Preparation of Neoglycoproteins

Although a variety of techniques is available for attaching carbohydrates to proteins, not all of them are suitable for work with biological systems. A number of criteria must be considered in developing and evaluating a chemical procedure for this type of modification. 

Preferably, the reagent should be soluble and stable in physiological solutions. If it is to be used for the modification of proteins in cells or such cellular components as membranes, the reagent should not be toxic or susceptible to metabolism that may transform it into other active substances. The modification reaction should proceed relatively 

The modified protein should be easy to purify from the reaction mixture, and there must be some way in which to quantitate the extent of modification. Most importantly, the modified protein should be soluble in physiological solutions, and retain full biological activity in such solutions. This requirement usually specifies that modification does not change the overall charge or the conformation of the protein, involve critical residues, or introduce bulky or hydrophobic groups. 

Diazonium ions react rapidly with histidyl, lysyl, and tyrosyl residues to form mono- and di-azo derivatives, and much more slowly with arginyl, cysteinyl, and tryptophanyl residues.18 The lack of specificity of the reaction can create problems in interpreting results, particularly if critical residues are modified. The azo-proteins formed may be readily purified, and are intensely colored this property may permit quantitation of the extent of the reaction by measurement of the absorbance. Not uncommonly, however, the products of the reaction are of a dark-brownish color that interferes with many of the standard assays for protein and carbohydrate. 

The application of diazo coupling is somewhat limited by the availability of the p-aminophenyl glycosides, particularly of those of the oligosaccharides. Their precursors, the p-nitrophenyl glycosides, are usually obtained by the condensation of a per-O-acetylated glycosyl halide with p-nitrophenol in ethanol (Michael reaction)19,20 or by the reaction of a per-O-acetylated sugar with p-nitrophenol in the presence of a Lewis acid catalyst (Helferich reaction).21 p-Nitrobenzyl 1-thioglycosides have also been prepared by the condensation of the 

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The choice of the modification reaction will be determined by the nature of the carbohydrate to be attached, as well as by the nature of the protein. The intended uses to which the neoglycoprotein will be put may also determine the suitability of a particular approach. The procedures described are varied, and offer a number of options for the preparation of neoglycoproteins. 

This approach is by far the most popular approach for the preparation of neoglycoproteins because of the ready surface availabihty of the e-amino group of Lys side chains and the higher reactivity of e-amino groups in most proteins. 

A transglutaminase reaction was found to be useful in the preparation of neoglycoproteins. For example, oo-amino glycosides were used as a substrate for transglutaminase for the addition of carbohydrates to glutamine side chains (35, 36). The success of this reaction, however, seems somewhat unpredictable. 

NMR analysis of the product indicate that Tyr and Lys were modified (53). A homobifunctionalp-nitrophenyl ester was proposed for the preparation of neoglycoproteins (26). In this method, one of the active ester groups is used to react with the co-aminoaUcyl glycoside and the product thereof then will be conjugated to a protein or other amino-bearing material. 

Lee RT, Lee YC. Preparation of neoglycoproteins using w-aldehydoalkyl 1-thioglycosides. Meth. Enzymol. 1982 83 289-294. 

Wu X, Ling C-C, Bundle DR. A new homobifunctional p-nitro 42. phenyl ester coupling reagent for the preparation of neoglycoproteins. Organic Lett. 2004 6 4407-4410. 

Szurmai, Z. Szabo, L. Liptak, A. Diethylene and triethylene glycol spacers for the preparation of neoglycoproteins. Acta Chim. 1989,126, 259. 

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