| Autor: |
Dingpeng Zhang, Zhen Wang, Side Hu, Ning-Yu Chan, Heng Tai Liew, Julien Lescar, James P. Tam, Chuan-Fa Liu |
| Přispěvatelé: |
School of Biological Sciences |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
Bioconjugate chemistry. 33(1) |
| ISSN: |
1520-4812 |
| Popis: |
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. Ministry of Education (MOE) This 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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
