Side-Chain Unprotected Fmoc-Arg/His/Tyr-OH Couplings and Their Application in Solid-Phase Peptide Synthesis through a Minimal-Protection/Green Chemistry Strategy

Conventional SPPS (solid-phase peptide synthesis) requires protecting the nucleophilic side-chains of amino acids to prevent undesired modifications. A large volume of TFA (trifluoroacetic acid) is generally needed to remove these protecting groups after the peptide assembly. Such a process significantly lowers the productivity of the subject peptide manufacture due to handling large volumes of TFA and the resultant anti-solvent for precipitating and isolating the crude products. Enabling the side-chain unprotected amino acid couplings should break through such a bottleneck in peptide production and drastically increase productivity. Fmoc-Arg-OH, Fmoc-His-OH, and Fmoc-Tyr-OH are utilized in this study as substrates to investigate their couplings to peptide molecules. Side reactions induced by these side-chain unprotected amino acids have been elucidated, and solutions addressing these side reactions are developed. The processes of the side-chain unprotected amino acid couplings have been successfully implemented in peptide synthesis through a minimal-protection SPPS (MP-SPPS) strategy. Significantly reduced TFA concentration was enabled to quantitatively detach the peptide from the solid support and obtain the crude peptide product in high yield. In this process, trifluorotoluene, an alternative solvent to DCM (dichloromethane) with low toxicity, was employed as the medium for TFA-mediated peptide cleavage and side-chain global deprotection. A crude peptide product with high purity was obtained from the MP-SPPS process, and a 5.3-fold productivity increase was accomplished.