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

Abstract

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 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 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. TKPs were 10⁶-fold more reactive than their DKP analogues towards deprotonation, which we attribute to the advanced development of aromaticity in the earlier transition state for proton transfer in these cases. A Brønsted linear free energy analysis of the reaction data was conducted to provide estimates of α-proton pK _as.

Fig. 1. Diketopiperazines (DKPs) and triketopiperazines (TKPs) used in our hydrogen–deuterium exchange study of α-protons. The exchangeable protons of interest are highlighted.

Fig. 2. (a) Proposed mechanism of hydrogen–deuterium exchange via enolate formation shown for Gly–L–Pro; (b) dependence of the observed rate constant of H/D exchange, k_ex, upon the concentration of deuteroxide for the DKPs studied at 25 °C and I = 1.0 (KCl). The red region encompasses the kinetic data for the majority of prolyl and glycyl residues whereas the blue region spans the data for all other amino acid residues. The slopes of linear fits of the H/D-exchange kinetic data are the second order rate constants for deuteroxide catalysed exchange, k_DO (M⁻¹ s⁻¹).

Fig. 3. Proposed stereoelectronic effect to account for the difference in acidities between the glycyl α-protons in c(Gly–l-Pro) and the enhanced acidity of the prolyl α-proton. The structure on the left indicates the line of sight for (a) glycyl α-protons, and (b) prolyl α-proton. The larger NOE interaction between H₁ and H₃ and the smaller NOE interaction between H₁ and H₂ are shown. Proposed stereoelectronic overlap between σ_C–H and indicated by dashed orange lines.

Fig. 4. Plot of the buffer independent first order rate constants of exchange against the concentration of deuteroxide for glycyl and prolyl TKPs in acetic acid buffer solutions with 40% d₃-MeCN co-solvent, I = 0.2 (KCl) for Pro-TKP and I = 0.06 (KCl) for Gly–TKP and 25 °C. The red region indicates the kinetic acidity for prolyl TKP and the blue region indicates the kinetic acidity of the glycyl residue.

Fig. 5. (a) Proposed mechanism of hydrogen–deuterium exchange via enolate formation for Gly TKP; (b) resonance structures and HOMOs for the fully delocalised TKP enolate and 2,3,6-trihydroxypyrazine.

Fig. 6. (a) α-Carbonyl compounds (□) used to construct the Brønsted LFER below with data from Richard and co-workers; (b) Brønsted linear free energy relationship between log(k_HO/p) and pK_a for the series of α-carbonyl carbon acids above (□). The data is fitted with log(k_HO/p) = −0.401pK_a + log(p) + 6.51 (—) where p = number of acidic α-CH protons. Kinetic data for the DKPs () and N-acyl glycyl amide () can be used to interpolate corresponding pK_a values using eqn (1), whereas kinetic data for TKPs () would require significant extrapolation.