Triazinylaniline derivatives as fluorescence probes. Part 4. Kinetics and selectivity in the reactions of N,N-diethyl-4-(dichloro-1,3,5-triazinyl)aniline with amines, amino acids and proteins relevant to fluorescence labelling

Abstract

The kinetics of reaction of the reactive triazinyl dye, N,N-diethyl-4-(dichloro-1,3,5-triazinyl)aniline with primary and secondary amines, amino acids, and the proteins bovine brain calmodulin and bovine serum albumin in aqueous media, as monitored by UV–VIS absorption spectroscopy, are reported. In general the reaction is first-order in the dye and in the substrate. Secondary aliphatic amines are more reactive than primary aliphatic amines, and a retarding influence of geminal hydroxy substituents is observed in many cases. The intrinsic reactivity of the dye towards the side chain ε-amino group of lysine is 10 times higher than that for a free N-terminal amino group. The influences of pH and temperature on reaction rates have been determined. Selectivity in reaction of the dye (TACl₂) with lysine residues of calmodulin and bovine serum albumin, of 1 : 1 and ≈2 : 1 dye-protein stoichiometry, respectively, is ascribed to the dominant role played by hydrophobic interactions of dye and protein surfaces. On the basis of the kinetic evidence a three-stage mechanism is proposed and numerically evaluated. Initial rapid diffusional encounter of the dye and protein is followed by a slower ‘internalization’ of the dye in a hydrophobic pocket of the protein (activation energy 65–77 kJ mol^–1). Finally, chemical adduct formation occurs in the protein interior having an activation energy of 21–36 kJ mol^–1, significantly lower than that found (42–48 kJ mol^–1) for reaction of dye and primary aliphatic amines in homogeneous solution.