Use of an Asparaginyl Endopeptidase for Chemo-enzymatic Peptide and Protein Labeling (original) (raw)

Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (e.g. depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By careful...