CONJUGATION OF UNPROTECTED TRISUCCIN, N-[TRIS[2-[(N-HYDROXYAMINO)CARBONYL]ETHYL]METHYL] SUCCINAMIC ACID, TO MONOCLONAL-ANTIBODY CC49 BY AN IMPROVED ACTIVE ESTER PROTOCOL

For the conjugation of the trihydroxamate bifunctional chelating agent -(benzyloxy)amino]carbonyl]ethyl]methyl]succinamic acid (trisuccin, 1 ) to antibodies, we originally used the corresponding 2,3,5,6-tetraflu orophenyl active ester followed by the postconjugation removal of the benzyl protecting groups by catalytic hydrogenation. It was of interes t to us to design a conjugation protocol capable of incorporating debl ocked hydroxamates into peptides and proteins. Reported procedures tha t were expected to be compatible with the functionalities present in t risuccin were used with no success, as judged by the lack of ability o f the products to radiolabel with Re-188. A simple conjugation method was then developed utilizing the o-nitrophenol (ONP) activated ester o f the unprotected trisuccin, -[(N-hydroxyamino)carbonyl]ethyl]methyl]s uccinamic acid, 3, which eliminates the need for the postconjugation d eblocking. An assay for indirect estimation of the active ester conten t, based on the concentration of its decomposition byproduct, ONP-OH, was developed. Comparison of the indirectly estimated concentrations w ith those obtained directly from purified products showed >90% accurac y for this assay. This procedure has the advantage of rapidly using th e unpurified active ester, eliminating the possibilities of its decomp osition through solvolysis or self-condensation by the unprotected hyd roxamate functions. A colorimetric assay was developed for estimation of the number of ligands per molecule of protein. This assay and the f act that all conjugates consistently radiolabeled with Re-188 show tha t this procedure conjugated the unprotected hydroxamate ligands to the CC49 monoclonal antibody. These results indicate the potential applic ability of this technique to conjugation of unprotected hydroxamate de rivatives with other proteins and peptides.