Advancing Sustainable Peptide Synthesis: Methanesulfonic Acid–Formic Acid as a Greener Substitute for TFA in Final Global Deprotection

Abstract

Solid-phase peptide synthesis is the preferred technique for producing peptides in both research and industrial applications.At the end of the synthetic process, the peptides are retrieved from the solid support by treatment with trifluoroacetic acid (TFA) along with scavengers. TFA, however, is a polyfluoroalkyl substance (PFAS) and poses a serious risk to human health and has a considerable ecological impact. Herein, the use of 2% methanesulfonic acid (MSA) with formic acid (FA) as solvent in the presence of TIS as scavenger has been employed for the global final deprotection. 2% MSA with FA is able to successfully cleave all protecting groups in 2-3 h. Peptides containing Ser, Thr, Trp, and Tyr showed formylation, which was successfully eliminated by treatment with 0.5 M NH 4 OH. After cleavage with MSA-TIS-FA (2:2.5:95.5), further tests revealed no traces of peptides remaining anchored to the RinkAmide resin, indicating in more cases quantitative cleavage of the peptide from the resin. This cleavage yield using MSA-FA is superior to that obtained with the classical TFA method.Furthermore, the use of MSA-FA reduces strong acid consumption by approximately 98% (from 95% when TFA is used to just 2% with MSA) and replaces the most harmful PFAS (TFA) with a far more environmentally friendly acid (MSA). This protocol has been successfully applied to the final global deprotection of several biologically important peptides, including tirzepatide, one of the most important active pharmaceutical ingredients (APIs) blockbusters currently on the market.