Solid-phase peptide synthesis (SPPS) enables routine synthesis of virtually any type of peptide sequence and is the preferred method for peptide synthesis in academia and the pharmaceutical industry alike. Still,...

Significant efforts have been made in recent years to identify more environmentally benign and less hazardous alternatives to N,N-dimethylformamide (DMF) in solid-phase peptide synthesis (SPPS). Several greener solvents have been...

Green binary solvent mixtures with a polarity and viscosity close to that of DMF perform similarly in solid-phase peptide synthesis (SPPS). However, while coupling reactions readily proceed in solvents of significantly lower polarity than that of DMF, a high solvent polarity is essential for Fmoc-removal using piperidine, which limits the options for green SPPS solvents. Herein, we report our efforts to expand the available solvent polarity space for green SPPS. We identified pyrrolidine as an efficient base to enable Fmoc-removal in less polar solvent mixtures that also favor coupling reactions, such as dimethyl sulfoxide/ethyl acetate (1:9) and N-butylpyrrolidone/1,3-dioxolane (2:8 and 4:6). Employing less polar binary solvent mixtures in combination with pyrrolidine gave crude peptide purities comparable to or better than for DMF with piperidine in the SPPS of challenging peptide targets. An evaluation of base-dependent side reactions such as diketopiperazine (DKP) and aspartimide formation showed increased side-product formation when using pyrrolidine on DKP- and aspartimide-prone sequences. However, the scaled-up syntheses (5 and 7.5 mmol, respectively) of the peptide therapeutics dasiglucagon (29-mer) and bivalirudin (20-mer) gave good crude peptide purities and purity profiles amenable to SPPS optimization. Pyrrolidine therefore represents a useful alternative to piperidine for Fmoc-removal in an expanded solvent space for green SPPS.

We have systematically explored three approaches based on Fmoc chemistry SPPS for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the ‘hybrid method’, in which maximally-protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[Oβ(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies should yield an effective total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.