Conjugation of N-acylated amino sugars to protein by reductive alkylation using sodium cyanoborohydride: application to an azo derivative of α-amanitin

Reductive alkylation mediated by cyanoborohydride is an attractive approach to the conjugation of small molecules, such as drugs, to proteins. This reaction is specific for protein amino groups and can be conducted under mild conditions with little risk of protein polymerization. However, the lability of the aldehyde function that is needed in such reactions presents a difficulty. We have investigated the use of derivatives of D-galactosamine and D-glucosamine in reductive alkylation, since these sugars contain aldehyde groups that are inherently protected and that may be readily linked to other molecules through their amino groups. The amino groups of these sugars were acylated with N-4-nitrobenzoylglycylglycine. Studies of the reductive coupling of the resultant adducts to bovine serum albumin revealed that conjugation to albumin is strongly dependent on cyanoborohydride, is much faster in the presence of borate, and shows a marked increase in rate between pH 7.0 and 9.0. In the presence of borate, the glucosamine derivative coupled much more rapidly than did the galactosamine derivative. The aryl nitro group of the glucosamine adduct was selectively reduced to an amine, diazotized, and reacted with α-amanitin to form an azo compound. This azo derivative was reductively coupled to form conjugates that inhibit calf thymus RNA polymerase II.Key words: α-amanitin, borate, bovine serum albumin, cyanoborohydride, protein modification, reductive alkylation.