Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions.

Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a ß-grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.

Aspartic Acid Forming α-Ketoacid-Hydroxylamine (KAHA) Ligations with (S)-4,4-Difluoro-5-oxaproline.

The α-ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. Currently, the most applied hydroxylamine is the 5-membered cyclic hydroxylamine (S)-5-oxaproline, which forms a homoserine ester as the primary ligation product. In order to access native aspartic acid residues at the ligation site, we synthesized a 4,4-difluoro version of this monomer. Upon KAHA ligation, the resulting difluoro alcohol hydrolyzes to an aspartic acid residue with little or no formation of aspartamide. We applied this monomer for the synthesis of the hormone peptides glucagon and an insulin variant, and as well for segment ligation of the peptides UbcH5a and SUMO3.

Facile folding of insulin variants bearing a prosthetic C-peptide prepared by α-ketoacid-hydroxylamine (KAHA) ligation.

The chemical synthesis of insulin is an enduring challenge due to the hydrophobic peptide chains and construction of the correct intermolecular disulfide pattern. We report a new approach to the chemical synthesis of insulin using a short, traceless, prosthetic C-peptide that facilitates the formation of the correct disulfide pattern during folding and its removal by basic treatment. The linear precursor is assembled by an ester forming α-ketoacid-hydroxylamine (KAHA) ligation that provides access to the linear insulin precursors in good yield from two readily prepared segments. This convergent and flexible route provides access to various human, mouse, and guinea pig insulins containing a single homoserine mutation that shows no detrimental effect on the biological activities.