Carbohydrates thiolation

PDPH also may be used as a thiolation reagent to add sulfhydryl functional groups to carbohydrate molecules. The reagent can be used in this sense similar to the protocol described for AMBH (Chapter 1, Section 4.1). After modification of an oxidized polysaccharide with the hydrazide end of PDPH, the pyridyl group is removed by treatment with DTT, leaving the exposed sulfhydryl (Figure 5.15). 

Prepare the protein or macromolecule to be thiolated in a non-amine-containing buffer at pH 8.0. For the modification of ribosomal proteins (often cited in the literature) use 50 mM triethanolamine hydrochloride, 1 mM MgCl2, 50 mM KC1, pH 8. The magnesium and potassium salts are for stabilization of some ribosomal proteins. If other proteins are to be thiolated, the same buffer may be used without added salts for stabilization. Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8, or 0.1 M sodium borate, pH 8.0 may be used. For the modification of polysaccharides, use 20 mM sodium borax, pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein to be modified at a concentration of 10 mg/ml in the reaction buffer of choice. Lower concentrations also may be used with a proportional scaling back of added 2-iminothiolane. 

Even though dendrimer surfaces can be constructed to exhibit all possible functionalities, amine-terminating groups are synthetically more appealing and have been used most extensively. The potentially useful thiolated dendrimers self-oxidize, while carboxylated dendrimers tend to form intramolecular anhydrides once activated. This last situation may cause defects upon carbohydrate attachment. Although alcohols seem also attractive, a priori, their direct use in glycosylation chemistry is hampered by potentially difficult complete anomeric stereocontrol. 

The following is the procedure that we have developed to thiolate IgG through modification of the carbohydrate functions. The procedure is very similar to the SPDP protocol after the initial thiolation process (see Note 3). 

Anitha, A. Deepa, N. Chennazhi, K.P. Nair, S.V. Tamura, H. Jayakumar, R. Development of mucoadhesive thiolated chitosan nanoparticles for biomedical applications. Carbohydr. Polym. 2011, 83 (1), 66-73. 

Prabaharan, M. and Gong, S. 2008. Novel thiolated carboxymethyl chitosan-g-P-cyclodextrin as mucoadhesive hydrophobic drug delivery carriers. Carbohydr. Polym. 73 117-125. 

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