KAHA Ligations That Form Aspartyl Aldehyde Residues as Synthetic Handles for Protein Modification and Purification

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.