Programming the Aqueous Reaction State for Carnosine Manufacture: An Ex Situ PFEP Framework for Biocatalysis, Workup, Salt Conversion, and Stability

Abstract

Article type: Perspective, mechanistic hypothesis, and prospective experimental framework. Aqueous peptide processing is often controlled by nominal pH even though pH records proton activity at the instant of measurement and does not specify titratable capacity, ionic composition, dissolved gases, reactive oxygen species, trace metals, conductivity, or the time elapsed since a process liquid was generated. This paper develops paired-fraction electrochemical programming (PFEP) as an ex situ method for treating that larger aqueous reaction state as a qualified, time-dependent process input. Carnosine, β-alanyl-L-histidine, is selected as a mechanistically useful model because it contains one noncanonical β,α peptide bond, a histidine imidazole group with pKₐ near the neutral range, established ATP-dependent and β-aminopeptidase synthesis routes, and documented hydrolytic, oxidative, and metal-sensitive solution liabilities. In the proposed configuration, anodic and cathodic liquids are produced in a divided cell, analytically released, time stamped, and metered into an electrode-free reactor. The peptide, enzyme, ATP, activated donor, and protected intermediates never contact the electrodes. PFEP is evaluated at five stage boundaries: aqueous medium preparation, biocatalytic bond formation, route-specific aqueous deprotection or quench, isolation and salt conversion, and post-synthesis stabilization. The central thesis is operational rather than structural: biochemical outcome may depend on a measurable nonequilibrium state vector comprising proton activity, acid/base capacity, major-ion inventory, redox-active species, dissolved gases, trace contaminants, temperature, and fraction age. The term does not imply persistent molecular memory of bulk water. A matched causal design compares PFEP fractions with optimized conventional buffer, sham treatment, pH-matched reconstruction, composition- and capacity-matched reconstruction, fresh and aged fractions, and paired delivery sequences. Primary endpoints are molar carnosine yield and closed material balance; secondary endpoints include donor hydrolysis, enzyme activity, ATP/ADP balance, β-alanine, histidine, 2-oxo-carnosine, unknown impurities, and metal burden. The framework identifies where PFEP is plausible, where strong buffering will erase its effect, and which results would reject a PFEP-specific interpretation. Evidence boundary. PFEP is an author-proposed, experimentally testable process-control theory. This article reports no new laboratory experiment, yield, purity, potency, safety, clinical, GMP, or shelf-life result. Calculated protonation fractions, buffer-capacity examples, and fraction-age curves are prospective design calculations. Published electrochemistry and carnosine biochemistry support individual premises but do not yet establish that ex situ paired electrochemical fractions improve carnosine manufacture or stability. The manuscript contains an English title and abstract together with a Russian title, author name, abstract, and keywords for bilingual indexing.