Unusually high α-proton acidity of prolyl residues in cyclic peptides

The acidity of the α-proton in peptides has an essential role in numerous biochemical reactions and underpins their stereochemical integrity which is critical to their biological function. We report here a detailed kinetic and computational study of the acidity of the α-proton in two cyclic peptide systems: diketopiperazine (DKP) and triketopiperazine (TKP). The kinetic acidity (protofugality) of the the α-protons were determined though hydrogen deuterium exchange studies in aqueous solutions. The acidities of the α-proton in Prolyl residues were increased by 3-89 fold relative to other amino acid residues (Prolyl > Glycyl >> Alanyl > Tyrosyl). Experimental and computational evidence for the stereoelectronic origins of this enhanced Prolyl reactivity is presented. The α-proton in TKPs were a remarkable 106-fold more reactive than their DKP analogues, which we attribute to the advanced development of aromaticity in the earlier transition state for proton transfer in these cases. A Bronsted linear free energy analysis of the reaction data was conducted to provide estimates of α-proton pKas.