α‐Methyl‐L‐proline is an α‐substituted analog of proline that has been previously employed to constrain prolyl peptide bonds in a trans conformation. Here, we revisit the cis‐trans prolyl peptide bond equilibrium in derivatives of α‐methyl‐L‐proline, such as N‐Boc‐protected α‐methyl‐L‐proline and the hexapeptide H‐Ala‐Tyr‐αMePro‐Tyr‐Asp‐Val‐OH. In Boc‐α‐methyl‐L‐proline, we found that both cis and trans conformers were populated, whereas, in the short peptide, only the trans conformer was detected. The energy barrier for the cis‐trans isomerization in Boc‐α‐methyl‐L‐proline was determined by line‐shape analysis of NMR spectra obtained at different temperatures and found to be 1.24 kcal/mol (at 298 K) higher than the corresponding value for Boc‐L‐proline. These findings further illuminate the conformationally constraining properties of α‐methyl‐L‐proline.

Cyclic peptides are promising next‐generation therapeutics with improved biological stability and activity. A catalyst‐free stapling method for cysteine‐containing peptides has been developed that enables fine‐tuning of the macrocycle by using the appropriate regioisomers of fluorobenzene linkers. Stapling was performed on the unprotected linear peptide or, more conveniently, directly on‐resin after peptide synthesis. NMR spectroscopy and circular dichroism studies demonstrate that the type of stapling can tune the secondary structures of the peptides. The method was applied to a set of potential agonists for melanocortin receptors, generating a library of macrocyclic potent ligands with ortho, meta or para relationships between the thioethers. Their small but significant differences in potency and efficacy demonstrate how the method allows facile fine‐tuning of macrocyclic peptides towards biological targets from the same linear precursor.

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