Paired-Fraction Electrochemical Programming of Ligase-Mediated Peptide Macrocyclization

A simple head-to-tail cyclic peptide is proposed as the fourth step of a PFEP validation ladder using Omniligase-1, a catalogued recombinant peptide ligase, subject to confirmation of current supply and lot activity. The model precursor is a 14-residue, side-chain-unprotected linear peptide with a free N terminus and an activated C-terminal carboxyamidomethyl ester. Its junction must avoid proline in the P1, P1-prime, and P2-prime positions and should provide a favourable hydrophobic P4 residue. The ligase therefore determines the precursor; PFEP cannot make an incompatible sequence cyclizable. The proposed role of paired electrochemical fractions is limited to ex situ control of enzyme-compatible pH, ionic strength, reducing environment, substrate solubility, and timed acidic quench while ligation occurs in an electrode-free vessel. Controls include optimized buffer, conventional pH-stat, each fraction alone, premixed fractions, and full chemical reconstruction. LC-MS, end-group analysis, and orthogonal structural evidence must distinguish cyclic monomer from linear isobaric material and oligomers. No cyclization result is claimed. Article type: Perspective, mechanistic hypothesis, and experimental framework. No experimental peptide yield, potency, clinical effect, or shelf-life evidence is reported.