Asparaginyl endopeptidase-mediated protein C-terminal hydrazinolysis for the synthesis of bioconjugates

• Main Authors: Zhang, Dingpeng, Wang, Zhen, Hu, Side, Chan, Ning-Yu, Liew, Heng Tai, Lescar, Julien, Tam, James P., Liu, Chuan-Fa
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2022
• Subjects: Science::Biological sciences; Asparaginyl; Bioconjugates
• Online Access: https://hdl.handle.net/10356/162124
• Institution: Nanyang Technological University

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.