2-Pyridone Formation: An Efficient Method for the Solid-Phase Synthesis of Homodimers.

This study presents an efficient method for on-resin dimer generation through self-condensation of 3,3-dimethoxypropionic acid-modified molecules, resulting in 2-pyridones. The approach demonstrated remarkable versatility by producing homodimers of peptides, peptoids, and non-peptidic ligands. Its ease of application, broad utility, and mild reaction conditions not only hold significance for peptide and peptoid research but also offer potential for the on-resin development of a wide range of bivalent ligands.

Enzyme-Cleavable Linkers for Protein Chemical Synthesis through Solid-Phase Ligations.

The total synthesis of long proteins requires the assembly of multiple fragments through successive ligations. The need for intermediate purification steps is a strong limitation, particularly in terms of overall yield. One solution to this problem would be solid-supported chemical ligation (SPCL), for which a first peptide segment must be immobilized on a SPCL-compatible solid support through a linker that can be cleaved under very mild conditions to release the assembled protein. The cleavage of SPCL linkers has previously required chemical conditions sometimes incompatible with sensitive protein targets. Herein, we describe an alternative enzymatic approach to trigger cleavage under extremely mild and selective conditions. Optimization of the linker structure and use of a small enzyme able to diffuse into the solid support were key to the success of the strategy. We demonstrated its utility by the assembly of three peptide segments on the basis of native chemical ligation to afford a 15 kDa polypeptide.

A straightforward methodology to overcome solubility challenges for N-terminal cysteinyl peptide segments used in native chemical ligation.

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2.

An optimized protocol for the synthesis of N-2-hydroxybenzyl-cysteine peptide crypto-thioesters.

We herein report a robust upgraded synthetic protocol for the synthesis of N-Hnb-Cys crypto-thioester peptides, useful building blocks for segment-based chemical protein synthesis through native chemical ligation. We recently observed the formation of an isomeric co-product when using a different solid support than the originally-reported one, thus hampering the general applicability of the methodology. We undertook a systematic study to characterize this compound and identify the parameters favouring its formation. We show here that epimerization from l- to d-cysteine occurred during the key solid-supported reductive amination step. We also observed the formation of imidazolidinones by-products arising from incomplete reduction of the imine. Structural characterization combined with the deciphering of underlying reaction mechanisms allowed us to optimize conditions that abolished the formation of all these side-products.