Site-Specific C-Terminal Labeling of Peptides and Proteins using Asparaginyl Endopeptidase in a Chemo-Enzymatic Sequence
2019-08-16T14:38:21Z (GMT) by
Asparaginyl endopeptides (AEP) are recognized for their catalytic efficiency, presenting as ideal tools for protein bioconjugation. However, the peptide ligation catalyzed by AEP is reversible. In an attempt to obtain high reaction yields, thiodepsipeptides have been used as substrates but found to be highly unstable, and labeling is only limited to the N-terminus. To maximize the potential use of AEP, here we developed a novel chemo-enzymatic sequence for protein bioconjugation at both the N- and C-termini. In this system, an alternative recognition sequence, Asn-Cys-Leu, was used. Upon ligation, the reaction yields Cys-Leu as leaving group, and its reactive 1,2-aminothiol functionality was quenched by an effective and affordable electrophile, 2-formyl phenylboronic acid (FPBA), to yield a non-reactive cyclic byproduct. In the presence of FPBA our model reaction proceeds with ~95% yield using only 1.2 equivalent of substrate, whereas the yield remains at ~50% in the absence of this additive. This “quenching” approach enables protein labeling at both the N- and C-termini ranging from 75 to 85% (five examples). The simplicity and versatility of this quenching approach will enhance the future use of AEPs in protein bioconjugation.