Structural Factors That Impact Thioimidate Isomerization and Peptide Backbone Conformation

Abstract

Thioimidates exhibit an unusual conformational equilibrium among peptide backbone functional groups, often populating their trans and cis isomers in nearly equal amounts. Despite their growing use as intermediates for peptide backbone editing and macrocyclization, the structural factors governing this equilibrium have not been systematically characterized. Here, we combine NMR spectroscopy and DFT calculations to define the thermodynamics and kinetics of thioimidate isomerization in a series of model peptides. Remarkably, the near-parity trans/cis equilibrium and barrier to interconversion are largely insensitive to the steric bulk and stereochemistry of the amino acids flanking the thioimidate. In contrast, increasing the steric demand at the thioimidate S substituent produces the largest observed shift in the conformational equilibrium. This trend is consistent with the enrichment of the tentatively assigned cis isomer and suggests that the S substituent may provide a means to perturb the equilibrium. Calculations further support an isomerization mechanism involving rehybridization at nitrogen through a linear transition state rather than direct rotation about the formal C═N bond. The resulting sequence independence provides substantial flexibility in the placement of thioimidates within peptides, enabling the broad application of their dynamic conformational behavior in peptide synthesis and macrocyclization.