A DMF-Free Large Scale Solid-Phase Synthesis of the Linear Precursor of Sunflower Trypsin Inhibitor-1 Guided by Sustainability Principles

Abstract Solid-phase peptide synthesis (SPPS) at commercial scale depends on three substances now subject to tightening regulatory pressure: dimethylformamide (DMF), classified as a reprotoxic substance of very high concern under EU REACH; dichloromethane (DCM), broadly prohibited for industrial use in the United States under the 2024 EPA TSCA rule; and piperidine, a DEA Schedule I precursor requiring licensed handling and disposal at manufacturing scale. We report a 50 mmol, DMF-free synthesis of the linear precursor of sunflower trypsin inhibitor-1 (SFTI-1) that simultaneously eliminates all three substances from a pharmaceutically relevant Fmoc/tBu SPPS process without compromising yield, purity, or stereochemical integrity. DCM-free resin loading and swelling were achieved using 1:1 EtOAc/ACN; coupling and Fmoc deprotection were conducted in 1:1 NBP/EtOAc throughout, with 2% DBU replacing 20% piperidine — reducing deprotection base consumption by 91%. Operating at 1.5 equivalents of amino acid and coupling reagents vs the conventional 3.0 equivalents delivered a 50% reduction in stoichiometric inputs. The principal synthetic challenge, arginine coupling, was addressed through high-throughput experimentation identifying 2:3 DMSO/1,3-dioxolane at 40 °C as a greener solvent system achieving 97% conversion. Head-to-head comparison against a conventional DMF benchmark confirmed equivalent potency-corrected peptide output, comparable crude purity, and no increase in epimerization across all nine chiral residues. Collectively, these improvements delivered an estimated 9% reduction in total process mass intensity (PMI) relative to the DMF process (847 vs 934 kg/kg crude peptide), with the dominant gains concentrated in deprotection base and reagent stoichiometry. Normalized per amino acid to enable comparison with reported industry benchmarks, both the conventional (66.7 kg/kg per amino acid) and green (60.5 kg/kg per amino acid) variants of this process already operate roughly an order of magnitude below the average PMI reported for commercial-scale SPPS in a recent multicompany benchmarking analysis (874.5 kg/kg per amino acid) (J. Org. Chem. 2024, 89, 4261−4282). These results provide a compliance-ready, industrially validated framework for commercial SPPS under current and forthcoming regulatory constraints.