The primary products of the chemical ligation of α-ketoacids and 5-oxaproline peptides are esters, rather than the previously reported amides. The depsipeptide product rapidly rearranges to the amide in basic buffers. The formation of esters sheds light on possible mechanisms for the type II KAHA ligations and opens an avenue for the chemical synthesis of depsiproteins.

The α-ketoacid–hydroxylamine (KAHA) ligation enables the direct cyclization of unprotected peptides upon cleavage, without coupling reagents or purification of precursors. We report the synthesis of a library of 24 cyclic peptides and a detailed mechanistic study.

An optimized protocol for the masking of α-ketoacids with acid-labile cyclic acetal protecting groups is reported. Unlike prior approaches, these new conditions allow the synthesis of protected α-ketoacids bearing aromatic, hindered alkyl, and protected polar side chains. Attachment to a Wang-type linker and solid support provides a resin that delivers fully unprotected C-terminal peptide α-ketoacids upon resin cleavage. These peptides are the key starting materials for chemical protein synthesis using the α-ketoacid-hydroxylamine ligation.

The development of ligation conditions suitable for highly hydrophobic peptide segments allowed the first chemical synthesis of the head-to-tail cyclized protein AS-48.

The convergent synthesis of proteins by multiple ligations requires segments protected at the N-and/or C-terminus with masking groups that are orthogonal to the acid-and base-labile protecting groups used in Fmoc-SPPS. They must be stable to solid-phase peptide synthesis, HPLC purification, and ligation conditions and easily removed in the presence of unprotected side chains. In this report, we document photolabile protecting groups for both a-ketoacids and hydroxylamines, the key functional groups employed in the a-ketoacid-hydroxylamine (KAHA) ligation. The novel photoprotected a-ketoacid is easily installed onto numerous different C-terminal peptide aketoacids and removed by UV light under aqueous conditions. These advances were applied to the one-pot synthesis of NEDD8, an important modifier protein, by three different convergent routes. These new protecting groups provide greater flexibility on the order of fragment assembly and reduce the number of reaction and purification steps needed for protein synthesis with the KAHA ligation.Scheme 1. Protection of a-ketoacids as photolabile cyclic acetals.Helv. Chim. Acta 2016, 99, 868 -894 In order to utilize the photoprotected a-ketoacids for iterative segment ligations, we applied them to the Fmoc-SPPS of C-terminal protected peptides. Using the same linker we previously reported for traceless preparation of C-terminal peptide a-ketoacids, we Scheme 2. Synthesis of enantiomerically enriched protecting group 2 and determination of the absolute configuration of acetal derivative 7 by single-crystal X-ray diffraction (thermal ellipsoids shown in the ORTEP model are set at a 50% probability level). 870Helv. Chim. Acta 2016, 99, 868 -894 www.helv.wiley.com a Determined by chiral SFC (column Chiralpak ADH) with detection at 220 nm. b After recrystallization from hexanes/AcOEt 25:1 (74% recrystallization yield). CatBH = catecholborane.Scheme 5. Synthesis of photoprotected (S)-5-oxaproline 21.Helv. Chim. Acta 2016, 99, 868 -894 873 Scheme 6. One-pot synthesis of NEDD8 in the N-to C-direction using a photoprotected tyrosine a-ketoacid.Helv. Chim. Acta 2016, 99, 868 -894 875Scheme 8. One-pot convergent synthesis of NEDD8 from four segments.Helv. Chim. Acta 2016, 99, 868 -894 877