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Title: Asparaginyl endopeptidase-mediated protein C-terminal hydrazinolysis for the synthesis of bioconjugates
Authors: Zhang, Dingpeng
Wang, Zhen
Hu, Side
Chan, Ning-Yu
Liew, Heng Tai
Lescar, Julien
Tam, James P.
Liu, Chuan-Fa
Keywords: Science::Biological sciences
Issue Date: 2022
Source: Zhang, D., Wang, Z., Hu, S., Chan, N., Liew, H. T., Lescar, J., Tam, J. P. & Liu, C. (2022). Asparaginyl endopeptidase-mediated protein C-terminal hydrazinolysis for the synthesis of bioconjugates. Bioconjugate Chemistry, 33(1), 238-247.
Project: MOE2016-T3-1-003 
Journal: Bioconjugate Chemistry
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.
ISSN: 1043-1802
DOI: 10.1021/acs.bioconjchem.1c00551
Rights: © 2022 American Chemical Society. All rights reserved.
Fulltext Permission: none
Fulltext Availability: No Fulltext
Appears in Collections:SBS Journal Articles

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