Stabilization arising from interactions due to (1) a specific amino acid residue (steric, electronic), (2) a particular solvent (organic, polar), andlor (3) the occurrence of specific low-energy conformation(s) ultimately determines the observed cisltrans ratio of a given X-imino acid peptide bond in a peptide chain. Examples of the influence of these factors may be seen in various linear'vz and cyclic p r ~l i n e -~-~ and sarcosine-containingS peptides, as well as polyproline itself?JO in any of which the cisltram distribution of isomers may vary from all-cis to all-trans depending upon the peptide and the surrounding medium.
Once these factors, whichever are present, have come into play, the observed cisltrans ratio of an isolated X-imino acid peptide bond should then depend essentially on the Boltzmann distribution derived from the total free-energy difference between the two conformers. Consistent with this, Wuthrich and coworkers1'J2 measured the tram content of the Ala-Pro bond in a series of Ala-and Pro-containing peptides (where varying numbers of Ala residues were employed to insulate the bond from chain ends) and found this value to be relatively constant at 90-95%. Interestingly, however, an increase from 10% cis to 55% cis on raising the pH from 1.5 to 5.5 was observed for the dipeptide Ala-Pro. This latter result suggested that a further specific factor must be considered: namely, the ionization state of the C-terminal carboxyl group of the peptide. Earlier e~perimental'~ and the~retical'~ work had also noted this phenomenon.
We sought to test the generality of these observations, and perhaps learn their origin, by examining in a systematic manner a group of peptides representative of the simplest examples of cisltrans peptide-bond isomerism: Gly-Sar-Gly, Gly-Gly-Sar, and Sar-Gly-Sar. These tripeptides have, among them, the following features: (1) they contain both C-terminal and N-terminal X-imino acid peptide bonds; (2) they contain no side chains, thus eliminating interactions arising from specific chemical moieties (e.g., aromatic rings, hydroxy groups);
(3) they have freedom of rotation about Sar N-Ca bonds (the position where rotation is restricted due to the pyrrolidine ring in proline residues); (4) they are totally achiral; and (5) they are sufficiently short in length to minimize the occurrence of ordered structures. These peptides exhibit characteristic carbon-13 nmr spectra in aqueous solutions, in which pairs of resonances due to cis and trans isomers of the Gly-Sar bond may be discerned. Typical spectra of Sar-Gly-Sar are presented in Fig. 1. Results of studies on these tripeptides a t pH 1 (protonation of amino and carboxyl termini), pH 5 (deprotonation of carboxyl terminus), and pH 10 (deprotonation of both termini) are summarized in Table I. Assignments of resonances as cis or trans, while not absolute, are in accord with empirical correlations for the ratios of these isomers in a number of Sar-containing peptides.15'7