Absorption and Fluorescence of Tyrosine Hydrogen-Bonded to Amide-like Ligands

The absorption and steady-state fluorescence of N-acetyl-l-tyrosinamide (NAYA), chosen to model the tyrosine in proteins, was measured in four solvents in the presence of N,N-dimethylacetamide, N-methylacetamide, and urea, chosen as ligands that model the amide of a protein backbone. The data were fit to a multilinear mathematical model to resolve the overlapping spectra of NAYA with and without ligand. The ligand binding constant was between 0.18 and 4 M-1, increasing with solvent polarity; lack of a strong dependence of binding constant on N-methylation suggests that the carbonyl oxygen is responsible for the hydrogen bonding. The emission spectrum of NAYA was essentially identical in all solvents and with and without added ligand. In contrast, the excitation spectrum shifted by up to 10 nm, depending on both solvent and ligand; this shift is described as a sum of three terms: a blue-shift due to hydrogen bonding to the phenolic oxygen which is proportional to ligand donor acidity, a red-shift due to hydrogen bonding to the phenolic hydrogen which is proportional to ligand acceptor basicity, and a blue-shift proportional to solvent dielectric effect. The extinction coefficient varies by up to 40%, depending on solvent and complex formation. The fluorescence quantum yield of the hydrogen-bonded complex varies between 0.102 and 0.044, increasing slightly with N-methylation, but more dependent on solvent. In methanol, acetone, and dioxane, the amide-model complex has a 3−10 times lower fluorescence quantum yield than that of NAYA in pure solvent; in water the complex has a higher quantum yield. Complex formation did not explain all of the fluorescence quenching by ligand in water and in dioxane, suggesting that the ligand also causes dynamic quenching of the excited state in these solvents. Many of the experimental findings are in good agreement with semiempirical molecular orbital calculations.