Alkyl 3-Position Substituents Retard the Isomerization of Prolyl and Hydroxyprolyl Amides in Water

Abstract

The influence of alkyl 3-position substituents on the rate of amide isomerization N-terminal to proline and hydroxyproline has been explored via the synthesis and analysis of (2S)-N-(acetyl)proline N‘-methylamide (1), (2S,4R)- and (2S,4S)-N-acetyl-4-hydroxyproline N‘-methylamides 2 and 3, and their respective 3,3-dimethyl analogues 4−6. The relative populations of the amide cis and trans isomers as well as the rates for cis-to-trans and trans-to-cis isomerization of 1−6 in water were ascertained by NMR spectroscopy and magnetization transfer experiments. The relative populations of free C-terminal and hydrogen-bonded amides in the γ-turn conformation were also estimated by integrating the N−H stretch absorbances in the FT-IR spectra of 1 and 4 in CHCl3. In addition, the structure of the amide trans isomer of (2S,4S)-N-acetyl-3,3-dimethyl-4-hydroxyproline N‘-methylamide (6) was determined in the solid state by X-ray crystallographic analysis. In prolyl peptides 1−6, the 3,3-dimethyl and hydroxyl substituents had little effect on the amide isomer equilibrium. A dramatic decrease in the rate of cis-to-trans amide isomerization was observed for N-acetyl-3,3-dimethylproline N‘-methylamide (4), which exhibited a kct nearly 7-fold slower than that of 1. Similar effects of the 3,3-dimethyl substituents were observed, albeit to a lesser degree, in the cases of the hydroxyprolyl peptides. The FT-IR data for 4 and X-ray data for 6 both demonstrated that the 3,3-dimethyl substituents restricted the proline ψ dihedral angle and prevented the formation of a γ-turn conformation, having a seven-membered hydrogen bond between the C-terminal amide NH and N-terminal amide carbonyl. Furthermore, restriction of the ψ dihedral angle by the methyl groups was observed in systematic computational conformational analyses of 1−6, in which the ψ and ω dihedral angles were rotated at 30° intervals and the energies of the local minima were determined. Retardation of the rate of cis-to-trans amide isomerization in the dimethyl analogues may be attributed to steric interactions favoring a ψ dihedral angle at which the C-terminal amide carbonyl destabilizes the transition state through Coulomb repulsion of either the developing nitrogen lone pair or the carbonyl oxygen of the pyramidalized N-terminal amide. The consequences of 3-alkyl and 4-hydroxyl substituents on the rate of proline amide isomerization in water, which was observed to decrease in the order 1 ≈ 3 > 2 > 6 > 5 > 4, may result from influences on the ψ dihedral angle geometry, inductive effects, and intramolecular hydrogen bonding.