Asparaginyl Endopeptidase-Mediated Protein C-Terminal Hydrazinolysis for the Synthesis of Bioconjugates
Dingpeng Zhang, Zhen Wang, Side Hu, Ning‐Yu Chan, Heng Tai Liew, Julien Lescar, James P. Tam, Chuan‐Fa Liu
Abstract
Asparaginyl endopeptidases (AEPs) are cysteinyl enzymes naturally catalyzing the hydrolysis and transpeptidation reactions at Asx–Xaa bonds. These reactions go by a common acyl-enzyme thioester intermediate, which is either attacked by water (for a protease-AEP) or by a peptidic amine nucleophile (for a ligase-AEP) to form the respective hydrolysis or aminolysis product. Herein, we show that hydrazine and hydroxylamine, two α-effect nucleophiles, are capable of resolving the thioester intermediate to yield peptide and protein products containing a C-terminal hydrazide and hydroxamic acid functionality, respectively. The hydrazinolysis reaction exhibits very high efficiency and can be completed in minutes at a low enzyme-to-substrate ratio. We further show the utility of the so-formed asparaginyl hydrazide in native chemical ligation and hydrazone conjugation. Using an EGFR-targeting affibody as a model protein, we have showcased our methodology in the preparation of a number of protein ligation or conjugation products, which are decorated with various functional moieties. The ZEGFR affibody-doxorubicin conjugate shows high selective binding and cytotoxicity toward the EGFR-positive A431 cells. Our results demonstrate the advantages of AEP-mediated protein hydrazinolysis as a simple and straightforward strategy for the precision manufacturing of protein bioconjugates.