Advancing Sustainable Peptide Synthesis by Solvent Quality Control: Understanding and Mitigating Hydroperoxide in 1-Butyl-2-pyrrolidinone (NBP)

While numerous studies aimed at producing peptides in a sustainable manner have recently been reported, the impact of the quality of starting materials on sustainability in peptide synthesis has been less investigated. Here we report that NBP, a greener dipolar aprotic solvent suitable for use in SPPS, readily undergoes air oxidation to the corresponding hydroperoxide (NBP-OOH), which adversely affects oxidation-sensitive amino acids and peptides. Air, light, elevated temperatures, and common peptide synthesis reagents such as DIC accelerated the rate of NBP oxidation. LC-HRMS analyses revealed that air-induced NBP degradation proceeds via a different mechanism than the previously elucidated degradation of the closely related NMP while GC–MS of NBP containing NBP-OOH showed that standard GC-based analytical methods are unsuitable for NBP-OOH detection, warranting implementation of improved methods for NBP analyses. Assessment of NBP-OOH-induced Met, Trp, Cys, and Tyr breakdown revealed not only that NBP-OOH degrades all these oxidation-prone substrates, but it was also discovered that DITU, previously reported to suppress N-oxyl radical-induced peptide breakdown, constitutes an efficient suppressant of hydroperoxide-induced degradation. Studies on aerial oxidation of additional greener dipolar aprotic solvents DMSO and DMPU revealed that while the former is stable and does not cause oxidation, the latter is oxidized extensively upon air exposure and degrades oxidation-prone substrates to a significant extent. On the other hand, air bubbling of NBP, DMSO, and DMPU proved to have an unexpected positive effect on the stability of the α-amino-bound Fmoc group, a finding that may be leveraged to minimize premature Fmoc loss during peptide synthesis. Overall, our studies on understanding and mitigating hydroperoxide formation in greener solvents for peptide synthesis illustrate the importance of a thorough quality assessment of starting materials during the development of sustainable synthetic methods.