1H‐ and 13C‐nmr investigations on cis–trans isomerization of proline peptide bonds and conformation of aromatic side chains in H‐Trp‐(Pro)n‐Tyr‐OH peptides

Abstract

¹H and ¹³C high‐resolution nmr spectra of cationic, zwitterionic, and anionic forms of the peptides: H‐Trp‐(Pro)_n‐Tyr‐OH, n = 0‐5, and H‐Trp‐Pro‐OCH₃ were obtained in D₂O solution. Analysis of H^α(Pro₁), H^α(Trp), C^γ(Pro), H^ε(Tyr), and H^δ(Trp) resonances provided evidence for the presence of two predominant backbone isomers: the all‐trans one and another with the Trp‐Pro peptide bond in cis conformation; the latter constituted about 0.8 molar fraction of the total peptide (n > 1) concentration. Relative content of these isomers varied in a characteristic way with the number of Pro residues and the ionization state of the peptides. The highest content of the cis (Trp‐Pro) isomer, 0.74, was found in the anionic form of H‐Trp‐Pro‐Tyr‐OH; it decreased in the order of: anion ≫ zwitterion ≈ cation, and with the number of Pro residues to reach the value of 0.42 in the cationic form of H‐Trp‐ (Pro)₅‐Tyr‐OH. Isomerization equilibria about Pro‐Pro bond(s) were found to be shifted far (⩾0.9) in favor of the trans conformation. Interpretation of the measured vicinal coupling constants J_α−β′ and J_α−β″ for C^αH‐C^βH₂ proton systems of Trp and Tyr side chains in terms of relative populations of g⁺, g⁻, and t staggered rotamers around the χ₁ dihedral angle indicated that in all the peptides studied (a) rotation of Trp indole ring in cis (Trp‐Pro) isomers is strongly restricted, and (b) rotation of Tyr phenol ring is relatively free. The most preferred χ₁ rotamer of Trp (0.8‐0.9 molar fraction) was assigned as the t one on the basis of a large value of the vicinal coupling constant between the high‐field H^β and carbonyl carbon atoms of Trp, estimated for the cis (Pro₁) form of H‐Trp‐Pro‐Tyr‐OH from a ¹H, ¹³C correlated spectroscopy ¹H detected multiple quantum experiment. This indicates that cis ↔ trans equilibrium in the Trp‐Pro fragment is governed by nonbonding interactions between the pyrrolidine (Pro) and indole (Trp) rings. A molecular model of the terminal cis Trp‐Pro dipeptide fragment is proposed, based on the presented nmr data and the results of our molecular mechanics modeling of low‐energy conformers of the peptides, reported elsewhere. © 1993 John Wiley & Sons, Inc.