Glycan components of glycoproteins play key roles in many biological processes as recognition determinants and modulators of intrinsic properties including folding and stability of proteins. In N-glycoproteins, the core pentasaccharide, Man3GlcNAc2, is linked to the side chain amide nitrogen of Asn in the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro. Glycoproteins obtained from natural sources are very low in occurrence and isolation of microgram quantities requires enormous efforts. Furthermore, glycan components of the glycoproteins vary structurally depending on their degree of branching, the type of terminal residues and the substitution of the core pentasaccharide. A divergent combination of the possible linkages and substitution leads to thousands of related oligosaccharide structures and this phenomenon is called as microheterogeneity. Thus the expressed protein is most often a mixture of various glycoforms. These features pose a formidable challenge to the elucidation of structure-function correlations of glycoproteins.

Glycopeptides constitute structurally well-defined and homogeneous partial structures of glycoproteins and serve as valuable models that contribute to elucidate the functions of glycan chains of glycoproteins. Realizing the need for preparing glycopeptides in reasonable quantities to address the above-mentioned challenge, a number of methods based on chemical synthesis have been developed over the past several decades.

The first one more closely resembles the amide bond formation. The formation of the N-glycosidic bond has often been achieved by the coupling of a glycosylamine with an activated amino acid derivative that has been suitably protected to avoid side reactions, However, aspartic acid residue of peptides easily undergoes cyclization to afford the unwanted aspartimide as a side product. In addition, glycosylamines are relatively unstable resulting in not only anomerization but also hydrolysis to the hemiacetal during the coupling reaction. These problems have been overcome to some extent by the use glycosylamine equivalents such as glycosyl isothiocyanates and glycosyl azides. An alternative approach reported from the laboratory of Fraser-Reid involved the trapping of a β-nitrilium ion, generated by the reaction of n-pentenyl

2-deoxy-2-acetamido-3,4,6-tri-O-acetyl-β-D-glucopyranoside with acetonitrile using NBS as a promoter, with an aspartic acid derivative followed by selective N-deacetylation using piperidine.

The second strategy, based on the biosynthesis of N-glycoproteins, involves N-glycosylation of protected asparagine and Asn containing di- and tripeptides using appropriately protected glycosyl trifluoroacetimidates as donors and TMSOTf as a catalyst. The β-D-glucosyl imidate with an acyl protecting group at C2 position underwent conversion to the corresponding N-((β-D-glycosyl)asparagine with complete P-stereoselectivity in 98 % yield, whereas the imidate derived from N-Troc glucosamine was obtained in moderate yield. The perbenzylated β-D-galactosyl trifluoroacetimidate, on the other hand, furnished the corresponding N-(β-D-galactosyl)asparagine with poor P-selectivity (23 %) albeit in moderate yield. This method had not been applied to N-glycosylation of protected glutamine and Gln containing di- and tripeptides Therefore, there is a need to develop general, versatile and efficient methodologies for the synthesis of N-glycoamino acids and N-glycopeptides.

Disclosed herein is a conceptually novel methodology based on the retrosynthetic cleavage of Cα - Cβ bond for the synthesis of N-glycoamino acids. To begin with, ethyl nitroacetate and fully protected N-(β-glycosyl)haloacetamides were identified as synthons. The use of nitro group as latent functionality for the amino group has been well known in organic synthesis. However, this concept is yet to be exploited for the synthesis of N-glycoamino acids. We reasoned that the newer strategy would represent a general approach to glycoamino acid synthesis, as the key reaction is essentially alkylation of active methylene compounds. Furthermore, the newer strategy has the inherent potential to afford either L- or D-asparagine conjugate by the appropriate modulation of diastereoselectivity of alkylation reaction. The current strategy also gains an advantage by employing fully protected N-(β-glycosyl)haloacetamides as alkylating agents, which are readily prepared, stable and easy to handle crystalline solids unlike glycosylamines used in the above-mentioned first strategy.

The initial alkylation was performed by reacting per-O-acetylated N-(β-D-glucopyranosyl)iodoacetamide with ethyl nitroacetate using K2CO3 as the base in dry DMF at room temperature. After complete consumption of the starting material, the reaction mixture was worked-up and the crude product obtained was purified by column chromatography to afford the desired product as a diastereomeric mixture (55:45) in 62 % yield.

Prompted by the recent progress in asymmetric alkylation, a diastereoselective synthesis of per-O-acetylated N-(β-glycosyl)asparagine precursors was then explored. Anthracenylmethyl ammonium salts derived from cinchonine and cinchonidine have been shown to be useful chiral organocatalysts in the enantioselective alkylation of benzophenone-imine derived from tert-butyl glycinate under phase-transfer conditions. The feasibility of the diastereoselective alkylation was first examined by reacting per-0-acetylated N-(P-D-glucopyranosyl)iodoacetamide with ethyl nitroacetate in the presence of K2CO3 as a base and N-(9-anthracenylmethyl)cinchoninium chloride as the chiral catalyst in dry DMF. After worked-up, the crude product was purified by column chromatography over silica gel to give the desired product as a mixture of diastereomers (82:18) in 60% yield.

The generality of the above-described diastereoselective alkylation was then examined by using several fully acetylated N-(β-glycosyl)iodoacetamides derived from free monosaccharide viz., D-GlcNAc, D-Gal, D-Man & L-Rha and the disaccharide, cellobiose as sugar substrates. The corresponding fully acetylated N-(- β glycosyl)asparagine precursors were obtained in fairly good yields (52 - 68 %) and moderate to good (24 - 64 %) diastereoselectivity. These new products were fully characterized based on physical and spectral methods including two-dimensional NMR and high resolution ESI mass spectrometry.

EXAMPLE

A typical procedure for the synthesis of per-O-acetylated N-(β-D-glucopyranosyl)asparagine precursor:
To a stirred solution of anhydrous K2CO3 (207 mg, 1.5 mmol) and chiral catalyst, N-(9-anthracenylmethyl)cinchoninium chloride (10 mol%) in dry DMF (5 mL), ethyl nitroacetate (138 mg, 1 mmol) was added slowly at room temperature under nitrogen atmosphere. After stirring the reaction mixture for about 0.5 h, a solution of fully acetylated N-(β-D-glucopyranosyl)iodoacetamide (515 mg, 1 mmol) in dry DMF (3 mL) was added and stirring continued. The progress of the reaction was monitored by TLC analysis. Following completion of reaction in 6 h, the reaction mixture was diluted with ethyl acetate (50 mL). The resulting solution was washed with water (30 mL x 2), then with brine solution (30 mL), dried over anhydrous sodium sulfate and concentrated to dryness to get a syrup that was purified by column chromatography over silica gel (100-200 mesh) to obtain the product as a mixture (82:18) of diastereomers.

Single crystal structure analysis of the purified major diastereomer of the Glc derivative revealed an absolute configuration of S at the a-carbon of the monosubstituted ethyl nitroacetate which is a precursor of the L-asparagine conjugate.
A newer and general synthetic methodology for N'-(β-glycosyl)asparagine precursors has been developed based on the alkylation of ethyl nitroacetate using six different per-O-acetylated N-(β-glycosyl)iodoacetamides in good yield and with moderate diastereoselectivity. There is scope for improving the diastereoselectivity by screening a large number of newly emerging organocatalysts. Modulation of diastereoselectivity would enable the conjugation of the sugar to either L- or D-asparagine precursor thus facilitating the synthesis of natural as well as unnatural glycoaminoacids and glycopeptides. Recent years have witnessed enhanced interest in the development of unnatural glycopeptide-based drugs in view of their pharmaceutical importance.

We claim:

1. A process for the synthesis of N-(β-glycosyl)asparagine precursors comprising the reaction of fully acetylated N-(β-glycosyl)iodoacetamides as alkylating agents with ethyl nitroacetate as an amino acid precursor using K2CO3 as base in dimethylformamide medium and in the presence of anthracenylmethyl ammonium salts derived from cinchonine and cinchonidine as organocatalysts; the desired products, fully acetylated N-(β-glycosyl)asparagine precursors, being obtained in good yield with moderate to good diastereoselectivity.

2. A process for the synthesis of N-(β-glycosyl)asparagine precursors substantially as herein described.