Metal Complexes with Macrocyclic Ligands. Part XLIV Kinetics of the Cu 2+ incorporation into a macrocyclic ligand conjugated to proteins: Model studies for the d‘post‐labeling’ technique

The kinetics of the Cu 2+ 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 Cu 2+ complex. From the pH dependence of k obs , the rate law is v = [Cu 2+ ] ( k LH [LH] + k   LH   2[LH 2 ]), where k LH and k   LK   2are the bimolecular rate constants for Cu 2+ with the diprotonated (LH 2 ) and monoprotonated (LH 1 ) form of the ligand, respectively. The values are k   LH   2= 1.7( 1 ) M −1 s −1 and k LH = 2.3(1) 10 5 M −1 s −1 for 1 , and k   LH   2, = 0.28(9) M −1 s −1 and k LH = 2.0(1) 10 5 M −1 s −1 for 2. The kinetics of the Cu 2+ 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 Cu 2+ 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.