Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/162124
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dc.contributor.authorZhang, Dingpengen_US
dc.contributor.authorWang, Zhenen_US
dc.contributor.authorHu, Sideen_US
dc.contributor.authorChan, Ning-Yuen_US
dc.contributor.authorLiew, Heng Taien_US
dc.contributor.authorLescar, Julienen_US
dc.contributor.authorTam, James P.en_US
dc.contributor.authorLiu, Chuan-Faen_US
dc.date.accessioned2022-10-04T08:25:06Z-
dc.date.available2022-10-04T08:25:06Z-
dc.date.issued2022-
dc.identifier.citationZhang, 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. https://dx.doi.org/10.1021/acs.bioconjchem.1c00551en_US
dc.identifier.issn1043-1802en_US
dc.identifier.urihttps://hdl.handle.net/10356/162124-
dc.description.abstractAsparaginyl 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.en_US
dc.description.sponsorshipMinistry of Education (MOE)en_US
dc.language.isoenen_US
dc.relationMOE2016-T3-1-003en_US
dc.relation2019-T1-002-100en_US
dc.relationNGF-2019-07-029en_US
dc.relation.ispartofBioconjugate Chemistryen_US
dc.rights© 2022 American Chemical Society. All rights reserved.en_US
dc.subjectScience::Biological sciencesen_US
dc.titleAsparaginyl endopeptidase-mediated protein C-terminal hydrazinolysis for the synthesis of bioconjugatesen_US
dc.typeJournal Articleen
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.identifier.doi10.1021/acs.bioconjchem.1c00551-
dc.identifier.pmid34985285-
dc.identifier.scopus2-s2.0-85122824481-
dc.identifier.issue1en_US
dc.identifier.volume33en_US
dc.identifier.spage238en_US
dc.identifier.epage247en_US
dc.subject.keywordsAsparaginylen_US
dc.subject.keywordsBioconjugatesen_US
dc.description.acknowledgementThis research was supported by Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) to the J.P.T., J.L., and C.-F.L. laboratories and by AcRF Tier 1 (2019-T1-002-100) and NTUitive Gap grant (NGF-2019-07-029) from MOE to the C.-F.L. laboratory.en_US
item.grantfulltextnone-
item.fulltextNo Fulltext-
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