Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation
Many clinically relevant therapeutic agents are formed from the conjugation of small molecules to biomolecules through conjugating linkers. In this study, two novel conjugating linkers were prepared, comprising a central coumarin core, functionalized with a dimaleimide moiety at one end and a termin...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-02-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/10/3/369 |
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| author | Kelvin K. Tsao Ann C. Lee Karl É. Racine Jeffrey W. Keillor |
| author_facet | Kelvin K. Tsao Ann C. Lee Karl É. Racine Jeffrey W. Keillor |
| author_sort | Kelvin K. Tsao |
| collection | DOAJ |
| description | Many clinically relevant therapeutic agents are formed from the conjugation of small molecules to biomolecules through conjugating linkers. In this study, two novel conjugating linkers were prepared, comprising a central coumarin core, functionalized with a dimaleimide moiety at one end and a terminal alkyne at the other. In our first design, we developed a protein labelling method that site-specifically introduces an alkyne functional group to a dicysteine target peptide tag that was genetically fused to a protein of interest. This method allows for the subsequent attachment of azide-functionalized cargo in the facile synthesis of novel protein-cargo conjugates. However, the fluorogenic aspect of the reaction between the linker and the target peptide was less than we desired. To address this shortcoming, a second linker reagent was prepared. This new design also allowed for the site-specific introduction of an alkyne functional group onto the target peptide, but in a highly fluorogenic and rapid manner. The site-specific addition of an alkyne group to a protein of interest was thus monitored <i>in situ</i> by fluorescence increase, prior to the attachment of azide-functionalized cargo. Finally, we also demonstrated that the cargo can also be attached first, in an azide/alkyne cycloaddition reaction, prior to fluorogenic conjugation with the target peptide-fused protein. |
| first_indexed | 2024-12-12T23:41:01Z |
| format | Article |
| id | doaj.art-8c2845231cb940c881e947909e9c2b59 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-12-12T23:41:01Z |
| publishDate | 2020-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-8c2845231cb940c881e947909e9c2b592022-12-22T00:07:10ZengMDPI AGBiomolecules2218-273X2020-02-0110336910.3390/biom10030369biom10030369Site-Specific Fluorogenic Protein Labelling Agent for BioconjugationKelvin K. Tsao0Ann C. Lee1Karl É. Racine2Jeffrey W. Keillor3Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, CanadaDepartment of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, CanadaDepartment of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, CanadaDepartment of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, CanadaMany clinically relevant therapeutic agents are formed from the conjugation of small molecules to biomolecules through conjugating linkers. In this study, two novel conjugating linkers were prepared, comprising a central coumarin core, functionalized with a dimaleimide moiety at one end and a terminal alkyne at the other. In our first design, we developed a protein labelling method that site-specifically introduces an alkyne functional group to a dicysteine target peptide tag that was genetically fused to a protein of interest. This method allows for the subsequent attachment of azide-functionalized cargo in the facile synthesis of novel protein-cargo conjugates. However, the fluorogenic aspect of the reaction between the linker and the target peptide was less than we desired. To address this shortcoming, a second linker reagent was prepared. This new design also allowed for the site-specific introduction of an alkyne functional group onto the target peptide, but in a highly fluorogenic and rapid manner. The site-specific addition of an alkyne group to a protein of interest was thus monitored <i>in situ</i> by fluorescence increase, prior to the attachment of azide-functionalized cargo. Finally, we also demonstrated that the cargo can also be attached first, in an azide/alkyne cycloaddition reaction, prior to fluorogenic conjugation with the target peptide-fused protein.https://www.mdpi.com/2218-273X/10/3/369protein labellingheterobifunctionalfluorogeniccuaac |
| spellingShingle | Kelvin K. Tsao Ann C. Lee Karl É. Racine Jeffrey W. Keillor Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation Biomolecules protein labelling heterobifunctional fluorogenic cuaac |
| title | Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation |
| title_full | Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation |
| title_fullStr | Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation |
| title_full_unstemmed | Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation |
| title_short | Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation |
| title_sort | site specific fluorogenic protein labelling agent for bioconjugation |
| topic | protein labelling heterobifunctional fluorogenic cuaac |
| url | https://www.mdpi.com/2218-273X/10/3/369 |
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