Simple purification of small-molecule-labelled peptides via palladium enolate formation from -ketoamide tags

Palladium enolates derived from & beta;-ketocarbonyl compounds serve as key intermediates in various catalytic asymmetric reactions. We found that the palladium enolate formed from & beta;-ketoamide is stable in air and moisture and we applied this property to develop a peptide purification system using & beta;-ketoamide as a small affinity tag in aqueous media. A solid-supported palladium complex successfully captured & beta;-ketoamide-tagged molecules as palladium enolates and released them in high yield upon acid treatment. Optimum conditions for the catch and release of tagged peptides from a mixture of untagged peptides were established. To demonstrate the value of this methodology in identifying the binding site of a ligand to its target protein, we purified and identified a peptide containing the ligand-binding site from the tryptic digest of cathepsin B labelled with a covalent cathepsin B inhibitor containing a & beta;-ketoamide tag.