Metal Complexes with Macrocyclic Ligands. Part XLIV Kinetics of the Cu2+ incorporation into a macrocyclic ligand conjugated to proteins: Model studies for the d‘post‐labeling’ technique

The kinetics of the Cu2+ complexation by macrocycles 1 (4‐[(l,4,8,11‐tetraazacyclotetradec‐1‐yl)methyl]‐benzoic acid) and 2 (N‐propyl‐4‐[(1,4,8,11‐tetraazacyclotetradec‐1‐yl)methyl]‐benzamide) as well as by macrocycle 1 conjugated to bovine serum albumin (bsa) and to ribonuclease A (rnase) were studied by stopped 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 kobs, the rate law is v = [Cu2+] (kLH[LH] + k [LH2]), where kLH and k are the bimolecular rate constants for Cu2+ with the diprotonated (LH2) and monoprotonated (LH1) form of the ligand, respectively. The values are k = 1.7(1) M−1s−1 and kLH = 2.3(1) 105 M−1s−1 for 1, and k , = 0.28(9) M−1s−1 and kLH = 2.0(1) 105 M−1s−1 for 2. The kinetics of the Cu2+ incorporation into 1,2 and 1 conjugated to bsa and rnase, i.e., 3 and 4, respectively, were also followed using nitroso‐R salt as a metal indicator in the μM range, i.e., under conditions typical for the ‘post‐labeling’ technique to give radiolabeled monoclonal antibodies. In these cases, the reaction takes place between the 1:1 complex of 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.