Choice of reagent used for conjugation

Acylating reagents form stable derivatives primarily with amino groups (deprotonated lysine) and tyrosyl residues, and unstable derivatives with cysteyl or histidyl residues under alkaline conditions, due to the pAa of the groups involved (Section 12.1). Modified tyrosine can be regenerated by treatment with hydroxylamine (Rior-dan and Vallee, 1963). 

The general formula of acylating reagents is R-C( = 0)-X, obtained by activation of carboxyl groups. Examples are acetic or mixed anhydride, iV-hydroxysuccinimide esters (NHS Fig. 11.11 and 11.14) and 5-acetylmercaptosuccinic anhydride (Fig. 11.10). In general, anhydrides are rapidly hydrolyzed and acylation is, therefore, in competition with the hydrolysis of the reagent. 

Important alkylating reagents, used under mildly alkaline conditions (pH 7-8.5), are the maleimides (Section 11.2.3.1.3), the prototype of which is A -ethylmaleimide, and the aryl halides, such as DNFB or TDIC. Here again, hydrolysis competes with the alkylation reaction. 

Redox reactions, which take advantage of the ease of reduction and oxidation of disulfides, are rarely used for the conjugation. 

The best known among the electrophilic reagents are the diazonium salts. 

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