16] Phthalylation of amino groups

Abstract

This chapter presents a procedure for the phthalylation of amino groups. It has been found that phthalic anhydride reacts rapidly and specifically at pH 8.5 with the free amino groups of proteins and peptides, thereby converting them quantitatively to o -carboxybenzoyl derivatives. This reaction is also quantitatively reversible by the incubation of the phthalyl derivative at pH 3.5 and 50° for 48 hr. Spectral changes occur during the reaction of phthalic anhydride with free amino groups, allowing the conclusion that open o -carboxybenzoyl derivatives, and not cyclic imides, are formed exclusively under slightly alkaline pH reaction conditions. The important contribution to the absorption, at 270 nm, of the incorporated benzene rings provides a sensitive way of ascertaining the extent of the reaction. Two methods have been found useful for checking that the reaction has taken place quantitatively. The first method relies on the spectral changes associated with the reaction. The second method makes use of the reaction of dansyl chloride with free amino groups.