1,3-diacytized compound having structure (II). Step (a) is typically carried out by introducing a 4,6-benzylidene group, then acylating the remaining two hydroxyl groups at positions 1 and 3 of the monosaccharide, followed by removal of the benzylidene groups at positions 4 and 6 by acid hydrolysis or hydrogenation. These two hydroxyl groups are therefore unprotected, allowing compound (II) to also be referred to as a diol. The reaction scheme corresponding to step (a) is as follows:

[0035]

[0036] In a preferred embodiment, the reaction scheme corresponding to step (a) is as follows:

[0037]

[0038] The benzylidene protection of 2-N-acetyl-monosaccharides is a well-known method in the art, typically involving treatment with benzaldehyde (or its substituted or acetal form) in a polar aprotic solvent in the presence of an acid, as described, for example, by Yule et al., Tetrahedr. Lett., 36, 1995, 6839 (which is incorporated herein by reference). Acylation of the remaining alcohol functional groups at positions 1 and 3 is a well-known method in the art. Removal of the benzylidene group by acidic hydrolysis or hydrogenation is also a well-known method in the art, as described, for example, by Jiaang et al., Synlett., 2000, 6, 797–800 and Nishimura et al., Angew. Chem. Int. Ed., 2012, 51, 3386–3390 (which is incorporated herein by reference). Typically, the reaction in step (a) is carried out by treating the monosaccharide with benzaldehyde (or an acetal derivative) in a polar aprotic solvent such as DMF or acetonitrile in the presence of a catalytic sulfonic acid (p-TsOH or CSA). The next step of acylation can be carried out in pyridine by treatment with an acid anhydride or in a non-basic organic solvent (e.g., dichloromethane, acetonitrile, ethyl acetate) by treatment with an acyl chloride in the presence of a tertiary amine (e.g., triethylamine or DIPEA). Finally, removal of the benzylidene group can be carried out by acid hydrolysis in an aqueous solution of an acid or by Pd-C hydrogenation in a suitable solvent (e.g., MeOH, i-PrOH, or THF). The compound of structure (II) can be used as is in the next step, or it can be purified and / or isolated by methods known in the art.

[0039] R 1 Represents the acyl group introduced in step (a). R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In one embodiment, R 1 Selected from C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. Given the reaction conditions, the two R groups that appear... 1 They are usually the same. In a preferred embodiment, each R that appears is... 1 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It is C(O)Me, in which case "acylation" can be called "acetylation". R 1 This definition applies to all aspects of the invention. In some aspects, R 1 It can also be hydrogen.

[0040] [A.2] Step (b)

[0041] In the method of the present invention, step (b) involves reacting a diol having structure (II) with a sulfite-lating agent to form a cyclic sulfite having structure (IIIa). The reaction scheme corresponding to step (b) is as follows:

[0042]

[0043] The formation of sulfite compounds from diols is well known in the art, for example, according to Megia-Fernandez, Curr. Org. Chem. 2010, 14, 401, the entire contents of which are incorporated herein by reference. Typically, the reaction in step (b) is carried out by treating the diol in dichloromethane or ethyl acetate with a thionyl chloride and a tertiary base (e.g., triethylamine or DIPEA). The sulfitizing agent is known in the art and refers to a compound capable of introducing a sulfurous moiety. In a preferred embodiment, the sulfitizing agent is a thionyl halide or 1,1'-thionylimidazole, preferably a thionyl chloride. The diol having structure (II) is preferably prepared from N-acetyl-2-glucosamine (GlcNAc) or N-acetyl-2-galactosamine (GalNAc), most preferably obtained according to step (a) as defined above.

[0044] Compounds of structure (IIIa) can be used as is in the next step, or can be purified and / or isolated by means known in the art.

[0045] [A.3] Step (c)

[0046] In the method of this invention, step (c) involves reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfite having structure (IIIb). The reaction scheme corresponding to step (c) is as follows:

[0047]

[0048] The oxidation of sulfite compounds to sulfate compounds is well known in the art, for example, according to Megia-Fernandez, Curr. Org. Chem. 2010, 14, 401, the entire contents of which are incorporated herein by reference. Typically, the reaction in step (c) is carried out by treating the crude cyclic sulfite product in dichloromethane, THF, AcOH, or acetonitrile with a strong oxidizing agent (e.g., m-CPBA, MnO2, KMnO4, TEMPO / NaOCl, H2O2, RuO4). Suitable oxidizing agents are known in the art and are generally selected from organic and inorganic oxidizing agents. In a preferred embodiment, the oxidizing agent is an inorganic reagent, more preferably RuO4. The oxidizing agent can be regenerated in situ, for example by adding a catalytic amount of RuCl3 and a stoichiometric amount of NaIO4.

[0049] Compounds of structure (IIIb) can be used as is in the next step, or can be purified and / or isolated by means known in the art.

[0050] [A.4] Step (d)

[0051] In the method of the present invention, step (d) involves a cyclic sulfate having structure (IIIb) and an inorganic azide (i.e., N3). – The anion reacts to form a 6-azido-6-deoxy monosaccharide with structure (I). The reaction scheme corresponding to step (d) is as follows:

[0052]

[0053] The introduction of an azide moiety via nucleophilic ring-opening of a sulfate ester is well known in the art, for example, according to Megia-Fernandez, Curr. Org. Chem. 2010, 14, 401 and van der Klein et al., J. Carbohydr. Chem. 1992, 11, 837, the full text of which is incorporated herein by reference. Typically, the reaction in step (d) is carried out by stirring the cyclic sulfate ester with the azide in a polar solvent such as DMF, THF, or acetonitrile, preferably DMF. The reaction can be accelerated by increasing the temperature (50–80 °C). The sulfate monoester formed after ring-opening is usually obtained by catalytic reaction. Hydrolysis is achieved by short-term treatment (1 hour) with an acid (e.g., sulfuric acid). Suitable inorganic azides are known in the art and are typically selected from sodium azide, lithium azide, or tetrabutylammonium azide. In a preferred embodiment, the inorganic azide is sodium azide.

[0054] Compounds of structure (I) can be directly proceeded to the next step as is, or can be purified and / or isolated by means known in the art.

[0055] Preferably, the compound of structure (I) is further converted into a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or a salt thereof having structure (IX). This conversion can be carried out in any suitable manner. Preferably, the conversion involves converting the 6-azido-6-deoxymonosaccharide compound having structure (I) into a 1-monophosphate monosaccharide compound, which reacts with the nucleoside monophosphate to form the compound having structure (IX). Such a reaction sequence includes a deprotection step before or after the reaction of the 1-monophosphate monosaccharide compound with the nucleoside monophosphate.

[0056] In a preferred embodiment, the conversion of the compound of structure (I) to the compound of structure (IX) is carried out via one of the following reaction sequences:

[0057] - Steps (e), (f), (g), (i), and (j);

[0058] - Steps (e), (f), (g), (j1), and (i1);

[0059] - Steps (e), (x1), (x2), (x3), (x4), (i), and optionally (j);

[0060] -Steps (e), (x1), (x2), (x3), (x4), (j1), (i1);

[0061] - Steps (y1), (y2), and (i).

[0062] Each of these steps is further defined below.

[0063] [A.5] Step (e)

[0064] In the method of this invention, step (e) involves protecting the 6-azido-6-deoxy monosaccharide having structure (I) to form a peracylated 6-azido-6-deoxy monosaccharide compound having structure (VI). The reaction scheme corresponding to step (e) is as follows:

[0065]

[0066] The introduction of the acyl moiety is well known in the art. Typically, the reaction in step (e) is carried out in dichloromethane in the presence of a tertiary base (such as triethylamine) with an activated form of an acid (e.g., an anhydride in pyridine), by treatment of the alcohol with an acyl halide, or by in-situ activation of the acid, for example, with a carbodiimide reagent. The compound of structure (VI) can be used as is in the next step, or it can be purified by methods known in the art.

[0067] R 2 Represents the acyl group introduced in step (e). R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In one embodiment, R 2 Selected from C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In a preferred embodiment, R 2 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, R... 2 It is C(O)Me, in which case "acylated" can be called "acetylated". R 1 and R 2 They may be the same or different, regardless of further transformation of the compound having structure (IV) in the method of the present invention. R 2This definition applies to all aspects of the invention. In some aspects, R 2 It can also be hydrogen.

[0068] [A.6] Step (f)

[0069] In the method of this invention, step (f) involves converting a compound having structure (VI) into an oxazoline compound having structure (VII) in the presence of one or more Lewis acids. The reaction scheme corresponding to step (f) is as follows:

[0070]

[0071] The formation of the oxazoline ring is well known in the art, for example, according to Matta et al., Carbohydr. Res. 1973, 26, 215 and Srivastava et al., Carbohydr. Res. 1982, 103, 286 and Nakabayashi et al., Carbohydr. Res. 1986, 150, C7 and Colon et al., Tetrahedron Lett. 1991, 32, 4447 and Rising et al., Carbohydr. Res. 2006, 341, 1574, all of which are incorporated herein by reference in their entirety. Typically, the reaction in step (f) is carried out by treating the acylated hexosamine monosaccharide with a Lewis acid in a chlorinated solvent such as dichloromethane, dichloroethane, or chloroform. Suitable Lewis acids are known in the art and are generally selected from ferric chloride (III), tin chloride (IV), boron trifluoride, zinc iodide (II) trimethylchlorosilane, trimethylbromosilane, and trimethylsilyl trifluoromethanesulfonate, or combinations thereof. In a preferred embodiment, the Lewis acid is a combination of trimethylsilyl trifluoromethanesulfonate or BF3 (e.g., BF3 diethyl ether complex) and a trimethylsilyl halide (e.g., TMSBr). Alternatively, the formation of oxazoline can also be achieved by anomeric deprotected acetylated hexosamine through treatment with a chloroformamidine reagent, as reported by Noguchi et al., J. Org. Chem. 2009, 74, 2210, which is incorporated herein by reference.

[0072] Compounds of structure (VII) can be used as is in the next step, or can be purified and / or isolated by means known in the art.

[0073] [A.7] Step (g)

[0074] In the method of this invention, step (g) involves reacting the compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va). The reaction scheme corresponding to step (g) is as follows:

[0075]

[0076] The formation of monophosphate monosaccharides from peracylated hexosamines is well known in the art, for example, according to MacDonald et al., J. Org. Chem. 1966, 31, 513 and Masuko et al., J. Org. Chem. 2012, 77, 1449, the entire contents of which are incorporated herein by reference. Typically, the reaction in step (g) is carried out by treating the peracylated hexosamine with pure phosphoric acid at a temperature of 0-120°C, preferably 20-90°C, more preferably 40-80°C, even more preferably 60-80°C. In contrast, the conversion of oxazolines to anophosphates under these conditions (pure phosphoric acid) has not been previously reported. Typically, the conversion of oxazoles is carried out in DMF at a temperature of 0-120°C, preferably 20-90°C, more preferably 40-80°C, with 1-100 equivalents, preferably 2-8 equivalents, most preferably 5-7 equivalents of phosphoric acid. Similarly, the conversion of oxazolines to anophosphates by treatment with phosphoric acid in DMF has not been previously reported.

[0077] Compounds with structure (Va) can be used as is in the next step, or can be purified and / or isolated by means known in the art.

[0078] [A.8] Step (i)

[0079] In the method of this invention, step (i) involves reacting a compound having structure (Va) with a nucleoside monophosphate to form an acylated nucleoside diphosphate having structure (VIII). Thus, the phosphoric acid compound having structure (Va) is coupled to the nucleoside monophosphate, thereby forming the diphosphate moiety. During the reaction with (Va), it is possible that at the 2' and 3' positions (R... 4 Acylation of the nucleoside monophosphate occurs at the 2' and 3' positions, for example, carbonylation may occur upon activation with 1,1'-carbonyldiimidazole (CDI). Alternatively, the 2' and 3' positions of the nucleoside monophosphate can be acylated before coupling to (Va), for example, by acetylation or benzoylation. The reaction scheme corresponding to step (i) is as follows:

[0080]

[0081] In a preferred embodiment, the reaction scheme corresponding to step (i) is as follows:

[0082]

[0083] The formation of nucleoside diphosphate monosaccharides is well known in the art and can be achieved through a variety of synthetic strategies, such as those summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which are incorporated herein by reference. Most commonly, the reaction in step (i) is carried out by, for example, in situ activation of a mixture of sugar-1-phosphate and UMP using a carbonylating agent such as carbonyl diimidazole (CDI), as reported by Illarionov et al., Russ. Chem. Bull. 2001, 50, 1303 and Loureiro Morais, Can. J. Chem. 2006, 84, 587, which are incorporated herein by reference. Alternatively, sugar-1-phosphate can be coupled with a morpholino ester derivative of UMP, as reported by Moffatt, J. Am. Chem. Soc. 1958, 80, 3756 (which is incorporated herein by reference), optionally in the presence of 1-H-tetrazole, as reported by Wittmann et al., J. Org. Chem. 1997, 62, 2144, or by using a sulfonyl imidazolium salt, as reported by Mohamady et al., Curr. Prot. Nucl. Acid Chem. 2012, DOI: 10.1002 / 0471142700.nc1311s51 (which is incorporated herein by reference), or in the presence of 4,5-dicyanimidazolium (DCI), as reported by Vargeese et al., Nucleic Acids Res. 1998, 26, 1046 (which is incorporated herein by reference). The coupling of sugar-1-phosphate with imidazoline-activated NMP in the presence of ZnCl2 or MgCl2 (as reported by Dabrowski-Tumanski, Eur. J. Org. Chem. 2013, 11, 2147, which is incorporated herein by reference) may be particularly effective. Alternatively, enzymatic methods may be employed, for example by reacting the compound of structure (Va) with a nucleoside triphosphate in the presence of pyrophosphatase (PPA) (which converts nucleoside triphosphates to nucleoside monophosphates) and pyrophosphatase (e.g., UDP-GalNAc pyrophosphatase AGX1) (which transfers nucleoside monophosphates to sugar derivatives (Va)), see, for example, Guan et al., Chem. Eur. J. 2010, 16, 13343-13345, which is incorporated herein by reference. The compound of structure (VIII) may be used as is in the next step, or may be purified and / or isolated by means known in the art.

[0084] R 4 The groups at the 2' and 3' positions of the ribose ring representing the nucleoside moiety. When two R groups appear... 4 Both are hydrogen, and there are two R's. 4They are linked together by the carbonyl group, thus connecting to the two R groups. 4 The best results were obtained when the two oxygen atoms of the group formed a carbonate. The optimal result was achieved when the two R groups appeared. 4 The use of hydrogen as the sole element yields the most efficient synthesis and the highest yield, and is therefore preferred in the context of this invention.

[0085] [A.9] Step (j)

[0086] In the method of this invention, step (j) involves deprotecting the acylated nucleoside diphosphate having structure (VIII) to obtain the nucleoside diphosphate having structure (IX) or a salt thereof. The reaction scheme corresponding to step (j) is as follows:

[0087]

[0088] Deprotection of acylated compounds is well known in the art. A wide range of reaction conditions are applicable to it; for example, the reaction in step (j) is carried out by treating the acylated compound in methanol with a (catalytic) amount of sodium methoxide or by treatment with a mixture of Et3N, MeOH, and H2O. The compound of structure (IX) can be used as is in the next step, or it can be purified and / or isolated by methods known in the art.

[0089] Here, B is a nucleobase. Although any nucleobase can be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0090] In the method of this invention, the coupling in step (i) and the deprotection in step (j) can also be performed in reverse without any negative impact on process efficiency and yield. These reactions are referred to herein as steps (i1) and (j1).

[0091] [A.10] Step (j1)

[0092] In the method of this invention, step (j1) involves deprotecting the acylated 1-monophosphate monosaccharide having structure (Va) to obtain the 1-monophosphate monosaccharide having structure (Vb). The reaction scheme corresponding to step (j1) is as follows:

[0093]

[0094] Deprotection of acylated compounds is well known in the art. A wide range of reaction conditions are applicable to it; for example, the reaction in step (j) is carried out by treating the acylated compound in methanol with (catalytic) amounts of sodium methoxide or by treating the compound with triethylamine in a water / methanol mixture. The compound of structure (Vb) can be used as is in the next step, or it can be purified and / or isolated by methods known in the art.

[0095] [A.11] Step (i1)

[0096] In the method of this invention, step (i1) involves reacting a compound having structure (Vb) with a nucleoside monophosphate to form a nucleoside diphosphate having structure (IX). Therefore, the 1-monophosphate monosaccharide compound with structure (Vb) is coupled with the nucleoside monophosphate to form the diphosphate moiety. The reaction scheme corresponding to step (i1) is as follows:

[0097]

[0098] In a preferred embodiment, the reaction scheme corresponding to step (i1) is as follows:

[0099]

[0100] The formation of nucleoside diphosphate monosaccharides is well known in the art and can be achieved through a variety of synthetic strategies, such as those summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which are incorporated herein by reference. Most commonly, the reaction in step (i1) is carried out by, for example, in situ activation of a mixture of sugar-1-phosphate and UMP using a carbonylating agent such as carbonyl diimidazole (CDI), as reported by Illarionov et al., Russ. Chem. Bull. 2001, 50, 1303 and Loureiro Morais, Can. J. Chem. 2006, 84, 587, which are incorporated herein by reference. Alternatively, sugar-1-phosphate can be coupled with morpholine ester derivatives of UMP, as reported by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756 (incorporated herein by reference), optionally in the presence of 1-H-tetrazole, as reported by Wittmann et al., J. Org. Chem. 1997, 62, 2144 (incorporated herein by reference), or in the presence of 4,5-dicyanimidazole (DCI), as reported by Vargeese et al., Nucleic Acids Res. 1998, 26, 1046 (incorporated herein by reference). Coupling of sugar-1-phosphate with imidazoline-activated NMP (as described in Wagner et al., Nat. Prod. Rep. 2009, 26, 1172, incorporated herein by reference) may be particularly effective. Alternatively, an enzymatic method may be used, for example, by reacting the compound of structure (Va) with a nucleoside triphosphate in the presence of a pyrophosphatase (PPA) (which converts the nucleoside triphosphate to a nucleoside monophosphate) and a pyrophosphatase (e.g., UDP-GalNAc pyrophosphatase AGX1) (which transfers the nucleoside monophosphate to the sugar derivative (Va)), see, for example, Guan et al., Chem. Eur. J. 2010, 16, 13343-13345, which is incorporated herein by reference. The compound of structure (IX) may be used as is in the next step, or may be purified and / or isolated by means known in the art.

[0101] Here, B is a nucleobase. Although any nucleobase can be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0102] [A.12] Step (z)

[0103] Both steps (i) and (i1) can be carried out by reacting a compound having structure (Va) or (Vb) with a nucleoside monophosphate having structure (X). Since this applies to both steps, this reaction is also independently referred to herein as step (z). In the method of the present invention, step (z) is the reaction of a compound having structure (V) with a nucleoside monophosphate having structure (X) to form a nucleoside diphosphate having structure (IX). Thus, the phosphate ester compound of structure (V) is coupled to the nucleoside monophosphate, thereby forming the diphosphate moiety. The reaction scheme corresponding to step (z) is as follows:

[0104]

[0105] Here, R 1 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl, and R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl, wherein R 1 and R 2 Both are H (compound (Vb) or (IX)) or R 1 and R 2 Neither is H (compound (Va) or (VIII)). B is a nucleobase. Although any nucleobase can be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0106] R 4 The groups at the 2' and 3' positions of the riboyl ring representing the nucleoside moiety, and both being hydrogen atoms or two R atoms appearing. 4 They are linked together by a carbonyl group. Preferably, the two R groups are... 4 They're all hydrogen.

[0107] The formation of nucleoside diphosphate monosaccharides is well known in the art and can be achieved through a variety of synthetic strategies, such as those summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which are incorporated herein by reference. Most commonly, the reaction in step (i) is carried out by in situ activation of the sugar-1-phosphate in the presence of UMP, for example, using a carbonylating agent such as carbonyl diimidazole (CDI), as reported by Illarionov et al., Russ. Chem. Bull. 2001, 50, 1303 and Loureiro Morais, Can. J. Chem. 2006, 84, 587, which are incorporated herein by reference. Alternatively, sugar-1-phosphate can be coupled with morpholino ester derivatives of UMP, as reported by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756 (which is incorporated herein by reference), optionally in the presence of 1-H-tetrazole, or by using sulfonyl imidazolium salts, as reported by Mohamady et al., Curr. Prot. Nucl. Acid Chem. 2012, DOI: 10.1002 / 0471142700.nc1311s51 (which is incorporated herein by reference), or in the presence of 4,5-dicyanimidazolium (DCI), as reported by Vargeese et al., Nucleic Acids Res. 1998, 26, 1046 (which is incorporated herein by reference). In one embodiment, step (z) is carried out in the presence of an organic base (e.g., 1-methylimidazolium chloride or 1-H-tetrazole) or a Lewis acid (e.g., MgCl2 or ZnCl2). The coupling of sugar-1-phosphate with imidazoline-activated NMP in the presence of ZnCl2 or MgCl2 (as reported by Dabrowski-Tumanski, Eur. J. Org. Chem. 2013, 11, 2147, and as reviewed in Wagner et al., Nat. Prod. Rep. 2009, 26, 1172, which is incorporated herein by reference) may be particularly effective. Compounds of structure (VIII) or (IX) may be used as is in the next step, or may be purified and / or isolated by means known in the art.

[0108] In an alternative embodiment, the compound with structure (VI) obtained in step (e) is not converted into an oxazoline compound with structure (VII) via step (f), but instead forms a compound with structure (Va) via steps (x1)-(x4). This compound can then be converted into a compound with structure (IX) via steps (i)+(j) or via steps (j1)+(i1).

[0109] [A.13] Step (x1)

[0110] In the method of this invention, step (x1) involves deprotecting the hydroxyl moiety attached to the anomeric carbon of the compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI). The reaction scheme corresponding to step (x1) is as follows:

[0111]

[0112] The selective deprotection of anomeric hydroxyl groups from peracylated monosaccharides is well known in the art, for example as reported by Johnson et al., Synlett, 2005, 2939 and Baumik et al., Aus. J. Chem. 2003, 56, 909, the full text of which is incorporated herein by reference. For example, the reaction in step (x1) is carried out by treating the peracylated monosaccharide in an organic solvent such as THF or acetonitrile with a small molar excess of benzylamine, dimethylamine, or hydrazine acetate. The compound of structure (xi) can be used as is in the next step, or it can be purified and / or isolated by methods known in the art.

[0113] [A.14] Step (x2)

[0114] In the method of this invention, step (x2) involves converting a 1-hydroxy-monosaccharide compound having structure (XI) into a monophosphite diester having structure (XII). The reaction scheme corresponding to step (x2) is as follows:

[0115]

[0116] Phosphorylation of hydroxyl groups is well known in the art, for example as summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, the entire text of which is incorporated herein by reference. Typically, the reaction in step (x2) is carried out by treating the anolyte-deprotected monosaccharide with a phosphorylating agent (e.g., chlorophosphonite or phosphoramide), which can be activated in the presence of a proton scavenger or a weak acid to react with the anolyte hydroxyl group. The compound of structure (XII) can be used as is in the next step, or it can be purified and / or isolated by methods known in the art.

[0117] Here, R 3 Represents the portion bonded to the two oxygen atoms of the phosphite diester (i.e., excluding the anomeric oxygen atom of the monosaccharide portion). R 3 Selected from C 1-6 Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH2OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C 1-2-alkyl, wherein the phenyl group is optionally substituted with one or more halides or nitro or methoxy groups. Here, phenyl-C 1-2 -alkyl refers to 2-phenylethyl or benzyl. Nitro or methoxy substituents, especially methoxy substituents, are preferred at the para position. Although R can be selected individually... 3 However, R is preferred. 3 The two parts are the same. In R 3 In the case of 2-alkylsulfonylethyl or CH2OC(O)alkyl, the preferred alkyl group is C. 1-6 Alkyl, more preferably C 1-2 Alkyl group, with methyl group being the most preferred. In R 3 In the case of 2-arylsulfonylethyl, the aryl group is preferably phenyl. 3 Each option can be easily removed in the subsequent step (x4), and this deprotection can optionally be combined with R in step (j1). 1 and R 3 Deprotection occurs simultaneously. Therefore, in a particularly preferred embodiment, R 3 It is a CH2OC(O) alkyl group, wherein the alkyl group is preferably C. 1-6 Alkyl, more preferably C 1-2 Alkyl, most preferably methyl, and steps (x4) and (j1) are carried out in a single step (one-pot process).

[0118] [A.15] Step (x3)

[0119] In the method of this invention, step (x3) involves oxidizing the monophosphite diester having structure (XII) to form a 1-monophosphite diester having structure (XIII). The reaction scheme corresponding to step (x3) is as follows:

[0120]

[0121] Oxidation of the phosphite moiety is well known in the art, for example as summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, the entire contents of which are incorporated herein by reference. Typically, the reaction in step (x3) is carried out by contacting the phosphite (XII) with an oxidizing agent such as iodine, m-CPBA, t-BuOOH, or H2O2 in a solution of dichloromethane or acetonitrile. The compound of structure (XIII) can be used as is in the next step, or it can be purified and / or isolated by methods known in the art.

[0122] [A.16] Step (x4)

[0123] In the method of this invention, step (x4) involves deprotecting the monophosphate diester having structure (XIII) to form a 1-monophosphate monosaccharide having structure (Va). The reaction scheme corresponding to step (x4) is as follows:

[0124]

[0125] Step (x4) can also completely deprotect compound (XIII), wherein the acyl group R 1 and R 2 Accompanied by the group R 3 The substance is removed. The reaction scheme corresponding to this implementation scheme of step (x4) is as follows:

[0126]

[0127] Overall, the reaction scheme for step (x4) is as follows:

[0128]

[0129] The following applies here:

[0130] -For compound (XIII): R 1 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl, and R 2 Selected from optionally substituted C(O)-alkyl, optionally substituted C(O)-aryl and C(O)-arylalkyl;

[0131] -For compound (V), R 1 Selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl, R 2 Selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl, wherein R 1 and R 2 All are H (compound (Vb)) or R 1 and R 2 None of them are H (compound (Va)).

[0132] Deprotection of acylated monosaccharides is well known in the art. Compounds with structure (Va) or (Vb) can be used as is in the next step, or can be purified and / or isolated by means known in the art.

[0133] In an alternative embodiment, the compound with structure (I) obtained in step (d) is not converted to a triacylated compound with structure (VI) via step (e), but instead undergoes steps (y1)–(y2) to form a compound with structure (Vb). This compound can then be converted to a compound with structure (IX) via step (i1).

[0134] [A.17] Step (y1)

[0135] In the method of this invention, step (y1) involves deprotecting the 6-azido-6-deoxy monosaccharide having structure (I) to form a 1,3,4-trihydroxy-6-azido-monosaccharide having structure (XIV). The reaction scheme corresponding to step (y1) is as follows:

[0136]

[0137] Deprotection of acylated monosaccharides is well known in the art. Compounds with structure (XIV) can be used as is in the next step, or can be purified and / or isolated by methods known in the art.

[0138] [A.18] Step (y2)

[0139] In the method of this invention, step (y2) involves reacting a 1,3,4-trihydroxy-6-azido-monosaccharide having structure (XIV) with a phosphate ester source to form a 1-monophosphate monosaccharide compound having structure (Vb). The reaction scheme corresponding to step (y2) is as follows:

[0140]

[0141] Regioselective phosphorylation of monosaccharides is well known in the art, as summarized by Bülter et al. in Glycoconj. J. 1999, 16, 147, the entire text of which is incorporated herein by reference. The reaction in step (y2) is carried out in the presence of a phosphorylase such as N-acetylglucosamine 1-kinase (NahK), see, for example, Cai et al. Chem. Commun. 2009, 2944-2946, which is incorporated herein by reference. The compound of structure (Vb) can be used in the next step on its own, or can be purified and / or isolated by methods known in the art. If the compound of structure (Vb) is enzymatically converted to a nucleoside diphosphate having structure (IX) by step (i1) as the next step, the entire reaction sequence can be carried out in a one-pot manner, see, for example, Heinzler et al. Adv. Synth. Catal. 2019, 361, 4506-4516, which is incorporated herein by reference.

[0142] [B] Compounds according to the present invention

[0143] The inventors have identified several key intermediates in the synthesis method according to the present invention. The present invention also relates to these intermediates.

[0144] [B.1] Compound (III)

[0145] Therefore, the present invention relates to cyclic sulfate monosaccharide compounds having structure (III):

[0146]

[0147] In this article, R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl groups. The carbon atom with the wavy bond can be in either the S-configuration or the R-configuration; all four diastereomers are covered.

[0148] R 1 Represents the acyl group introduced in step (a). R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl groups. Under given reaction conditions, the two R groups that appear... 1 They are usually the same. In a preferred embodiment, each R that appears is... 1 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It’s C(O)Me.

[0149] [B.2] Compound (I)

[0150] The present invention also relates to 6-azido-6-deoxy monosaccharide compounds having structure (I):

[0151]

[0152] Here, R 1 The same definition applies to compounds with structure (III). The carbon atom with the wavy bond can be in either the S- or R-configuration; this covers all four diastereomers.

[0153] [B.3] Compound (VII)

[0154] This invention also relates to oxazoline compounds having structure (VII):

[0155]

[0156] Here, R 1Same as defined for compounds with structure (III). The carbon atom with the wavy bond can be in either the S- or R-configuration; encompassing both diastereomers. The oxygen atom in the oxazoline ring at carbon 1 of the GlcNAc or GalNAc moiety is the only α-anomer.

[0157] R 2 Represents the acyl group introduced in step (e). R 2 Represents the acyl group introduced in step (e). R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In one embodiment, R 2 Selected from C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In a preferred embodiment, R 2 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, R... 2 It is C(O)Me, in which case "acylated" can be called "acetylated". R 1 and R 2 They may be the same or different, regardless of further transformation of the compound having structure (IV) in the method of the present invention.

[0158] [B.4] Compound (Va)

[0159] The present invention also relates to a mixture of α-anodic and β-anodic forms of a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having the structure (Va), or a salt thereof:

[0160]

[0161] Here, R 1 and R 2 The same definition applies to compounds with structure (VII). The carbon atom with the wavy bond can be either S- or R-configuration; this covers all four diastereomers, provided that the molar ratio between the α- and β-anomers in the mixture is between 3 / 1 and 10 / 1. Both anomeric forms of the compound (Va) present in the mixture can be in salt form.

[0162] Known chemical methods for preparing 6-azido-6-deoxy-1-monophosphate monosaccharide compounds having structure (Va) typically yield a mixture of α- and β-anomers (through chemical conversion, i.e., reaction with a phosphorylated agent, such as phosphoramide). While these conventional methods are also applicable in the context of this invention, it is preferred to introduce a phosphate group via compound (VII), in which case a mixture of α- and β-anomers is formed, which predominantly contains, but not entirely contains, the α-anomer. For subsequent reactions toward compound (IX), the α-anomer form is preferred because it is the sole substrate for a transferase that can be used to incorporate the azide-containing monosaccharide moiety into the glycan chain. To date, enantiomeric excess of the α-anomer has only been obtained via enzymatic methods. Compound (Va), as a mixture of isomers but predominantly containing the α-anomer, is obtained via a chemical synthesis step without enzymatic conversion.

[0163] [B.5] Compound (VIII)

[0164] This invention also relates to nucleoside diphosphates having structure (VIII):

[0165]

[0166] Here, R 1 and R 2 The same definition applies to compounds with structure (VII). The carbon atom with the wavy bond can be in either the S- or R-configuration; this covers both diastereomers.

[0167] R 4 The groups at the 2' and 3' positions of the riboyl ring representing the nucleoside moiety, and both being hydrogen atoms or two R atoms appearing. 4 They are linked together by a carbonyl group. Preferably, the two R groups are... 4 They're all hydrogen.

[0168] B is a nucleobase. Although any nucleobase can be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0169] [B.6] Compound (XI)

[0170] The present invention also relates to 1-hydroxy-monosaccharide compounds having structure (XI).

[0171]

[0172] Here, R 1 and R 2 Same as defined for compounds with structure (VI). The carbon atom with the wavy bond can be in either the S- or R-configuration; covering all four diastereomers.

[0173] [B.7] Compound (XII)

[0174] The present invention also relates to phosphites having the structure (XII):

[0175]

[0176] Here, R 1 and R 2 Same as defined for compounds with structure (VI). The carbon atom with the wavy bond can be in either the S- or R-configuration; covering all four diastereomers.

[0177] Here, R 3 Selected from C 1-6 Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH2OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C 1-2 -alkyl, wherein the phenyl group is optionally substituted with one or more halides or nitro or methoxy groups. Here, phenyl-C 1-2 -alkyl refers to 2-phenylethyl or benzyl. Nitro or methoxy substituents, especially methoxy substituents, are preferred at the para position. Although R can be selected individually... 3 However, two Rs are preferred. 3 The parts are the same. In R 3 In the case of 2-alkylsulfonylethyl or CH2OC(O)alkyl, the preferred alkyl group is C. 1-6 Alkyl, more preferably C 1-2 Alkyl group, with methyl group being the most preferred. In R 3 In the case of 2-arylsulfonylethyl, the aryl group is preferably phenyl. In a particularly preferred embodiment, R 3 It is a CH2OC(O) alkyl group, wherein the alkyl group is preferably C. 1-6 Alkyl, more preferably C 1-2 Alkyl group, with methyl group being the most preferred.

[0178] [B.8] Compound (XIII)

[0179] The present invention also relates to phosphodiesters having structure (XIII):

[0180]

[0181] Here, R 1 R 2 and R 3 The same definition applies to compounds with structure (XII). The carbon atom with the wavy bond can be in either the S- or R-configuration; this covers all four diastereomers.

[0182] [C] Synthesis Method

[0183] In pursuit of the overall objective of this invention, namely, to provide an efficient synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX), the inventors have identified several key synthetic steps. This invention also relates to these methods for the partial synthesis of compounds having structure (IX). The methods according to these aspects of the invention are well-suited for achieving the overall objective of synthesizing 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX).

[0184] [C.1] Synthesis of compound (I)

[0185] In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy monosaccharide compound having structure (I) according to the following scheme:

[0186]

[0187] The method includes:

[0188] (b) Reacting a diol having structure (II) with a sulfite oxidizing agent to form a cyclic sulfite having structure (IIIa);

[0189] (c) Reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfite having structure (IIIb);

[0190] (d) Reaction of a cyclic sulfate ester with structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide with structure (I).

[0191] Steps (b), (c), and (d) are as defined above. R 1 The acyl group represents a protecting group that is attached to the hydroxyl groups at the carbon atoms at positions 1 and 3 of the monosaccharide moiety. These acyl protecting groups can be introduced in step (a) prior to step (b). R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. Preferably, the two Rs appearing are... 1 They are the same. In a preferred embodiment, each R that appears... 1 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It is C(O)Me, in which case "acylated" can be called "acetylated".

[0192] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the 4-carbon atom of the monosaccharide moiety can be axial (galactose configuration) or flat (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, compounds having structure (II) are preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), with GalNAc being the most preferred. This preparation is preferably carried out by step (a) as defined above.

[0193] The method according to this aspect is very suitable for the overall objective of the present invention, namely, the synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Therefore, it is preferable to further convert the compound having structure (I) obtained in step (d) into 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or a salt thereof having structure (IX). This conversion can be carried out in any suitable manner. Preferably, the conversion comprises converting the 6-azido-6-deoxymonosaccharide compound having structure (I) into a 1-monophosphate monosaccharide compound, which reacts with a nucleoside monophosphate to form a compound having structure (IX), which generally has structure (V). This reaction sequence includes a deprotection step before or after the reaction of the 1-monophosphate monosaccharide compound with the nucleoside monophosphate.

[0194] 1-Monophosphate monosaccharides with structure (V) are defined as follows:

[0195]

[0196] Here, R 1 and R 2 Independently selected from H and optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. Additionally, the following applies:

[0197] -For compound (Va): R 1 and R 2 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl;

[0198] -For compound (Vb): R 1 and R 2 All are H.

[0199] In a preferred embodiment, the conversion of the compound of structure (I) to the compound of structure (V) is carried out via one of the following reaction sequences:

[0200] - Steps (e), (f) and (g) typically provide compounds having a structure (Va);

[0201] - Steps (e), (x1), (x2), (x3) and (x4), which typically provide compounds with structure (Va);

[0202] Steps (y1) and (y2) typically provide compounds with the structure (Vb).

[0203] Preferably, the compound having structure (V) is converted into the compound having structure (IX), preferably via steps (i) and (j) or via steps (j1) and (i1). In the case of obtaining a compound having structure (Vb), it is preferred to convert it into the compound having structure (IX) via step (i1). In this case, deprotection in step (j) or (j1) is not required. If the reaction sequence toward the compound having structure (IX) involves steps (x4) and (j1), these can optionally be carried out simultaneously to obtain the compound having structure (Vb). Here, R is preferred. 3 It is a CH2OC(O) alkyl group, wherein the alkyl group is preferably C. 1-6 Alkyl, more preferably C 1-2 Alkyl group, with methyl group being the most preferred.

[0204] In a preferred embodiment, the conversion of a compound of structure (I) to a compound of structure (IX) from a compound having structure (V) is carried out via one of the following reaction sequences:

[0205] - Steps (e), (f), (g), (i), and (j);

[0206] - Steps (e), (f), (g), (j1), and (i1);

[0207] - Steps (e), (x1), (x2), (x3), (x4), (i), and optionally (j);

[0208] -Steps (e), (x1), (x2), (x3), (x4), (j1), (i1);

[0209] - Steps (y1), (y2), and (i).

[0210] Steps (e), (f), (g), (i), (j), (i1), (j1), (x1), (x2), (x3), (x4), (y1), and (y2) are defined as above.

[0211] [C.2] Synthesis of compound (Va) via compound (VII)

[0212] In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having a structure (Va) according to the following scheme:

[0213]

[0214] The method includes:

[0215] (f) Transforming a 6-azido-6-deoxy monosaccharide having structure (VI) in the presence of one or more Lewis acids to form an oxazoline compound having structure (VII);

[0216] (g) Reaction of an oxazoline compound having structure (VII) with phosphoric acid to form a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va).

[0217] Steps (f) and (g) are as defined above. R 1 The acyl group represents a protecting group that is attached to a hydroxyl group at the 3-position (3- and 1-positions of carbon atom in (VI)) of the monosaccharide moiety. The acyl protecting group can be introduced in step (a) prior to step (f). R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In one embodiment, R 1 Selected from C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. Preferably, the two Rs appearing are... 1 They are the same. In a preferred embodiment, each R that appears... 1 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It is C(O)Me, in which case "acylated" can be called "acetylated".

[0218] R 2 The acyl group represents the acyl group, which exists as a protecting group for the hydroxyl group attached to the 4-carbon atom of the monosaccharide moiety. The acyl protecting group can be introduced in step (e) prior to step (f). R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In one embodiment, R 2 Selected from C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In a preferred embodiment, R 2 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, R... 2 It is C(O)Me, in which case "acylated" can be called "acetylated". R 1 and R 2 They may be the same or different, regardless of further transformation of the compound having the structure (Va) in the method of the present invention.

[0219] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the 4-carbon atom of the monosaccharide moiety can be axial (galactose configuration) or flat (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, the compound having structure (VI) is preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), more preferably from GalNAc. This preparation is preferably carried out by step (e) as defined above, more preferably by steps (b), (c), (d) and (e) as defined above, and most preferably by steps (a), (b), (c), (d) and (e) as defined above.

[0220] The method according to this aspect is very suitable for the overall objective of the present invention, namely, the synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Therefore, it is preferable to further convert the compound having structure (Va) obtained in step (g) into 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or a salt thereof having structure (IX). This conversion can be carried out in any suitable manner. Preferably, the conversion comprises steps (i) and (j) or steps (j1) and (i1) as defined above.

[0221] [C.3] Synthesis of compound (Va) from compound (XII)

[0222] In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having a structure (Va) according to the following scheme:

[0223]

[0224] The method includes:

[0225] (x1) Deprotecting the anodic sites of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI);

[0226] (x2) A 1-hydroxy-monosaccharide compound having structure (XI) is converted into a 6-azido-6-deoxy-1-monophosphite having structure (XII);

[0227] (x3) In the presence of an oxidizing agent, a monophosphite diester with structure (XII) is oxidized to form a 1-monophosphite diester compound with structure (XIII).

[0228] (x4) Deprotecting the phosphate diester with structure (XIII) to form a 1-monophosphate monosaccharide compound with structure (Va).

[0229] Steps (x1), (x2), (x3), and (x4) are defined as above. R 1 The acyl group represents a protecting group that is attached to a hydroxyl group at the 3-position (3 and 1 positions of carbon in (VI)) of the monosaccharide moiety. The acyl protecting group can be introduced in step (a) prior to step (x1). R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. Preferably, the two Rs appearing are... 1 They are the same. In a preferred embodiment, each R that appears... 1 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It is C(O)Me, in which case "acylated" can be called "acetylated".

[0230] R 2 The acyl group represents the hydroxyl group, which exists as a protecting group attached to the hydroxyl group at the 4-carbon position of the monosaccharide moiety. The acyl protecting group can be introduced in step (e) prior to step (x1). R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In a preferred embodiment, R 2 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, R... 2 It is C(O)Me, in which case "acylated" can be called "acetylated". R 1 and R 2 They may be the same or different, regardless of further transformation of the compound having the structure (Va) in the method of the present invention.

[0231] R 3 Represents the phosphite substituent introduced in step (x2). R 3 Selected from C 1-6 Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH2OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C 1-2 -alkyl (2-phenylethyl or benzyl), wherein the phenyl group is optionally substituted with one or more halides or nitro or methoxy groups. In R 3 In the case of 2-alkylsulfonylethyl or CH2OC(O)alkyl, the preferred alkyl group is C. 1-6 Alkyl, more preferably C 1-2 Alkyl group, with methyl group being the most preferred. In R 3 In the case of 2-arylsulfonylethyl, the aryl group is preferably phenyl.

[0232] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the 4-carbon atom of the monosaccharide moiety can be axial (galactose configuration) or flat (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, the compound having structure (VI) is preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), more preferably from GalNAc. This preparation is preferably carried out by step (e) as defined above, more preferably by steps (b), (c), (d) and (e) as defined above, and most preferably by steps (a), (b), (c), (d) and (e) as defined above.

[0233] The method according to this aspect is very suitable for the overall objective of the present invention, namely, the synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Therefore, it is preferable to further convert the compound having structure (Va) obtained in step (x4) into 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or a salt thereof having structure (IX). This conversion can be carried out in any suitable manner. Preferably, the conversion includes steps (i) and (j) or steps (j1) and (i1) as defined above.

[0234] [C.4] Synthesis of compound (Vb) from compound (XII)

[0235] In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having a structure (Vb) according to the following scheme:

[0236]

[0237] The method includes:

[0238] (x1) Deprotecting the anodic sites of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI);

[0239] (x2) A 1-hydroxy-monosaccharide compound having structure (XI) is converted into a 6-azido-6-deoxy-1-monophosphite having structure (XII);

[0240] (x3) In the presence of an oxidizing agent, a monophosphite diester with structure (XII) is oxidized to form a 1-monophosphite diester compound with structure (XIII).

[0241] (x4) Simultaneously, the phosphate diester and monosaccharide with structure (XIII) are deprotected to form a 1-monophosphate monosaccharide compound with structure (Vb).

[0242] Steps (x1), (x2), (x3), and (x4) are defined as above. R 1 The acyl group represents a protecting group that is attached to a hydroxyl group at the 3-position (3 and 1 positions of carbon atom in (VI)) of the monosaccharide moiety. The acyl protecting group can be introduced in step (a) prior to step (x1). R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. Preferably, the two Rs appearing are... 1 They are the same. In a preferred embodiment, each R that appears... 1 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It is C(O)Me, in which case "acylated" can be called "acetylated".

[0243] R 2 The acyl group represents the hydroxyl group, which exists as a protecting group attached to the hydroxyl group at the 4-carbon position of the monosaccharide moiety. The acyl protecting group can be introduced in step (e) prior to step (x1). R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In a preferred embodiment, R 2 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, R... 2 It is C(O)Me, in which case "acylated" can be called "acetylated". R 1 and R 2 They may be the same or different, which is irrelevant to the applicability of the method of this aspect of the invention.

[0244] R 3 Represents the phosphite substituent introduced in step (x2). R 3 Selected from C 1-6 Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH2OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C 1-2 -alkyl (2-phenylethyl or benzyl), wherein the phenyl group is optionally substituted with one or more halides or nitro or methoxy groups. In R 3 In the case of 2-alkylsulfonylethyl or CH2OC(O)alkyl, the preferred alkyl group is C. 1-6 Alkyl, more preferably C 1-2Alkyl group, with methyl group being the most preferred. In R 3 In the case of 2-arylsulfonylethyl, the aryl group is preferably phenyl.

[0245] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the 4-carbon atom of the monosaccharide moiety can be axial (galactose configuration) or flat (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, the compound having structure (VI) is preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), more preferably from GalNAc. This preparation is preferably carried out by step (e) as defined above, more preferably by steps (b), (c), (d) and (e) as defined above, and most preferably by steps (a), (b), (c), (d) and (e) as defined above.

[0246] The method according to this aspect is very suitable for the overall objective of the present invention, namely, the synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Therefore, it is preferable to further convert the compound having structure (Vb) obtained in step (x4) into 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or a salt thereof having structure (IX). This conversion can be carried out in any suitable manner. Preferably, the conversion includes step (i) as defined above.

[0247] [C.5] Synthesis of compound (Vb) via compound (XIV)

[0248] In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having a structure (Vb) according to the following scheme:

[0249]

[0250] The method includes the following steps:

[0251] (y1) Deprotection of 6-azido-6-deoxy monosaccharide with structure (I) to form 1,3,4-trihydroxy-6-azido-monosaccharide compound with structure (XIV);

[0252] (y2) In the presence of a phosphate ester source, a compound with structure (XIV) is contacted with a phosphorylase to form a 1-monophosphate monosaccharide compound with structure (Vb).

[0253] Steps (y1) and (y2) are defined as above. R 1The acyl group represents the hydroxyl group, which exists as a protecting group for the hydroxyl groups attached to the carbon atoms at positions 1 and 3 of the monosaccharide moiety of compound (I). The acyl protecting group can be introduced in step (a) prior to step (y1). R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. Preferably, the two Rs appearing are... 1 They are the same. In a preferred embodiment, each R that appears... 1 It is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It is C(O)Me, in which case "acylated" can be called "acetylated".

[0254] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the 4-carbon atom of the monosaccharide moiety can be axial (galactose configuration) or flat (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, compounds having structure (I) are preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), more preferably from GalNAc. This preparation is preferably carried out by steps (b), (c), and (d) as defined above, and more preferably by steps (a), (b), (c), and (d) as defined above.

[0255] The method according to this aspect is very suitable for the overall objective of the present invention, namely, the synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Therefore, it is preferable to further convert the compound having structure (Vb) obtained in step (x4) into 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or a salt thereof having structure (IX). This conversion can be carried out in any suitable manner. Preferably, the conversion includes step (i1) as defined above.

[0256] Synthesis of compound (VIII) [C.6]

[0257] In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (VIII) according to the following scheme:

[0258]

[0259] The method includes:

[0260] (z) Reacting a compound having structure (Va) with a nucleoside monophosphate having structure (X) to form a nucleoside diphosphate having structure (VIII), optionally wherein the reaction is carried out in the presence of an organic base, MgCl2 or ZnCl2.

[0261] Step (z) is as defined above. In the method according to this aspect, R 1 It can be an H or an acyl group, wherein the acyl group exists as a protecting group for a hydroxyl group attached to the 3-carbon atom of the monosaccharide moiety. The acyl protecting group can be introduced in step (a) prior to step (z). R 1 Selected from H and optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In a preferred embodiment, R 1 It is H, C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, each R that appears... 1 It is C(O)Me, in which case "acylated" can be called "acetylated". In one embodiment, the compound having structure (V) is the compound having structure (Va) and R 1 Selected from H, C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In an alternative embodiment, the compound having structure (V) is a compound having structure (Vb) and R 1 It's H.

[0262] In the method according to this aspect, R 2 It can be an H or an acyl group, wherein the acyl group exists as a protecting group for a hydroxyl group attached to the 4-carbon atom of the monosaccharide moiety. The acyl protecting group can be introduced in step (e) prior to step (z). R 2 Selected from H and optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In a preferred embodiment, R 2 It is H, C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph. Most preferably, R 2 It is C(O)Me, in which case "acylated" can be called "acetylated". In one embodiment, the compound having structure (V) is the compound having structure (Va) and R 2 Selected from H, optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In an alternative embodiment, the compound having structure (V) is the compound having structure (Vb) and R 2 It's H.

[0263] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the 4-carbon atom of the monosaccharide moiety can be axial (galactose configuration) or flat (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, compounds having structure (V) are preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), with GalNAc being more preferred. This preparation is preferably carried out by the following method:

[0264] - Steps (f) and (g) in this case yield a compound having structure (Va);

[0265] - Steps (e), (f) and (g) yield a compound having structure (Va) in this case;

[0266] - Steps (b), (c), (d), (e), (f) and (g) yield a compound having structure (Va) in this case;

[0267] - Steps (a), (b), (c), (d), (e), (f) and (g) yield a compound having structure (Va) in this case;

[0268] - Steps (x1), (x2), (x3) and (x4) yield compounds with structures (Va) or (Vb) in this case;

[0269] - Steps (e), (x1), (x2), (x3) and (x4) yield compounds having structures (Va) or (Vb);

[0270] - Steps (b), (c), (d), (e), (x1), (x2), (x3) and (x4) yield compounds having structures (Va) or (Vb);

[0271] - Steps (a), (b), (c), (d), (e), (x1), (x2), (x3) and (x4) yield compounds having structures (Va) or (Vb);

[0272] - Steps (y1) and (y2) yield a compound with structure (Vb) in this case;

[0273] - Steps (b), (c), (d), (y1) and (y2) yield a compound with structure (Vb) in this case;

[0274] - Steps (a), (b), (c), (d), (y1) and (y2) yield a compound with structure (Vb) in this case.

[0275] Steps (a), (b), (c), (d), (e), (f), (g), (x1), (x2), (x3), (x4), (y1), and (y2) are defined as above.

[0276] The method according to this aspect is very suitable for the overall objective of the present invention, namely, the synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having the structure (IX). In one embodiment, R 1 and R 2 All are H and such compounds have been provided according to the methods of this aspect. Alternatively, it is preferable to further convert the compound having structure (VIII) obtained in step (z) into 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or a salt thereof having structure (IX). This conversion can be carried out in any suitable manner. Preferably, the conversion includes step (j) as defined above.

[0277] Synthesis of [D] compound (IX)

[0278] This invention provides methods for the total synthesis of several target compounds (IX) utilizing the steps, methods, and intermediates defined above. Each of these methods offers one or more unique advantages over prior art methods.

[0279] [D.1] Synthesize compound (IX) using compounds (VII) and (VIII).

[0280] In one aspect, the present invention relates to a method for preparing nucleoside diphosphates having structure (IX) or salts thereof according to the following scheme:

[0281]

[0282] The method includes:

[0283] (a) Converting N-acetylglucosamine or N-acetylglucosamine into a 1,3-diacytized compound having structure (II);

[0284] (b) Reacting a diol having structure (II) with a sulfite oxidizing agent to form a cyclic sulfite having structure (IIIa);

[0285] (c) Reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfite having structure (IIIb);

[0286] (d) Reacting a cyclic sulfate ester with structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide with structure (I);

[0287] (e) Protect 6-azido-6-deoxy monosaccharides having structure (I) to form 6-azido-6-deoxy monosaccharide compounds having structure (VI);

[0288] (f) Transforming a compound having structure (VI) in the presence of one or more Lewis acids to form an oxazoline compound having structure (VII);

[0289] (g) Reacting a compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va);

[0290] (i) reacting a compound having structure (Va) with a nucleoside monophosphate to form an acylated nucleoside diphosphate having structure (VIII); and

[0291] (j) Deprotecting an acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate or a salt having structure (IX).

[0292] Here, R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 4 Both are hydrogen or two R appear. 4 They are linked together by a carbonyl group; B is a nucleobase. Preferred embodiments of each step and compound in this method are defined above.

[0293] [D.2] Synthesis of compound (IX) via compounds (VII) and (Vb)

[0294] In one aspect, the present invention relates to a method for preparing nucleoside diphosphates having structure (IX) or salts thereof according to the following scheme:

[0295]

[0296] The method includes:

[0297] (a) Converting N-acetylglucosamine or N-acetylglucosamine into a 1,3-diacytized compound having structure (II);

[0298] (b) Reacting a diol with structure (I) with a sulfite oxidizing agent to form a cyclic sulfite with structure (IIIa);

[0299] (c) Reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfite having structure (IIIb);

[0300] (d) Reacting a cyclic sulfate ester with structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide with structure (I);

[0301] (e) Protect 6-azido-6-deoxy monosaccharides having structure (I) to form 6-azido-6-deoxy monosaccharide compounds having structure (VI);

[0302] (f) Transforming a compound having structure (VI) in the presence of one or more Lewis acids to form an oxazoline compound having structure (VII);

[0303] (g) Reacting a compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va);

[0304] (j1) Deprotecting a compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb);

[0305] (i1) React a compound with structure (Vb) with a nucleoside monophosphate to form a nucleoside diphosphate with structure (IX).

[0306] Here, R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2 The compound is selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl groups; B is a nucleobase. Preferred embodiments of each step and compound in this method are as defined above.

[0307] In one embodiment of this aspect of the invention, step (f) is omitted and the compound having structure (VI) is subjected to step (g1) to provide a compound having structure (Va).

[0308] [D.3] Synthesize compound (IX) using compounds (XII) and (VIII).

[0309] In one aspect, the present invention relates to a method for preparing nucleoside diphosphates having structure (IX) or salts thereof according to the following scheme:

[0310]

[0311] The method includes:

[0312] (a) Converting N-acetylglucosamine or N-acetylglucosamine into a 1,3-diacytized compound having structure (II);

[0313] (b) Reacting a diol having structure (II) with a sulfite oxidizing agent to form a cyclic sulfite having structure (IIIa);

[0314] (c) Reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfite having structure (IIIb);

[0315] (d) Reacting a cyclic sulfate ester with structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide with structure (I);

[0316] (e) Protect 6-azido-6-deoxy monosaccharides having structure (I) to form 6-azido-6-deoxy monosaccharide compounds having structure (VI);

[0317] (x1) Deprotecting the anodic sites of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI);

[0318] (x2) A 1-hydroxy-monosaccharide compound having structure (XI) is converted into a 6-azido-6-deoxy-1-monophosphite having structure (XII);

[0319] (x3) In the presence of an oxidizing agent, a monophosphite diester with structure (XII) is oxidized to form a 1-monophosphite diester compound with structure (XIII).

[0320] (x4) Deprotecting the phosphate diester with structure (XIII) to form a 1-monophosphate monosaccharide compound with structure (Va);

[0321] (i) reacting a compound having structure (Va) with a nucleoside monophosphate to form an acylated nucleoside diphosphate having structure (VIII); and

[0322] (j) Deprotecting an acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate or a salt having structure (IX).

[0323] Here, R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 3 Selected from C 1-6Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C 1-2 -alkyl (2-phenylethyl or benzyl), wherein the phenyl group is optionally substituted with one or more halides or nitro or methoxy groups; R 4 Both are hydrogen or two R appear. 4 They are linked together by a carbonyl group; B is a nucleobase. Preferred embodiments of each step and compound in this method are defined above.

[0324] [D.4] Synthesis of compound (IX) via compounds (XII) and (Vb)

[0325] In one aspect, the present invention relates to a method for preparing nucleoside diphosphates having structure (IX) or salts thereof according to the following scheme:

[0326]

[0327]

[0328] The method includes:

[0329] (a) Converting N-acetylglucosamine or N-acetylglucosamine into a 1,3-diacytized compound having structure (II);

[0330] (b) Reacting a diol having structure (II) with a sulfite oxidizing agent to form a cyclic sulfite having structure (IIIa);

[0331] (c) Reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfite having structure (IIIb);

[0332] (d) Reacting a cyclic sulfate ester with structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide with structure (I);

[0333] (e) Protect 6-azido-6-deoxy monosaccharides having structure (I) to form 6-azido-6-deoxy monosaccharide compounds having structure (VI);

[0334] (x1) Deprotecting the anodic sites of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI);

[0335] (x2) A 1-hydroxy-monosaccharide compound having structure (XI) is converted into a 6-azido-6-deoxy-1-monophosphite having structure (XII);

[0336] (x3) Oxidation of a monophosphite diester with structure (XII) in the presence of an oxidizing agent to form a 1-monophosphite diester compound with structure (XIII);

[0337] (x4) Deprotecting the phosphate diester with structure (XIII) to form a 1-monophosphate monosaccharide compound with structure (Va);

[0338] (j1) Deprotecting a compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb);

[0339] (i1) React a compound with structure (Vb) with a nucleoside monophosphate to form an acylated nucleoside diphosphate with structure (IX).

[0340] Here, R 1 Independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2 Selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 3 Selected from C 1-6 Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C 1-2 -alkyl (2-phenylethyl or benzyl), wherein the phenyl group is optionally substituted with one or more halides or a nitro or methoxy group; B is a nucleobase. Preferred embodiments of each step and compound in this method are as defined above.

[0341] [D.5] Synthesis of compound (IX) from compound (XIV)

[0342] In one aspect, the present invention relates to a method for preparing nucleoside diphosphates having structure (IX) or salts thereof according to the following scheme:

[0343]

[0344] The method includes:

[0345] (a) Converting N-acetylglucosamine or N-acetylglucosamine into a 1,3-diacytized compound having structure (II);

[0346] (b) Reacting a diol having structure (II) with a sulfite oxidizing agent to form a cyclic sulfite having structure (IIIa);

[0347] (c) Reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfite having structure (IIIb);

[0348] (d) Reacting a cyclic sulfate ester with structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide with structure (I);

[0349] (y1) Deprotection of 6-azido-6-deoxy monosaccharide with structure (I) to form 1,3,4-trihydroxy-6-azido-monosaccharide compound with structure (XIV);

[0350] (y2) In the presence of a phosphate source, a compound with structure (XIV) is contacted with a phosphorylase to form a 1-monophosphate monosaccharide compound with structure (Vb).

[0351] (i) Reacting a compound having structure (Vb) with a nucleoside monophosphate to form a nucleoside diphosphate or a salt thereof having structure (IX).

[0352] Here, R 1 The compounds are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl groups; B is a nucleobase. Preferred embodiments of each step and compound in this method are as defined above.

[0353] Example

[0354] Chemicals were purchased from commonly used suppliers (Sigma-Aldrich, Acros, Alfa Aesar, Fluorochem, ApolloScientific Ltd, and TCI) and were ready for use without further purification. Solvents (including dry solvents) used for chemical transformation, post-processing, and chromatography were purchased from Aldrich (Dorset, UK) at HPLC grade and were ready for use without further distillation. Silica gel 60F254 analytical thin-layer chromatography (TLC) plates were purchased from Merck (Darmstadt, Germany) and visualized under UV light using potassium permanganate or anisaldehyde staining. Chromatographic purification was performed using Acros silica gel (0.06–0.200, 60A) or pre-packed columns (Screening Devices) with a Buchi Sepacor C660 fraction collector (Flawil, Switzerland). Deuterated solvents used for NMR spectroscopy were purchased from Cambridge Isotope Laboratories.

[0355] The conditions in Examples 1-1 to 1-3 are based on those of Yule et al., Tet. Lett., 36, 1995, 6839–6842, Jiaang et al., Synlett., 2000, 6, 797–800, and Nishimura et al., Angew. Chem. Int. Ed., 2012, 51, 3386–3390, all of which are incorporated herein by reference.

[0356] Example 1-1: Synthesis of 4,6-O-benzylidene-N-acetyl-D-galactosamine

[0357]

[0358] DL-camphorsulfonic acid (81 g, 352 mmol) and benzaldehyde dimethyl acetal (2190 g, 2160 mL, 14.4 mol) were added to a suspension of N-acetyl-D-galactosamine (1299 g, 5.9 mol) in MeCN (13 L). The reaction mixture was stirred at room temperature and filtered through a Buchner funnel. The white filter cake was washed with MeCN (10 × 1 L) and dried on the filter overnight. The product was dried in a circulating oven at 35 °C for four days to give 1938 g, 107%. A pure product as a white crystalline solid was obtained by crystallization from EtOH / H₂O (13:1–9:1) at 5–8 °C for 16–24 hours. 1 H NMR(400MHz,DMSO)δ(ppm)7.49-7.46(m,2H),7.38-7.34(m,3H),5.56(s,1H),5.04- 5.03(m,1H),4.14-4.13(m,1H),4.08-3.94(m,3H),3.85-3.78(m,2H),1.82(s,3H).

[0359] Examples 1-2: Synthesis of 1,3-di-O-acetyl-4,6-O-benzylidene-N-acetyl-D-galactosamine

[0360]

[0361] The starting material 4,6-O-benzyl-N-acetyl-D-galactosamine (1938 g, 6.3 mmol) was dissolved in pyridine (7750 mL), and acetic anhydride (1919 g, 1.765 L, 18.8 mol) was added dropwise over 10 minutes. The reaction mixture was stirred overnight at room temperature, and then ice water (19.5 L) was added. After stirring for 15 minutes, a spoonful of ice was added, and after another 15 minutes, the mixture was filtered. The filter cake was washed with ice water (3 × 6 L) and dried on a filter overnight, then dried overnight in a circulating oven at 45 °C. The crude product was dissolved in methanol (13.7 L), and the mixture was heated to reflux to obtain a clear solution. The solution was cooled to room temperature overnight, then cooled to 0 °C in an ice bath and held for 4 hours, and then filtered. The filter cake was washed three times with the filtrate and dried on a filter for 1.5 hours. The product was dried in a circulating oven at 45°C for 3.5 days to obtain product 2 (1450 g, 3.7 mol, 59%). 1 HNMR (400MHz, CDCl3) δ (ppm) 7.53-7.50 (m, 2H), 7.39-7.53 (m, 3H), 6.34 (d, J = 4.8Hz, 1H), 5.57 (d, J = 12Hz, 1H), 5.24 (dd, J = 11.2, 4.4Hz,1H),4.92-4.85(m,1H),4.34(d,J=4Hz,1H),4.29-4.24(m,1H),4.05-4.00(m,1H),2.15(s,3H),2.10(s,3H),1.93(s,3H).

[0362] Examples 1-3: Synthesis of 1,3-di-O-acetyl-N-acetyl-D-galactosamine (2)

[0363]

[0364] The starting material 1,3-di-O-acetyl-4,6-O-benzyl-N-acetyl-D-galactosamine (230 g, 585 mmol) was dissolved in a mixture of MeOH and dioxane (1:1, 3 L), placed in a Parr container, and AcOH (1.9 g, 1.77 mL, 31 mmol) was added. Subsequently, Pd-C (37.3 g, 175 mmol) was added, and the container was attached to the Parr apparatus. The reaction mixture was stirred overnight under a H2 atmosphere (5 bar). The mixture was then filtered through a diatomaceous earth mat and washed with 1,4-dioxane (1200 mL). Acetic acid (12 mL) was added to the filtrate, and the mixture was concentrated under reduced pressure to give product 3 (224 g, 126%). 1H NMR (400MHz, DMSO) δ (ppm) 7.91 (d, J = 8.4Hz, 1H), 5.96 (d, J = 3.6Hz, 1H), 5.20 (d, J = 5.2Hz, 1H), 4.84 (dd, J = 8.8, 2.8Hz, 1H ),4.49-4.43(m,1H),3.79(t,J=6.4Hz,1H),3.55-3.50(m,1H),3.44-3.40(m,1H),2.11(s,3H),2.01(s,3H),1.78(s,3H).

[0365] Examples 1-4: Synthesis of 4,6-O-sulfoxyl-1,3-di-O-acetyl-N-acetyl-D-galactosamine (3a)

[0366]

[0367] Method A Compound 2 (190 g, 622 mmol) was dissolved in DCM (11.5 L) and placed under a nitrogen atmosphere. Next, SOCl2 (182 mL, 933 mmol) was added dropwise, and the reaction mixture was cooled to 0 °C. Then, Et3N (500 mL, 1306 mmol) was slowly added. After complete addition, the reaction mixture was heated to room temperature and stirred for 1 hour. The reaction was quenched by carefully adding water (2.3 L). The layers were separated, and the organic layer was extracted twice more with water (2 × 2.3 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product (4a) was ready for the next step without further purification. 1 H NMR(400MHz,MeOD)δ(ppm)6.23(d,J=3.6Hz,1H),5.34-5.35(m,1H),5.24(dd,J=8.4,3.2Hz,1H),4.94-.495(m,1H ), 4.63(dd,J=8.4,3.2Hz,1H),4.11(s,1H),3.96(dd,J=10.8,1.6Hz,1H),2.17(s,3H),2.07(s,3H),1.95(s,3H).

[0368] Method BCompound 2 (1 equivalent) was dissolved in EtOAc (0.15 M) and placed under a N2 atmosphere. SOCl2 (1.05 equivalent) was added dropwise, followed by pyridine (2.1 equivalent). The reaction progress was monitored by TLC analysis (100% EtOAc), and complete conversion was achieved after 30 minutes. The reaction was quenched by adding H2O (0.07 M) and EtOAc (0.03 M). The layers were separated, and the aqueous layer was extracted again with EtOAc (0.03 M). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product (3a) could be used in the next step without further purification. Examples 1-5: Synthesis of 4,6-O-sulfonyl-1,3-di-O-acetyl-N-acetyl-D-galactosamine (3b)

[0369]

[0370] Crude product 3a (176 g, 501 mmol) was dissolved in a mixture of DCM (1460 mL) and MeCN (1460 mL). NaIO4 (214 g, 1002 mmol) in water (2199 mL) was then added, and the mixture was stirred vigorously. A solution of RuCl3·xH2O (2.08 g, 10.0 mmol) in water (106 mL) was added dropwise, and the reaction mixture was cooled in an ice bath to prevent further exothermic reaction. The reaction mixture was stirred at room temperature for approximately 30 minutes (the reaction progress was tracked by TLC analysis (100% EtOAc)). After the reaction was complete (1 hour), the reaction mixture was cooled to 5 °C in an ice / NaCl bath. Then, a solution of sodium metabisulfite (438 g, 2304 mmol) in water (704 mL) was added dropwise over 45 minutes. The reaction mixture was transferred to a separatory funnel, and the layers were separated. The aqueous layer was extracted twice more with DCM (850 mL). The combined organic layers were washed with a semi-saturated NaCl solution and dried over Na₂SO₄, filtered, and concentrated under vacuum. The white solid was ground overnight in diisopropyl ether (substitute: diethyl ether) (700 mL), then filtered and washed with another 3 × 200 mL of diisopropyl ether (substitute: diethyl ether). The solid was dried under a gentle nitrogen stream to give product 3b (153 g, 416 mmol, 83%) as a grayish-white solid.

[0371] Post-processor B After the reaction was complete, the mixture was diluted with DCM (0.2 M). The reaction mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted once with DCM (0.2 M). The combined organic layers were dried over Na2SO4, filtered (optionally, filtered with diatomaceous earth), and concentrated under vacuum. Gradient rapid chromatography (DCM:MeOH; 100:0 → 90:10) was performed to give purified product 3b (79-82%). 1¹H NMR (400MHz, DMSO) δ (ppm) 8.07 (d, J = 8.8Hz, 1H), 6.07 (d, J = 3.2Hz, 1H), 5.45 (d, J = 3.2Hz, 1H), 5.25 (dd, J = 8.0, 2.8Hz, 1H), 4.83 (AB system, 36.4, 12.8Hz, 2H), 4.42-4.37 (m, 2H), 2.15 (s, 3H), 2.09 (s, 3H), 1.83 (s, 3H).

[0372] Examples 1-6: Synthesis of 6-azido-6-deoxy-1,3-di-O-acetyl-N-acetyl-D-galactosamine (1)

[0373]

[0374] Method A Crude product 3b (1 equivalent) was dissolved in DMF (0.1–0.2 M) and NaN3 (1.2–5 equivalents) was added. The reaction mixture was stirred at room temperature and concentrated upon completion (1–24 hours, depending on the amount of NaN3 used). The residue was dissolved in THF (0.2 M) and H2SO4 (1.2 equivalents) and H2O (1.2 equivalents) were added. The reaction mixture was stirred at room temperature (1–4 hours) and analyzed by TLC (DCM:MeOH 9:1).

[0375] Post-processing Add EtOAc (0.2 M), saturated NaHCO3 aqueous solution (0.4 M), and water (0.4 M). Extract the aqueous layer with EtOAc (2 × 0.2 M) and dry to Na2SO4, filter, and concentrate under reduced pressure. Crude product 1 was obtained in 80-100% yield. NMR analysis showed partial acetyl migration from 3-OH to 4-OH, but crude product 1 was used for the next step. The two regioisomers were separated on a small scale by silica gel column chromatography to obtain pure samples of compound 1 and its regioisomer compound 1' (with 3-OH and 4-OAc groups), and NMR was performed.

[0376] purification After concentration, gradient rapid chromatography (DCM:MeOH; 100:0→90:10) was performed to obtain product 1 (61%).

[0377] 1¹H-NMR (400MHz, CDCl₃) 3-OAc (Compound 1): δ (ppm) 6.19 (d, J = 3.6 Hz, 1H), 5.67 (d, J = 9.3 Hz, 1H), 5.17 (dd, J = 3.2, 8.4 Hz, 1H), 4.84–4.76 (m, 1H), 4.08 (s, 1H), 3.99 (t, J = 6.4 Hz, 1H), 3.53 (ddd, J = 6.4, 6.4, 8.8 Hz, 2H), 2.88 (d, J = 3.6 Hz, 1H), 2.18 (s, 3H), 2.14 (s, 3H), 1.95 (s, 3H).

[0378] 1 ¹H-NMR (400MHz, CDCl₃)⁴-OAc (compound 1'): δ (ppm) 6.24 (d, J = 3.6 Hz, 1H), 6.05 (d, J = 8.0 Hz, 1H), 5.35 (d, J = 2.8 Hz, 1H), 4.52 (ddd, J = 3.2, 3.6, 4.8 Hz, 1H), 4.07 (dd, J = 1.6, 5.2 Hz, 1H), 4.01–3.95 (m, 1H), 3.43–3.38 (m, 1H), 3.28–3.24 (m, 2H), 2.24 (s, 3H), 2.18 (s, 3H), 2.04 (s, 3H).

[0379] Method B Crude product 3b (150 g, 408 mmol, 1 equivalent) was dissolved in DMF (1500 mL) and NaN3 (26.8 g, 412 mmol) was added. The reaction mixture was stirred overnight at room temperature, and then 2-(bromomethyl)naphthalene (4.51 g, 20.4 mmol) was added. The reaction mixture was stirred for another 1 hour, followed by concentration under reduced pressure. The residue was dissolved in THF (900 mL) and water (8.9 mL, 490 mmol) by heating in a water bath (40 °C). The reaction mixture was then cooled in an ice bath and sulfuric acid (26.1 mL, 490 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour, and then a saturated aqueous solution of NaHCO3 (1.2 L) was added, and the mixture was extracted with EtOAc (7 × 1 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was dissolved in EtOAc (1 L) and dried over Na2SO4, filtered, and concentrated to obtain crude product 1, which was a yellow oil. NMR analysis showed partial acetyl migration from 3-OH to 4-OH, but crude product 1 was used for the next step. The two regioisomers were separated on a small scale by silica gel column chromatography to obtain pure samples of compound 1 and its regioisomer compound 1' (containing 3-OH and 4-OAc groups), and NMR was performed.

[0380] 1 ¹H-NMR (400MHz, CDCl₃)⁻³-OAc (Compound 1): δ (ppm) 6.19 (d, J = 3.6 Hz, 1H), 5.57 (d, J = 9.2 Hz, 1H), 5.18 (dd, J = 3.2, 8.4 Hz, 1H), 4.84–4.78 (m, 1H), 4.08 (s, 1H), 3.98 (t, J = 6.4 Hz, 1H), 3.53 (ddd, J = 6.4, 6.4, 8.8 Hz, 2H), 2.81 (d, J = 3.6 Hz, 1H), 2.18 (s, 3H), 2.14 (s, 3H), 1.95 (s, 3H).

[0381] 1 ¹H-NMR (400MHz, CDCl₃) 4-OAc (compound 1'): δ (ppm) 6.23 (d, J = 3.6 Hz, 1H), 5.61 (d, J = 8.0 Hz, 1H), 5.35 (d, J = 2.8 Hz, 1H), 4.52 (ddd, J = 3.2, 3.6, 4.8 Hz, 1H), 4.07 (dd, J = 1.6, 5.2 Hz, 1H), 4.01-3.95 (m, 1H), 3.43-3.38 (m, 1H), 3.28-3.24 (m, 2H), 2.24 (s, 3H), 2.18 (s, 3H), 2.04 (s, 3H). Examples 1-7: Synthesis of 6-O-toluenesulfonyl-1,3,4-tri-O-acetyl-N-acetyl-D-galactosamine (3c)

[0382]

[0383] Compound 2 (1.3 g, 4.3 mmol) was dissolved in pyridine (20 mL), cooled to 0 °C, and p-toluenesulfonyl chloride (989 mg, 5.2 mmol) was added. After 2 hours, p-toluenesulfonyl chloride (380 mg, 2.4 mmol) was added, and the reaction mixture was stirred for another hour. The reaction mixture was concentrated under reduced pressure, and then DCM (50 mL) and 1 M HCl (30 mL) were added. After separating the layers, the organic layer was washed with 1 M HCl aqueous solution (2 × 30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification was performed by rapid chromatography (DCM:MeOH = 100:0 → 94:6) to give product 3c (1.45 g, 3.1 mmol, 72%) as a white solid. 1¹H-NMR (400MHz, CDCl₃): δ 7.77 (d, J = 8.8Hz, 2H), 7.34 (d, J = 8.8Hz, 2H), 6.11 (d, J = 4Hz, 1H), 5.54 (d, J = 9.6Hz, 1H), 5.17 (dd, J = 2.8, 8.4Hz, 1H), 4.77-4.71 (m, 1H), 4.27 (AB-system, J = 3.2, 6.4Hz, 1H), 4.06 (AB-system, J = 3.2, 6.4Hz, 1H), 4.16-4.13 (m, 2H), 2.46 (s, 3H), 2.17 (s, 3H), 2.11 (s, 3H), 1.93 (s, 3H). Examples 1-8: Synthesis of 1 by nucleophilic substitution of 3c with an azide followed by acetylation.

[0384] To compound 3c (1.4 g, 3 mmol) in DMF (15 mL), NaN3 (1 g, 15 mmol), tetrabutylammonium iodide (575 mg, 1.5 mmol), and 15-crown-5 (341 mg, 1.5 mmol) were added. The suspension was heated to 70 °C and maintained for 48 hours, then concentrated under reduced pressure. The crude product mixture was dissolved in pyridine (20 mL), and then acetic anhydride (3 mL, 30 mmol) was added. The reaction mixture was stirred overnight at room temperature. Next, DCM (30 mL) was added, and the reaction mixture was washed with 1 M HCl aqueous solution (3 × 20 mL) and saturated NaHCO3 aqueous solution (2 × 20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by rapid chromatography (DCM:MeOH = 100:0 → 95:5) gave 1 (515 mg, 1.4 mmol, 47%). 1 ¹H-NMR (400MHz, CDCl₃): δ 6.24 (d, J = 4Hz, 1H), 5.48–5.46 (m, 1H), 5.41–5.40 (m, 1H), 5.24–5.21 (m, 1H), 4.77–4.71 (m, 1H), 4.12–4.09 (m, 1H), 3.44 (AB-system, J = 7.2, 5.6Hz, 1H), 3.22 (AB-system, J = 7.2, 5.6Hz, 1H), 2.20 (s, 3H), 2.18 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H).

[0385] Examples 1-9: Synthesis of 6-O-methanesulfonyl-1,3,4-tri-O-acetyl-N-acetyl-D-galactosamine (3d)

[0386]

[0387] To a solution of compound 2 (100 mg, 0.327 mmol, 1.00 equivalent) in MeCN (0.5 mL), DCM (1.0 mL), Et3N (63.0 μL, 0.452 mmol, 1.4 equivalent), and MsCl (27 μL, 0.349 mmol, 1.07 equivalent) were added. The resulting reaction mixture was stirred at room temperature and monitored by TLC analysis. After stirring the reaction mixture for 1 hour, TLC analysis showed incomplete conversion. Following this analysis, MsCl (10 μL) was added, and after another 1 hour, MsCl (10 μL) and Et3N (20 μL) were added. The reaction mixture was stirred for another 30 minutes, and then DCM and a saturated aqueous solution of NH4Cl were added. The resulting mixture was shaken vigorously and the resulting biphasic system was separated. The organic layer was washed with a saturated aqueous solution of NH4Cl (3×), dried (Na2SO4), filtered, and concentrated under vacuum. Furthermore, the combined aqueous layers were extracted with EtOAc (3×), and the combined organic layers were dried (Na2SO4), filtered, and concentrated under vacuum. The resulting residues were combined with the residues from the DCM extraction and purified by rapid column chromatography (0-5% MeOH in DCM solution) to give compound 3d (40 mg, 32% yield). For C 13 H 21 NNaO 10 S + (M+Na + The calculated value of LCMS(ESI+) is 406.08. The measured value is 405.95.

[0388] Example 1-10a: Synthesis of 1 by nucleophilic substitution of 3d with NBu4N3

[0389] NBu4N3 (18 mg, 63 μmol, 1.3 equivalent) was added to a mixture of 3d (20 mg, 49 μmol, 1.0 equivalent) in MeCN (200 μL), and the mixture was then heated to reflux. The mixture was refluxed over the entire weekend. Refluxing the resulting mixture over the entire weekend indicated the formation of compound 1. For C 10 H 15 N4O5 + (M-OAc - The calculated value of LCMS(ESI+) is 271.1. The measured value is 271.23.

[0390] Example 1-10b: Synthesis of 1 by nucleophilic substitution of 3d with NaN3

[0391] To a mixture of 3d (20 mg, 49 μmol, 1.0 equivalent) in DMF (200 μL), NaN3 (16 mg, 5 equivalent) was added, and the mixture was heated to reflux. The mixture was refluxed over the entire weekend, indicating the formation of compound 1. For C 10 H 15 N4O5 + (M-OAc - The calculated value of LCMS(ESI+) is 271.1. The measured value is 271.23.

[0392] Examples 1-11: Synthesis of 1 by replacing 3d with azide under Mitsunobu conditions

[0393]

[0394] Compound 2 (50 mg, 0.16 mmol) was mixed with PPh3 (58 mg, 0.22 mmol, 1.4 equivalences), diphenyl azidophosphate (47 μL, 0.22 mmol, 1.4 equivalences), and diethyl azodicarbonate (100 μL, 40 wt% toluene solution, 0.22 mmol, 1.4 equivalences), dissolved in cold THF (1.0 mL), and cooled in an ice bath. The reaction mixture was placed at 0 °C for 4 hours, then warmed to room temperature. TLC analysis showed limited conversion. The reaction mixture was cooled to 0 °C, and PPh3 (30 mg, 0.13 mmol, 0.83 equivalences), diphenyl azidophosphate (24 μL, 0.11 mmol, 0.71 equivalences), and diethyl azodicarbonate (49 μL, 40 wt% toluene solution, 0.11 mmol, 0.68 equivalences) were added. The resulting mixture was warmed to room temperature and stirred overnight. Next, the reaction mixture was partially concentrated under reduced pressure and purified by silica gel column chromatography (DCM:MeOH = 100:0 → 95:5) to give crude compound 1 (38 mg). For C 10 H 15 N4O5 + (M-OAc - The calculated value of LCMS(ESI+) is 271.1. The measured value is 271.23.

[0395] Examples 1-12: Synthesis of 6-azido-6-deoxy-1,3,4-tri-O-acetyl-N-acetyl-D-galactosamine (6)

[0396]

[0397] Method ACrude product 1 (152 g, 416 mmol) was dissolved in pyridine (750 mL) and acetic anhydride (236 mL, 2496 mmol) was added. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. Then, EtOAc (1 L) was added, and the suspension was added to a stirred 1 M HCl aqueous solution (2.16 L). After vigorous stirring for 15 minutes, the layers were separated, and the aqueous layer was extracted again with EtOAc (1 L). The combined organic layers were washed with brine (2 × 1 L), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Then, diethyl ether (1.2 L) was added and the mixture was heated to reflux. The mixture was mechanically stirred while cooling to room temperature. After stirring overnight, the solid was filtered and washed with diethyl ether (3 × 200 mL). After drying under a nitrogen stream, product 6 (123 g, 333 mmol, 80%, in 2 steps) was obtained as a white solid. Product 6 (77%, obtained by 5 steps) was purified by rapid chromatography (EtOAc:heptane; 50:50→100:0) to give a white solid product 6. 1 ¹H NMR (400MHz, CDCl₃) δ (ppm) 6.24 (d, J = 3.6 Hz, 1H), 5.57 (d, J = 9.2 Hz, 1H), 5.41–5.40 (m, 1H), 5.21 (dd, J = 8.4, 3.2 Hz, 1H), 4.76–4.70 (m, 1H), 4.12 (t, J = 6.8 Hz, 1H), 3.43 (AB system, J = 7.2, 5.6 Hz, 1H), 3.22 (AB system, J = 7.2, 5.6 Hz, 1H), 2.20 (s, 3H), 2.19 (s, 3H), 2.04 (s, 3H), 1.96 (s, 3H).

[0398] Method B Crude product 1 (9 mg, 0.03 mmol) was dissolved in DCM (0.02 M) and acetic anhydride (5 equivalents), DMAP (0.1 equivalents), and Et3N (5 equivalents) were added. The reaction mixture was stirred at room temperature for 3 hours. Then, EtOAc (0.03 M) was added, and the reaction mixture was washed with 1 M HCl (0.03 M) and saturated aqueous NaHCO3 solution (0.03 M). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give product 6 (71%). 1¹H NMR (400MHz, CDCl₃) δ (ppm) 6.24 (d, J = 3.6 Hz, 1H), 5.57 (d, J = 9.2 Hz, 1H), 5.41–5.40 (m, 1H), 5.21 (dd, J = 8.4, 3.2 Hz, 1H), 4.76–4.70 (m, 1H), 4.12 (t, J = 6.8 Hz, 1H), 3.43 (AB system, J = 7.2, 5.6 Hz, 1H), 3.22 (AB system, J = 7.2, 5.6 Hz, 1H), 2.20 (s, 3H), 2.19 (s, 3H), 2.04 (s, 3H), 1.96 (s, 3H).

[0399] Example 2-1: Synthesis of 6-azido-6-deoxy-3,4-tri-O-acetyl-N-acetyl-D-galactosamine (11)

[0400]

[0401] Method A A solution of compound 6 (420 mg, 1.13 mmol) in anhydrous THF (11 mL) was cooled to 0 °C, and then benzylamine (130 μL, 1.18 mmol, 1.04 equivalents) was added. The resulting mixture was stirred at 0 °C for 2 hours, and then concentrated under vacuum. The residue was purified by rapid chromatography (EtOAc:heptane; 60:40 → 100:0) to give 353 mg (95%) of compound 11. 1 H NMR (400MHz, CDCl3) δ (ppm) 6.24 (d, J = 3.6Hz, 1H), 5.74 (d, J = 9.1Hz, 1H), 5.44-5.37 (m, 1H), 5.25-5.18 (m, 1H), 4. 77-4.65(m,1H),4.16-4.08(m,1H),3.49-3.38(m,1H),3.28-3.15(m,1H),2.18(s,3H),2.03(s,3H),1.95(s,3H).

[0402] Method BA stirred solution of compound 6 (2.3 g, 6.18 mmol) in anhydrous THF (40 mL) was treated with BnNH2 (0.95 mL, 8.65 mmol, 1.4 equivalences) and the resulting mixture was stirred until LCMS indicated complete reaction, typically for 18 hours. The mixture was diluted with DCM (150 mL) and washed successively with saturated NH4Cl aqueous solution and brine (100 mL each), dried (MgSO4), filtered, and concentrated. The residue was subjected to rapid column chromatography (80 g SiO2 column; 50-100% EtOAc in petroleum ether 40-60 solution) to give compound 11 (1.64 g, 4.96 mmol, 83% yield) as a white foam. NMR showed that compound 11 was a 4:1 α / β anomeric mixture and also contained BnNHAc (>5%).

[0403] Method C The solution of compound 6 (1.80 g, 4.83 mmol) in anhydrous THF (24 mL) was treated with dimethylaminopropylamine (DMAPA, 0.9 mL, 7.2 mmol, 1.5 equivalences) and stirred until LC-MS indicated completion, typically 18 hours. The yellow mixture was diluted with MeOH (75 mL) and tested with IR120 (H). + (Form) Resin treatment until LCMS showed the disappearance of DMAPA and its acetamide byproducts (along with the yellow color). Filter, wash with MeOH, and concentrate the filtrate to give compound 11 (1.64 g, 4.96 mmol, 92% yield) as a white foam. NMR showed that compound 11 was a 4:1 α / β anomeric mixture.

[0404] Method D The stirred solution of compound 6 (200 mg, 0.54 mmol) in anhydrous THF (2.75 mL) was treated with DMAPA (0.1 mL, 0.81 mmol, 1.5 equivalences) and stirred until LCMS showed complete reaction, typically 18 hours. The yellow mixture was diluted with DCM (40 mL) and washed successively with 1 M HCl and brine (15 mL each) and dried (MgSO4). Filtration and concentration gave compound 11 (130 mg, 0.39 mmol, 73% yield) as a white foam. NMR showed that compound 11 was a 4:1 α / β anomeric mixture and contained THF residue (~10%).

[0405] Example 2-2: Synthesis of (3aR,5R,6S,7R,7aR)-5-(azidomethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6,7-dimethyldiacetate (7)

[0406] Method A

[0407]

[0408] The reaction was carried out in a 1000 mL three-necked flask, which was dried overnight in an oven at >100 °C and purged with nitrogen before use. In this flask, compound 6 (16.6 g, 44.6 mmol) was dissolved in anhydrous dichloromethane (500 mL). 2,4,6-Trimethylpyridine (10 mL, 80 mmol) was slowly added to this solution via a syringe. The reaction mixture was stirred for 10 minutes, and bromo-trimethylsilane (10 mL, 76 mmol) was slowly added to the clear solution via a dropping funnel. After 5 minutes, the boron trifluoride diethyl ether complex (approximately 48% BF3·Et2O 20 mL, 158 mmol) was added via a dropping funnel. After 10 minutes, the mixture was heated to 35 °C and stirred for 5 hours. TLC analysis (DCM:MeOH 93:7) then showed almost complete conversion. The heating was turned off, and the mixture was allowed to reach room temperature overnight. TLC analysis showed that the starting material was completely consumed. The mixture was cooled to 0°C in an ice bath and slowly poured into 1000 mL of ice-cooled saturated NaHCO3 solution with gentle stirring. Significant CO2 emissions were observed. The layers were separated, and the aqueous layer was extracted twice more (2 × 500 mL DCM). The organic layers were combined, dried over Na2SO4, and concentrated under reduced pressure. The mixture was coated onto hydromatrix (inert diatomaceous earth adsorbent, 40 g) and purified by rapid column chromatography (heptane:EtOAc = 70:30 → 0:100, containing 1% Et3N) to give compound 7 as a grayish-white solid (13.1 g, 39.2 mmol, 88% yield). 1 ¹H-NMR (400MHz, CDCl₃): δ 6.02 (d, J = 7.2Hz, 1H), 5.40 (t, J = 3.2Hz, 1H), 4.95 (dd, J = 3.6, 3.6Hz, 1H), 4.13–4.09 (m, 1H), 4.06–4.03 (m, 1H), 3.48 (AB system, J = 5.2, 7.6Hz, 1H), 3.27 (AB system, J = 5.2, 8Hz, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.07–2.04 (m, 3H).

[0409] Alternative post-processing proceduresThe reaction mixture was cooled in an ice bath, followed by the addition of Et3N (5 equivalents). After stirring for 5 minutes, the resulting reaction mixture was placed directly onto a silica gel column and purified by rapid column chromatography (heptane:EtOAc 50:50→10:90). After concentrating the appropriate column fraction, the residue was dissolved in DCM (0.1M) and washed with 2% citric acid aqueous solution (2×0.2M). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 7 as a yellow oil (87-89% yield).

[0410] Extraction process for selective removal of trimethylpyridine The crude product reaction mixture was concentrated under vacuum and redissolved in EtOAc (100 mL). The resulting suspension was filtered, and the filtrate was washed with 5% CuSO4 aqueous solution (3 × 100 mL). The organic layer was washed with brine, dried (Na2SO4), and concentrated under vacuum. The residue could be used as the crude product in the next step, or purified by silica gel column chromatography (heptane:EtOAc = 70:30 → 0:100, containing 1% Et3N) to give compound 7 (1.31 g, 95% qNMR purity, 58% yield).

[0411] Method B

[0412]

[0413] Compound 6 (201 mg, 0.540 mmol, 1.00 equivalent) was added to a flame-dried round-bottom container containing a stirrer and molecular sieve, followed by dichloromethane (6.0 mL). The resulting solution was cooled to 0 °C, and 2,4,6-trimethylpyridine (220 μL, 1.62 mmol, 3.0 equivalent), trimethylchlorosilane (206 μL, 1.62 mmol, 3.0 equivalent), and boron trifluoride diethyl ether complex (210 μL, 1.62 mmol, 3 equivalent) were added sequentially. The resulting mixture was heated to 35–40 °C and maintained for 23 hours. The reaction was then cooled to 0 °C and quenched with Et3N (375 μL, 2.70 mmol, 5.0 equivalent). The resulting mixture was stirred for 5 minutes, and then purified directly by silica gel column chromatography (pentane: EtOAc = 50:50 → 10:90) to give compound 7 (137 mg, 93%), which was a yellow oil. 1 H-NMR purity, 76% yield). 1H NMR(CDCl3): 6.02(d,J=7.1Hz,1H),5.40(t,J=3.2Hz,1H),5.02-4.89(m,1H),4.19-4.07(m,1H),4. 07-3.93(m,1H),3.55-3.43(m,1H),3.32-3.20(m,1H),2.14(s,3H),2.08(s,3H),2.07-2.03(m,3H).

[0414] Method C

[0415]

[0416] At room temperature and under a nitrogen atmosphere, TMSOTf (1.5 equivalents) was added to a solution of compound 6 (1 equivalent) in dry 1,2-dichloroethane (0.1 M). The resulting reaction mixture was heated to 50 °C and the reaction was monitored by TLC (DCM:MeOH 9:1) until completion (optionally, TMSOTf was added up to a maximum of 3 equivalents). After TLC analysis indicated complete conversion (4–16 h), the reaction mixture was cooled to 0 °C, and Et3N (1.5–5 equivalents) was added dropwise over 2 min with stirring for another 10 min. The resulting reaction mixture was purified directly by silica gel column chromatography (heptane:EtOAc = 50:50 → 10:90, containing 1% Et3N) to give compound 7 (62–90% yield) as a pale yellow oil. 1 H NMR(CDCl3): 6.02(d,J=7.1Hz,1H),5.40(t,J=3.2Hz,1H),5.02-4.89(m,1H),4.19-4.07(m,1H),4. 07-3.93(m,1H),3.55-3.43(m,1H),3.32-3.20(m,1H),2.14(s,3H),2.08(s,3H),2.07-2.03(m,3H).

[0417] Example 3-1a: Synthesis of dihydrophosphoryl6-azido-6-deoxy-3,4-tri-O-acetyl-N-acetyl-d-galactosaminopyranose in pure phosphoric acid (5a)

[0418]

[0419] Compound 7 (4 g, 10.7 mmol) was placed under a nitrogen atmosphere, followed by the addition of phosphoric acid (8.5 g, 91 mmol, pre-dried in P2O5). The reaction mixture was placed on P2O5 under vacuum and heated to 60 °C. After 5 hours, the reaction mixture was cooled to room temperature and THF (50 mL) was added. The mixture was then cooled to 0 °C and neutralized with aqueous NH4OH solution until pH 7. The solid was removed by filtration, washed with THF, and the filtrate was concentrated under reduced pressure. Purification was performed by rapid chromatography (MeCN:MeOH = 100:0 → 50:50) to give product 5a (888 mg, 2.2 mmol, 20%) with an α:β ratio of 1:1. 1 H-NMR (400MHz, CDCl3): δ5.47-5.44(m,1H),5.35-5.34(m,1H),5.25-5.24(m,1H),5.11(dd,J=3.2,8.7Hz,1H),5.03(t,J=8.4Hz,1H),4.97(dd,J=3 .2,8Hz,1H),4.41-4.36(m,1H),4.27-4.30(m,1H),4.09-4.04(m,1H),3.8 4-3.80(m,1H),3.49-3.38(m,4H),2.07-2.06(m,6H),1.88-1.85(m,12H).

[0420] Table 1. α:β ratios of 5 formed in pure phosphoric acid at different temperatures and reaction times.

[0421]

[0422] Example 3-1b: Synthesis of dihydrophosphoryl6-azido-6-deoxy-3,4-tri-O-acetyl-N-acetyl-d-galactosaminopyranose in DMF using phosphoric acid (5a)

[0423]

[0424] In a 250 mL flask, dissolve 7 (12.8 g, 38.9 mmol) in anhydrous DMF (80 mL) and add phosphoric acid (2.58 M solution in DMF, 91 mL, 234 mmol) via syringe. Allow the solution to react overnight at 80 °C. 1¹H-NMR analysis (in CD3OD) showed complete consumption of the starting material and the formation of the desired α-anomeric 5a. The reaction mixture was concentrated under reduced pressure and coated onto hydromatrix (inert diatomaceous earth adsorbent, 40 g) and purified on a C18 silica gel column (water:MeCN 100:0 → 90:10, containing 0.1% HCOOH). Combined batches containing the product were concentrated under reduced pressure and stripped three times with toluene to give product 5a as a white solid (10.1 g, 23.3 mmol, 60% yield). 1 ¹H-NMR (400MHz, MeOD): δ 5.68–5.65 (m, 1H), 5.48–5.47 (m, 1H), 5.18 (dd, J = 8.8, 2.8 Hz, 1H), 4.48–4.45 (m, 1H), 4.34–4.33 (m, 1H), 3.48 (AB system, J = 6.8, 6 Hz, 1H), 3.34–3.30 (m, 1H, partially covered by solvent peaks), 2.16 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H).

[0425] Alternative post-processing The reaction mixture was concentrated under reduced pressure, and then DCM (0.03 M), MeOH (0.15 M), and 4-methylmorpholine (3.5 equivalents) were added. Water (0.03 M) was added, and the organic layer was extracted with water (3 × 0.03 M). The combined aqueous layers were washed with EtOAc (3 × 0.03 M), concentrated, and the residue was dissolved in a small amount of MeOH, followed by the addition of MeCN (0.05 M) to form a precipitate. The mixture was centrifuged at 10,000 rpm for 5 minutes. The liquid was concentrated to give compound 5a (53-76%) as a grayish-white solid.

[0426] Alternative post-processing After heating the reaction mixture at 80°C for 24 hours, the reactants were cooled to room temperature, Et3N (6.2 equivalents) was added, and the solution was concentrated under vacuum. The residue was dissolved in a 5% aqueous (0.1 M) solution of MeCN and purified on silica-C18 (gradient 16-34% B, A = water, B = 30% aqueous solution of MeCN) to give compound 5a (56%) as a fluffy white solid.

[0427] Alternative post-processingThe reaction mixture was heated at 80°C for 24 hours, and then concentrated under vacuum. A thick brown syrup was dissolved in a mixture of DCM and MeOH (82:18 ratio, 0.07 M), and then 4-methylmorpholine (6.4 equivalents) was added. The mixture was then concentrated under vacuum, and the resulting residue was dissolved in a minimal amount of EtOAc / MeOH / H2O (6 / 2 / 1). The suspension was transferred to a silica gel column and purified by rapid column chromatography (using a step gradient of EtOAc / MeOH / H2O from 6 / 2 / 1 to 2 / 2 / 1). Compound 5a was given as a brown solid (59% yield). 1 H NMR (400MHz, CD3OD) δ (ppm): 5.70-5.60 (m, 1H), 5.49 (d, J = 2.3Hz, 1H), 5.23 (dd, J = 11.4Hz, 3.0Hz, 1H), 4. 54-4.45(m,1H),4.45-4.35(m,1H),3.57-3.46(m,1H),3.40-3.34(m,1H),2.20(s,3H),2.03-1.96(m,6H).

[0428] Method B (reduced phosphate and IEX purification in DMF) Compound 7 (2.1 g, 6.37 mmol, 1 equivalent) was added to a flame-dried, round-bottomed container with a stir bar, followed by anhydrous DMF (2.72 mL). The resulting pale yellow solution was stirred at room temperature, and then a solution of 6.6 M H3PO4 in anhydrous DMF (1.93 mL, 12.73 mmol, 2.0 equivalent) was added. The resulting reaction mixture was heated to 80 °C under N2 (by placing it in a preheated oil bath) and stirred for 135 minutes. Note: Before post-treatment, the mixture should be... 1 ¹H NMR analysis of the sample was performed to assess whether the α:β ratio was ≥10:1, which is typically achieved after 135 minutes. For this purpose, ~20 μL of the reaction mixture was dissolved in 0.5 mL of MeOD-d³. When the aforementioned ratio was achieved, the reaction vessel was cooled to room temperature and then quenched by adding the reaction mixture to a stirred 20 mM aqueous solution of NH₄HCO₃ (1.27 L, 4 equivalents). Any remaining product was removed from the reaction vessel by washing with anhydrous DMF (a few mL), which was then added to the aqueous solution of NH₄HCO₃. The resulting solution was then loaded into a 300 mL Q-Sepharose Fast PCR vessel at a rate of 10 mL / min. The product was loaded onto an ion-exchange column (A: 10 mM ammonium bicarbonate, B: 250 mM ammonium bicarbonate). The column was then washed with 10 mM ammonium bicarbonate solution, followed by a gradient to 40% B to elute the product. The fraction containing the product was concentrated under vacuum to remove most of the buffer, and the remaining buffer was then lyophilized overnight to give 6-N3-GalNAc-monophosphate as a grayish-white solid (5a, 1.85 g, corrected 1.56 g (based on qNMR), 0.41 mmol, 58.1% yield). 1 H NMR (400MHz, CD3OD) δ (ppm): 5.70-5.60 (m, 1H), 5.49 (d, J = 2.3Hz, 1H), 5.23 (dd, J = 11.4Hz, 3.0Hz, 1H), 4. 54-4.45(m,1H),4.45-4.35(m,1H),3.57-3.46(m,1H),3.40-3.34(m,1H),2.20(s,3H),2.03-1.96(m,6H).

[0429] Example 3-2: Preparation of 5a' (5a's NBu3 salt) by quenching with NBu3

[0430]

[0431] Method A After heating the reaction mixture at 80°C for 24 hours, the reactants were cooled to room temperature, Bu3N (6.2 equivalents) was added, and the solution was concentrated under vacuum. The residue was dissolved in a 5% MeCN aqueous (0.1M) solution and purified on silica-C18 (gradient 16-100% B, A = water, B = 30% MeCN aqueous solution) to give compound 5a' (117 mg, 44% MeCN) as a yellow oil. 1 H-NMR purity, 37% yield).

[0432] Similarly, in the same manner as described in Example 3-1, quenching with Et3N, 4-methylmorpholine or NH4HCO3 yielded the corresponding salts of compound 5a.

[0433] Example 3-3: Deacetylation of compound 5a yielded compound 5b.

[0434]

[0435] A 25% aqueous solution of NH4OH (60 mL) was added to a suspension of acetylated sugar 5a (4.9 g, 11.9 mmol) in MeOH (15 mL). The reaction mixture was stirred at room temperature, and the conversion was monitored by LCMS. After 4 hours, the mixture was concentrated under reduced pressure and stored at -20 °C for 2 days. The solid was then dissolved in a 25% aqueous solution of NH4OH (75 mL), stirred at room temperature, and MS showed complete conversion after 3 hours. Concentration of the solvent gave crude product 5b (3.2 g, 9.9 mmol, 83%) as a yellow solid. 1 H-NMR (400MHz, D2O): δ 5.28 (dd, J = 7.2, 3.2Hz, 1H), 4.12 (dd, J = 6.8, 6.4Hz, 1H), 4.06 (ddd, J = 10.8, 3.2, 2.0Hz, 1H), 3.92–3.81 (m, 2H), 3.47 (AB system, J = 12.8, 7.2Hz, 1H), 3.40 (AB system, J = 12.8, 6.4Hz, 1H), 1.88 (s, 3H).

[0436] Example 4-1: Conversion of UMP disodium salt to UMP tributylammonium salt (UMP.NBu3)

[0437]

[0438] Will 50WX8 50-100 mesh (250g) was placed in a 500mL glass filter and washed with softened water (3×, 500mL). It was then mixed with a solution of UMP disodium salt (40.0g, 108mmol) in water (250mL), and the resulting suspension was stirred at room temperature for 2 hours. 50WX8 was filtered through a 50-100 mesh filter and rinsed with softened water (3×, 30 ml). Tributylamine (25.9 ml, 108 mmol) was added to the resulting filtrate, and the mixture was stirred vigorously for 60 minutes. The clear solution was lyophilized overnight and then dried overnight with P2O5. The resulting UMP tributylammonium salt batch (25.9 g, 91%)... 1 The 1H-qNMR (purity, 80% yield) was obtained as a white solid. 1H NMR (400MHz, D2O) δ (ppm) 7.79 (d, 1H, J = 8Hz), 5.81 (d, 1H, J = 4.4Hz), 5.77 (d, 1H, J = 8Hz), 4.6-4.22 (m, 2H), 4.07-4. 02(m,1H),3.97-3.93(m,1H),3.00-2.95(m,6H),1.55-1.47(m,6H),1.22(sex,6H,J=7.2Hz),0.77(t,9H,J=7.2Hz).

[0439] Example 4-2: Synthesis of a mixture of UDP derivatives 8 and 8' from 5a

[0440]

[0441] Preparation of anhydrous phosphate: UMP tributylamine salt (12.5 g, 22.9 mmol) was suspended in toluene (100 ml) and concentrated to dryness on a rotary evaporator filled with nitrogen. This process was repeated three times. The same procedure was performed for 5 (10.0 g, 23.1 mmol).

[0442] Anhydrous UMP tributylamine salt (12.5 g, 22.9 mmol) was dissolved in anhydrous DMF (100 mL) under a nitrogen atmosphere, and CDI (9.4 g, 57.9 mmol) was added (once). The yellow mixture was stirred at room temperature for 1 hour. A solution of dried 5a (10.0 g, 23.2 mmol) in anhydrous DMF (40 mL) was added to the reaction mixture using a syringe. Then, 1-methylimidazolium chloride (2 M solution in anhydrous DMF, 24 mL, 48.0 mmol) was added, and the reaction mixture was stirred at room temperature under an argon atmosphere. After a reaction time of 4 hours, the crude product reaction mixture was analyzed by LCMS, and the results showed 10% unconsumed starting material. Therefore, 0.1 equivalents of CDI-activated UMP tributylamine salt were prepared as follows. In a 100 mL flask under a nitrogen atmosphere, UMP tributylamine salt (3.0 g, 5.5 mmol) was dissolved in anhydrous DMF (20 mL), and CDI (1.88 g, 11.58 mmol) was added. The mixture was stirred for 30 minutes and injected into the main reaction mixture via a syringe. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. This product (the mixture of 8 / 8') was ready for use in the next step without further purification. Examples 4-3: Silica gel separation of the mixture of 8 and 8' to obtain a pure sample of 8'.

[0443] The crude product reaction mixture was resuspended in a minimal amount of eluent (EtOAc:MeOH:water = 8:2:1). A minimal amount of methanol was added to dissolve the product, and the mixture was purified by rapid column chromatography (EtOAc:MeOH:water 8:2:1 → 2:2:1) to give compound 8' as a grayish-white solid (14.2 g, 15.5 mmol, 67% yield). 1 ¹H-NMR (400MHz, D₂O): δ 7.63 (d, J = 8Hz, 1H), 5.91 (s, 1H), 5.76–5.74 (m, 1H), 5.55–5.50 (m, 2H), 5.38–5.34 (m, 2H), 5.12–5.09 (m, 1H), 4.38–4.35 (m, 2H), 4.16–4.09 (m, 3H), 3.43 (AB system, J = 6.4, 6.4Hz, 1H), 3.26 (AB system, J = 6.4, 6.4Hz, 1H), 2.10 (s, 3H), 1.88 (s, 3H), 1.87 (s, 3H).

[0444] Example 4-4: Selective hydrolysis and rapid C18 purification of mixtures 8 and 8' to obtain 8, followed by deprotection to obtain pure 9a.

[0445]

[0446] H₂O (37 mL) was added to a round-bottom flask containing crude product 8' (808 μmol, based on the amount of 5a used in the previous step), and the mixture was allowed to stand for a few minutes to allow the H₂O to react with any remaining CDI. NH₄HCO₃ (436 mg, 5.51 mmol, 6.8 equivalence) was added to the resulting suspension to produce a 150 mM aqueous solution of NH₄HCO₃. The reaction mixture was then heated to 40 °C and maintained for 20 hours. The reaction progress was monitored by HPLC, which showed complete conversion after stirring overnight (20 hours). After complete conversion, RM was concentrated under vacuum to give crude product 8, which was then purified using the rapid C18 column chromatography protocol described below.

[0447] Crude product 8 (0.834 mmol, based on the amount during 5 years of UMP coupling) was dissolved in 4 mL of 5% MeCN in 200 mM Et3N.HOAc (actually 190 mM) aqueous solution. The resulting solution was then concentrated under vacuum, and the residue was redissolved in 4 mL of 5% MeCN in 200 mM Et3N.HOAc aqueous solution. This solution was purified by rapid C18 column chromatography using a 25 g C18 column (5% MeCN in 190 mM Et3N.HOAc aqueous solution → 30% MeCN in H2O). The corresponding pure fractions were combined and concentrated under vacuum to give an orange oil. This product (8 and a small amount of 8” and 8”’ mixture) was ready for use in the next step without further purification.

[0448] To a round-bottom flask containing rapidly purified C18 solution (2.87 g, 269 mg corrected (qNMR), 0.375 mmol) in H₂O (12 mL), add MeOH (12 mL) and Et₃N (12 mL). The resulting solution was heated to 45 °C and maintained for 22 hours, then concentrated under vacuum to give 2.748 g of a yellow oily substance. The residue was then subjected to the Dowex treatment as described below.

[0449] Add crude product 9a (2.75 g, ~9% 9a and ~40% AcOH) to H2O (27.5 mL) 1 16.5 g of Dowex MAC-3 hydrogen form was added to a round-bottom flask for 1H-NMR. The resulting suspension was stirred at room temperature for 90 minutes and filtered through a P3 glass filter. The residue was washed with H2O (3 × 25 mL). The filtrate was concentrated under vacuum. The resulting residue (984 mg) was co-evaporated with H2O (1 × 20 mL) to give 9a (703 mg, 32.2% qNMR purity) as a yellow oil. 1 ¹H-NMR (400MHz, D₂O): δ 7.82 (d, J = 8.4 Hz, 1H), 5.85–5.78 (m, 2H), 5.40 (dd, J = 3.6, 3.6 Hz, 1H), 4.25–4.18 (m, 3H), 4.16–4.02 (m, 5H), 3.89–3.86 (m, 1H), 3.86–3.80 (m, 1H), 3.45 (AB system, J = 7.6, 5.2 Hz, 1H), 3.34 (AB system, J = 7.2, 5.6 Hz, 1H), 1.94 (s, 3H).

[0450] Example 4-4: Synthesis of 9a (triethylammonium salt) by deacetylation of a mixture of 8 and 8'

[0451]

[0452] Method A The crude product 8 / 8' was dissolved in water (0.15 M), methanol (0.15 M), and triethylamine (0.15 M) and stirred overnight. The mixture was then concentrated under reduced pressure and purified on Q agarose. First, the mixture was diluted with water (0.02 M) and buffer A (NH4HCO3, 10 mM, 0.005 M), and then loaded onto Q agarose (100 mL resin / mmol raw material). The product was separated from the byproducts by a step gradient (first to 10% B (250 mM NH4HCO3) over 20 min, then to 40% B over 120 min). The product fraction was lyophilized from the obtained UDP-6-azido-GalNAc triethylammonium salt (9a). The intermediate was dissolved in H2O (0.2M) and purified by C18-HPLC using a Phenomenex Luna 10u C18 (2) column (A: 50mM Et3N.HOAc, pH 6.8, B: MeCN). The collected fractions were combined and co-evaporated with H2O several times, and finally lyophilized to give UDP-6-N3-GalNAc (9a) as a brittle, grayish-white solid (53%, starting from the mixture of 8 and 8').

[0453] Method B: In a 1 L single-necked flask, 8' (14.0 g, 15.3 mmol) was dissolved in an Et3N / MeOH / water (300 mL, 1:1:1) solution and stirred overnight at room temperature. The crude product mixture was partially concentrated under reduced pressure to a maximum of 23 g of 9 (37%). 1 H-q NMR purity, 88% yield), and dissolved it in 140 mL of water. This 9a solution was subjected to preparative LC-MS (elution buffer 0-4% ACN in 50 mM Et3N.HOAc aqueous solution, pH = 6.8). The fractions containing the product were combined to obtain 9 (9 L in 50 mM Et3N.HOAc buffer). The combined fraction (9 L) was divided into two parts, and one part (4.5 L) was loaded at 30 mL / min into 1 L of Q-Sepharose Fast PCR. Ion exchange column (A: 25 mM ammonium bicarbonate, B: 25 mM ammonium bicarbonate). The column was then washed with 25 mM ammonium bicarbonate solution, followed by a gradient to 100% B to elute the product. The product fractions were combined and concentrated under reduced pressure to give compound 9. This procedure was repeated to give UDP-6"-N3-6"-deoxy-GalNAc(9, ammonium salt) (8.79 g, 92%). 1 H-qNMR purity, 12.8 mmol, 84% yield. 1¹H-NMR (400MHz, D₂O): δ 7.82 (d, J = 8.4 Hz, 1H), 5.84–5.81 (m, 2H), 5.39 (dd, J = 3.6, 3.6 Hz, 1H), 4.26–4.20 (m, 3H), 4.16–4.08 (m, 5H), 3.89–3.85 (m, 1H), 3.85–3.81 (m, 1H), 3.44 (AB system, J = 7.6, 5.2 Hz, 1H), 3.33 (AB system, J = 7.2, 5.6 Hz, 1H), 1.93 (s, 3H).

[0454] Example 4-4: Coupling of 5b with UMP tributylammonium salt in the presence of CDI

[0455]

[0456] UMP-NBu3 (2.22 g, 4.36 mmol) was dissolved in DMF (25 mL) and CDI (1.17 g, 7.2 mmol) was added. The reaction mixture was stirred for 30 minutes. MeOH (177 μL, 4.36 mmol) was added and the reaction mixture was stirred for 15 minutes. The reaction mixture was placed under vacuum for 15 minutes, followed by the addition of a DMF (25 mL) solution of 6-azido-6-deoxy-GalNAc-1-monophosphate 5b (1.60 g, 4.9 mmol), and then NMI·HCl (2.25 g, 14.4 mmol). After stirring overnight, additional activated UMP-NBu4 was added. Therefore, UMP-NBu3 (700 mg, 1.4 mmol) was dissolved in DMF (10 mL) in a separate flask and CDI (371 mg, 2.3 mmol) was added. This mixture was stirred for 5 minutes and added to the reaction mixture. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product mixture was dissolved in water and divided into three fractions. One fraction was loaded into 300 mL of Q-Sepharose Fast at a rate of 10 mL / min. On an ion exchange column (A: 25 mM ammonium bicarbonate, B: 250 mM ammonium bicarbonate). The column was then washed with 25 mM ammonium bicarbonate solution, followed by a gradient to 40% B to elute the product. The product fractions were combined and concentrated under reduced pressure to give compound 9. This procedure was repeated twice to give UDP-6"-N3-6"-deoxy-GalNAc(9, ammonium salt) (2.72 g, 47%). 1 H-qNMR purity, 2.0 mmol, 41% yield. 1¹H-NMR (400MHz, D₂O): δ 7.82 (d, J = 8.4 Hz, 1H), 5.84–5.81 (m, 2H), 5.42 (dd, J = 3.6, 3.6 Hz, 1H), 4.26–4.20 (m, 3H), 4.16–4.08 (m, 5H), 3.90–3.89 (m, 1H), 3.86–3.83 (m, 1H), 3.47 (AB system, J = 7.6, 5.2 Hz, 1H), 3.36 (AB system, J = 7.2, 5.6 Hz, 1H), 1.95 (s, 3H).

[0457] Examples 4-5: Synthesis of UMP imidazole phosphate 10 from UMP.NBu3

[0458]

[0459] Anhydrous DMF (55.0 mL) was added to a round-bottomed container containing UMP tributylammonium salt (UMP.NBu3, 6.05 g, 91.4% (QNMR), 10.84 mmol, 1.00 equivalent) and imidazole (7.38 g, 108.4 mmol, 10.0 equivalent), followed by 2,2-dithiopyridine (3.36 g, 15.25 mmol, 1.41 equivalent). Et3N (6.05 mL, 43.4 mmol, 4 equivalent) was added to the resulting colorless solution, followed by PPh3 (8.53 g, 32.5 mmol, 3.00 equivalent), producing a yellow solution. The mixture was stirred under N2 for 2 hours and then cooled to 0°C in an ice bath. 553 mL of a 0.106 M solution of NaClO4 in acetone was added to the cooled reaction mixture. The resulting yellow suspension was cooled to 0°C in an ice bath, then filtered through a glass filter and washed with cold-dried acetone (3×). The residue was concentrated under vacuum and dried over P2O5 to give 4.68 g of a grayish-white solid (88.7% qNMR purity, 4.15 g (corrected), 10.47 mmol, 96.7% yield). 1 H NMR (400MHz, D2O) δ (ppm): 7.93 (s, 1H), 7.84 (d, J = 8.2Hz, 1H), 7.32 (q, J = 1.4Hz, 1H), 7.13-7.01 (m, 1H), 5.96 (d, J = 5.3Hz, 1H), 5.79 (d, J = 8.1Hz, 1H), 4.18 (t, J = 5.0Hz, 1H), 4.14-3.94 (m, 4H), 3.07-2.97 (m, 1H), 2.93-2.87 (m, 1H).

[0460] Examples 4-6: Synthesis of 8 from 5a and 10

[0461]

[0462] Add 2.346 g (77.6% (qNMR), 4.437 mmol, 1.00 equivalent) and 27.5 mL of anhydrous DMF to a flame-dried round-bottom flask equipped with a stir bar. Cool the resulting solution in an ice bath and add MgCl2 (486 mg, 5.105 mmol, 1.15 equivalent). Stir the resulting mixture for 13 minutes to produce a fine suspension. Remove the ice bath and allow the reaction mixture to slowly warm to room temperature. Then, add 10 (2.282 g, 88.7% (qNMR), 1.15 equivalent) in portions over 30 seconds while stirring. Stir the resulting pale yellow suspension at room temperature for 18 hours. Then concentrate the reaction mixture under vacuum to give 9.20 g of a turbid yellow oil, which can be used for the next step without further purification.

[0463] Examples 4-7: Synthesis of UDP-6-azido-GalNAc(9) via deprotection of 8

[0464]

[0465] Crude product 8 was dissolved in H2O (32 mL) and MeOH (32 mL), and then Et3N (32 mL) was added. The resulting turbid yellow suspension was stirred at room temperature for 22 hours, and then concentrated under vacuum to obtain a turbid yellow oil. The residue was dissolved in H2O (222 mL) and slowly pulled over into DEAE Sephadex A-25 packing. DEAE packing was prepared as follows: DEAE Sephadex A-25 (12.5 g) was loaded into a glass filter P3 (6 cm diameter) and suspended in H2O, washed with 1 M NH4HCO3, and then washed with H2O. Thus, a 3.5 cm high packing (approximately 100 mL) of DEAE Sephadex was obtained. Then, over a period of 15 minutes, a solution of crude product 9 (approximately 355 μmol product / g resin) in H2O (222 mL) was loaded into the DEAE packing using minimal vacuum suction. The packing material was washed with softened water (60 mL, 3×), then with 20 mM NH4HCO3 (100 mL, 4×). Finally, the product was eluted with 1 M NH4HCO3 (100 mL, 3×). The eluted product was concentrated under vacuum, co-evaporated once with H2O, and then lyophilized to give 9 (3.986 g, 69.2% (qNMR), 4.36 mmol, 98.3% yield) as a grayish-white solid. 1H NMR(400MHz,D2O)δ(ppm):7.88(d,J=8.2Hz,1H),5.94-5.84(m,2H),5.46(dd,J=7.2,3.4Hz,1H),4.33-4.24(m,2H ),4.24-4.06(m,5H),3.97-3.84(m,2H),3.51(dd,J=12.8,7.3Hz,1H),3.41(dd,J=12.8,7.3Hz,1H),2.00(s,3H).

[0466] Example 5-1: Synthesis of 6-azido-1-(di-2-cyanoethyl) phosphate - GalNAc(12)

[0467]

[0468] Method A A solution containing hemiacetal compound 11 (1.20 g, 3.63 mmol) and 0.45 M 1-H-tetrazole in acetonitrile (32 mL, 14.5 mmol, 4 equivalents) was concentrated in a round-bottom flask and co-evaporated from anhydrous toluene (3 × 20 mL). The residue was dissolved in anhydrous DCM (40 mL), cooled (-5 °C), and treated with a solution of 2-cyanoethyl-N,N'-diisopropyl-phosphorochloro-amidite (2.0 mL, 7.27 mmol, 2 equivalents) in anhydrous DCM (4.0 mL), added dropwise over 15 minutes with stirring at Ar (g). The reactants were slowly heated to room temperature and stirred for another 80 minutes, then cooled (-40 °C) and treated with mCPBA (2.10 g, 9.08 mmol, 2.5 equivalents). The reactants were slowly heated to room temperature and stirred for another 30 minutes, then partitioned between DCM (100 mL) and 10% sodium thiosulfate solution (100 mL). The organic layer was washed sequentially with saturated NaHCO3 solution and brine (100 mL each), dried (MgSO4), filtered, and concentrated. The residue was subjected to rapid column chromatography (80 g SiO2 column; 70-100% EtOAc in petroleum ether 40-60 solution, then 0-25% MeCN in EtOAc solution), first yielding oxazoline (F2) (100 mg, 0.32 mmol, 8.8% yield), followed by compound 12 (F1) as a white foam (1.10 g, 2.13 mmol, 59% yield).

[0469] Method BUnder Ar(g) and with stirring, over 15 minutes, a solution of 2-cyanoethyl-N,N'-diisopropylphosphonic chloramide (0.9 mL, 3.31 mmol, 2 equivalents) in anhydrous DCM (2.0 mL) was added dropwise to a mixture of hemiacetal compound 11 (547 mg, 1.65 mmol) and 4,5-dicyanimidazole (781 mg, 6.62 mmol, 4 equivalents) in anhydrous DCM (15 mL). The reaction mixture was slowly heated to room temperature and stirred for another 80 minutes, then cooled (-40 °C) and treated with mCPBA (927 mg, 4.14 mmol, 2.5 equivalents). The reaction mixture was slowly heated to room temperature and stirred for another 30 minutes, then a 10% sodium thiosulfate solution (10 mL) was added. The organic layer was washed with saturated NaHCO3 solution (10 mL), dried (MgSO4), filtered, and concentrated. The residue was subjected to rapid column chromatography (40 g SiO2 column; 70-100% EtOAc in petroleum ether 40-60 solution, then 0-50% MeCN in EtOAc solution) to give compound 3 as a white foam (540 mg, 1.05 mmol, 63% yield). NMR showed that compound 12 was 98% pure; LCMS (ELSD) showed that compound 12 was 95% pure.

[0470] Method C Under Ar(g) and with stirring, over 15 minutes, a solution of 2-cyanoethyl-N,N'-diisopropylphosphonic chloramide (0.9 mL, 3.31 mmol, 2 equivalents) in anhydrous DCM (2.0 mL) was added dropwise to a mixture of hemiacetal compound 11 (547 mg, 1.65 mmol) and 4,5-dicyanimidazole (781 mg, 6.62 mmol, 4 equivalents) in anhydrous DCM (15 mL). The reaction mixture was slowly heated to room temperature and stirred for another 80 minutes, then cooled (-40 °C) and treated with mCPBA (927 mg, 4.14 mmol, 2.5 equivalents). The reaction mixture was slowly heated to room temperature and stirred for another 30 minutes, then a 10% sodium thiosulfate solution (10 mL) was added. The organic layer was washed with saturated NaHCO3 solution (10 mL), dried (MgSO4), filtered, and concentrated. The residue was subjected to rapid column chromatography (40 g SiO2 column; 70-100% EtOAc in petroleum ether 40-60 solution, then 0-50% MeCN in EtOAc solution) to give compound 3 as a white foam (540 mg, 1.05 mmol, 63% yield). NMR showed that compound 12 was 98% pure; LCMS (ELSD) showed that compound 12 was 95% pure.

[0471] Method D Under Ar(g) and stirring, over 15 minutes, a solution of 2-cyanoethyl-N,N'-diisopropylphosphonic chloride amide (0.9 mL, 3.31 mmol, 2 equivalents) in anhydrous DCM (2.0 mL) was added dropwise to a mixture of hemiacetal compound 11 (547 mg, 1.65 mmol) and BnS-1-H-tetrazole (1.27 g, 6.62 mmol, 4 equivalents) cooled (-5 °C) and stirred. The reactants were slowly heated to room temperature and stirred for another 80 minutes, then cooled (-40 °C) and treated with mCPBA (927 mg, 4.14 mmol, 2.5 equivalents). The reactants were slowly heated to room temperature and stirred for another 30 minutes, then a 10% sodium thiosulfate solution (10 mL) was added. The organic layer was washed with saturated NaHCO3 solution (10 mL), dried (MgSO4), filtered, and concentrated. The residue was subjected to rapid column chromatography (40 g SiO2 column; 70-100% EtOAc in petroleum ether 40-60 solution, then 0-50% MeCN in EtOAc solution) to give compound 12 as a white foam (362 mg, 0.70 mmol, 43% yield). NMR and LCMS (ELSD) both showed that the purity of compound 12 was >90%.

[0472] Example 5-2: Synthesis of 6-azido-1-phosphate-GalNAc(5b)

[0473]

[0474] Compound 12 (1.60 g, 3.10 mmol) was treated at 35 °C with a mixture of TEA / MeOH / water in an 8:35:57 ratio (100 mL) until LCMS showed the reaction was complete (3 days). The mixture was partially concentrated to an aqueous fraction on a rotary evaporator and then lyophilized. The lyophilization was repeated (twice) until excess triethylamine was removed (as determined by NMR). The residue was ground with acetone (2 × 100 mL), the acetone was removed by decantation, and the residue was dried under high vacuum to give compound 5b (1.33 g, 2.53 mmol, 82% yield) as a clear foam.

[0475] Example 5-3: Coupling of 5b with UMP-morpholidate

[0476]

[0477] Method A: Under argon (g), compound 5b (81 mg, 0.15 mmol, 1.00 equivalent), anhydrous DMF (2.0 mL), UMP-morpholine (116 mg, 0.17 mmol, 1.1 equivalent), and 4,5-dicyanimidazole (DCI, 56 mg, 0.48 mmol, 3.2 equivalent) were added to a flame-dried round-bottom flask equipped with a stir bar. The resulting solution was stirred at 35 °C for 20 hours; LCMS of the crude product mixture showed that the starting material 5b was not consumed. Further addition of the starting material UMP-morpholine (35 mg, 0.3 equivalent) was added, and the reaction mixture was stirred for another 6 hours; LCMS showed that the level of 5b was 2.5%. The mixture was treated with MeCN (10 mL), then centrifuged and the liquid was decanted (this process was repeated again) to obtain a white solid, which was dissolved in water and added to DEAE-agarose medium (2.4 × 13 cm). The solid was eluted sequentially with 50 mL of water and increasing concentrations of triethylammonium bicarbonate (TEAB) at concentrations of 100 mM, 200 mM, 300 mM, and 400 mM. Most of the compound eluted in the 300 mM TEAB fraction and lyophilized to give 120 mg of compound 9. LCMS analysis showed that compound 9 contained compound 5b (based on the presence of the mass ion of compound 5b in the -ve mode). The residue was dissolved in water and subjected to C18 chromatography (30 g column; eluent = 100% 10 mM TEAB). First, a 2:1 mixture of UMP and compound 5b (11 mg) was obtained; second, pure (NMR and LCMS) compound 9 (65 mg, 51%) was obtained.

[0478] Method B Compound 5b (53 mg, 0.10 mmol, 1.00 equivalent), anhydrous DMF (1.5 mL), UMP-morpholine (90 mg, 0.13 mmol, 1.3 equivalent), and DCI (42 mg, 0.36 mmol, 3.6 equivalent) were added to a flame-dried round-bottom flask equipped with a stir bar under argon (g) atmosphere. The resulting solution was stirred at 35 °C for 20 h; LCMS of the crude product mixture indicated that the starting material UMP-morpholine had been consumed. The mixture was treated with acetone (10 mL), then centrifuged and the liquid was decanted (this process was repeated again) to obtain a white solid, which was dissolved in water and added to DEAE-agarose medium (2.4 × 13 cm). The solid was eluted sequentially with 50 mL of water and TEAB at increasing concentrations of 100 mM, 200 mM, 300 mM, and 400 mM. Most of the compounds were eluted in a 300 mM TEAB fraction and freeze-dried to give 102 mg of compound 9. By LCMS (ELSD), compound 9 contained 15% of compound 5b, while NMR showed that the amount of compound 5b was 46%.

[0479] Method C Under argon (g) atmosphere, compound 5b (53 mg, 0.10 mmol, 1.00 equivalent), anhydrous DMF (1.5 mL), UMP-morpholine (90 mg, 0.13 mmol, 1.3 equivalent), and EtS-1-H-tetrazole (47 mg, 0.36 mmol, 3.6 equivalent) were added to a flame-dried round-bottom flask equipped with a stir bar. The resulting solution was stirred at 35 °C for 40 h; LCMS of the crude product mixture indicated that the starting material UMP-morpholine was still present. The temperature was increased to 40 °C and stirring was continued for 20 h. The mixture was treated with acetone (10 mL) and centrifuged (repeated once) to give a white solid, 9, containing 8% compound 5b (LCMS (ELSD)), while NMR showed that the amount of compound 5b was 40%.

[0480] Method D Under argon (g) atmosphere, compound 5b (53 mg, 0.10 mmol, 1.00 equivalent), anhydrous DMF (1.5 mL), UMP-morpholine (90 mg, 0.13 mmol, 1.3 equivalent), and BnS-1-H-tetrazole (69 mg, 0.36 mmol, 3.6 equivalent) were added to a flame-dried round-bottom flask equipped with a stir bar. The resulting solution was stirred at 35 °C for 40 hours; LCMS of the crude product mixture indicated that the starting material UMP-morpholine was still present. The temperature was increased to 40 °C and stirring was continued for 6 hours. The mixture was treated with acetone (10 mL) and centrifuged (repeated once) to give a white solid, 9, containing 13% compound 5b (LCMS (ELSD)), while NMR showed that the amount of compound 5b was 39%.

Claims

1. A method of preparing a 6-azido-6-deoxymonosaccharide compound having structure (I) according to the following scheme: The method comprises: (b) reacting the diol having structure (II) with a sulphiting agent to form a cyclic sulphite having structure (Ilia); (c) reacting the cyclic sulphite having structure (Ilia) with an oxidizing agent to form a cyclic sulphate having structure (Illb); (d) reacting the cyclic sulphate having structure (Illb) with an inorganic azide to form a 6-azido-6-deoxymonosaccharide having structure (I), wherein R 1 is independently selected from C(O)-C 1-6 alkyl, C(O)-C 5-6 aryl and C(O)-C 6-12 arylalkyl, and wherein the monosaccharide is N-acetyl-D-galactosamine.

2. The method according to claim 1, wherein the sulphiting agent is a sulfinyl halide or a 1,1’-sulfinyl imidazole.

3. The method according to claim 2, wherein the sulphiting agent is sulfinyl chloride.

4. The method according to claim 1, wherein the oxidizing agent is selected from an organic oxidizing agent or an inorganic oxidizing agent.

5. The method according to claim 4, wherein the oxidizing agent is an inorganic reagent.

6. The method according to claim 5, wherein the oxidizing agent is Ru04.

7. The method according to claim 1, wherein the diol having structure (II) is prepared from GalNAc.

8. The method of claim 1, wherein each occurrence of R 1 is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph.

9. The method of claim 1, wherein each occurrence of R 1 is C(O)Me.

10. A method of preparing a nucleoside diphosphate having structure (IX) or a salt thereof, wherein the method is the further conversion of a 6-azido-6-deoxymonosaccharide compound having structure (I) obtained according to any one of claims 1-9 to a nucleoside diphosphate having structure (IX) or a salt thereof: wherein B is a nucleobase.

11. The method according to claim 10, wherein the 6-azido-6-deoxymonosaccharide compound having structure (I) is converted to a 1-monophosphate monosaccharide compound which is reacted with a nucleoside monophosphate to form a compound having structure (IX) involving a deprotection step before or after the reaction of the 1-monophosphate monosaccharide compound with the nucleoside monophosphate.

12. The method according to claim 11, wherein the 6-azido-6-deoxymonosaccharide compound having structure (I) is first converted to a 1-monophosphate monosaccharide compound having structure (Va) or a salt form thereof according to the following scheme: The method comprises: (e) protecting the 6-azido-6-deoxymonosaccharide having structure (I) to form a 6-azido-6-deoxymonosaccharide compound having structure (VI); (f) converting the compound having structure (VI) in the presence of one or more Lewis acids to form an oxazoline compound having structure (VII); (g) reacting the compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va), wherein R 2 is selected from optionally substituted C(O)-C 1-6 alkyl, C(O)-C 5-6 aryl and C(O)-C 6-12 arylalkyl.

13. The method according to claim 11, wherein the 6-azido-6-deoxymonosaccharide compound having structure (I) is first converted to a 1-monophosphate monosaccharide compound having structure (Va) or a salt form thereof according to the following scheme: The method comprises: (e) protecting a 6-azido-6-deoxymonosaccharide having structure (I) to form a 6-azido-6-deoxymonosaccharide compound having structure (VI); (x1) deprotecting the anomeric position of the compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI); (x2) converting the 1-hydroxy-monosaccharide compound having structure (XI) to a 6-azido-6-deoxy-1-monophosphite diester having structure (XII); (x3) oxidizing the 1-monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphate diester compound having structure (XIII); (x4) deprotecting the 1-monophosphate diester having structure (XIII) to form a 1-monophosphomono saccharide compound having structure (Va), wherein R 2 is selected from optionally substituted C(O)-C 1-6 alkyl, C(O)-C 5-6 aryl, and C(O)-C 6-12 arylalkyl, and R 3 is selected from C 1-6 alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonyl ethyl, 2-arylsulfonyl ethyl, 2,2,2-trichloroethyl, CH2OC(O)alkyl, fluorenylmethyl, 2-pyridinioethyl, phenyl-C 1-2 -alkyl (2-phenylethyl or phenylmethyl), wherein the phenyl is optionally substituted with one or more halides or nitro or methoxy groups.

14. The method of claim 12 or 13, wherein the 1-monophosphonic monosaccharide compound having structure (Va) is converted to the nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: the method comprising: (i) reacting the compound having structure (Va) with a nucleoside monophosphate to form an acylated nucleoside diphosphate having structure (VIII); and (j) deprotecting the acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate having structure (IX) or a salt thereof, wherein R 4 are each hydrogen, or two R 4 groups present are joined together via the carbonyl moieties, thereby forming a carbonate with the two oxygen atoms attached to the two R 4 groups.

15. The method of claim 12 or 13, wherein the 1-monophosphonic monosaccharide compound having structure (Va) is converted to the nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: the method comprising: (j1) deprotecting the compound having structure (Va) to obtain a 1-monophosphomono saccharide compound having structure (Vb), (i1) reacting the compound having structure (Vb) with a nucleoside monophosphate to form a nucleoside diphosphate having structure (IX).

16. The method of claim 11, converting a 6-azido-6-deoxymonosaccharide compound having structure (I) to a nucleoside diphosphate having structure (IX) or a salt form thereof according to the following scheme: the method comprising: (e) protecting a 6-azido-6-deoxymonosaccharide having structure (I) to form a 6-azido-6-deoxymonosaccharide compound having structure (VI); (x1) deprotecting the anomeric position of the compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI); (x2) converting the 1-hydroxy-monosaccharide compound having structure (XI) to a 6-azido-6-deoxy-1-monophosphite diester having structure (XII); (x3) oxidizing the 1-monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphate diester compound having structure (XIII); (x5) deprotecting the 1-monophosphate diester having structure (XIII) to obtain a 1-monophosphomono saccharide compound having structure (Vb); (i1) reacting the compound having structure (Vb) with a nucleoside monophosphate to form a nucleoside diphosphate having structure (IX). wherein R 2 is selected from optionally substituted C(O)-C 1-6 alkyl, C(O)-C 5-6 aryl, and C(O)-C 6-12 arylalkyl, and R 3 is selected from C 1-6 alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonyl ethyl, 2-arylsulfonyl ethyl, 2,2,2-trichloroethyl, CH2OC(O)alkyl, fluorenylmethyl, 2-pyridinylethyl, phenyl-C 1-2 -alkyl (2-phenylethyl or phenylmethyl), wherein the phenyl is optionally substituted with one or more halides or nitro or methoxy groups.

17. The method of claim 11, wherein the 6-azido-6-deoxymonosaccharide compound having structure (I) is first converted to a 1-monophosphomono saccharide compound having structure (Vb) according to the following scheme: the method comprising: (y1) deprotecting the 6-azido-6-deoxymonosaccharide having structure (I) to form a 1,3,4-trihydroxy-6-azido-monosaccharide compound having structure (XIV); (y2) contacting the compound having structure (XIV) with a phosphorylase in the presence of a source of phosphate to form a 1-monophosphomono saccharide compound having structure (Vb).

18. A cyclic sulfate N-acetyl-2-galactosamine monosaccharide compound having structure (III): ###0003### wherein R 1 is independently selected from the group consisting of optionally substituted C(O)-C 1-6 alkyl, C(O)-C 5-6 aryl and C(O)-C 6-12 arylalkyl.

19. The compound of claim 18, wherein each occurrence of R 1 is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph.

20. The compound of claim 19, wherein each occurrence of R 1 is (CO)Me.

21. A 6-azido-6-deoxy N-acetyl-2-galactosamine monosaccharide compound having structure (I): ###0005### (I) wherein R 1 is independently selected from the group consisting of optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl.

22. The compound of claim 21, wherein each occurrence of R 1 is C(O)Me, C(O)tBu, C(O)Ph, or C(O)CH2Ph.

23. The compound of claim 22, wherein each occurrence of R 1 is C(O)Me.

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