Metal complexes with macrocyclic ligands .44. Kinetics of the Cu2+ incorporation into a macrocyclic ligand conjugated to proteins: Model studies for the 'post-labeling' technique

The kinetics of the Cu2+ complexation by macrocycles 1(4-[(1.4,5,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid) and 2 (N-propyl-4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzamide) as by macrocycle 1 conjugated to bovine serum albumin (bsa) and to ribonuclease A (mase) were studied by slopped flow techniques. For 1 and 2 the kinetics were followed in the mM range monitoring the d-d* absorption band of the Cu2+ complex. From the pH dependence of k(obs), the rate law is v = [Cu2+] (k(LH)[LH] + k(LH2)[LH2]), where k(LH) and k(LH2) are the bimolecular rate constants for Cu2+ with the diprotonated (LH2) and monoprotonated (LH1) form of the ligand, respectively. The values are k(LH2) = 1.7(1) M(-1)s(-1) and k(Lh) = 2.3(1).10(5)M(-1)s(-1) for 1, and k(LH2) = 0.28(9)M(-1)s(-1) and k(LH) = 2.01(1).10(5)M(-1)s(-1) for 2. The kinetics of the Cu2+ incorporation into 1,2 and 1 conjugated to bsa and mase i.e, 3 and 4, respectively, were also followed using nitroso-R sail as a metal indicator in the mu M range, i.e., under conditions typical fur the 'post-labeling' technique tn give radiolabeled monoclonal antibodies. In these cases, the reaction takes place between the 1:1 complex Cu2+ with nitroso-R-salt and the macrocycle. At pH 6.5, the rates are very similar to each other indicating that the complexation properties of the macrocycle attached to a protein are not very different from those of the free ligand under comparable conditions.