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.
Aldehydes are widely recognized as valuable synthetic handles for the chemoselective manipulation of peptides and proteins. In this report, we show that peptides and small proteins containing the aspartic acid semialdehyde (Asa) side chain can be easily prepared by a chemoselective amide-forming ligation that results in the formation of the Asa residue at the ligation site. This strategy employs the α-ketoacid-hydroxylamine (KAHA) ligation in combination with a new isoxazolidine monomer that forms a side-chain aldehyde upon ligation. This monomer is easily prepared on a preparative scale by a catalytic, enantioselective approach and is readily introduced onto the N-terminus of a peptide segment by solid phase peptide synthesis. The ligated product can be further functionalized by bioorthogonal reactions between the aldehyde residue and alkoxyamines or hydrazides. We demonstrated that glucagon aldehyde, an unprotected 29-mer peptide prepared by KAHA ligation, can be site specifically and chemoselectively modified with biotin, dyes, aliphatic oximes, and hydroxylamines. We further describe a simple and high recovery one-step purification process based on the capture of a 29-mer glucagon aldehyde and a 76-mer ubiquitin aldehyde by an alkoxyamine-functionalized polyethylene glycol resin. The peptide or protein was released from the resin by addition of a hydroxylamine to provide the corresponding oximes.
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
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