Scope and Limitations of Fmoc Chemistry SPPS‐Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin

Abstract

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.

Total synthesis of bovine pancreatic trypsin inhibitor and the protein diastereomer [Gly37D‐Ala]BPTI using Boc chemistry solid phase peptide synthesis

“…19 Embodied in Fmoc chemistry SPPS, 20 it is now almost universally used for the chemical synthesis of peptides and, in combination with modern chemical ligation methods, is becoming widely used for the total chemical synthesis of protein molecules. [21][22][23] ---------------------------------…”

Recent Advances in the Chemical Synthesis of Insulin and Related Peptides

“…Following native folding the two-chain lispro insulin was obtained by selective alkaline saponification of the single ester. Recently, Kent's group further evaluated several methods in constructing the GluA4-ThrB30 ester intermediate to demonstrate that native chemical ligation was the optimal synthetic route [21]. Bode and coworkers have applied their KAHA (α-ketoacidhydroxylamine) ligation chemistry to the syntheses of the M2 metabolite of glargine as well as mouse, guinea pig and human insulin [22].…”

Nagoya Gold Medal: E. W. Meijer / Nagoya Silver Medal: H. Suga / Prelog Medal and Lectureship: S. B. H. Kent / Tajima Prize: X. Hu

“…Kent and his group are interested in the application of chemistry to reveal the molecular basis of protein function. He has reported in Chemistry—A European Journal on using solid‐phase peptide synthesis for the total synthesis of insulin lispro, and in Angewandte Chemie on inversion of the configuration of peptide‐residue side chains in a protein molecule . Kent is on the Editorial Advisory Board of ChemBioChem .…”