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Hydrazone formation and similar reactions are highly versatile and specific, but their application to biological systems has been limited by their characteristically slow reaction kinetics at neutral pH. Catalysis of these reactions through imine formation with aromatic amines such as aniline has broadened the applicability of these reactions to biomolecular labeling. High concentrations of the catalyst are necessary, which may be incompatible with the native structure of certain proteins. In this study, we investigated the utility of 4-aminophenylalanine (4a-Phe) as a catalyst for these reactions. We find that 4a-Phe is nearly as effective as aniline in catalyzing hydrazone formation between the reactive amino acid 3-formyltyrosine (3f-Tyr) and hydrazine-containing fluorophores, both free in solution and incorporated into the protein tubulin. The catalyst 4a-Phe maintains ∼70% of the catalytic efficacy of aniline and is less detrimental to the native structure of tubulin. Examination of the temperature dependence of imine formation between 3f-Tyr and 4a-Phe shows an increase in imine concentration accompanying a decrease in temperature, confirming the exothermic nature of the equilibrium reaction. Interestingly, decreasing the temperature of the 4a-Phe-catalyzed hydrazone reaction between 3f-Tyr and the fluorophore 7-hydrazinyl-4-methylcoumarin increases the overall rate of the reaction. This result indicates that the temperature dependence of the catalyst-aldehyde equilibrium is greater than the temperature dependence of the rate constant for hydrazone formation from this intermediate, and that the rate of hydrazone formation a direct function of the concentration of the intermediate imine. These results provide a platform for conducting nucleophilic catalysis under conditions that are more compatible with biomolecular targets than previously demonstrated, thereby expanding the utility of hydrazone ligations in biological systems.

4-Aminophenylalanine as a Biocompatible Nucleophilic Catalyst for Hydrazone Ligations at Low Temperature and Neutral pH