Synthesis of 6-azido-6-deoxy-2-n-acetyl-hexosamine-nucleoside diphosphate

Abstract

To provide an improved method for producing a sugar nucleoside diphosphate in the field of a functionalized nucleoside sugar.SOLUTION: A method for the chemical conversion of GalNAc or GlcNAc to its respective 6-azido derivative via a cyclic sulfate, followed by conversion to the respective UDP derivative via an anomeric phosphorylation step and a UMP coupling step, wherein a 6-azido-6-deoxy monosaccharide compound having the structure (I) is prepared according to the following scheme: SELECTED DRAWING: None

Description

[Technical Field]

[0001]

[0001] The present invention is in the field of functionalized nucleoside sugars and relates to an improved production of sugar nucleoside diphosphates. More specifically, the present invention relates to a method for the chemical conversion of GalNAc or GlcNAc to their respective 6-azido derivatives via a cyclic sulfate, followed by an anomeric phosphorylation step and a UMP coupling step to the respective UDP derivative. The present invention also relates to various specific intermediates and purification steps. [Background technology]

[0002] Carbohydrates play a vital role in cell biology due to their function in energy metabolism and storage, and are also important components of genetic material and other structural elements. Furthermore, carbohydrates attached to proteins or lipids, also called glycans, are crucial in cell differentiation and cellular communication during development. Glycans are defined as the oligosaccharide moiety of glycoconjugates, such as glycoproteins, which can be attached 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 repeated components, which can be directly attached to serine or threonine, as in O-glycoproteins (GalNAc), or to asparagine, as in N-glycoproteins (GlcNAc). GalNAc or GlcNAc can also be part of a larger oligosaccharide chain within a glycoprotein, either internally or as the most distant monosaccharide, most often connected to another sugar via a β-glycosidic bond. To incorporate GalNAc moieties into proteins or oligosaccharide chains, a series of N-acetyl-galactosaminyltransferases (GalNAc-transferases) have evolved naturally that can transfer the monosaccharide GalNAc from UDP-GalNAc (donor) to the alcohol moiety of serine / threonine or another sugar (acceptor). Similarly, N-acetyl-glucosaminyltransferases (GlcNAc-transferases) can attach GlcNAc to an alcohol acceptor. For this purpose, the UDP-GalNAc donor substrate is generated from glucose-6-phosphate and glutamate in a pathway known as the hexosamine pathway. Five subsequent enzymatic transformations form UDP-GlcNAc, which is then converted to UDP-GalNAc by the action of UDP-galactose-4-epimerase, as disclosed, for example, in Yamamoto et al., Applied Environ. Microbiol. 1981, 41, 392, incorporated by reference.

[0003] To investigate in detail the mechanism and functional role of GalNAc incorporation into naturally occurring glycans, or to identify novel galactosaminyltransferases, large amounts of (labeled) UDP-GalNAc are required, as disclosed in Maley et al., Biochem. Biophys. Res. Commun. 1970, 39, 371, which is incorporated by reference. As a result, various methodologies for the preparation of UDP-GalNAc (and its analogs) have been devised over the years, typically following chemical procedures, enzymatic conversions, or a combination thereof.

[0004]

[0004] Chemical synthesis of sugar nucleotides generally follows one of two routes: (a) pyrophosphorylation using a sugar-1-phosphate and an activated nucleoside monophosphate (NMP), or (b) direct glycosylation of a glycosyl donor using a nucleoside diphosphate (NDP), as summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which is incorporated by reference; the pyrophosphorylation route is more commonly applied. Thus, pyrophosphorylation first requires the synthesis of a sugar-1-phosphate derivative, for which numerous methods have been published, as summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which is incorporated by reference. One difficulty in these procedures is obtaining the required sugar-1-phosphate with exclusive α-selectivity, the anomeric configuration of the majority of sugar nucleotides. The second step of the pyrophosphorylation method involves coupling of the sugar-1-phosphate with activated NMP. The seminal work on coupling with phosphomorpholidates by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756, incorporated by reference, remains widely applied, along with the 1-H-tetrazole modification reported by Wittmann et al., J. Org. Chem. 1997, 62, 2144. However, progress beyond this classic phosphomorpholidate strategy has led to a series of other approaches summarized in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, incorporated by reference. One particularly useful approach to sugar nucleotides involves coupling of the sugar-1-phosphate with imidazolide-activated NMP, as discussed in Wagner et al., Nat. Prod. Rep. 2009, 26, 1172, incorporated by reference. The required imidazolides can be readily generated from nucleoside monophosphates and activated with ZnCl or MgCl by the procedure reported by Dabrowski-Tumanski et al., Eur. J. Org. Chem. 2013, 2147, which is incorporated by reference. However, no generally accepted chemical route to sugar nucleotides has been found.In particular, most of the methods are centered around common hexose sugars, with only a small subset being suitable for N-acetylated hexosamines, e.g., due to the use of strongly basic conditions, and / or for azide-modified sugars, e.g., due to the use of azide-incompatible phosphate deprotection conditions.

[0005]

[0005] Enzymatic synthesis of sugar nucleotides avoids the use of protection and deprotection steps required during chemical synthesis. Furthermore, enzymatic formation of pyrophosphate bonds typically proceeds with greater efficiency and stereoselectivity than chemical bond formation. Various enzymatic methods have been reported, varying in the number of enzymes and type of starting material used, as summarized in Buter et al., Glycoconj. J. 1999, 16, 147, incorporated by reference, and reported in Cai et al., J. Carbohydr. Chem. 2012, 31, 535, incorporated by reference. For example, Piller et al., Anal. Biochem. 1982, 127, 171, incorporated by reference, reported that the UDP derivative of N-acetylglucosamine (GlcNAc) can be converted to UDP-GalNAc by mammalian Gal-4 epimerase. The main drawbacks of this method are the low yield (30% equilibrium GalNAc:GlcNAc) combined with the difficulty of separating UDP-GalNAc from excess UDP-GlcNAc. The enzymatic pathway reported by Carlson et al., Biochemistry 1964, 3, 402, incorporated by reference, starts with D-galactosamine and utilizes yeast Mort galactokinase 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-glucose uridyl transferase, as reported by Heidlas et al., J. Org. Chem. 1992, 57, 152, incorporated by reference. In both cases, UDP-GalNH2 is chemically N-acetylated in the final step, resulting in low overall yields after purification (typically 20% or less).An alternative enzymatic synthesis, such as that described by Buter et al., Carbohydr. Res. 1997, 305, 469, incorporated by reference, facilitates the production of UDP-GalNAc using a coupled seven-enzyme system that converts UMP, sucrose, and GalNH2-1-P to UDP-GalNH2, which is ultimately chemically converted to the N-acetylated product in a respectable overall yield of 34%. Zou et al., Carb. Res. 2013, 373, 76, incorporated by reference, devised an elegant one-pot, three-enzyme protocol for producing UDP-GalNAc and derivatives, utilizing enzymes obtained from Streptococcus pneumoniae: UTP-glucose-1-phosphate uridylyltransferase (SpGalU), galactokinase (SpGalK), and inorganic phosphatase (PPase). In the presence of ATP, SpGalK converts GalNAc to GalNAc-1-P, which, in combination with uridine triphosphate (UTP), is the substrate for SpGalK to produce UDP-GalNAc in reasonable yield (32%). The third enzyme in this reaction, yeast inorganic pyrophosphatase (PPase), drives UDP-GalNAc production by preventing the reverse reaction, which cleaves PPi into two monophosphate (Pi) molecules. Following previous work, Liu et al., Bioorg. Med. Chem. Lett. 2013, 23, 3764, incorporated by reference, applied the same one-pot, three-enzyme method, substituting UDP-sugar pyrophosphorylase (AtUSP) from Arabidopsis thaliana for SpGalU. A similar three-enzyme method was applied to produce UDP-GalNAc by Bourgeaux et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459, which is incorporated by reference. Starting from GalNAc, UTP, and ATP, UDP-GalNAc was synthesized in high yield (68%) using recombinant human GalNAc kinase (GK2) and UDP-GalNAc pyrophosphorylase (AGX1).Mammalian GK2 catalyzes the phosphorylation of GalNAc using ATP as the phosphate donor. Mammalian AGX1 then converts GalNAc-1-P to UDP-GalNAc using UTP, and PPase is used to increase product formation and obtain significant amounts of UDP-GalNAc. Pouilly et al., ACS Chem. Biol. 2012, 7, 753 (incorporated by reference), subsequently demonstrated the versatility of this method by generating several UDP-GalNAc analogs for use as substrates for polypeptide GalNAc transferase T1 (ppGalNAcT1). In addition to reported enzymatic protocols for the preparation of UDP-GalNAc, several chemoenzymatic procedures, i.e., the use of a combination 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, which are incorporated by reference, describe two strategies for synthesizing UDP-GalNAc and some of its analogs starting from N-acetylgalactosamine.

[0006]

[0006] Despite the sophistication of (chemo)enzymatic UDP-sugar synthesis, particularly by eliminating lengthy syntheses involving numerous (de)protection steps, scalability of enzymatic UDP-sugar synthesis has proven difficult. Furthermore, various enzymes need to be recombinantly expressed, making protocols involving multiple enzymes expensive. Obviously, costs increase even further when such UDP-sugars are produced in GMP for the manufacture of clinical-grade materials, as disclosed, for example, by Warneck et al., Biotechnol. Bioengin. 2005, 92, 831, which is incorporated by reference. Finally, most of the required enzymes preclude the use of alternative N-substituted galactosamine variants, making the enzymatic synthesis of unnatural UDP-GalNAc analogs a challenging, if not impossible, task. In this regard, there has been strong interest in the use of azide-modified sugars in metabolic reporter strategies, as reported by Hang et al., PNAS 2003, 100, 14846, which are incorporated by reference, or in 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, which are incorporated by reference. In the latter area, van Geel et al., Bioconj. Chem. 2015, 26, 2233, which are incorporated by reference, show that UDP-GalNAz can be cleanly installed onto monoclonal antibodies to generate stable, homogeneous antibody-drug conjugates after metal-free click conjugation of toxic payloads. More recently, Verkade et al., Antibodies, 7, 12, incorporated by reference, showed that migration of azide to the 6-position of GalNAc produces antibody-drug conjugates with reduced aggregation propensity relative to GalNAz-containing analogs. However, it is clear that production of any ADC based on 6-azido-GalNAc incorporation would require the availability of multi-gram to multi-kilogram quantities of UDP-6-azido sugars, which cannot be achieved by known routes.

[0007]

[0007] Several complete synthetic strategies for 6-azido-GalNAc have been reported and are disclosed herein. Without exception, the introduction of the azide group begins with the S-reaction of a 6-O-sulfonylated derivative of N-protected D-galactosamine. N The galactosamine derivative is achieved by a nucleophilic substitution. However, the efficiency of the latter substitution is highly dependent on the specific protecting groups at the O-3 and O-4 positions, with a strong correlation between reaction rate and the order diacetyl < isopropylidene < no protection. As a result of such a strong structure-reactivity relationship, the nucleophilic substitution is slow, requires treatment with azide anion at high temperatures for extended periods (e.g., 100 °C for 5 days), and results in low yields (<50%) or requires lengthy synthetic routes (up to 10 synthetic steps) to achieve the properly protected galactosamine derivative. Furthermore, certain synthetic routes may require expensive, odorous, and / or dangerous reagents (e.g., thiophenol, triflate anhydride, 15-crown-5, ceric ammonium nitrate), ultimately requiring cumbersome amine protecting group exchange and anomeric deprotection protocols (e.g., phthalimide removal with hydrazine or allyl removal with palladium reagents). Finally, one procedure reported by Hang et al., PNAS 2003, 100, 14846, incorporated by reference, reports a rapid route (three synthetic steps) from GalNAc to 6-azido-GalNAc; however, due to a lack of selectivity in the tosylation step, the desired product is obtained as a mixture of components that can be difficult to handle, thereby requiring expensive and laborious silica gel purification. From a manufacturing perspective, neither of these features is desirable; therefore, a short, high-yielding route to a suitably protected 6-azido-GalNAc derivative (formally 6-azido-6-deoxy-N-acetyl-D-galactosamine) is needed.

[0008] The second challenge to obtaining UDP 6-azido-GalNAc on a suitable scale lies in the subsequent laborious step, i.e., the conversion of the 6-azido-GalNAc monosaccharide to the uridine diphosphate derivative (UDP). While various routes can be considered, they typically involve a first phosphorylation at the anomeric position followed by a coupling step with UMP, both of which can be carried out chemically or using enzyme catalysis. For chemical phosphorylation at the anomeric position, neat phosphoric acid has been used, for example, by MacDonald et al., J. Org. Chem. 1966, 31, 513 and Masuko et al., J. Org. Chem. 2012, 77, 1449, both of which are incorporated by reference. However, the product is obtained as an anomeric α / β mixture, with low yields (<50%), thus requiring laborious purification and substantial loss of valuable material at a later stage. Alternatively, the anomeric, selectively deprotected 6-N3-GalNAc derivative can be reacted with a phosphitylation reagent, such as a chlorophosphinate or a phosphoramidite, which can be activated for reaction with the anomeric hydroxyl group in the presence of a proton scavenger or a weak acid, respectively. Both of these phosphitylation reagents have a protecting group that is removed after the phosphitylation step and subsequent oxidation of the intermediate phosphite triester to a phosphate triester using mCPBA, HO, iodine, or other oxidizing agents. The use of phosphoramidite reagents for anomeric phosphorylation has been demonstrated, for example, by Hang et al., J. Am. Chem. Soc. 2004, 126, 6, which is incorporated by reference. The drawbacks of such a route are the high cost and sensitivity of the phosphoramidite reagent, and phosphorylation at O-1 typically produces a mixture of the α- and β-anomeric forms.

[0009]

[0009] With regard to the coupling step between sugar-monophosphate and UMP, known procedures involve activation of UMP with a nucleoside 5'-phosphoramidate, based on 1-H-tetrazole activation, as described by Moffatt et al., J. Am. Chem. Soc. 1961, 83, 649, which is incorporated by reference, and later improved by Wittmann et al., J. Org. Chem. 1997, 62, 2144, which is incorporated by reference. Another common strategy for coupling sugar-monophosphate and UMP involves the use of a carbonylation-type reagent, as reported, for example, by Illarionov et al., Russ. Chem. Bull. 2001, 50, 1303 and Loureiro Morais, Can. J. Chem. 2006, 84, 587, which are incorporated by reference. Alternatively, sugar-1-phosphates can be coupled with morpholidate derivatives of UMP as reported by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756, which is incorporated by reference, optionally in the presence of 1-H-tetrazole, as reported by Wittmann et al., J. Org. Chem. 1997, 62, 2144, which is incorporated by reference. Coupling of sugar-1-phosphates with imidazolide-activated NMP can be particularly effective, as discussed in Wagner et al., Nat. Prod. Rep. 2009, 26, 1172, which is incorporated by reference. However, despite the availability of numerous methods, there is no generally accepted, high-yielding, and scalable route to obtain UDP derivatives of monosaccharides, particularly N-acetylated hexosamines. Furthermore, the presence of the 6-azido group in UDP 6-azido-N-acetyl-hexosamine further limits the choice of conditions due to its electron-withdrawing properties and incompatibility with a range of (reducing) conditions. Therefore, there is a great need for improved protocols for producing UDP 6-azido-6-deoxy-N-acetyl-hexosamines, such as UDP 6-azido-6-deoxy-GalNAc and UDP 6-azido-6-deoxy-GlcNAc. Summary of the Invention

[0010] The present inventors have developed a method for synthesizing 6-azido-6-deoxy-2-N-acetyl-monosaccharide-nucleoside diphosphates, 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 various salt forms thereof. This target compound of the present invention is represented herein by the following structure (IX). [ka]

[0011]

[0011] where B is a nucleobase.

[0012] The present invention relates to methods for the (partial) synthesis of the target compounds according to the invention, and to key intermediates in these methods. The present invention also relates to several approaches towards the total synthesis of compounds having structure (IX).

[0013] The synthesis method according to the present invention is characterized by high efficiency and high yield. In particular, it obviates the drawbacks of the prior art methods identified above. Thanks to 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 DESCRIPTION OF THE INVENTION

[0014] definition

[0014] The verb "to comprise" and its conjugations, when used in this specification and claims, are used in their open-ended sense, meaning that items following the word are included but not items not specifically mentioned are excluded. Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of that element is present, unless the context clearly dictates that only one of that element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0015] The compounds disclosed herein may contain one or more asymmetric centers, and various diastereomers and / or enantiomers of the compounds may exist. The description of any compound herein is intended to include all diastereomers and mixtures thereof, unless otherwise specified. Furthermore, the description of any compound herein is intended to include both the individual enantiomers and any mixtures, racemic or otherwise, of enantiomers, unless otherwise specified. When the structure of a compound is depicted as a specific enantiomer, it should be understood that the invention of this application is not limited to that specific enantiomer.

[0016]

[0016] Compounds can occur in various tautomeric forms. The compounds according to the present invention are intended to include all tautomeric forms unless otherwise specified. When the structure of a compound is depicted as a particular tautomer, it should be understood that the invention of this application is not limited to that particular tautomer.

[0017] The compounds according to the present invention may exist in the form of salts, which are also included in the present invention. Salts are typically pharmaceutically acceptable salts containing a pharmaceutically acceptable anion. The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced by a cation, such as 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 with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt. Pharmaceutically acceptable salts are acceptable for administration to patients, e.g., mammals (salts with counterions having acceptable mammalian safety for a given dosing 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 pharmaceutically acceptable salts of a compound, which can be derived from a variety of organic and inorganic counterions known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, alkylammonium, dialkylammonium, trialkylammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc. In preferred embodiments, the counterion of the salt according to the present invention is selected from trialkylammonium, ammonium, and sodium, more preferably selected from ammonium and sodium, and most preferably sodium.

[0018] The term "monosaccharide" is used herein in its ordinary scientific sense to refer to an oxygen-containing heterocycle resulting from intramolecular hemiacetal formation after cyclization of a chain containing 5 to 9 (hydroxylated) carbon atoms, most commonly 5 carbon atoms (pentose) or 6 carbon atoms (hexose). Typical monosaccharides are glucose (Glu), galactose (Gal), and mannose (Man).

[0019]

[0019] The term "hexosamine" is used herein to refer to a monosaccharide having an amino group at the second position of the carbon chain. Exemplary hexosamines are D-galactosamine (GalNH2) and D-glucosamine (GlcNH2). Hexosamines may be acetylated. Exemplary acetylated hexosamines are N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc).

[0020]

[0020] The terms "substantial" or "substantially" are defined herein as the majority, i.e., >50%, of a population, mixture, or sample, preferably greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the population.

[0021]

[0021] Alkyl groups may be substituted or unsubstituted, may be linear or branched, and may optionally contain cyclic moieties. Optionally, alkyl groups are substituted with one or more substituents. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, t-butyl, etc. In the context of this invention, R 1 and R 2 In the definition of, preferably, alkyl is C 1~6 Alkyl, more preferably C 1~2 Alkyl, most preferably methyl.

[0022]

[0022] Aryl groups can include monocyclic, bicyclic and polycyclic structures. Optionally, aryl groups can be substituted. Examples of aryl groups include groups such as phenyl, naphthyl, anthracyl, etc. In the context of the present invention, R 1 and R 2 In the definition of 5~6 Aryl, most preferably phenyl.

[0023]

[0023] Arylalkyl groups include alkyl (or alkylene) and aryl (or arylene) moieties and can be considered as substituted alkyl or substituted aryl moieties. The aryl (or arylene) moieties can include monocyclic and bicyclic structures. Optionally, the arylalkyl group can be substituted with one or more substituents. Examples of arylalkyl groups are benzyl, naphthylmethyl, 4-t-butylphenyl, etc. In the context of the present invention, R 1 and R 2 In the definition of, preferably, arylalkyl is C 6~12 Aryl alkyl, more preferably C 6~8 Arylalkyl, most preferably benzyl.

[0024] The present invention The present inventors have developed an improved, high-yield method for the synthesis of 6-azido-2-N-acetyl-hexosamine-nucleoside diphosphates or salts thereof, particularly where the hexosamine is galactosamine or glucosamine and the corresponding acetylated hexosamine is N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc). This target compound of the present invention is represented herein by the following structure (IX): [ka]

[0025]

[0025] wherein B is a nucleobase. Although any nucleobase may be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0026]

[0026] Here, the wavy bond at the carbon atom at position 4 of the monosaccharide moiety may be axial (galactose configuration) or equatorial (glucose configuration). Both of these products can be readily obtained by the method of the present invention. In a preferred embodiment, a product with a galactose configuration is prepared, since this compound (GalNAc) can find use in its transfer to terminal GlcNAc moieties of glycans of glycoproteins; this reaction is readily carried out in the presence of a mutant galactosyltransferase (GalT) or N-acetylgalactosaminyltransferase (GalNAcT) enzyme. This use of the compounds of the present invention is known in the art, for example, from Ramakrishnan et al., J. Biol. Chem. 2002, 277, 20833 and WO2016170186, the entire contents of which are incorporated herein.

[0027] [A] Reaction step Methods according to various aspects of the present invention involve one or more of steps (a), (b), (c), (d), (e), (f), (g), (i), (j), (i1), (j1), (x1), (x2), (x3), (x4), (y1), (y2) and (z), which steps are defined below.

[0028] [A.1] Step (a) In the process according to the invention, step (a) is the conversion of N-acetylglucosamine or N-acetylgalactosamine to a 1,3-diacylated compound having structure (II). Step (a) is typically carried out by the introduction of a 4,6-benzylidene group, followed by acylation of 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. In this way, these two hydroxyl groups are unprotected, and compound (II) may therefore also be referred to as a diol. The reaction scheme corresponding to step (a) is as follows: [ka]

[0029] In a preferred embodiment, the reaction scheme corresponding to step (a) is as follows: [ka]

[0030] Benzylidene protection of 2-N-acetyl-monosaccharides is a procedure well known in the art and typically involves treatment of the 2-N-acetyl-monosaccharide with benzaldehyde (or a substituted or acetal form thereof) 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 are incorporated by reference. Acylation of the remaining alcohol functionalities at the 1- and 3-positions is also a procedure well known in the art. Removal of the benzylidene by acidic hydrolysis or hydrogenation is also a procedure well known 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 are incorporated by reference. Typically, the reaction in step (a) is carried out by treatment of the monosaccharide with benzaldehyde (or acetal derivative) and catalytic sulfonic acid (p-TsOH or CSA) in a polar aprotic solvent such as DMF or acetonitrile; the next step, 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 acid chloride in the presence of a tertiary amine (e.g., triethylamine or DIPEA); and finally, removal of the benzylidene can be carried out by acid hydrolysis in aqueous acid or by hydrogenation with Pd—C in a suitable solvent (e.g., MeOH, i-PrOH, or THF). The compound of structure (II) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0031]

[0031] R 1 represents the acyl group introduced in step (a). 1 is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In one embodiment, R 1 is selected from C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Taking into account the reaction conditions, R 1 Both occurrences of R are typically the same. 1 Each occurrence of R is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 1 Each occurrence of R is C(O)Me, in which case "acylation" can be called "acetylation." 1 The definition of R applies to all aspects of the present invention. 1 may also be hydrogen.

[0032] [A.2] Step (b) In the process according to the invention, step (b) is reacting a diol having structure (II) with a sulfitylating agent to form a cyclic sulfite having structure (IIIa). The reaction scheme corresponding to step (b) is as follows: [ka]

[0033] The formation of sulfite compounds from diols is well known in the art, for example, from Megia-Fernandez, Curr. Org. Chem. 2010, 14, 401, which is incorporated herein in its entirety. Typically, the reaction of step (b) is carried out by treating the diol with thionyl chloride and a tertiary base (e.g., triethylamine or DIPEA) in dichloromethane or ethyl acetate. Sulfitating agents are known in the art and refer to compounds capable of introducing sulfite moieties. In a preferred embodiment, the sulfitating agent is a thionyl halide or 1,1'-thionyl imidazole, preferably thionyl chloride. Diols having structure (II) are preferably prepared from N-acetyl-2-glucosamine (GlcNAc) or N-acetyl-2-galactosamine (GalNAc), most preferably obtained according to step (a) defined above.

[0034] The compound of structure (IIIa) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0035] [A.3] Step (C) In the method according to the present invention, step (c) is reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb). The reaction scheme corresponding to step (c) is as follows: [ka]

[0036]

[0036] The oxidation of sulfite compounds to sulfate compounds is well known in the art, for example, from Megia-Fernandez, Curr. Org. Chem. 2010, 14, 401, which is incorporated herein in its entirety. Typically, the reaction of step (c) is carried out by treatment of the crude cyclic sulfate with a strong oxidizing agent (e.g., m-CPBA, MnO2, KMnO4, TEMPO / NaOCl, H2O2, RuO4) in dichloromethane, THF, AcOH, or acetonitrile. Suitable oxidizing agents are known in the art and are typically selected from organic and inorganic oxidizing agents. In a preferred embodiment, the oxidizing agent is inorganic, and more preferably, the oxidizing agent is RuO4. The oxidizing agent can be regenerated in situ, for example, by the addition of a catalytic amount of RuCl3 and a stoichiometric amount of NaIO4.

[0037] The compound of structure (IIIb) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0038] [A.4] Step (d) In the process according to the invention, step (d) comprises reacting a cyclic sulfate having structure (IIIb) with an inorganic azide (i.e., N3 - anion) to form a 6-azido-6-deoxy monosaccharide having structure (I). The reaction scheme corresponding to step (d) is as follows: [ka]

[0039] The introduction of an azide moiety by nucleophilic ring-opening of a sulfate is well known in the art, e.g., from Megia-Fernandez, Curr. Org. Chem. 2010, 14, 401 and van der Klein et al., J. Carbohydr. Chem. 1992, 11, 837, which are incorporated herein in their entireties. Typically, the reaction in step (d) is carried out by stirring the cyclic sulfate with the azide in a polar solvent such as DMF, THF, or acetonitrile, preferably DMF. The reaction can be accelerated by performing it at elevated temperatures (50-80°C). The sulfate monoester formed after ring-opening is typically hydrolyzed by short-term treatment (1 hour) with a catalytic Bronsted 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.

[0040] The compound of structure (I) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0041] The compound of structure (I) is preferably further converted to a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate or salt thereof having structure (IX). Such conversion can be accomplished in any suitable manner. Preferably, this conversion involves converting the 6-azido-6-deoxy monosaccharide compound having structure (I) to a 1-monophosphate monosaccharide compound, which is reacted with a nucleoside monophosphate to form a compound having structure (IX). Such a reaction sequence involves a deprotection step before or after the reaction of the 1-monophosphate monosaccharide compound with the nucleoside monophosphate.

[0042] In a preferred embodiment, the conversion of a compound of structure (I) to a compound of structure (IX) is carried out by one of the following reaction sequences: Steps (e), (f), (g), (i), and (j); Steps (e), (f), (g), (j1), and (i1); Steps (e), (x1), (x2), (x3), (x4), (i), and optionally (j); Step (e), (x1), (x2), (x3), (x4), (j1), (i1); Steps (y1), (y2), and (i).

[0043] Each of these steps is further defined below.

[0044] [A.5] Step (e) In the method according to the present invention, step (e) is 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: [ka]

[0045]

[0045] The introduction of an acyl moiety is well known in the art. Typically, the reaction of step (e) is carried out by treating the alcohol with an activated form of the acid, such as an acid anhydride in pyridine, with an acid halide in dichloromethane in the presence of a tertiary base, such as triethylamine, or by in situ activation of the acid, for example with a carbodiimide reagent. The compound of structure (VI) may be used as is in the next step or may be purified by means known in the art.

[0046]

[0046] R 2 represents the acyl group introduced in step (e). 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In one embodiment, R 2is selected from C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In a preferred embodiment, R 2 is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 2 is C(O)Me, in which case "acylation" can be called "acetylation". 1 and R 2 may be the same or different, regardless of the further transformation of the compound having structure (IV) in the process of the present invention. 2 The definition of R applies to all aspects of the present invention. 2 may also be hydrogen.

[0047] [A.6] Step (f) In the process according to the invention, step (f) is the conversion of a compound having structure (VI) to 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: [ka]

[0048] Formation of the oxazoline ring is well known in the art, for example from 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, which are incorporated herein in their entireties. Typically, the reaction of step (f) is carried out by treatment of the peracylated 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 typically selected from iron(III) chloride, tin(IV) chloride, boron trichloride, zinc(II) iodide, chlorotrimethylsilane, bromotrimethylsilane, and trimethylsilyl triflate, or combinations thereof. In a preferred embodiment, the Lewis acid is trimethylsilyl triflate, or a combination of BF (e.g., BF etherate) and a trimethylsilyl halide (e.g., TMSBr). Alternatively, oxazoline formation can also be achieved from the anomeric deprotected acetylated hexosamine by treatment with a chloroformamidinium reagent, as reported, for example, by Noguchi et al., J. Org. Chem. 2009, 74, 2210, which is incorporated by reference.

[0049] The compound of structure (VII) may be used as is in the next step or may be purified and / or isolated by means known in the art.

[0050] [A.7] Step (g) In the method according to the present invention, step (g) is reacting a 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: [ka]

[0051] The formation of monophosphate monosaccharides from peracylated hexosamines is well known in the art, e.g., from MacDonald et al., J. Org. Chem. 1966, 31, 513 and Masuko et al., J. Org. Chem. 2012, 77, 1449, which are incorporated herein in their entireties. Typically, the reaction in step (g) is carried out by treatment of the peracylated hexosamine with neat phosphoric acid at a temperature between 0 and 120°C, preferably between 20 and 90°C, more preferably between 40 and 80°C, and more preferably between 60 and 80°C. In contrast, the conversion of oxazolines to anomeric phosphoric acids under these conditions (neat phosphoric acid) has not previously been reported. Typically, conversion of the oxazole is carried out by treatment with 1 to 100 equivalents, preferably 2 to 8 equivalents, and most preferably 5 to 7 equivalents of phosphoric acid in DMF at temperatures between 0 and 120° C., preferably between 20 and 90° C., more preferably between 40 and 80° C., more preferably between 60 and 80° C. Similarly, conversion of oxazolines to anomeric phosphoric acids by treatment with phosphoric acid in DMF has not previously been reported.

[0052] The compound of structure (Va) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0053] [A.8] Step (i) In the method according to the invention, step (i) is reacting a compound having structure (Va) with a nucleoside monophosphate to produce an acylated nucleoside diphosphate having structure (VIII). Thus, the phosphate compound of structure (Va) is coupled to the nucleoside monophosphate, thus forming the diphosphate moiety. The 2' and 3' positions (R 4Acylation of the nucleoside monophosphate in (Va) can occur during the reaction process with (Va), for example, carbonylation can occur after 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: [ka]

[0054] In a preferred embodiment, the reaction scheme corresponding to step (i) is as follows: [ka]

[0055] The formation of nucleoside diphosphate monosaccharides is well known in the art and can be achieved by numerous synthetic strategies summarized, for example, in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which are incorporated herein in their entireties. Most commonly, the reaction of step (i) is carried out by in situ activation of a mixture of sugar-1-phosphate and UMP with a carbonylating reagent, such as carbonyldiimidazole (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-phosphates can be coupled with morpholidate derivatives of UMP as reported by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756, which is incorporated 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 sulfonylimidazolium salts as reported by Mohamady et al., Curr. Prot. Nucl. Acid Chem. 2012, DOI:10.1002 / 0471142700.nc1311s51, which is incorporated by reference, or in the presence of 4,5-dicyanoimidazole (DCI) as reported by Vargeese et al., Nucleic Acids Res. 1998, 26, 1046, which is incorporated by reference. Coupling of sugar-1-phosphates with imidazolide-activated NMPs under the action of ZnCl or MgCl can be particularly effective, as reported by Dabrowski-Tumanski, Eur. J. Org. Chem. 2013, 11, 2147, which is incorporated by reference. Alternatively, an enzymatic approach can be performed, for example, by reacting a compound of structure (Va) with a nucleoside triphosphate in the presence of a pyrophosphatase (PPA) that converts the nucleoside triphosphate to a nucleoside monophosphate, and in the presence of a pyrophosphorylase (e.g., UDP-GalNAc pyrophosphorylase AGX1) that 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 by reference. The compound of structure (VIII) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0056]

[0056] R 4 represent groups at the 2' and 3' positions of the ribosyl ring of the nucleoside moiety. 4 If both occurrences of are hydrogen, then R 4 Both occurrences of are connected to each other through the carbonyl moiety, so the two oxygen atoms are in both R 4 This is obtained when a carbonate ester is formed connected to the R group. 4 The most efficient synthesis and highest yields are obtained when both occurrences of are hydrogen, and this is therefore preferred in the context of the present invention.

[0057] [A.9] Step (j) In the method according to the present invention, step (j) is deprotecting the acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate having structure (IX) or a salt thereof. The reaction scheme corresponding to step (j) is as follows: [ka]

[0058]

[0058] The deprotection of acylated compounds is well known in the art. Numerous reaction conditions are suitable for this, for example, the reaction of step (j) is carried out by treating the acylated compound with a (catalytic) amount of sodium methoxide in methanol, or with a mixture of Et3N, MeOH and HO. 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.

[0059]

[0059] Wherein B is a nucleobase. Although any nucleobase may be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0060] In the process according to the invention, the coupling of step (i) and the deprotection of step (j) can also be reversed without any adverse effect on the efficiency and yield of the process. These reactions are referred to herein as step (i1) and step (j1).

[0061] [A.10] Step (j1) In the method according to the present invention, step (j1) is the deprotection of an acylated 1-monophosphate monosaccharide having structure (Va) to obtain a 1-monophosphate monosaccharide having structure (Vb). The reaction scheme corresponding to step (j1) is as follows: [ka]

[0062]

[0062] The deprotection of acylated compounds is well known in the art. Numerous reaction conditions are suitable for this, for example, the reaction of step (j) is carried out by treating the acylated compound with a (catalytic) amount of sodium methoxide in methanol, or by triethylamine in a water / methanol mixture. The compound of structure (Vb) may be used as is in the next step, or may be purified and / or isolated by means known in the art.

[0063] [A.11] Step (i1) In the method according to the present invention, step (i1) is reacting a compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX). Thus, the 1-monophosphate monosaccharide compound of structure (Vb) is coupled to the nucleoside monophosphate, thus forming the diphosphate moiety. The reaction scheme corresponding to step (i1) is as follows: [ka]

[0064] In a preferred embodiment, the reaction scheme corresponding to step (i1) is as follows: [ka]

[0065] The formation of nucleoside diphosphate monosaccharides is well known in the art and can be achieved by numerous synthetic strategies summarized, for example, in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which are incorporated herein in their entireties. Most commonly, the reaction of step (i1) is carried out by in situ activation of a mixture of sugar-1-phosphate and UMP with a carbonylating reagent, such as carbonyldiimidazole (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 by reference. Alternatively, sugar-1-phosphates can be coupled with morpholidate derivatives of UMP as reported by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756, which is incorporated by reference, optionally in the presence of 1-H-tetrazole, as reported by Wittmann et al., J. Org. Chem. 1997, 62, 2144, which is incorporated by reference, or in the presence of 4,5-dicyanoimidazole (DCI), as reported by Vargeese et al., Nucleic Acids Res. 1998, 26, 1046, which is incorporated by reference. Coupling of sugar-1-phosphates with imidazolide-activated NMP can be particularly effective, as discussed in Wagner et al., Nat. Prod. Rep. 2009, 26, 1172, which is incorporated by reference. Alternatively, an enzymatic approach may be performed, for example, by reacting a compound of structure (Vb) with a nucleoside triphosphate in the presence of a pyrophosphatase (PPA) that converts the nucleoside triphosphate to a nucleoside monophosphate, and in the presence of a pyrophosphorylase (e.g., UDP-GalNAc pyrophosphorylase AGX1) that transfers the nucleoside monophosphate to a sugar derivative (Vb). See, e.g., Guan et al., Chem. Eur. J. 2010, 16, 13343-13345, which is incorporated by reference. The compound of structure (IX) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0066]

[0066] wherein B is a nucleobase. Although any nucleobase may be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0067] [A.12] Step (z)

[0067] Both step (i) and step (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 referred to herein independently as step (z). In the method according to the present invention, step (z) is reacting a compound having structure (V) with a nucleoside monophosphate having structure (X) to form a nucleoside diphosphate having structure (IX). Thus, the phosphate compound of structure (V) is coupled to the nucleoside monophosphate, thus forming a diphosphate moiety. The reaction scheme corresponding to step (z) is as follows: [ka]

[0068]

[0068] where R 1 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 1 and R 2 are both H (compounds (Vb) or (IX)), or R 1 and R 2 is not H (compound (Va) or (VIII)). B is a nucleobase. Although any nucleobase may be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0069]

[0069] R 4represent groups at the 2' and 3' positions of the ribosyl ring of the nucleoside moiety, both hydrogen, or R 4 Both occurrences of R are connected to each other via the carbonyl moiety. 4 Both occurrences of are hydrogen.

[0070] The formation of nucleoside diphosphate monosaccharides is well known in the art and can be achieved by numerous synthetic strategies summarized, for example, in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which are incorporated herein in their entireties. Most commonly, the reaction of step (i) is carried out by in situ activation of the sugar-1-phosphate with a carbonylating reagent, such as carbonyldiimidazole (CDI), in the presence of UMP, as reported by Illarionov et al., Russ. Chem. Bull. 2001, 50, 1303 and Loureiro Morais, Can. J. Chem. 2006, 84, 587, which are incorporated by reference. Alternatively, the sugar-1-phosphate can be coupled with a morpholidate derivative of UMP as reported by Moffatt et al., J. Am. Chem. Soc. 1958, 80, 3756, incorporated by reference, optionally in the presence of 1-H-tetrazole, or by using a sulfonylimidazolium salt, as reported by Mohamady et al., Curr. Prot. Nucl. Acid Chem. 2012, DOI:10.1002 / 0471142700.nc1311s51, incorporated by reference, or in the presence of 4,5-dicyanoimidazole (DCI), as reported by Vargeese et al., Nucleic Acids Res. 1998, 26, 1046, incorporated by reference. In one embodiment, step (z) is carried out in the presence of an organic base, such as 1-methylimidazolium chloride or 1-H-tetrazole, or a Lewis acid, such as MgCl or ZnCl. Coupling of sugar-1-phosphates with imidazolide-activated NMP under the action of ZnCl or MgCl can be particularly effective, as reported by Dabrowski-Tumanski, Eur. J. Org. Chem. 2013, 11, 2147, which is incorporated by reference, and discussed in Wagner et al., Nat. Prod. Rep. 2009, 26, 1172. Compounds of structure (VIII) or (IX) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0071] In an alternative embodiment, the compound having structure (VI) as obtained in step (e) is not converted to an oxazoline compound having structure (VII) by step (f), but is instead subjected to steps (x1) to (x4) to form a compound having structure (Va), which can then be converted to a compound having structure (IX) by steps (i)+(j) or steps (j1)+(i1).

[0072] [A.13] Step (x1) In the method according to the present invention, step (x1) is the deprotection of the hydroxyl moiety attached to the anomeric carbon of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI). The reaction scheme corresponding to step (x1) is as follows: [ka]

[0073] Selective deprotection of anomeric hydroxyl groups from peracylated monosaccharides is well known in the art, as reported, for example, by Johnsson et al., Synlett, 2005, 2939, and Baumik et al., Aus. J. Chem. 2003, 56, 909, which are incorporated herein in their entireties. For example, the reaction of step (x1) is carried out by treatment of the peracylated monosaccharide with a slight molar excess of benzylamine, dimethylamine, or hydrazine acetate in an organic solvent such as THF or acetonitrile. The compound of structure (XI) may then be used in the next step or may be purified and / or isolated by means known in the art.

[0074] [A.14] Step (x2) In the process according to the invention, step (x2) is the conversion of a 1-hydroxy-monosaccharide compound having structure (XI) to a monophosphite diester having structure (XII). The reaction scheme corresponding to step (x2) is as follows: [ka]

[0075] Phosphitylation of hydroxyl groups is well known in the art, as summarized, for example, in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which is incorporated herein in its entirety. Typically, the reaction of step (x2) is carried out by treatment of the anomerically deprotected monosaccharide with a phosphitylation reagent, such as a chlorophosphinate or a phosphoramidite, which can be activated for reaction with the anomeric hydroxyl group in the presence of a proton scavenger or a weak acid, respectively. The compound of structure (XII) may then be used in the next step or may be purified and / or isolated by means known in the art.

[0076]

[0076] where R 3 represents the moiety attached to both oxygen atoms of the phosphityl diester (i.e., does not include the anomeric oxygen atom of the monosaccharide moiety). 3 is 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 phenyl is optionally substituted with one or more halide, nitro or methoxy groups; 1~2 -alkyl refers to 2-phenylethyl or phenylmethyl. Nitro or methoxy substituents, especially methoxy substituents, are preferably in the para position. R 3 can be selected individually, but R 3It is preferred that both parts of R are the same. 3 When is 2-alkylsulfonylethyl or CHOC(O)alkyl, preferably alkyl is C 1~6 Alkyl, more preferably C 1~2 alkyl, most preferably methyl. R 3 When R is 2-arylsulfonylethyl, preferably the aryl is phenyl. 3 Each of the options is easily removed in a subsequent step (x4), and this deprotection can optionally be followed by the removal of R 1 and R 3 Therefore, in a particularly preferred embodiment, the deprotection of R 3 is CH2OC(O)alkyl, where alkyl is preferably C 1~6 Alkyl, more preferably C 1~2 In the case of alkyl, most preferably methyl, steps (x4) and (j1) are carried out in a single step (one pot).

[0077] [A.15] Step (x3) In the method according to the present invention, step (x3) is the oxidation of a monophosphite diester having structure (XII) to form a 1-monophosphate diester having structure (XIII). The reaction scheme corresponding to step (x3) is as follows: [ka]

[0078] Oxidation of the phosphite moiety is well known in the art and is summarized, for example, in Ahmadipour et al., Carbohydr. Res. 2017, 451, 95, which is incorporated herein in its entirety. Typically, the reaction of step (x3) is carried out by contacting the phosphite ester (XII) with an oxidizing agent such as iodine, m-CPBA, t-BuOOH, or H2O2 in a dichloromethane or acetonitrile solution. The compound of structure (XIII) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0079] [A.16] Step (x4) In the method according to the present invention, step (x4) is the deprotection of the monophosphate diester having structure (XIII) to form the 1-monophosphate monosaccharide having structure (Va). The reaction scheme corresponding to step (x4) is as follows: [ka]

[0080] Step (x4) also involves the addition of an acyl group R 1 and R 2 R 3 This may involve complete deprotection of compound (XIII) which is simultaneously removed with the removal of the group. The reaction scheme corresponding to this embodiment of step (x4) is as follows: [ka]

[0081] Generally, the reaction scheme for step (x4) is as follows: [ka]

[0082]

[0082] Here, the following holds: In the case of compound (XIII), R1 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 2 is selected from optionally substituted C(O)-alkyl, optionally substituted C(O)-aryl and C(O)-arylalkyl; In the case of compound (V), R 1 is selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 2 is selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 1 and R 2 are both H (compound (Vb)), or R 1 and R 2 None of the groups is H (compound (Va)).

[0083]

[0083] Deprotection of acylated monosaccharides is well known in the art. Compounds of structure (Va) or (Vb) may be used directly in the next step or may be purified and / or isolated by means known in the art.

[0084] In an alternative embodiment, the compound having structure (I) as obtained in step (d) is not converted to a triacylated compound having structure (VI) by step (e), but instead is subjected to steps (y1)-(y2) to form a compound having structure (Vb), which can then be converted to a compound having structure (IX) by step (i1).

[0085] [A.17] Step (y1) In the method according to the present invention, step (y1) is the deprotection of a 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: [ka]

[0086]

[0086] Deprotection of acylated monosaccharides is well known in the art. Compounds of structure (XIV) may then be used in the next step or may be purified and / or isolated by means known in the art.

[0087] [A.18] Step (y2) In the method according to the present invention, step (y2) is reacting a 1,3,4-trihydroxy-6-azido-monosaccharide having structure (XIV) with a phosphate source to form a 1-monophosphate monosaccharide compound having structure (Vb). The reaction scheme corresponding to step (y2) is as follows: [ka]

[0088]

[0088] Regioselective phosphorylation of monosaccharides is well known in the art and is summarized, for example, in Buter et al., Glycoconj. J. 1999, 16, 147, which is incorporated herein in its entirety. The reaction of step (y2) is carried out in the presence of a phosphorylating enzyme such as N-acetylhexosamine 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) may then be used in the next step, or may be purified and / or isolated by means known in the art. If the compound of structure (Vb) is enzymatically converted to a nucleoside diphosphate having structure (IX) via step (i1) as the next step, the entire reaction sequence may be carried out in one pot. See, e.g., Heinzler et al., Adv. Synth. Catal. 2019, 361, 4506-4516, which is incorporated by reference.

[0089] [B] Compound according to the present invention The present inventors have identified several key intermediates in the synthetic method according to the present invention, and the present invention also relates to these intermediates.

[0090] [B.1] Compound (III)

[0090] Accordingly, the present invention relates to a cyclic sulfate monosaccharide compound having the structure (III): [ka]

[0091]

[0091] where R 1 is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. The carbon atom bearing the wavy bond may be in the S or R configuration, and all four diastereomers are included.

[0092]

[0092] R 1 represents the acyl group introduced in step (a). 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Considering the reaction conditions, R 1 Both occurrences of R are typically the same. 1 Each occurrence of R is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 1 Each occurrence of is C(O)Me.

[0093] [B.2] Compound (I) The present invention further relates to a 6-azido-6-deoxy monosaccharide compound having the structure (I): [ka]

[0094]

[0094] where R 1is the same as defined for compounds having structure (III). The carbon atom bearing the wavy bond may be in the S or R configuration, and all four diastereomers are included.

[0095] [B.3] Compound (VII) The present invention further relates to oxazoline compounds having the structure (VII). [ka]

[0096]

[0096] where R 1 is the same as defined for compounds having structure (III). The carbon atom bearing the wavy bond may be in the S or R configuration, and both diastereomers are included. The oxygen of the oxazoline ring located at carbon 1 of the GlcNAc or GalNAc moiety is the α-anomer only.

[0097]

[0097] R 2 represents the acyl group introduced in step (e). 2 represents the acyl group introduced in step (e). 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In one embodiment, R 2 is selected from C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In a preferred embodiment, R 2 is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 2 is C(O)Me, in which case "acylation" can be called "acetylation". 1 and R 2 may be the same or different, regardless of the further transformation of the compound having structure (IV) in the process of the present invention.

[0098] [B.4] Compound (Va) The present invention further relates to a mixture comprising the α- and β-anomeric forms of the 6-azido-6-deoxy-1-monophosphate monosaccharide compound having the structure (Va), or a salt thereof. [ka]

[0099]

[0099] where R 1 and R 2 is the same as defined for the compound having structure (VII). The carbon atom bearing the wavy bond may be in the S or R configuration, and all four diastereomers are included, provided that the molar ratio of the α-anomeric form to the β-anomeric form in the mixture is within the range of 3 / 1 to 10 / 1. Both anomeric forms of compound (Va) present in the mixture may be in the form of a salt.

[0100] Known chemical methods for preparing 6-azido-6-deoxy-1-monophosphate monosaccharide compounds having structure (Va) typically produce a mixture of α- and β-anomeric forms (by chemical conversion, i.e., reaction with a phosphitylating agent such as a phosphoramidite). While these conventional methods are also suitable in the context of the present invention, it is preferred that the phosphate group be introduced via compound (VII), in which case a mixture of α- and β-anomers is formed, containing primarily, but not exclusively, the α-anomer. The α-anomeric form is preferred for the subsequent reaction to compound (IX), because this anomeric form is the only substrate for transferase enzymes that can be used to incorporate the azide-containing monosaccharide moiety into the glycan chain. Until now, obtaining an enantiomeric excess of the α-anomer was only achievable by enzymatic methods. Compound (Va) can be obtained as a mixture of isoforms, but containing primarily the α-anomer, by chemical synthesis steps and without the need for enzymatic conversion.

[0101] [B.5] Compound (VIII) The present invention further relates to a nucleoside diphosphate having the structure (VIII). [ka]

[0102]

[0102] where R 1 and R 2 is the same as defined for compounds having structure (VII). The carbon atom bearing the wavy bond may be in the S or R configuration, and both diastereomers are included.

[0103]

[0103] R 4 represent groups at the 2' and 3' positions of the ribosyl ring of the nucleoside moiety, both hydrogen, or R 4 Both occurrences of R are connected to each other via the carbonyl moiety. 4 Both occurrences of are hydrogen.

[0104] B is a nucleobase. Although any nucleobase can be used, B is preferably a pyrimidine nucleobase, and most preferably B is uracil.

[0105] [B.6] Compound (XI) The present invention further relates to 1-hydroxy-monosaccharide compounds having the structure (XI): [ka]

[0106]

[0106] where R 1 and R 2 is the same as defined for compounds having structure (VI). The carbon atom bearing the wavy bond may be in the S or R configuration, and all four diastereomers are included.

[0107] [B.7] Compound (XII) The present invention further relates to a phosphite having the structure (XII). [ka]

[0108]

[0108] where R 1 and R 2 is the same as defined for compounds having structure (VI). The carbon atom bearing the wavy bond may be in the S or R configuration, and all four diastereomers are included.

[0109]

[0109] where R 3 is 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 phenyl is optionally substituted with one or more halide, nitro or methoxy groups; 1~2 -alkyl refers to 2-phenylethyl or phenylmethyl. Nitro or methoxy substituents, especially methoxy substituents, are preferably in the para position. R 3 can be selected individually, but R 3 It is preferred that both parts of R are the same. 3 When is 2-alkylsulfonylethyl or CHOC(O)alkyl, preferably alkyl is C 1~6 Alkyl, more preferably C 1~2 alkyl, most preferably methyl. R 3 When R is 2-arylsulfonylethyl, preferably, aryl is phenyl. In a particularly preferred embodiment, R 3 is CH2OC(O)alkyl, where alkyl is preferably C 1~6 Alkyl, more preferably C 1~2 Alkyl, most preferably methyl.

[0110] [B.8] Compound (XIII) The present invention further relates to a phosphoric acid diester having the structure (XIII). [ka]

[0111]

[0111] where R 1 , R 2 and R 3 is the same as defined for compounds having structure (XII). The carbon atom bearing the wavy bond may be in the S or R configuration, and all four diastereomers are included.

[0112] [C] Synthesis method In pursuit of the overall objective of the present invention, namely, to provide an efficient synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphates having structure (IX), the inventors have identified several key synthetic steps. The present invention also relates to these methods for the partial synthesis of compounds having structure (IX). The methods according to these aspects of the present invention are ideally suited in the context of the overall objective of synthesizing 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphates having structure (IX).

[0113] [C.1] Synthesis of Compound (I) 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: [ka]

[0114] The method comprises: (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting a cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); Includes.

[0115] Steps (b), (c) and (d) are defined above. 1 R represents an acyl group present as a protecting group for the hydroxyl groups attached to the carbon atoms at positions 1 and 3 of the monosaccharide moiety. These acyl protecting groups can be introduced by step (a) before step (b). 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Preferably, R 1 In a preferred embodiment, both occurrences of R 1 Each occurrence of R is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 1 is C(O)Me, in which case "acylation" can be called "acetylation."

[0116]

[0116] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the carbon atom at position 4 of the monosaccharide moiety may be axial (galactose configuration) or equatorial (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, the compound having structure (II) is preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), preferably from GalNAc. This preparation is preferably achieved by step (a) as defined above.

[0117]

[0117] The process according to this embodiment is ideally suited in the context of the overall object of the present invention, namely, the synthesis of a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Accordingly, the compound having structure (I) obtained in step (d) is preferably further converted to a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX) or a salt thereof. Such conversion can be accomplished in any suitable manner. Preferably, this conversion involves the conversion of a 6-azido-6-deoxy monosaccharide compound having structure (I) to a 1-monophosphate monosaccharide compound, which reacts with a nucleoside monophosphate to form a compound having structure (IX), typically having structure (V). This reaction sequence involves a deprotection step before or after the reaction of the 1-monophosphate monosaccharide compound with a nucleoside monophosphate.

[0118]

[0118] The 1-monophosphate monosaccharide having the structure (V) is defined as follows: [ka]

[0119]

[0119] where R 1 and R 2 are independently selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Additionally, the following holds: In the case of compound (Va), R 1 and R 2 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In the case of compound (Vb), R 1 and R 2 are both H.

[0120] In a preferred embodiment, the conversion of a compound of structure (I) to a compound of structure (V) is carried out by one of the following reaction sequences: Steps (e), (f) and (g), typically producing a compound having structure (Va); Steps (e), (x1), (x2), (x3) and (x4), typically producing a compound having structure (Va); Steps (y1) and (y2) typically produce a compound having the structure (Vb).

[0121]

[0121] A compound having structure (V) is preferably converted to a compound having structure (IX), preferably by steps (i) and (j) or steps (j1) and (i1). When a compound having structure (Vb) is obtained, it is preferably converted to a compound having structure (IX) by step (i1). In that case, the deprotection of step (j) or (j1) is not necessary. When the reaction sequence to a compound having structure (IX) involves both step (x4) and step (j1), they may optionally be carried out simultaneously to produce a compound having structure (Vb). Where R 3 is preferably CH2OC(O)alkyl, where alkyl is preferably C 1~6 Alkyl, more preferably C 1~2 Alkyl, most preferably methyl.

[0122] In a preferred embodiment, the conversion of a compound of structure (I) to a compound of structure (IX) is carried out via a compound having structure (V) by one of the following reaction sequences: Steps (e), (f), (g), (i), and (j); Steps (e), (f), (g), (j1), and (i1); Steps (e), (x1), (x2), (x3), (x4), (i), and optionally (j); Step (e), (x1), (x2), (x3), (x4), (j1), (i1); Steps (y1), (y2), and (i).

[0123]

[0123] Steps (e), (f), (g), (i), (j), (i1), (j1), (x1), (x2), (x3), (x4), (y1) and (y2) are defined above.

[0124] [C.2] Synthesis of compound (Va) via compound (VII) In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va) according to the following scheme: [ka]

[0125] The method comprises: (f) converting 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); (g) reacting an oxazoline compound having structure (VII) with phosphoric acid to form a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va).

[0126] Steps (f) and (g) are defined above. 1 represents the acyl group(s) present as protecting group(s) for the hydroxyl group attached to the carbon atom at position 3 (and position 1 for (VI)) of the monosaccharide moiety. The acyl protecting group(s) may be introduced by step (a) prior to step (f). R 1 is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl. In one embodiment, R 1 is selected from C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Preferably, R 1 In a preferred embodiment, both occurrences of R 1 Each occurrence of R is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 1is C(O)Me, in which case "acylation" can be called "acetylation."

[0127]

[0127] R 2 represents an acyl group present as a protecting group for the hydroxyl group attached to the carbon atom at position 4 of the monosaccharide moiety. The acyl protecting group(s) may be introduced by step (e) prior to step (f). R 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In one embodiment, R 2 is selected from C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In a preferred embodiment, R 2 is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 2 is C(O)Me, in which case "acylation" can be called "acetylation". 1 and R 2 may be the same or different, regardless of the further transformation of the compound having structure (Va) in the process of the present invention.

[0128] 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 may be axial (galactose configuration) or equatorial (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), preferably from GalNAc. This preparation is preferably achieved 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.

[0129] The process according to this embodiment is ideally suited in the context of the overall object of the present invention, namely, the synthesis of a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Accordingly, it is preferred that the compound having structure (Va) obtained in step (g) is further converted to a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX) or a salt thereof. Such a conversion can be accomplished in any suitable manner. Preferably, this conversion involves steps (i) and (j) or steps (j1) and (i1) as defined above.

[0130] [C.3] Synthesis of compound (Va) via compound (XII) In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va) according to the following scheme: [ka]

[0131]

[0131] The method comprises: (x1) deprotecting the anomeric position of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI); (x2) converting a 1-hydroxy-monosaccharide compound having structure (XI) into a 6-azido-6-deoxy-1-monophosphite diester having structure (XII); (x3) oxidizing a 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 phosphate diester having structure (XIII) to form a 1-monophosphate monosaccharide compound having structure (Va); Includes.

[0132] Steps (x1), (x2), (x3) and (x4) are defined above. 1represents the acyl group(s) present as protecting group(s) for the hydroxyl group attached to the carbon atom at position 3 (and position 1 for (VI)) of the monosaccharide moiety. The acyl protecting group(s) may be introduced by step (a) prior to step (x1). R 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Preferably, R 1 In a preferred embodiment, both occurrences of R 1 Each occurrence of R is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 1 is C(O)Me, in which case "acylation" can be called "acetylation."

[0133]

[0133] R 2 represents an acyl group present as a protecting group for the hydroxyl group attached to the carbon atom at position 4 of the monosaccharide moiety. The acyl protecting group(s) may be introduced by step (e) prior to step (x1). R 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In a preferred embodiment, R 2 is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 2 is C(O)Me, in which case "acylation" can be called "acetylation". 1 and R 2 may be the same or different, regardless of the further transformation of the compound having structure (Va) in the process of the present invention.

[0134]

[0134] R 3 represents the phosphorous substituent introduced in step (x2). 3 is C 1~6 Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH2OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C1~2 -alkyl (2-phenylethyl or phenylmethyl), wherein the phenyl is optionally substituted with one or more halide or nitro or methoxy groups. 3 When is 2-alkylsulfonylethyl or CHOC(O)alkyl, preferably alkyl is C 1~6 Alkyl, more preferably C 1~2 alkyl, most preferably methyl. R 3 When is 2-arylsulfonylethyl, preferably aryl is phenyl.

[0135] 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 may be axial (galactose configuration) or equatorial (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), preferably from GalNAc. This preparation is preferably achieved 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.

[0136]

[0136] The process according to this embodiment is ideally suited in the context of the overall object of the present invention, namely, the synthesis of a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Accordingly, it is preferred that the compound having structure (Va) obtained in step (x4) is further converted to a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX) or a salt thereof. Such a conversion can be accomplished in any suitable manner. Preferably, this conversion involves steps (i) and (j) or steps (j1) and (i1) as defined above.

[0137] [C.4] Synthesis of compound (Vb) via compound (XII) In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Vb) according to the following scheme: [ka]

[0138] The method comprises: (x1) deprotecting the anomeric position of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI); (x2) converting a 1-hydroxy-monosaccharide compound having structure (XI) into a 6-azido-6-deoxy-1-monophosphite diester having structure (XII); (x3) oxidizing a monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphate diester compound having structure (XIII); (x4) simultaneously deprotecting the phosphate diester and monosaccharide having structure (XIII) to form a 1-monophosphate monosaccharide compound having structure (Vb); Includes.

[0139] Steps (x1), (x2), (x3) and (x4) are defined above. 1 represents the acyl group(s) present as protecting group(s) for the hydroxyl group attached to the carbon atom at position 3 (and position 1 for (VI)) of the monosaccharide moiety. The acyl protecting group(s) may be introduced by step (a) prior to step (x1). R 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Preferably, R 1 In a preferred embodiment, both occurrences of R 1Each occurrence of R is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 1 is C(O)Me, in which case "acylation" can be called "acetylation."

[0140]

[0140] R 2 represents an acyl group present as a protecting group for the hydroxyl group attached to the carbon atom at position 4 of the monosaccharide moiety. The acyl protecting group(s) may be introduced by step (e) prior to step (x1). R 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In a preferred embodiment, R 2 is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 2 is C(O)Me, in which case "acylation" can be called "acetylation". 1 and R 2 may be the same or different, and this is irrelevant to the suitability of the method of this aspect of the invention.

[0141]

[0141] R 3 represents the phosphorous substituent introduced in step (x2). 3 is 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 phenylmethyl), wherein the phenyl is optionally substituted with one or more halide or nitro or methoxy groups. 3 When is 2-alkylsulfonylethyl or CHOC(O)alkyl, preferably alkyl is C 1~6 Alkyl, more preferably C 1~2 alkyl, most preferably methyl. R 3When is 2-arylsulfonylethyl, preferably aryl is phenyl.

[0142] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the carbon atom at position 4 of the monosaccharide moiety may be axial (galactose configuration) or equatorial (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), preferably from GalNAc. This preparation is preferably achieved 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.

[0143] The process according to this embodiment is ideally suited in the context of the overall object of the present invention, namely, the synthesis of a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Accordingly, it is preferred that the compound having structure (Vb) obtained in step (x4) is further converted to a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX) or a salt thereof. Such a conversion can be accomplished in any suitable manner. Preferably, this conversion involves step (i) as defined above.

[0144] [C.5] Synthesis of compound (Vb) via compound (XIV) In one aspect, the present invention relates to a method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having the structure (Vb) according to the following scheme: [ka]

[0145]

[0145] The method is (y1) deprotecting a 6-azido-6-deoxy monosaccharide having structure (I) to form a 1,3,4-trihydroxy-6-azido-monosaccharide compound having structure (XIV); (y2) contacting a compound having structure (XIV) with a phosphorylating enzyme in the presence of a phosphate source to form a 1-monophosphate monosaccharide compound having structure (Vb); Includes.

[0146] Steps (y1) and (y2) are defined above. 1 represents an acyl group present 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(s) may be introduced by step (a) prior to step (y1). R 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. Preferably, R 1 In a preferred embodiment, both occurrences of R 1 Each occurrence of R is C(O)Me, C(O)tBu, C(O)Ph or C(O)CHPh. Most preferably, R 1 is C(O)Me, in which case "acylation" can be called "acetylation."

[0147]

[0147] The monosaccharide is preferably N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc). In other words, the wavy bond at the carbon atom at position 4 of the monosaccharide moiety may be axial (galactose configuration) or equatorial (glucose configuration). Preferably, the monosaccharide moiety is GalNAc. Similarly, the compound having structure (I) is preferably prepared from N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), preferably from GalNAc. This preparation is preferably achieved by steps (b), (c), and (d) as defined above, more preferably by steps (a), (b), (c), and (d) as defined above.

[0148] The process according to this embodiment is ideally suited in the context of the overall object of the present invention, namely, the synthesis of a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX). Accordingly, it is preferred that the compound having structure (Vb) obtained in step (x4) is further converted to a 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX) or a salt thereof. Such a conversion can be accomplished in any suitable manner. Preferably, this conversion involves step (i1) as defined above.

[0149] [C.6] Synthesis of compound (VIII) 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: [ka]

[0150]

[0150] The method is (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, MgCl or ZnCl.

[0151] Step (z) is defined above. In the method according to this aspect, R 1 R may be H or an acyl group present as a protecting group for the hydroxyl group attached to the carbon atom at position 3 of the monosaccharide moiety. The acyl protecting group may be introduced by step (a) prior to step (z). 1 is selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In a preferred embodiment, R 1 is H, C(O)Me, C(O)tBu, C(O)Ph, or C(O)CHPh. Most preferably, R 1 is C(O)Me, in which case "acylation" may be referred to as "acetylation." In one embodiment, the compound having structure (V) is a compound having structure (Va), and R 1 is 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), wherein R 1 is H.

[0152] In the method according to this aspect, R 2 R may be H or an acyl group present as a protecting group for the hydroxyl group attached to the carbon atom at position 4 of the monosaccharide moiety. The acyl protecting group may be introduced by step (e) prior to step (z). 2 is selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl. In a preferred embodiment, R 2 is H, C(O)Me, C(O)tBu, C(O)Ph, or C(O)CHPh. Most preferably, R 2is C(O)Me, in which case "acylation" may be referred to as "acetylation." In one embodiment, the compound having structure (V) is a compound having structure (Va), and R 2 is 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 a compound having structure (Vb), wherein R 2 is H.

[0153]

[0153] 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 may be axial (galactose configuration) or equatorial (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), preferably from GalNAc. This preparation is preferably achieved by: Steps (f) and (g), in which case a compound having structure (Va) is obtained; Steps (e), (f) and (g), in which case a compound having structure (Va) is obtained; Steps (b), (c), (d), (e), (f) and (g), in which case a compound having structure (Va) is obtained; Steps (a), (b), (c), (d), (e), (f) and (g), in which case a compound having structure (Va) is obtained; Steps (x1), (x2), (x3) and (x4), in which case a compound having structure (Va) or (Vb) is obtained; Steps (e), (x1), (x2), (x3) and (x4), in which case a compound having structure (Va) or (Vb) is obtained; Steps (b), (c), (d), (e), (x1), (x2), (x3) and (x4), where a compound having structure (Va) or (Vb) is obtained; Steps (a), (b), (c), (d), (e), (x1), (x2), (x3) and (x4), whereby a compound having structure (Va) or (Vb) is obtained; Steps (y1) and (y2), in which case a compound having structure (Vb) is obtained; Steps (b), (c), (d), (y1) and (y2), in which case a compound having structure (Vb) is obtained; Steps (a), (b), (c), (d), (y1) and (y2), in this case, a compound having structure (Vb) is obtained.

[0154]

[0154] Steps (a), (b), (c), (d), (e), (f), (g), (x1), (x2), (x3), (x4), (y1) and (y2) are defined above.

[0155] The method according to this aspect is ideally suited in the context of the overall object of the present invention, namely, the synthesis of 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphates having structure (IX). In one embodiment, R 1 and R 2 are both H, and the process according to this embodiment already produces this compound. Alternatively, the compound having structure (VIII) obtained in step (z) is preferably further converted to 6-azido-2-N-acetyl-monosaccharide-nucleoside diphosphate having structure (IX) or a salt thereof. Such conversion can be accomplished in any suitable manner. Preferably, this conversion involves step (j) as defined above.

[0156] [D] Synthesis of compound (IX)

[0156] The present invention provides several approaches towards the total synthesis of target compound (IX) using the steps, methods and intermediates defined above, each of which offers one or more specific advantages over prior art methods.

[0157] [D.1] Synthesis of compound (IX) via compounds (VII) and (VIII) In one aspect, the present invention relates to a method for preparing a nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: [ka]

[0158]

[0158] The method is (a) converting N-acetylglucosamine or N-acetylgalactosamine into a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting a cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting a 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (f) converting a compound having structure (VI) in the presence of one or more Lewis acids to form an oxazoline compound having structure (VII); (g) reacting a compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va); (i) reacting a compound having structure (Va) with a nucleoside monophosphate to produce an acylated nucleoside diphosphate having structure (VIII); (j) deprotecting the acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate having structure (IX) or a salt thereof; Includes.

[0159]

[0159] where R 1are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 4 are both hydrogen or R 4 are linked to each other via a carbonyl moiety; and B is a nucleobase. Preferred embodiments of each of the steps and compounds in this method are defined above.

[0160] [D.2] Synthesis of compound (IX) via compounds (VII) and (Vb) In one aspect, the present invention relates to a method for preparing a nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: [ka]

[0161]

[0161] The method is (a) converting N-acetylglucosamine or N-acetylgalactosamine into a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (I) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting a cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting a 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (f) converting a compound having structure (VI) in the presence of one or more Lewis acids to form an oxazoline compound having structure (VII); (g) reacting a compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va); (j1) deprotecting a compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb); (i1) reacting a compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX); Includes.

[0162]

[0162] where R 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; and B is a nucleobase. Preferred embodiments of each of the steps and compounds in this method are defined above.

[0163]

[0163] In one embodiment according to this aspect of the invention, step (f) is omitted and the compound having structure (VI) is subjected to step (g1) to produce a compound having structure (Va).

[0164] [D.3] Synthesis of compound (IX) via compounds (XII) and (VIII) In one aspect, the present invention relates to a method for preparing a nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: [ka]

[0165]

[0165] The method is (a) converting N-acetylglucosamine or N-acetylgalactosamine into a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting a cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting a 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (x1) deprotecting the anomeric position of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI); (x2) converting a 1-hydroxy-monosaccharide compound having structure (XI) into a 6-azido-6-deoxy-1-monophosphite diester having structure (XII); (x3) oxidizing a 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 phosphate diester having structure (XIII) to form a 1-monophosphate monosaccharide compound having structure (Va); (i) reacting a compound having structure (Va) with a nucleoside monophosphate to produce an acylated nucleoside diphosphate having structure (VIII); (j) deprotecting the acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate having structure (IX) or a salt thereof; Includes.

[0166]

[0166] where R 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 3 is 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 phenylmethyl), wherein phenyl is optionally substituted with one or more halide or nitro or methoxy groups; R 4 are both hydrogen or R 4 are linked to each other via a carbonyl moiety; and B is a nucleobase. Preferred embodiments of each of the steps and compounds in this method are defined above.

[0167] [D.4] Synthesis of compound (IX) via compounds (XII) and (Vb) In one aspect, the present invention relates to a method for preparing a nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: [ka]

[0168]

[0168] The method is (a) converting N-acetylglucosamine or N-acetylgalactosamine into a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting a cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting a 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (x1) deprotecting the anomeric position of a compound having structure (VI) to form a 1-hydroxy-monosaccharide compound having structure (XI); (x2) converting a 1-hydroxy-monosaccharide compound having structure (XI) into a 6-azido-6-deoxy-1-monophosphite diester having structure (XII); (x3) oxidizing a 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 phosphate diester having structure (XIII) to form a 1-monophosphate monosaccharide compound having structure (Va); (j1) deprotecting a compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb); (i1) reacting a compound having structure (Vb) with a nucleoside monophosphate to produce an acylated nucleoside diphosphate having structure (IX).

[0169]

[0169] where R 1 are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R 2 is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R 3 is 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 phenylmethyl), wherein phenyl is optionally substituted with one or more halide or nitro or methoxy groups; and B is a nucleobase. Preferred embodiments of each of the steps and compounds in this method are defined above.

[0170] [D.5] Synthesis of compound (IX) via compound (XIV) In one aspect, the present invention relates to a method for preparing a nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: [ka]

[0171]

[0171] The method is (a) converting N-acetylglucosamine or N-acetylgalactosamine into a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting a cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting a cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (y1) deprotecting a 6-azido-6-deoxy monosaccharide having structure (I) to form a 1,3,4-trihydroxy-6-azido-monosaccharide compound having structure (XIV); (y2) contacting a compound having structure (XIV) with a phosphorylating enzyme in the presence of a phosphate source to form a 1-monophosphate monosaccharide compound having structure (Vb); (i) reacting a compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX) or a salt thereof; Includes.

[0172]

[0172] where R 1 is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; and B is a nucleobase. Preferred embodiments of each of the steps and compounds in this method are defined above. [Example]

[0173] Chemicals were purchased from commonly used suppliers (Sigma-Aldrich, Acros, Alfa Aesar, Fluorochem, Apollo Scientific Ltd, and TCI) and used without further purification. Solvents (including anhydrous solvents) for chemical transformations, workup, and chromatography were purchased from Aldrich (Dorset, UK) as HPLC grade and used without further purification. Silica gel 60 F254 analytical thin-layer chromatography (TLC) plates were from Merck (Darmstadt, Germany) and visualized under UV light with potassium permanganate stain or anisaldehyde stain. Chromatographic purification was performed using Acros silica gel (0.06-0.200, 60A) or prepacked columns (Screening Devices) in combination with a Buchi Sepacor C660 fraction collector (Flawil, Switzerland). Deuterated solvents used for NMR spectroscopy were obtained from Cambridge Isotope Laboratories.

[0174]

[0174] The conditions of Examples 1-1 to 1-3 were based on 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, which are incorporated by reference.

[0175] Example 1-1: Synthesis of 4,6-O-benzylidene-N-acetyl-D-galactosamine [ka] To a suspension of N-acetyl-D-galactosamine (1299 g, 5.9 mol) in MeCN (13 L) was added DL-camphorsulfonic acid (81 g, 352 mmol) and benzaldehyde dimethyl acetal (2190 g, 2160 mL, 14.4 mol). The reaction was stirred at room temperature and filtered through a Buchner filter. The white filter cake was rinsed with MeCN (10 × 1 L) and allowed to dry on the filter overnight. Drying in a circulating oven at 35 °C for 4 days afforded the product (1938 g, 107%). Crystallization from EtOH / HO (13:1 to 9:1) at 5 to 8 °C for 16 to 24 hours afforded the pure product as a white crystalline solid. 1 H NMR (400 MHz, 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).

[0176] Example 1-2: Synthesis of 1,3-di-O-acetyl-4,6-O-benzylidene-N-acetyl-D-galactosamine [ka] The starting material, 4,6-O-benzylidene-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 at room temperature overnight, followed by the addition of ice water (19.5 L). After 15 minutes of stirring, an additional scoop of ice was added, and after another 15 minutes, the mixture was filtered. The cake was washed with ice water (3 × 6 L), dried on the filter overnight, and then dried overnight in a circulating oven at 45 °C. The crude material was dissolved in methanol (13.7 L), and the mixture was heated to reflux, resulting in a clear solution. The solution was cooled to room temperature overnight, then cooled to 0 °C in an ice bath for 4 hours, and then filtered. The cake was washed three times with the filtrate and dried on the filter for 1.5 hours. Drying in a circulating oven at 45° C. for 3.5 days gave product 2 (1450 g, 3.7 mol, 59%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 7.53-7.50 (m, 2H), 7.39-7.53 (m, 3H), 6.34 (d, J = 4.8 Hz,1H), 5.57 (d, J = 12 Hz, 1H), 5.24 (dd, J = 11.2, 4.4 Hz, 1H), 4.92-4.85 (m,1H), 4.34 (d, J = 4 Hz, 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).

[0177] Example 1-3: Synthesis of 1,3-di-O-acetyl-N-acetyl-D-galactosamine (2) [ka] The starting material, 1,3-di-O-acetyl-4,6-O-benzylidene-N-acetyl-D-galactosamine (230 g, 585 mmol), was dissolved in a mixture of MeOH / dioxane (1:1, 3 L) and placed in a Parr vessel. AcOH (1.9 g, 1.77 mL, 31 mmol) was added. Pd—C (37.3 g, 175 mmol) was then added, and the vessel was attached to a Parr apparatus. The reaction was stirred overnight under an H atmosphere (5 bar). The mixture was then filtered through a Celite pad and washed with 1,4-dioxane (1200 mL). Acetic acid (12 mL) was added to the filtrate, followed by concentration under reduced pressure to give product 3 (224 g, 126%). 1 H NMR (400 MHz, DMSO) δ (ppm) 7.91 (d, J = 8.4 Hz, 1H), 5.96 (d, J = 3.6 Hz, 1H), 5.20 (d,J = 5.2 Hz, 1H), 4.84 (dd, J = 8.8, 2.8 Hz, 1H), 4.49-4.43 (m, 1H), 3.79 (t, J= 6.4 Hz, 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).

[0178] Examples 1-4: Synthesis of 4,6-O-sulfoxyl-1,3-di-O-acetyl-N-acetyl-D-galactosamine (3a) [ka]

[0178] Method ACompound 2 (190 g, 622 mmol) was dissolved in DCM (11.5 L) and placed under a N2 atmosphere. Next, SOCl2 (182 mL, 933 mmol) was added dropwise, after which the reaction mixture was cooled to 0 °C, followed by the slow addition of Et3N (500 mL, 1306 mmol). After complete addition, the reaction was warmed to room temperature and stirred for 1 h. The reaction was quenched by the careful addition of 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 in vacuo. The crude product (4a) was used in the next step without further purification. 1 H NMR (400 MHz, MeOD) δ (ppm) 6.23 (d, J = 3.6 Hz, 1H), 5.34-5.35 (m, 1H), 5.24 (dd, J =8.4, 3.2 Hz, 1H), 4.94-.495 (m, 1H), 4.63 (dd, J = 8.4, 3.2 Hz, 1H), 4.11 (s,1H), 3.96 (dd, J = 10.8, 1.6 Hz, 1H), 2.17 (s, 3H), 2.07 (s, 3H), 1.95 (s, 3H).

[0179]

[0179] Method B Compound 2 (1 equiv.) was dissolved in EtOAc (0.15 M) and placed under a N2 atmosphere. SOCl2 (1.05 equiv.) was added dropwise, followed by pyridine (2.1 equiv.). The reaction progress was followed by TLC analysis (100% EtOAc), and complete conversion was obtained after 30 min. 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 once more with EtOAc (0.03 M). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude product (3a) was used in the next step without further purification.

[0180] Examples 1-5: Synthesis of 4,6-O-sulfonyl-1,3-di-O-acetyl-N-acetyl-D-galactosamine (3b) [ka] Crude 3a (176 g, 501 mmol) was dissolved in a mixture of DCM (1460 mL) and MeCN (1460 mL). Then, NaIO (214 g, 1002 mmol) in water (2199 mL) was added, and the mixture was stirred vigorously. A solution of RuCl.xH.sub.2O (2.08 g, 10.0 mmol) in water (106 mL) was added dropwise, and the reaction was cooled in an ice bath to prevent further exothermic reaction. The reaction was stirred at room temperature for approximately 30 minutes (reaction progress was followed by TLC analysis (100% EtOAc)). After completion of the reaction (1 h), the reaction mixture was cooled to 5° C. in an ice / NaCl bath. A solution of sodium metabisulfite (438 g, 2304 mmol) in water (704 mL) was then 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 half-saturated NaCl solution, dried over Na2SO4, filtered, and concentrated in vacuo. The white solid was triturated overnight in diisopropyl ether (alternative: diethyl ether) (700 mL), then filtered and rinsed with additional diisopropyl ether (3 x 200 mL) (alternative: diethyl ether). The solid was dried under a gentle stream of nitrogen to give product 3b (153 g, 416 mmol, 83%) as an off-white solid.

[0181]

[0181] Post-processing procedure B After completion of the reaction, 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 through Celite), and concentrated in vacuo. Gradient flash chromatography (DCM:MeOH; 100:0 to 90:10) afforded the purified product 3b (79-82%). 1H NMR (400 MHz, DMSO) δ (ppm) 8.07 (d, J = 8.8 Hz, 1H), 6.07 (d, J = 3.2 Hz, 1H), 5.45 (d,J = 3.2 Hz, 1H), 5.25 (dd, J = 8.0, 2.8 Hz, 1H), 4.83 (AB series, 36.4, 12.8 Hz, 2H), 4.42-4.37 (m, 2H), 2.15 (s, 3H), 2.09 (s, 3H),1.83 (s, 3H).

[0182] Examples 1-6: Synthesis of 6-azido-6-deoxy-1,3-di-O-acetyl-N-acetyl-D-galactosamine (1) [ka]

[0182] Method A Crude 3b (1 equiv.) was dissolved in DMF (0.1–0.2 M) and NaN3 (1.2–5 equiv.) was added. The reaction mixture was stirred at room temperature and, upon completion (1–24 h, depending on the amount of NaN3 used), concentrated. The residue was dissolved in THF (0.2 M) and H2SO4 (1.2 equiv.) and HO (1.2 equiv.) were added. The reaction was stirred at room temperature (1–4 h) and analyzed by TLC (DCM:MeOH 9:1).

[0183]

[0183] Post-processing steps 0.2 M EtOAc, 0.4 M saturated aqueous NaHCO3, and 0.4 M water were added. The aqueous layer was extracted with 2× 0.2 M EtOAc, dried over Na2SO4, filtered, and concentrated under reduced pressure. Crude 1 was obtained in 80–100% yield. NMR analysis indicated that partial acetyl migration from the 3-OH to the 4-OH occurred, but crude 1 was used in the next step. Small-scale separation of these two regioisomers by silica column chromatography provided pure samples of compound 1 and its regioisomer 1' (with the 3-OH and 4-OAc groups) for NMR analysis.

[0184]

[0184] purification:After concentrating, blending Frothofluster (DCM: MeOH; 100:0→90:10) to obtain product 1 (61%).

[0185]

[0185] 1 ¹H-NMR (400 MHz, 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).

[0186]

[0186] 1 ¹H-NMR (400 MHz, 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).

[0187]

[0187] Method BCrude 3b (150 g, 408 mmol, 1 equiv.) was dissolved in DMF (1500 mL) and NaN (26.8 g, 412 mmol) was added. The reaction mixture was stirred at room temperature overnight, followed by the addition of 2-(bromomethyl)naphthalene (4.51 g, 20.4 mmol). The reaction was stirred for an additional 1 h and then concentrated 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 was then cooled in an ice bath, and sulfuric acid (26.1 mL, 490 mmol) was added dropwise. The reaction was stirred for 1 h at room temperature, followed by the addition of saturated aqueous NaHCO (1.2 L), and the mixture was extracted with EtOAc (7 × 1 L). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The residue was dissolved in EtOAc (1 L), dried over NaSO, filtered, and concentrated to give crude 1 as a yellow oil. NMR analysis indicated that partial acetyl migration from the 3-OH to the 4-OH had occurred, but the crude product 1 was used in the next step. Small-scale separation of these two regioisomers by silica column chromatography provided pure samples of compound 1 and its regioisomer 1' (with the 3-OH and 4-OAc groups) for NMR.

[0188]

[0188] 1 H-NMR (400 MHz, CDCl3)3-OAc (compound 1): δ(ppm) 6.19 (d, J= 3.6 Hz, 1H), 5.57 (d, J = 9.2Hz, 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, 8.8Hz, 2H), 2.81 (d, J = 3.6 Hz, 1H), 2.18 (s, 3H), 2.14(s, 3H), 1.95 (s, 3H).

[0189]

[0189] 1H-NMR (400 MHz, 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).

[0190] Examples 1-7: Synthesis of 6-O-tosyl-1,3,4-tri-O-acetyl-N-acetyl-D-galactosamine (3c) [ka] 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 h, additional p-toluenesulfonyl chloride (380 mg, 2.4 mmol) was added, and the reaction was stirred for an additional 1 h. The reaction mixture was concentrated under reduced pressure, followed by the addition of DCM (50 mL) and 1 M HCl (30 mL). After layer separation, the organic layer was washed with 1 M aqueous HCl (2 × 30 mL), dried over NaSO, filtered, and concentrated under reduced pressure. Purification by flash chromatography (DCM:MeOH = 100:0 to 94:6) afforded product 3c (1.45 g, 3.1 mmol, 72%) as a white solid. 1H -NMR (400 MHz, CDCl3): δ 7.77 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 6.11 (d, J = 4Hz, 1H), 5.54 (d, J = 9.6 Hz, 1H), 5.17 (dd, J = 2.8, 8.4 Hz, 1H), 4.77-4.71(m, 1H), 4.27 (AB-system, J = 3.2, 6.4 Hz, 1H), 4.06 (AB-system, J = 3.2, 6.4 Hz, 1H) 4.16-4.13 (m, 2H), 2.46 (s, 3H), 2.17 (s,3H), 2.11 (s, 3H), 1.93 (s, 3H).

[0191] Examples 1-8: Synthesis of 1 by nucleophilic substitution of 3c with azide followed by acetylation To compound 3c (1.4 g, 3 mmol) in DMF (15 mL) was added NaN (1 gram, 15 mmol), tetrabutylammonium iodide (575 mg, 1.5 mmol), and 15-crown-5 (341 mg, 1.5 mmol). The suspension was heated to 70 °C for 48 hours and then concentrated under reduced pressure. The crude mixture was dissolved in pyridine (20 mL), followed by the addition of acetic anhydride (3 mL, 30 mmol). The reaction was stirred at room temperature overnight. Next, DCM (30 mL) was added, and the reaction mixture was washed with 1 M aqueous HCl (3 × 20 mL) and saturated aqueous NaHCO (2 × 20 mL). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. Purification by flash chromatography (DCM:MeOH = 100:0 to 95:5) gave 1 (515 mg, 1.4 mmol, 47%). 1H -NMR (400 MHz, CDCl3): δ 6.24 (d, J = 4 Hz, 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, 5.6 Hz, 1H), 3.22 (AB-system, J = 7.2,5.6 Hz, 1H), 2.20 (s, 3H), 2.18 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H).

[0192] Examples 1-9: Synthesis of 6-O-mesyl-1,3,4-tri-O-acetyl-N-acetyl-D-galactosamine (3d) [ka] To a solution of compound 2 (100 mg, 0.327 mmol, 1.00 equiv) in MeCN (0.5 mL) was added DCM (1.0 mL), EtN (63.0 μL, 0.452 mmol, 1.4 equiv), and MsCl (27 μL, 0.349 mmol, 1.07 equiv). The resulting reaction mixture was stirred at room temperature and monitored by TLC analysis. The reaction was stirred for 1 h, after which TLC analysis indicated incomplete conversion. After this analysis, additional MsCl (10 μL) was added, followed after another hour by the addition of a further additional MsCl (10 μL) along with EtN (20 μL). The reaction mixture was stirred for an additional 30 min, after which DCM and saturated aqueous NHCl were added. The resulting mixture was shaken vigorously, and the resulting two-phase system was separated. The organic layer was washed with saturated aqueous NH4Cl (3x), then dried (Na2SO4), filtered, and concentrated in vacuo. The combined aqueous layers were further extracted with EtOAc (3x), and the combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was combined with the residue from the DCM extraction and purified by flash column chromatography (0-5% MeOH in DCM) to give compound 3d (40 mg, 32% yield). LCMS (ESI+) C 13 H 21NNaO 10 S + (M+Na + ) calculated value 406.08, actual value 405.95.

[0193] Example 1-10a: Synthesis of 1 by nucleophilic substitution of 3d with NBu4N3 To a mixture of 3d (20 mg, 49 μmol, 1.0 equiv) in MeCN (200 μL) was added NBuN (18 mg, 63 μmol, 1.3 equiv) followed by heating to reflux. The mixture was refluxed over the weekend. The resulting mixture was refluxed over the weekend and showed the formation of compound 1. LCMS (ESI+) C 10 H 15 N4O5 + (M-OAc - ) calculated value 271.1, actual value 271.23.

[0194] Example 1-10b: Synthesis of 1 by nucleophilic substitution of 3d with NaN3 To a mixture of 3d (20 mg, 49 μmol, 1.0 equiv) in DMF (200 μL) was added NaN (16 mg, 5 equiv) followed by heating to reflux. The mixture was refluxed over the weekend, which indicated the formation of compound 1. LCMS (ESI+) C 10 H 15 N4O5 + (M-OAc - ) calculated value 271.1, actual value 271.23.

[0195] Example 1-11: Synthesis of 1 by displacement of 3d with azide under Mitsunobu conditions [ka] Compound 2 (50 mg, 0.16 mmol) was mixed with PPh3 (58 mg, 0.22 mmol, 1.4 equiv.), diphenylphosphoryl azide (47 μL, 0.22 mmol, 1.4 equiv.) and diethyl azodicarboxylate (100 μL, 40 wt.% solution in toluene, 0.22 mmol, 1.4 equiv.) and taken up in cold THF (1.0 mL) while cooling in an ice bath. The reaction was left at 0° C. for 4 h and 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 equiv.), diphenylphosphoryl azide (24 μL, 0.11 mmol, 0.71 equiv.), and diethyl azodicarboxylate (49 μL, 40 wt. % solution in toluene, 0.11 mmol, 0.68 equiv.) were added. The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was then partially concentrated under reduced pressure and purified by silica gel column chromatography (DCM:MeOH = 100:0 to 95:5) to give crude compound 1 (38 mg). LCMS (ESI+) C 10 H 15 N4O5 + (M-OAc - ) calculated value 271.1, actual value 271.23.

[0196] Examples 1-12: Synthesis of 6-azido-6-deoxy-1,3,4-tri-O-acetyl-N-acetyl-D-galactosamine (6) [ka] [0196 ]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 was stirred at room temperature overnight and concentrated under reduced pressure. EtOAc (1 L) was then added, and the suspension was poured into stirred 1 M aqueous HCl (2.16 L). After 15 min of vigorous stirring, the layers were separated, and the aqueous layer was extracted once more with EtOAc (1 L). The combined organic layers were washed with brine (2 × 1 L), dried over NaSO, filtered, and concentrated under reduced pressure. Diethyl ether (1.2 L) was then added and heated to reflux. The mixture was cooled to room temperature with mechanical stirring. After stirring overnight, the solid was filtered and rinsed with diethyl ether (3 × 200 mL). After drying under a stream of nitrogen, product 6 (123 g, 333 mmol, 80% over two steps) was obtained as a white solid. Purification by flash chromatography (EtOAc:heptane; 50:50 to 100:0) afforded the product 6 (77% over 5 steps) as a white solid. 1 H NMR (400 MHz, 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 = 6.8Hz, 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).

[0197]

[0197] Method BThe crude product 1 (9 mg, 0.03 mmol) was dissolved in DCM (0.02 M) and acetic anhydride (5 equiv.), DMAP (0.1 equiv.), and EtN (5 equiv.) were added. The reaction was stirred at room temperature for 3 h. EtOAc (0.03 M) was then added, and the reaction mixture was washed with 1 M HCl (0.03 M) and saturated aqueous NaHCO (0.03 M). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to give product 6 (71%). 1 H NMR (400 MHz, 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 = 6.8Hz, 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).

[0198] Example 2-1: Synthesis of 6-azido-6-deoxy-3,4-tri-O-acetyl-N-acetyl-D-galactosamine (11) [ka]

[0198] Method A A solution of compound 6 (420 mg, 1.13 mmol) in anhydrous THF (11 mL) was cooled to 0 °C, followed by the addition of benzylamine (130 μL, 1.18 mmol, 1.04 equiv.). The resulting mixture was stirred at 0 °C for 2 h and then concentrated in vacuo. The residue was purified by flash chromatography (EtOAc:heptane; 60:40 to 100:0) to give 353 mg (95%) of compound 11. 1H NMR (400 MHz, CDCl3) δ (ppm) 6.24 (d, J = 3.6 Hz, 1H), 5.74 (d, J = 9.1 Hz, 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).

[0199]

[0199] Method B A 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 equiv.), and the resulting mixture was stirred until LCMS indicated the reaction was complete, typically for 18 h. The mixture was diluted with DCM (150 mL), washed sequentially with saturated aqueous NH4Cl and brine (100 mL each), dried (MgSO4), filtered, and concentrated. Flash column chromatography of the residue (80 g SiO2 cartridge; 50–100% EtOAc in petroleum ether 40–60) afforded compound 11 as a white foam (1.64 g, 4.96 mmol, 83% yield). NMR analysis indicated that compound 11 was a 4:1 α / β anomeric mixture and contained BnNHAc (>5%).

[0200]

[0200] Method C A stirred solution of compound 6 (1.80 g, 4.83 mmol) in anhydrous THF (24 mL) was treated with dimethylamino-propylamine (DMAPA, 0.9 mL, 7.2 mmol, 1.5 equiv.), and the resulting mixture was stirred until LCMS indicated the reaction was complete, typically 18 h. The yellow mixture was diluted with MeOH (75 ml) and IR120 (H) was added until LCMS indicated the disappearance of DMAPA and its acetamide by-product (along with the yellow color). +The resulting mixture was treated with a (meth)acrylic acid (form) resin. Filtration, washing with MeOH, and concentration of the filtrate afforded compound 11 as a white foam (1.64 g, 4.96 mmol, 92% yield). NMR analysis indicated that compound 11 was a 4:1 mixture of α / β anomers.

[0201]

[0201] Method D A 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 equiv.), and the resulting mixture was stirred until LCMS indicated the reaction was complete, typically 18 h. The yellow mixture was diluted with DCM (40 mL), washed sequentially with 1 M HCl and brine (15 mL each), and dried (MgSO). Filtration and concentration afforded compound 11 as a white foam (130 mg, 0.39 mmol, 73% yield). NMR indicated that compound 11 was a 4:1 α / β anomeric mixture and contained residual THF (approximately 10%).

[0202] Example 2-2: Synthesis of (3aR,5R,6S,7R,7aR)-5-(azidomethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetate (7) Method A [ka] The reaction was carried out in a 1000 ml three-neck flask that had been dried overnight in an oven above 100°C and flushed with nitrogen before use. Compound 6 (16.6 g, 44.6 mmol) was dissolved in anhydrous dichloromethane (500 mL) in the flask. 2,4,6-collidine (10 ml, 80 mmol) was added slowly via syringe to the solution. The reaction mixture was stirred for 10 minutes, and bromotrimethylsilane (10 ml, 76 mmol) was added slowly via a dropping funnel to the clear solution. After 5 minutes, boron trifluoride etherate (20 mL of approximately 48% BF3.Et2O, 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 indicated nearly complete conversion. The heat was turned off, and the mixture was allowed to reach room temperature overnight. TLC analysis indicated complete consumption of the starting material. The mixture was cooled to 0 °C in an ice bath and slowly poured into 1000 mL of ice-cold saturated NaHCO solution under gentle stirring. Vigorous CO evolution was observed. The layers were separated, and the aqueous layer was extracted twice more (2 × 500 mL DCM). The organic layers were combined, dried over NaSO, and concentrated under reduced pressure. The mixture was coated onto a hydromatrix (inert diatomaceous earth adsorbent, 40 grams) and purified by flash column chromatography (heptane: EtOAc = 70:30 → 0:100, containing 1% EtN) to give compound 7 (13.1 g, 39.2 mmol, 88% yield) as an off-white solid. 1 H-NMR (400 MHz, CDCl3): δ 6.02 (d, J = 7.2 Hz, 1H), 5.40 (t, J = 3.2 Hz, 1H), 4.95 (dd, J =3.6, 3.6 Hz, 1H), 4.13-4.09 (m, 1H), 4.06-4.03 (m, 1H), 3.48 (AB system, J = 5.2, 7.6 Hz, 1H), 3.27 (AB system, J = 5.2,8 Hz, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.07-2.04 (m, 3H).

[0203]

[0203] Alternative post-processing proceduresThe reaction mixture was cooled in an ice bath, after which EtN (5 equiv.) was added. After stirring for 5 min, the resulting reaction mixture was directly loaded onto a silica column and purified by flash column chromatography (heptane:EtOAc 50:50 to 10:90). After concentration of the appropriate column fractions, the residue was dissolved in DCM (0.1 M) and washed with 2% aqueous citric acid (2 × 0.2 M). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to afford compound 7 (87–89% yield) as a yellow oil.

[0204]

[0204] Extraction procedure for selective removal of collidine The crude reaction mixture was concentrated in vacuo and redissolved in EtOAc (100 mL). The resulting suspension was filtered, and the filtrate was washed with 5% aqueous CuSO (3 × 100 mL). The organic phase was washed with brine, dried (Na SO ), and concentrated in vacuo. The residue could be used crude in the next step or purified by silica gel column chromatography (heptane: EtOAc = 70:30 → 0:100, containing 1% Et N) to give compound 7 (1.31 g, 95% purity by qNMR, 58% yield).

[0205] Method B [ka] To a flame-dried round-bottom flask containing a stir bar and molecular sieves was added compound 6 (201 mg, 0.540 mmol, 1.00 equiv.), followed by dichloromethane (6.0 mL). The resulting solution was cooled to 0 °C, and 2,4,6-collidine (220 μL, 1.62 mmol, 3.0 equiv.), chlorotrimethylsilane (206 μL, 1.62 mmol, 3.0 equiv.), and boron trifluoride etherate (210 μL, 1.62 mmol, 3 equiv.) were added sequentially. The resulting mixture was heated to 35–40 °C for 23 h. The reaction was then cooled to 0 °C and quenched with EtN (375 μL, 2.70 mmol, 5.0 equiv.). The resulting mixture was stirred for 5 min and then directly purified by silica gel column (pentane: EtOAc = 50:50 → 10:90) to give compound 7 (137 mg, 93% purity by H-NMR, 76% yield) as a yellow oil. 1 H NMR (CDCl3): 6.02(d, J = 7.1 Hz, 1H), 5.40 (t, J = 3.2 Hz, 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).

[0206] Method C [ka] To a solution of compound 6 (1 equiv.) in anhydrous 1,2-dichloroethane (0.1 M) was added TMSOTf (1.5 equiv.) at room temperature under a N atmosphere. The resulting reaction mixture was heated to 50°C and monitored by TLC (DCM:MeOH 9:1) until the reaction was complete (optionally, up to 3 equiv. of additional TMSOTf was added). After TLC analysis indicated complete conversion (4-16 h), the reaction mixture was cooled to 0°C, followed by the dropwise addition of EtN (1.5-5 equiv.) over 2 min and stirring for an additional 10 min. The resulting reaction mixture was directly purified by silica gel column chromatography (heptane:EtOAc = 50:50 → 10:90, containing 1% EtN) to afford compound 7 (62-90% yield) as a pale yellow oil. 1 H NMR (CDCl3): 6.02(d, J = 7.1 Hz, 1H), 5.40 (t, J = 3.2 Hz, 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).

[0207] Example 3-1a: Synthesis of dihydrophosphoryl 6-azido-6-deoxy-3,4-tri-O-acetyl-N-acetyl-d-galactosaminopyranose (5a) in neat phosphoric acid [ka] Compound 7 (4 g, 10.7 mmol) was placed under a N atmosphere, followed by the addition of phosphoric acid (8.5 g, 91 mmol, pre-dried over PO). The reaction was placed under vacuum over PO and heated to 60 °C. After 5 h, the reaction was cooled to room temperature, and THF (50 mL) was added. The mixture was then cooled to 0 °C and neutralized to pH 7 with NHOH. The solids were removed by filtration, rinsed with THF, and the filtrate was concentrated under reduced pressure. Purification by flash chromatography (MeCN:MeOH = 100:0 to 50:50) afforded product 5a (888 mg, 2.2 mmol, 20%) in a 1:1 α:β ratio. 1 H -NMR (400 MHz, CDCl3): δ 5.47-5.44 (m, 1H), 5.35-5.34 (m, 1H), 5.25-5.24 (m, 1H), 5.11 (dd,J = 3.2, 8.7 Hz, 1H), 5.03 (t, J = 8.4 Hz, 1H), 4.97 (dd, J = 3.2, 8 Hz, 1H),4.41-4.36 (m, 1H), 4.27-4.30 (m, 1H), 4.09-4.04 (m, 1H), 3.84-3.80 (m, 1H),3.49-3.38 (m, 4H), 2.07-2.06 (m, 6H), 1.88-1.85 (m, 12H). [Table 1]

[0208] Example 3-1b: Synthesis of dihydrophosphoryl 6-azido-6-deoxy-3,4-tri-O-acetyl-N-acetyl-d-galactosaminopyranose (5a) with phosphoric acid in DMF [ka] In a 250 ml flask, 7 (12.8 g, 38.9 mmol) was dissolved in anhydrous DMF (80 ml), and phosphoric acid (2.58 M solution in DMF, 91 ml, 234 mmol) was added via syringe. The solution was reacted at 80° C. overnight. 1H-NMR analysis (in CD3OD) indicated complete consumption of the starting material and the formation of primarily the desired α-anomer of 5a. The reaction mixture was concentrated under reduced pressure, coated onto a hydromatrix (inert diatomaceous earth adsorbent, 40 grams), and purified on a C18 silica gel column (water:MeCN 100:0 → 90:10, containing 0.1% HCOOH). The combined product-containing batches were concentrated under reduced pressure and stripped three times with toluene to give product 5a (10.1 g, 23.3 mmol, 60% yield) as a white solid. 1 H-NMR (400 MHz, 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 hidden by solvent peak), 2.16 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H).

[0209]

[0209] Alternative Post-Processing The reaction mixture was concentrated under reduced pressure, followed by the addition of DCM (0.03 M), MeOH (0.15 M), and 4-methylmorpholine (3.5 equiv.). 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), which resulted in the formation of a precipitate. The mixture was centrifuged at 10,000 rpm for 5 min. Concentration of the liquid afforded compound 5a (53–76%) as an off-white solid.

[0210]

[0210] Alternative Post-Processing After heating the reaction mixture at 80 °C for 24 h, the reaction was cooled to room temperature, EtN (6.2 equiv.) was added, and the solution was concentrated in vacuo. The residue was dissolved in 5% MeCN in water (0.1 M) and purified on silica-C18 (gradient 16-34% B, A = water, B = 30% MeCN in water) to give compound 5a (56%) as a fluffy white solid.

[0211]

[0211] Alternative Post-Processing After heating the reaction mixture at 80 °C for 24 h, the reaction mixture was concentrated in vacuo. The thick brown syrup was taken up in a mixture of DCM and MeOH (82:18 ratio, 0.07 M), followed by the addition of 4-methylmorpholine (6.4 equiv.). The mixture was then concentrated in vacuo, and the resulting residue was taken up in a minimal amount of EtOAc / MeOH / H2O (6 / 2 / 1). The suspension was transferred to a silica column and subsequently purified by flash column chromatography (using a stepwise gradient from 6 / 2 / 1 to 2 / 2 / 1 EtOAc / MeOH / H2O). Compound 5a (59% yield) was obtained as a brown solid. 1 H NMR (400 MHz, CD3OD) δ (ppm): 5.70-5.60 (m, 1H), 5.49 (d, J = 2.3 Hz, 1H), 5.23 (dd, J =11.4 Hz, 3.0 Hz, 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).

[0212]

[0212] Method B (less phosphoric acid in DMF and IEX-purified) To a flame-dried round-bottom flask equipped with a stir bar, compound 7 (2.1 g, 6.37 mmol, 1 equiv.) was added, followed by anhydrous DMF (2.72 mL). The resulting pale yellow liquid was stirred at room temperature, followed by the addition of a 6.6 M solution of H3PO4 in anhydrous DMF (1.93 mL, 12.73 mmol, 2.0 equiv.). The resulting reaction mixture was heated to 80 °C under N2 (by placing in a preheated oil bath) and stirred for 135 min. Note: Prior to workup, the sample was 1The α:β ratio should be evaluated by H NMR to determine whether it is ≥10:1 (typically achieved after 135 min). To this end, approximately 20 μL of the reaction mixture was dissolved in 0.5 mL of MeOD-d3. Once this ratio was achieved, the reaction mixture was cooled to room temperature and then quenched by adding it to a stirred aqueous solution of 20 mM NH4HCO3 (1.27 L, 4 equiv.). Any residual product can be removed from the reaction vessel by washing with several mL of anhydrous DMF, which can then be added to the aqueous NH4HCO3 solution. The resulting solution was then loaded onto a 300 mL Q-Sepharose Fast Flow® ion-exchange column (A: 10 mM ammonium bicarbonate, B: 250 mM ammonium bicarbonate) at 10 mL / min. The column was then washed with 10 mM ammonium bicarbonate, followed by a gradient to 40% B to elute the product. The product-containing fractions were concentrated under vacuum to remove most of the buffer, followed by overnight lyophilization of the remaining buffer to give 6-N3-GalNAc-monophosphate (5a, 1.85 g, corrected 1.56 g (based on qNMR), 0.41 mmol, 58.1% yield) as an off-white solid. 1 H NMR (400 MHz, CD3OD) δ (ppm): 5.70-5.60 (m, 1H), 5.49 (d, J = 2.3 Hz, 1H), 5.23 (dd, J =11.4 Hz, 3.0 Hz, 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).

[0213] Example 3-2: Preparation of 5a' (NBu3 salt of 5a) by quenching with NBu3 [ka]

[0213] Method AAfter heating the reaction mixture at 80 °C for 24 h, the reaction was cooled to room temperature, BuN (6.2 equiv.) was added, and the solution was concentrated in vacuo. The residue was dissolved in 5% MeCN in water (0.1 M) and purified on silica-C18 (gradient 16-100% B, A = water, B = 30% MeCN in water) to give compound 5a' (117 mg, 44% pure by H-NMR, 37% yield) as a yellow oil.

[0214] Similarly, quenching with EtN, 4-methylmorpholine, or NHHCO affords Each salt of compound 5a was obtained in the same manner as described in Example 3-1.

[0215] Example 3-3: Deacetylation of Compound 5a to Form Compound 5b [ka] To a suspension of the acetylated sugar 5a (4.9 g, 11.9 mmol) in MeOH (15 mL) was added 25% NH4OH (60 mL). The reaction was stirred at room temperature and the conversion was monitored by LCMS. After 4 h, the mixture was concentrated under reduced pressure and stored at -20 °C for 2 days. The solid was then dissolved in 25% aqueous NH4OH (75 mL) and stirred at room temperature; after 3 h, MS showed complete conversion. Concentration of the solvent gave crude product 5b (3.2 g, 9.9 mmol, 83%) as a yellow solid. 1 H-NMR (400 MHz, D2O): δ 5.28 (dd, J = 7.2, 3.2 Hz, 1H), 4.12 (dd, J = 6.8, 6.4 Hz, 1H),4.06 (ddd, J = 10.8, 3.2, 2.0 Hz, 1H), 3.92-3.81 (m, 2H), 3.47 (AB system, J = 12.8, 7.2 Hz, 1H), 3.40 (AB system, J =12.8, 6.4 Hz, 1H), 1.88 (s, 3H).

[0216] Example 4-1: Exchange of UMP disodium salt with UMP tributylammonium salt (UMP.NBu3) [ka] Dowex® 50WX8 50-100 mesh (250 g) was placed in a 500 mL glass filter and washed with demineralized water (3×, 500 mL). It was then mixed with UMP disodium salt (40.0 g, 108 mmol) in water (250 mL), and the resulting suspension was stirred at room temperature for 2 hours. The Dowex® 50WX8 50-100 mesh was removed by filtration and rinsed with demineralized water (3×, 30 mL). To the resulting filtrate, tributylamine (25.9 mL, 108 mmol) was added, and the mixture was stirred vigorously for 60 minutes. The clear solution was lyophilized overnight and then dried over PO overnight. The resulting UMP tributylammonium salt (25.9 g, 1 The batch was obtained as a white solid with a purity of 91% by 1H-qNMR and a yield of 80%. 1 H NMR (400 MHz, D2O) δ (ppm) 7.79 (d, 1H, J = 8 Hz), 5.81 (d, 1H, J = 4.4 Hz), 5.77 (d,1H, J = 8 Hz), 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 (sextet, 6H, J= 7.2 Hz), 0.77 (t, 9H, J = 7.2 Hz).

[0217] Example 4-2: Synthesis of a mixture of UDP derivatives 8 and 8' from 5a [ka] Preparation of the anhydrous phosphoric acid salt: UMP tributylamine salt (12.5 g, 22.9 mmol) was suspended in toluene (100 mL) and concentrated to dryness on a rotary evaporator under nitrogen. This procedure was repeated three times. The same procedure was carried out with 5 (10.0 g, 23.1 mmol).

[0218] Under a nitrogen atmosphere, anhydrous UMP tributylamine salt (12.5 g, 22.9 mmol) was dissolved in anhydrous DMF (100 mL) and CDI (9.4 g, 57.9 mmol) was added (in one portion). The yellow mixture was stirred at room temperature for 1 h. A solution of anhydrous 5a (10.0 g, 23.2 mmol) in anhydrous DMF (40 mL) was added to the reaction mixture via syringe. 1-Methylimidazolium chloride (2 M solution in anhydrous DMF, 24 mL, 48.0 mmol) was then added, and the reaction mixture was stirred at room temperature under a nitrogen atmosphere. After 4 h of reaction time, LCMS analysis of the crude reaction mixture indicated 10% unconsumed starting material. Therefore, 0.1 equivalents of CDI-activated UMP tributylamine salt was prepared as follows: In a 100 ml flask under nitrogen, 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 then injected into the main reaction mixture via syringe. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The material (8 / 8 mixture) was used in the next step without further purification.

[0219] Example 4-3: Silica gel separation of a mixture of 8 and 8' to obtain a pure sample of 8' The crude reaction mixture was resuspended in a minimal amount of eluent (8:2:1 EtOAc:MeOH:water), to which a minimal amount of methanol was added to dissolve the product, and the mixture was purified by flash column chromatography (8:2:1 EtOAc:MeOH:water → 2:2:1) to give compound 8' (14.2 g, 15.5 mmol, 67% yield) as an off-white solid. 1H-NMR (400 MHz, D2O): δ 7.63 (d, J = 8 Hz, 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, 6.4 Hz, 1H), 3.26 (AB system, J = 6.4, 6.4 Hz, 1H), 2.10 (s, 3H), 1.88 (s, 3H), 1.87 (s, 3H).

[0220] Example 4-4: Selective hydrolysis of mixture 8 and 8' and flash C18 purification to give 8, followed by deprotection to give pure 9a. [ka] To a round-bottom flask containing crude 8' (808 μmol based on the amount of 5a used in the previous step), HO (37 mL) was added and left for several minutes to allow the HO to react with any remaining CDI. To the resulting suspension, NH4HCO3 (436 mg, 5.51 mmol, 6.8 equiv) was added to produce a 150 mM aqueous solution of NH4HCO3. The reaction mixture was then heated to 40 °C for 20 h. The progress of the reaction was monitored by HPLC, which showed complete conversion after stirring overnight (20 h). After complete conversion, the reaction mixture was concentrated in vacuo to give crude 8, which was then purified according to the flash C18 column chromatography protocol discussed below.

[0221] Crude 8 (0.834 mmol based on the amount of 5a in the UMP coupling) was dissolved in 4 mL of 5% MeCN in 200 mM aqueous EtN.HOAc (190 mM effective). The resulting solution was then concentrated in vacuo, and the residue was redissolved in 4 mL of 5% MeCN in 200 mM aqueous EtN.HOAc. This solution was purified by flash C18 column chromatography using a 25 g C18 cartridge (5% MeCN in 190 mM aqueous EtN.HOAc → 30% MeCN in HO). The corresponding pure fractions were combined and concentrated in vacuo to give an orange oil. The material (8 and a small mixture of 8″ and 8′″) was used in the next step without further purification.

[0222] To a round-bottom flask containing flash C18 purified 8 (2.87 g, corrected 269 mg (qNMR), 0.375 mmol) in HO (12 mL) was added MeOH (12 mL) and EtN (12 mL). The resulting solution was heated to 45 °C for 22 h and then concentrated in vacuo to give 2.748 g of a yellow oil. The residue was then subjected to Dowex treatment as described below.

[0223] Crude 9a (2.75 g, ca. 9% 9a and ca. 40% AcOH ( 1 To a round-bottom flask containing 9a (H-NMR) was added Dowex MAC-3 hydrogen form (16.5 g). The resulting suspension was stirred at room temperature for 90 min and filtered through a P3 glass filter. The residue was washed with HO (3 × 25 mL). The filtrate was concentrated in vacuo. The resulting residue (984 mg) was co-evaporated with HO (1 × 20 mL) to give 9a (703 mg, 32.2% pure by qNMR) as a yellow oil. 1H-NMR (400 MHz, D2O): δ 7.82 (d, J = 8.4 Hz, 1H), 5.85-5.78 (m, 2H), 5.40 (dd, J = 3.6, 3.6Hz, 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).

[0224] Example 4-4: Synthesis of 9a (triethylammonium salt) by deacetylation of a mixture of 8 and 8' [ka]

[0224] 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. The mixture was first 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 starting material) with Buffer A. The product was separated from the by-products by a stepwise gradient (first to 10% B (250 mM NH4HCO3) in 20 min, then to 40% B in 120 min). Lyophilization of the product fractions afforded UDP-6-azidoGalNAc triethylammonium salt (9a). This intermediate was taken up in HO (0.2 M) and purified by C18-HPLC using a Phenomenex Luna 10u C18 (2), 250 × 50 mm column (A: 50 mM EtN.HOAc, pH 6.8, B: MeCN). The collected fractions were combined, co-evaporated with HO several times, and finally lyophilized to give UDP-6-N3-GalNAc (9a) as a brittle off-white solid (53%, starting from a mixture of 8 and 8'). Method BIn a 1 L single-neck flask, 8' (14.0 g, 15.3 mmol) was dissolved in EtN / MeOH / water (300 mL, 1:1:1) and stirred overnight at room temperature. The crude mixture was partially concentrated under reduced pressure to 23 grams of 9 (37% purity by H-qNMR, 88% yield) and dissolved in 140 mL of water. This solution of 9a was subjected to preparative LCMS (eluent: 0-4% ACN in 50 mM EtN.HOAc in water, pH = 6.8). The product-containing fractions were combined to give 9 (9 L of solution in 50 mM EtN.HOAc buffer). The combined fractions (9 L) were split into two portions, and one portion (4.5 L) was loaded onto a 1 L Q-Sepharose Fast Flow® ion-exchange column (A: 25 mM ammonium bicarbonate, B: 250 mM ammonium bicarbonate) at 30 mL / min. The column was then washed with 25 mM ammonium bicarbonate solution, followed by elution of the product with a gradient to 100% B. The product fractions were pooled 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, 1 H-qNMR revealed a purity of 92%, 12.8 mmol, and a yield of 84%. 1 H-NMR (400 MHz, D2O): δ 7.82 (d, J = 8.4 Hz, 1H), 5.84-5.81 (m, 2H), 5.39 (dd, J = 3.6, 3.6Hz, 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).

[0225] Example 4-4. Coupling of 5b with UMP tributylammonium salt by CDI [ka] UMP-NBu3 (2.22 g, 4.36 mmol) was dissolved in DMF (25 mL), CDI (1.17 g, 7.2 mmol) was added, and the reaction was stirred for 30 min. MeOH (177 μL, 4.36 mmol) was added, and the reaction was stirred for 15 min. The reaction was placed under vacuum for 15 min. 6-Azido-6-deoxy-GalNAc-1-monophosphate 5b (1.60 g, 4.9 mmol) in DMF (25 mL) was then added, followed by NMI.HCl (2.25 g, 14.4 mmol). After stirring overnight, an additional amount of activated UMP-NBu4 was added. Therefore, in a separate flask, UMP-NBu3 (700 mg, 1.4 mmol) was dissolved in DMF (10 mL), and CDI (371 mg, 2.3 mmol) was added. This mixture was stirred for 5 minutes and added to the reaction. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude mixture was dissolved in water and divided into three portions. One portion was loaded onto a 300 mL Q-Sepharose Fast Flow® ion exchange column (A: 25 mM ammonium bicarbonate, B: 250 mM ammonium bicarbonate) at 10 ml / min. The column was then washed with 25 mM ammonium bicarbonate solution, followed by elution of the product with a gradient to 40% B. The product fractions were pooled 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, 1 H-qNMR revealed a purity of 47%, 2.0 mmol, and a yield of 41%. 1 H-NMR (400 MHz, D2O): δ 7.82 (d, J = 8.4 Hz, 1H), 5.84-5.81 (m, 2H), 5.42 (dd, J = 3.6, 3.6Hz, 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).

[0226] Examples 4-5: Synthesis of UMP-imidazole phosphate ester 10 from UMP.NBu3 [ka] To a round-bottom flask containing UMP tributylammonium salt (UMP.NBu3, 6.05 g, 91.4% by QNMR, 10.84 mmol, 1.00 equiv.) and imidazole (7.38 g, 108.4 mmol, 10.0 equiv.) was added anhydrous DMF (55.0 mL), followed by 2,2-dithiopyridine (3.36 g, 15.25 mmol, 1.41 equiv.). To the resulting colorless solution was added Et3N (6.05 mL, 43.4 mmol, 4 equiv.), followed by PPh3 (8.53 g, 32.5 mmol, 3.00 equiv.), resulting in the formation of a yellow solution. The mixture was stirred under N2 for 2 h and then cooled to 0 °C in an ice bath. To the cold reaction mixture was added 553 mL of a 0.106 M solution of NaClO4 in acetone. The resulting yellow suspension was cooled to 0 °C in an ice bath, then filtered through a glass filter and washed with cold anhydrous acetone (3x). The residue was concentrated in vacuo and dried over P2O5 to give 4.68 g of an off-white solid (88.7% pure by qNMR, corrected 4.15 g, 10.47 mmol, 96.7% yield). 1 H NMR (400 MHz, D2O) δ (ppm): 7.93 (s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.32 (q, J = 1.4 Hz,1H), 7.13-7.01 (m, 1H), 5.96 (d, J = 5.3 Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H),4.18 (t, J = 5.0 Hz, 1H), 4.14-3.94 (m, 4H), 3.07-2.97 (m, 1H), 2.93-2.87 (m,1H).

[0227] Examples 4-6: Synthesis of 8 from 5a and 10 [ka] To a flame-dried round-bottom flask equipped with a stir bar was added 6-N3-sugar monophosphate 5a (2.346 g, 77.6% by qNMR, 4.437 mmol, 1.00 equiv) and anhydrous DMF (27.5 mL). The resulting solution was cooled in an ice bath, and MgCl (486 mg, 5.105 mmol, 1.15 equiv) was added. The resulting mixture was stirred for 13 min, forming a fine suspension. The ice bath was removed, and the reaction mixture was allowed to warm gradually to room temperature. 10 (2.282 g, 88.7% by qNMR, 1.15 equiv) was then added portionwise over 30 s with stirring. The resulting pale yellow suspension was stirred at room temperature for 18 h. The reaction mixture was then concentrated in vacuo to give 9.20 g of a cloudy yellow oil, which was used in the next step without further purification.

[0228] Example 4-7: Synthesis of UDP 6-azido-GalNAc (9) by deprotection of 8 [ka] Crude 8 was dissolved in HO (32 mL) and MeOH (32 mL), followed by the addition of EtN (32 mL). The resulting cloudy yellow suspension was stirred at room temperature for 22 h and then concentrated in vacuo to give a cloudy yellow oil. The residue was dissolved in HO (222 mL) and slowly aspirated onto a plug of DEAE Sephadex A-25. The DEAE plug was prepared as follows: a glass filter P3 (6 cm diameter) was loaded with DEAE Sephadex A-25 (12.5 g), suspended in HO, and washed with 1 M NHHCO, followed by HO. This resulted in a 3.5 cm-tall plug (approximately 100 mL) of DEAE Sephadex. The DEAE plug was then loaded with a solution of crude 9 (approximately 355 μmol product / g resin) in HO (222 mL) using minimal vacuum over a 15 min period. The plug was washed with demineralized water (60 mL, 3×), followed by 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 HO, and then lyophilized to give 9 (3.986 g, 69.2% by qNMR, 4.36 mmol, 98.3% yield) as an off-white solid. 1 H NMR (400 MHz, D2O) δ (ppm): 7.88 (d, J = 8.2 Hz, 1H), 5.94-5.84 (m, 2H), 5.46 (dd, J =7.2, 3.4 Hz, 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, 7.3 Hz, 1H), 3.41 (dd, J = 12.8, 7.3 Hz, 1H), 2.00 (s, 3H).

[0229] Example 5-1: Synthesis of 6-azido-1-(di-2-cyanoethyl)phosphate-GalNAc (12) [ka]

[0229] Method AA round-bottom flask containing hemiacetal compound 11 (1.20 g, 3.63 mmol) and a 0.45 M solution of 1-H-tetrazole in acetonitrile (32 mL, 14.5 mmol, 4 equiv.) was concentrated and coevaporated from anhydrous toluene (3 × 20 mL). The residue was taken up in anhydrous DCM (40 mL), cooled (−5 °C), and treated dropwise with a solution of 2-cyanoethyl-N,N′-diisopropyl-phosphorochloroamidite (2.0 mL, 7.27 mmol, 2 equiv.) in anhydrous DCM (4.0 mL) over 15 min with stirring under Ar(g). The reaction was gradually warmed to room temperature, and stirring was continued for an additional 80 min before being cooled (−40 °C) and treated with mCPBA (2.10 g, 9.08 mmol, 2.5 equiv.). The reaction was allowed to warm gradually to room temperature, and stirring was continued for an additional 30 min before being 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. Flash column chromatography of the residue (80 g SiO2 cartridge; 70–100% EtOAc in petroleum ether 40–60, then 0–25% MeCN in EtOAc) gave first oxazoline (F2) (100 mg, 0.32 mmol, 8.8% yield) and then compound 12 (F1) as a white foam (1.10 g, 2.13 mmol, 59% yield).

[0230]

[0230] Method BTo a cooled (-5 °C) and stirred mixture of hemiacetal compound 11 (547 mg, 1.65 mmol) and 4,5-dicyanoimidazole (781 mg, 6.62 mmol, 4 equiv.) in anhydrous DCM (15 mL) under Ar(g), a solution of 2-cyanoethyl-N,N'-diisopropyl-phosphorochloroamidite (0.9 mL, 3.31 mmol, 2 equiv.) in anhydrous DCM (2.0 mL) was added dropwise with stirring over 15 min. The reaction was gradually warmed to room temperature and stirring was continued for an additional 80 min before being cooled (-40 °C) and treated with mCPBA (927 mg, 4.14 mmol, 2.5 equiv.). The reaction was gradually warmed to room temperature and stirring was continued for an additional 30 min before 10% sodium thiosulfate solution (10 mL) was added. The organic layer was washed with saturated NaHCO3 solution (10 mL), dried (MgSO4), filtered, and concentrated. Flash column chromatography of the residue (40 g SiO2 cartridge; 70-100% EtOAc in petroleum ether 40-60, then 0-50% MeCN in EtOAc) afforded compound 3 as a white foam (540 mg, 1.05 mmol, 63% yield). NMR indicated compound 12 was 98% pure, and LCMS (ELSD) indicated compound 12 was 95% pure.

[0231]

[0231] Method CTo a cooled (-5 °C) and stirred mixture of hemiacetal compound 11 (547 mg, 1.65 mmol) and 4,5-dicyanoimidazole (781 mg, 6.62 mmol, 4 equiv.) in anhydrous DCM (15 mL) under Ar(g), a solution of 2-cyanoethyl-N,N'-diisopropyl-phosphorochloroamidite (0.9 mL, 3.31 mmol, 2 equiv.) in anhydrous DCM (2.0 mL) was added dropwise with stirring over 15 min. The reaction was gradually warmed to room temperature and stirring was continued for an additional 80 min before being cooled (-40 °C) and treated with mCPBA (927 mg, 4.14 mmol, 2.5 equiv.). The reaction was gradually warmed to room temperature and stirring was continued for an additional 30 min before 10% sodium thiosulfate solution (10 mL) was added. The organic layer was washed with saturated NaHCO3 solution (10 mL), dried (MgSO4), filtered, and concentrated. Flash column chromatography of the residue (40 g SiO2 cartridge; 70-100% EtOAc in petroleum ether 40-60, then 0-50% MeCN in EtOAc) afforded compound 3 as a white foam (540 mg, 1.05 mmol, 63% yield). NMR indicated compound 12 was 98% pure, and LCMS (ELSD) indicated compound 12 was 95% pure.

[0232]

[0232] Method DTo a cooled (-5 °C) and stirred mixture of hemiacetal compound 11 (547 mg, 1.65 mmol) and BnS-1-H-tetrazole (1.27 g, 6.62 mmol, 4 equiv.) in anhydrous DCM (15 mL) under Ar(g), a solution of 2-cyanoethyl-N,N'-diisopropyl-phosphorochloroamidite (0.9 mL, 3.31 mmol, 2 equiv.) in anhydrous DCM (2.0 mL) was added dropwise with stirring over 15 min. The reaction was gradually warmed to room temperature and stirring was continued for an additional 80 min before being cooled (-40 °C) and treated with mCPBA (927 mg, 4.14 mmol, 2.5 equiv.). The reaction was gradually warmed to room temperature and stirring was continued for an additional 30 min before 10% sodium thiosulfate solution (10 mL) was added. The organic layer was washed with saturated NaHCO3 solution (10 mL), dried (MgSO4), filtered, and concentrated. Flash column chromatography of the residue (40 g SiO2 cartridge; 70-100% EtOAc in petroleum ether 40-60, then 0-50% MeCN in EtOAc) afforded compound 12 as a white foam (362 mg, 0.70 mmol, 43% yield). Both NMR and LCMS (ELSD) indicated that compound 12 was >90% pure.

[0233] Example 5-2: Synthesis of 6-azido-1-phosphate-GalNAc (5b) [ka] Compound 12 (1.60 g, 3.10 mmol) was treated with an 8:35:57 mixture of TEA / MeOH / water (100 mL) at 35 °C until LCMS showed the reaction was complete (3 days). The mixture was concentrated to a partially aqueous fraction on a rotary evaporator and then freeze-dried. The freeze-drying was repeated (twice) until excess triethylamine was removed (as determined by NMR). The residue was triturated 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.

[0234] Example 5-3: Coupling of 5b with UMP-morpholidate [ka]

[0234] Method A To a flame-dried round-bottom flask equipped with a stir bar under argon (g) was added compound 5b (81 mg, 0.15 mmol, 1.00 equiv.), anhydrous DMF (2.0 mL), UMP-morpholidate (116 mg, 0.17 mmol, 1.1 equiv.), and 4,5-dicyanoimidazole (DCI, 56 mg, 0.48 mmol, 3.2 equiv.). The resulting solution was stirred at 35° C. for 20 h. LCMS of the crude mixture indicated that starting material 5b had not been consumed. Additional starting material UMP-morpholidate (35 mg, 0.3 equiv.) was added, and the reaction was stirred for an additional 6 h. LCMS indicated the level of 5b was 2.5%. The mixture was treated with MeCN (10 mL), then centrifuged and the liquid decanted (this process was repeated once more), yielding a white solid, which was absorbed in water and added to DEAE-Sepharose media (2.4 × 13 cm), which was eluted sequentially with 50 mL each of water and increasing concentrations of triethylammonium bicarbonate (TEAB) at 100 mM, 200 mM, 300 mM, and 400 mM. The main bulk of the compound eluted in the 300 mM TEAB fraction and freeze-dried to give 120 mg of compound 9, which contained compound 5b by LCMS (based on the presence of the compound 5b mass ion in -ve mode). The residue was absorbed in water and subjected to C18-chromatography (30 g cartridge; eluent = 100% 10 mM TEAB). First, a 2:1 mix of UMP and compound 5b (11 mg) was obtained, followed by compound 9 (65 mg, 51%), which was pure by NMR and LCMS.

[0235]

[0235] Method BTo a flame-dried round-bottom flask equipped with a stir bar under argon (g) was added compound 5b (53 mg, 0.10 mmol, 1.00 equiv.), anhydrous DMF (1.5 mL), UMP-morpholidate (90 mg, 0.13 mmol, 1.3 equiv.), and DCI (42 mg, 0.36 mmol, 3.6 equiv.). The resulting solution was stirred at 35 °C for 20 h. LCMS of the crude mixture indicated that the starting UMP-morpholidate had been consumed. The mixture was treated with acetone (10 mL), followed by centrifugation and decantation of the liquid (this process was repeated once more) to give a white solid, which was taken up in water and added to DEAE-Sepharose medium (2.4 × 13 cm), which was sequentially eluted with 50 mL each of water and increasing concentrations of TEAB (100 mM, 200 mM, 300 mM, and 400 mM). The main bulk of the compound was eluted in the 300 mM TEAB fraction and freeze-dried to give 102 mg of compound 9, which contained 15% of compound 5b by LCMS (ELSD), while NMR indicated that the amount of compound 5b was 46%.

[0236]

[0236] Method C To a flame-dried round-bottom flask equipped with a stir bar under argon (g) was added compound 5b (53 mg, 0.10 mmol, 1.00 equiv.), anhydrous DMF (1.5 mL), UMP-morpholidate (90 mg, 0.13 mmol, 1.3 equiv.), and EtS-1-H-tetrazole (47 mg, 0.36 mmol, 3.6 equiv.). The resulting solution was stirred at 35 °C for 40 h. LCMS of the crude mixture indicated that starting material UMP-morpholidate still remained. The temperature was raised to 40 °C, and stirring was continued for 20 h. The mixture was treated with acetone (10 mL) and centrifuged (repeated once more) to give 9 as a white solid, which contained 8% compound 5b by LCMS (ELSD), while NMR indicated the amount of compound 5b was 40%.

[0237]

[0237] Method DTo a flame-dried round-bottom flask equipped with a stir bar under argon (g) was added compound 5b (53 mg, 0.10 mmol, 1.00 equiv.), anhydrous DMF (1.5 mL), UMP-morpholidate (90 mg, 0.13 mmol, 1.3 equiv.), and BnS-1-H-tetrazole (69 mg, 0.36 mmol, 3.6 equiv.). The resulting solution was stirred at 35 °C for 40 h. LCMS of the crude mixture indicated that starting material UMP-morpholidate still remained. The temperature was raised to 40 °C, and stirring was continued for 6 h. The mixture was treated with acetone (10 mL) and centrifuged (repeated once more) to give 9 as a white solid, which contained 13% compound 5b by LCMS (ELSD), while NMR indicated the amount of compound 5b was 39%.

Claims

1. 1. A method for preparing a 6-azido-6-deoxy monosaccharide compound having structure (I) according to the following scheme: 【Chemistry 1】 (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting said cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting said cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); wherein R １ is independently selected from C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

2. 2. The method of claim 1, wherein the monosaccharide is N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc), preferably GalNAc.

3. The method according to claim 1 or 2, wherein the sulfitating agent is a thionyl halide or 1,1'-thionyl imidazole, preferably thionyl chloride.

4. The oxidizing agent is selected from the group of organic or inorganic oxidizing agents, preferably the oxidizing agent is inorganic, more preferably the oxidizing agent is RuO ４ The method according to any one of claims 1 to 3, wherein

5. 5. The method of any one of claims 1 to 4, wherein the diol having structure (II) is prepared from GlcNAc or GalNAc.

6. R １ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ The method of any one of claims 1 to 5, wherein each occurrence of is C(O)Me.

7. the 6-azido-6-deoxy monosaccharide compound having structure (I) is further converted to a nucleoside diphosphate or salt thereof having structure (IX); 【Chemistry 2】 7. The method of claim 1, wherein B is a nucleic acid base.

8. 8. The method of claim 7, wherein the 6-azido-6-deoxy monosaccharide compound having structure (I) is converted to a 1-monophosphate monosaccharide compound, which reacts with a nucleoside monophosphate to form the compound having structure (IX), and wherein the compound having structure (IX) participates in a deprotection step before or after the reaction of the 1-monophosphate monosaccharide compound with the nucleoside monophosphate.

9. The 6-azido-6-deoxy monosaccharide 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: 【Transformation 3】 The method comprises: (e) protecting said 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (f) converting said compound having structure (VI) in the presence of one or more Lewis acids to form an oxazoline compound having structure (VII); (g) reacting said compound having structure (VII) with phosphoric acid to form said 1-monophosphate monosaccharide compound having structure (Va); wherein R ２ The method of claim 8, wherein is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

10. The 6-azido-6-deoxy monosaccharide 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: 【Chemistry 4】 The method comprises: (e) protecting said 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (x1) deprotecting the anomeric position of a 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 said 1-monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphite compound having structure (XIII); (x4) deprotecting said 1-monophosphate diester having structure (XIII) to form said 1-monophosphate monosaccharide compound having structure (Va); wherein R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ３ is C １～６ Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH ２ OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C １～２ -alkyl (2-phenylethyl or phenylmethyl), wherein phenyl is optionally substituted with one or more halide or nitro or methoxy groups.

11. A 1-monophosphate monosaccharide compound having structure (Va) is converted to a nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: 【Transformation 5】 The method comprises: (i) reacting said compound having structure (Va) with a nucleoside monophosphate to produce an acylated nucleoside diphosphate having structure (VIII); (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 both hydrogen, or R ４ 11. The method of claim 9 or 10, wherein both occurrences of are linked to each other via a carbonyl moiety.

12. A 1-monophosphate monosaccharide compound having structure (Va) is converted to a nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: 【Transformation 6】 The method comprises: (j1) deprotecting said compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb); (i1) reacting said compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX); 11. The method of claim 9 or 10, comprising:

13. 13. The method of claim 12, wherein steps (x4) and (j1) are performed in a single deprotection step to produce the compound having structure (Vb).

14. The 6-azido-6-deoxy monosaccharide compound having structure (I) is first converted to a 1-monophosphate monosaccharide compound having structure (Vb) according to the following scheme: 【Transformation 7】 The method comprises: (y1) deprotecting the 6-azido-6-deoxy monosaccharide having structure (I) to form a 1,3,4-trihydroxy-6-azido-monosaccharide compound having structure (XIV); (y2) contacting said compound having structure (XIV) with a phosphorylating enzyme in the presence of a phosphate source to form a 1-monophosphate monosaccharide compound having structure (Vb); The method of claim 8, comprising:

15. having the structure (III): 【Transformation 8】 In the formula, R １ are cyclic sulfate monosaccharide compounds independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

16. R １ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ 16. The compound of claim 15, wherein each occurrence of is (CO)Me.

17. having the structure (I): 【Chemistry 9】 In the formula, R １ is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

18. R １ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ 18. The compound of claim 17, wherein each occurrence of is C(O)Me.

19. The compound according to any one of claims 15 to 18, wherein the monosaccharide is N-acetyl-2-galactosamine (GalNAc) or N-acetyl-2-glucosamine (GlcNAc), preferably GalNAc.

20. 1. A method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va) according to the following scheme: 【Chemistry 10】 The method comprises: (f) converting 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); (g) reacting the oxazoline compound having structure (VII) with phosphoric acid to form the 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va) or a salt thereof; wherein R １ and R ２ is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

21. 21. The process of claim 20, wherein step (g) is carried out in DMF as solvent and the temperature is preferably from 0°C to 120°C, preferably from 20°C to 90°C, more preferably from 40°C to 80°C, even more preferably from 60°C to 80°C, most preferably 75°C.

22. 22. The method of claim 20 or 21, wherein the 6-azido-6-deoxy monosaccharide having structure (VI) is prepared from the 6-azido-6-deoxy monosaccharide compound having structure (I) of claim 17.

23. the 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va) is further converted to a nucleoside diphosphate or salt thereof having structure (IX); 【Chemistry 11】 23. The method of any one of claims 20 to 22, wherein B is a nucleobase.

24. The compound having structure (Va) is converted to the nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: 【Chemistry 12】 The method comprises: (i) converting said compound having structure (Va) with a nucleoside monophosphate to an acylated nucleoside diphosphate having structure (VIII); (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 both hydrogen, or R ４ 24. The method of claim 23, wherein both occurrences of are linked to each other via a carbonyl moiety.

25. The compound having structure (Va) is converted to the nucleoside diphosphate having structure (IX) or a salt thereof according to the following scheme: 【Chemistry 13】 The method comprises: (j1) deprotecting said compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb); (i1) reacting said compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX); 24. The method of claim 23, comprising:

26. having the structure (VII): 【Chemistry 14】 In the formula, R １ and R ２ is independently selected from H, optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

27. R １ and R ２ is H, C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, preferably R １ and R ２ 27. The compound of claim 26, wherein is H or C(O)Me.

28. 28. The compound according to claim 26 or 27, obtained from the monosaccharide N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc), preferably GalNAc.

29. 1. A mixture comprising the α- and β-anomeric forms of a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va): 【Chemistry 15】 The molar ratio of the α-anomeric form to the β-anomeric form is in the range of 3 / 1 to 10 / 1, and both anomeric forms of compound (Va) may be in the salt form, wherein R １ and R ２ is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

30. R １ and R ２ is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, preferably R １ and R ２ 30. The compound of claim 29, wherein is C(O)Me.

31. 31. The compound according to claim 29 or 30, wherein the monosaccharide is N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc), preferably GalNAc.

32. 1. A method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Va) according to the following scheme: 【Chemistry 16】 The method comprises: (x1) deprotecting the anomeric position of a 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 said monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphate diester compound having structure (XIII); (x4) deprotecting said phosphate diester having structure (XIII) to form said 1-monophosphate monosaccharide compound having structure (Va); wherein R １ and R ２ are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ３ is C １～６ Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH ２ OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C １～２ -alkyl (2-phenylethyl or phenylmethyl), wherein phenyl is optionally substituted with one or more halide or nitro or methoxy groups.

33. 1. A method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Vb) according to the following scheme: 【Chemistry 17】 The method comprises: (x1) deprotecting the anomeric position of a 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 said monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphate diester compound having structure (XIII); (x4) simultaneously deprotecting the phosphate diester and monosaccharide having structure (XIII) to form the 1-monophosphate monosaccharide compound having structure (Vb); wherein R １ and R ２ are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ３ is C １～６ Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH ２ OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C １～２ -alkyl (2-phenylethyl or phenylmethyl), wherein phenyl is optionally substituted with one or more halide or nitro or methoxy groups.

34. 1. A method for preparing a 6-azido-6-deoxy-1-monophosphate monosaccharide compound having structure (Vb) according to the following scheme: [Chemistry 18] The method comprises: (y1) deprotecting the 6-azido-6-deoxy monosaccharide having structure (I) to form a 1,3,4-trihydroxy-6-azido-monosaccharide compound having structure (XIV); (y2) contacting said compound having structure (XIV) with a phosphorylating enzyme in the presence of a phosphate source to form a 1-monophosphate monosaccharide compound having structure (Vb); wherein R １ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

35. 1. A method for preparing a nucleoside diphosphate having structure (VIII) according to the following scheme: 【Chemistry 19】 The method comprises: (z) reacting a compound having structure (Va) with a nucleoside monophosphate having structure (X) to form a nucleoside diphosphate having structure (VIII), optionally comprising the step of: ２ or ZnCl ２ in the presence of In the formula, R １ and R ２ is independently selected from H and optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl, and B is a nucleobase.

36. R １ and R ２ is H, C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ and R ２ 36. The method of claim 35, wherein is C(O)Me.

37. 37. The method of claim 35 or 36, wherein B is uracil.

38. having the structure (VIII): 【Chemistry 20】 During the ceremony, B is a nucleobase; R １ are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ４ are both hydrogen, or R ４ A nucleoside diphosphate in which both occurrences are linked to each other via a carbonyl moiety.

39. R １ and R ２ is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ and R ２ 39. The compound of claim 38, wherein is C(O)Me.

40. R ４ 40. The compound of claim 38 or 39, wherein both occurrences of are hydrogen.

41. 41. The compound of any one of claims 38 to 40, wherein B is uracil.

42. 42. The compound according to any one of claims 38 to 41, wherein the monosaccharide is N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc), preferably GalNAc.

43. having the structure (XI): 【Chemistry 21】 During the ceremony, R １ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl.

44. R １ and R ２ is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, preferably R １ and R ２ is C(O)Me.

45. 45. The compound according to claim 43 or 44, wherein the monosaccharide is N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc), preferably GalNAc.

46. having the structure (XII): 【Chemistry 22】 During the ceremony, R １ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ３ is C １～６ Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH ２ OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C １～２ - alkyl (2-phenylethyl or phenylmethyl), wherein the phenyl is optionally substituted with one or more halide or nitro or methoxy groups.

47. R １ and R ２ is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, preferably R １ and R ２ 47. The compound of claim 46, wherein is C(O)Me.

48. 48. The compound according to claim 46 or 47, wherein the monosaccharide is N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc), preferably GalNAc.

49. having the structure (XIII): 【Chemistry 23】 During the ceremony, R １ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ３ is C １～６ Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH ２ OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C １～２ - alkyl (2-phenylethyl or phenylmethyl), wherein the phenyl is optionally substituted with one or more halide or nitro or methoxy groups.

50. R １ and R ２ is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, preferably R １ and R ２ 50. The compound of claim 49, wherein is C(O)Me.

51. 51. The compound according to claim 49 or 50, wherein the monosaccharide is N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc), preferably GalNAc.

52. 1. A method for preparing a nucleoside diphosphate or a salt thereof having structure (IX) according to the following scheme: 【Chemistry 24】 During the ceremony, R １ are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ４ are both hydrogen, or R ４ both occurrences of are connected to each other via a carbonyl moiety; B is a nucleobase, The method is: (a) converting N-acetylglucosamine or N-acetylgalactosamine to a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting said cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting said cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting said 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (f) converting said compound having structure (VI) to an oxazoline compound having structure (VII) in the presence of one or more Lewis acids; (g) reacting said compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va) or a salt thereof; (i) reacting said compound having structure (Va) with a nucleoside monophosphate to form an acylated nucleoside diphosphate having structure (VIII); (j) deprotecting the acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate having structure (IX) or a salt thereof; A method comprising:

53. R １ and R ２ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ and R ２ 53. The method of claim 52, wherein each occurrence of is C(O)Me.

54. R ４ 54. The method of claim 52 or 53, wherein both occurrences of are hydrogen.

55. 55. The method of any one of claims 52 to 54, wherein B is uracil.

56. 1. A method for preparing a nucleoside diphosphate or a salt thereof having structure (IX) according to the following scheme: 【Chemistry 25】 During the ceremony, R １ are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; B is a nucleobase, The method is: (a) converting N-acetylglucosamine or N-acetylgalactosamine to a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (I) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting said cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting said cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting said 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (f) converting said compound having structure (VI) to an oxazoline compound having structure (VII) in the presence of one or more Lewis acids; (g) reacting said compound having structure (VII) with phosphoric acid to form a 1-monophosphate monosaccharide compound having structure (Va) or a salt thereof; (j1) deprotecting said compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb); (i1) reacting said compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX); A method comprising:

57. R １ and R ２ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ and R ２ 57. The method of claim 56, wherein each occurrence of is C(O)Me.

58. 58. The method of claim 56 or 57, wherein B is uracil.

59. 1. A method for preparing a nucleoside diphosphate or a salt thereof having structure (IX) according to the following scheme: 【Chemistry 26】 During the ceremony, R １ are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ３ is C １～６ Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH ２ OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C １～２ - alkyl (2-phenylethyl or phenylmethyl), wherein phenyl is optionally substituted with one or more halide or nitro or methoxy groups; R ４ are both hydrogen, or R ４ both occurrences of are connected to each other via a carbonyl moiety; B is a nucleobase, The method is: (a) converting N-acetylglucosamine or N-acetylgalactosamine to a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting said cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting said cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting said 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (x1) deprotecting the anomeric position of a 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 said monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphate diester compound having structure (XIII); (x4) deprotecting said phosphate diester having structure (XIII) to form said 1-monophosphate monosaccharide compound having structure (Va) or a salt thereof; (i) reacting said compound having structure (Va) with a nucleoside monophosphate to produce an acylated nucleoside diphosphate having structure (VIII); (j) deprotecting said acylated nucleoside diphosphate having structure (VIII) to obtain a nucleoside diphosphate having structure (IX) or a salt thereof.

60. R １ and R ２ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ and R ２ 60. The method of claim 59, wherein each occurrence of is C(O)Me.

61. R ４ 61. The method of claim 59 or 60, wherein both occurrences of are hydrogen.

62. 62. The method of any one of claims 59 to 61, wherein B is uracil.

63. 1. A method for preparing a nucleoside diphosphate or a salt thereof having structure (IX) according to the following scheme: 【Chemistry 27】 During the ceremony, R １ are independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; R ２ is selected from optionally substituted C(O)-alkyl, C(O)-aryl and C(O)-arylalkyl; R ３ is C １～６ Alkyl, allyl, 2-cyanoethyl, 2-alkylsulfonylethyl, 2-arylsulfonylethyl, 2,2,2-trichloroethyl, CH ２ OC(O)alkyl, fluorenylmethyl, 2-pyridylethyl, phenyl-C １～２ - alkyl (2-phenylethyl or phenylmethyl), wherein phenyl is optionally substituted with one or more halide or nitro or methoxy groups; B is a nucleobase, The method is: (a) converting N-acetylglucosamine or N-acetylgalactosamine to a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting said cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting said cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (e) protecting said 6-azido-6-deoxy monosaccharide having structure (I) to form a 6-azido-6-deoxy monosaccharide compound having structure (VI); (x1) deprotecting the anomeric position of a 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 said monophosphite diester having structure (XII) in the presence of an oxidizing agent to form a 1-monophosphate diester compound having structure (XIII); (x4) deprotecting said phosphate diester having structure (XIII) to form said 1-monophosphate monosaccharide compound having structure (Va) or a salt thereof; (j1) deprotecting said compound having structure (Va) to obtain a 1-monophosphate monosaccharide compound having structure (Vb) or a salt thereof; (i1) reacting said compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX); A method comprising:

64. R １ and R ２ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ and R ２ 64. The method of claim 63, wherein each occurrence of is C(O)Me.

65. 65. The method of claim 63 or 64, wherein B is uracil.

66. 66. The method of any one of claims 63-65, wherein steps (x4) and (j1) are performed in a single deprotection step to produce the compound having structure (Vb).

67. 1. A method for preparing a nucleoside diphosphate or a salt thereof having structure (IX) according to the following scheme: 【Chemistry 28】 In the formula, R １ is independently selected from optionally substituted C(O)-alkyl, C(O)-aryl, and C(O)-arylalkyl; B is a nucleobase; The method is: (a) converting N-acetylglucosamine or N-acetylgalactosamine to a 1,3-di-acylated compound having structure (II); (b) reacting a diol having structure (II) with a sulfitating agent to form a cyclic sulfite having structure (IIIa); (c) reacting said cyclic sulfite having structure (IIIa) with an oxidizing agent to form a cyclic sulfate having structure (IIIb); (d) reacting said cyclic sulfate having structure (IIIb) with an inorganic azide to form a 6-azido-6-deoxy monosaccharide having structure (I); (y1) deprotecting the 6-azido-6-deoxy monosaccharide 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 phosphorylating enzyme in the presence of a phosphate source to form a 1-monophosphate monosaccharide compound having structure (Vb) or a salt thereof; (i) reacting said compound having structure (Vb) with a nucleoside monophosphate to produce a nucleoside diphosphate having structure (IX) or a salt thereof; A method comprising:

68. R １ each occurrence of is C(O)Me, C(O)tBu, C(O)Ph or C(O)CH ２ Ph, and preferably R １ 68. The method of claim 67, wherein each occurrence of is C(O)Me.

69. 69. The method of claim 67 or 68, wherein B is uracil.

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