Application of a new green protocol in solid-phase peptide synthesis: identification of a new green solvent mixture compatible with TBEC/ETT

Solid Phase Peptide Synthesis (SPPS) is the preferred technique for synthesizing bioactive peptides. However, traditional SPPS generates significant waste and employs hazardous solvents like DMF and DCM. The aim of this research is to investigate solvents and agents of coupling that align with the green chemistry and are suitable for all stages of SPPS. Some solvents, such as p-cymene and anisole, taken into consideration in this work, can be derived from renewable sources like plants and biomass, rendering them environmentally sustainable choices. However, many of these alternative solvents possess different physicochemical properties compared to DMF. To overcome this challenge, solvent mixtures are employed. In this study, we identified a novel green solvent mixture by combining anisole with NOP; its ability to swell different resins and its capability to solubilize all Fmoc protected amino acids was investigated. The same mixture was also assessed with a green coupling agent, TBEC, in combination with ETT as additive. Model peptides Aib-enkephalin and Aib-ACP were synthesized resulting in favorable outcomes in terms of peptide synthesis efficiency, 97.81% and 98.86%, respectively. Abbreviations: SPPS: Solid Phase Peptide Synthesis; DMF: N, N-dimethylformamide; DCM: dichloromethane; APIs: Active Pharmaceutical Ingredients; 2-MeTHF: 2-methyltetrahydrofuran; THF: tetrahydrofuran; CPME: cyclopentyl methyl ether; GVL: gamma-valerolactone; NFM: N-formylmorpholine; PC: propylene carbonate; DMI: 1,3-Dimethyl-2-imidazolidinone; NBP: N-butyl pyrrolidone; DMC: dimethyl carbonate; DMM: dipropylene glycol dimethyl ether; NOP: N-octylpyrrolidone; ETT: 5-(ethylthio)-1H-tetrazole; TBEC: N-tert-butyl-N'-ethylcarbodiimide; TFA: trifluoroacetic acid; DIC: N,N '-Diisopropilcarbodiimmide; CM: ChemMatrix; PS: polystyrene; PEG: polyethylene glycol; TG: TentaGel; SEM: scanning electron microscopy; HOBt: hydroxybenzotriazole; HBTU: N,N,N ',N '-Tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate.