High Efficiency Coupling of Diazonium Ions to Proteins and Amino Acids.

Azoproteins, obtained by the coupling of diazonium ions with the histidyl, tyrosyl, and lysyl groups of proteins, have continued to be materials of utility since their early investigation (1). The ease with which proteins can be modified by this procedure, the variety of aromatic amines that can be used, and the correlation that can be made between the chemical structure of the introduced group and the properties of the resulting azoprotein are responsible for the continued interest in these substances for studies of the relationship between chemical structure and biological activity of proteins. However, side reactions have been responsible for a low over-all efficiency of the coupling reaction, i.e. percentage of diazonium ion coupled. This is due primarily to a susceptibility of the diazonium ion to hydrolysis. The methods generally employed do not readily lend themselves to use with tracer amounts of aromatic amines containing 14C or tritium. In this paper a method is presented that permits the incorporation of radioactively labeled aromatic amines with high efficiency. One of the reactions with a-amino acids and diazonium ions leads to the formation of an unstable diazoamino compound with the a-amino group. Because this reaction results in loss of diasonium ion and deamination of the amino acid, chloracetyl derivatives of the amino acid have been used for the preparation of azo derivatives (2). A method in which unmodified amino acids bound to cation exchange resins are used for the preparation of azo derivatives is also presented in this paper.