No abstract

Studies of ion-specific effects on oligopeptides have aided our understanding of Hofmeister effects on proteins, yet the use of different model peptides and different experimental sensitivities have led to conflicting conclusions. To resolve these controversies, we study a small model peptide, L-Alanyl-L-alanine (2Ala), carrying all fundamental chemical protein motifs: C-terminus, amide bond, and N-terminus. We elucidate the effect of GdmCl, LiCl, KCl, KI, and KSCN by combining dielectric relaxation, nuclear magnetic resonance (1H-NMR), and (two-dimensional) infrared spectroscopy. Our dielectric results show that all ions reduce the rotational mobility of 2Ala, yet the magnitude of the reduction is larger for denaturing cations than for anions. The NMR chemical shifts of the amide group are particularly sensitive to denaturing anions, indicative of anion-amide interactions. Infrared experiments reveal that LiCl alters the spectral homogeneity and dynamics of the carboxylate, but not the amide group. Interaction of LiCl with the negatively charged pole of 2Ala, the COO− group, can explain the marked cationic effect on dipolar rotation, while interaction of anions between the poles, at the amide, only weakly perturbs dipolar dynamics. As such, our results provide a unifying view on ions’ preferential interaction sites at 2Ala and help rationalize Hofmeister effects on proteins.

Substituted diphenylthioureas (DPTUs) are efficient hydrogen-bonding organo-catalysts, and substitution of DPTUs has been shown to greatly affect catalytic activity. Yet, both the conformation of DPTUs in solution and the conformation and hydrogen-bonded motifs within catalytically active intermediates, pertinent to their mode of activation, have remained elusive. By combining linear and ultrafast vibrational spectroscopy with spectroscopic simulations and calculations, we show that different conformational states of thioureas give rise to distinctively different N−H stretching bands in the infrared spectra. In the absence of hydrogen-bond-accepting substrates, we show that vibrational structure and dynamics are highly sensitive to the substitution of DPTUs with CF 3 groups and to the interaction with the solvent environment, allowing for disentangling the different conformational states. In contrast to bare diphenylthiourea (0CF-DPTU), we find the catalytically superior CF 3 -substituted DPTU (4CF-DPTU) to favor the trans−trans conformation in solution, allowing for donating two hydrogen bonds to the reactive substrate. In the presence of a prototypical substrate, DPTUs in trans−trans conformation hydrogen bond to the substrate's C�O group, as evidenced by a red-shift of the N−H vibration. Yet, our time-resolved infrared experiments indicate that only one N−H group forms a strong hydrogen bond to the carbonyl moiety, while thiourea's second N−H group only weakly interacts with the substrate. Our data indicate that hydrogen-bond exchange between these N−H groups occurs on the timescale of a few picoseconds for 0CF-DPTU and is significantly accelerated upon CF 3 substitution. Our results highlight the subtle interplay between conformational equilibria, bonding states, and bonding lifetimes in reactive intermediates in thiourea catalysis, which help rationalize their catalytic activity.