Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity Assays
A Black Hole Quencher-3 (BHQ-3) derivative was synthesized through an azo-coupling reaction between Methylene Violet 3RAX and a tertiary aniline functionalized with a pendant primary amine, allowing subsequent peptide conjugation. The synthesized compounds were characterized using NMR, UV–vis absorp...
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MDPI AG
2024-11-01
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| author | Cátia D. F. Martins Maria Manuela M. Raposo Susana P. G. Costa |
| author_facet | Cátia D. F. Martins Maria Manuela M. Raposo Susana P. G. Costa |
| author_sort | Cátia D. F. Martins |
| collection | DOAJ |
| description | A Black Hole Quencher-3 (BHQ-3) derivative was synthesized through an azo-coupling reaction between Methylene Violet 3RAX and a tertiary aniline functionalized with a pendant primary amine, allowing subsequent peptide conjugation. The synthesized compounds were characterized using NMR, UV–vis absorption, fluorescence spectroscopy, and mass spectrometry. The spectral properties of a Cy5/BHQ-3 amine pair were investigated through titration experiments in PBS (pH 7.4). The results confirmed Förster Resonance Energy Transfer (FRET), along with additional dynamic quenching, as evidenced by the Stern–Volmer analysis. The Stern–Volmer constant (<i>K<sub>SV</sub></i>) was determined to be 1.40 × 10<sup>5</sup> M<sup>−1</sup>. These findings confirm the potential of this system for use in molecular probes and bioimaging applications. |
| format | Article |
| id | doaj-art-4eea43ee6102493da8dd5d7b7390e97e |
| institution | OA Journals |
| issn | 2673-4583 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
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| series | Chemistry Proceedings |
| spelling | doaj-art-4eea43ee6102493da8dd5d7b7390e97e2025-08-20T02:24:25ZengMDPI AGChemistry Proceedings2673-45832024-11-011614810.3390/ecsoc-28-20170Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity AssaysCátia D. F. Martins0Maria Manuela M. Raposo1Susana P. G. Costa2Centre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, PortugalCentre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, PortugalCentre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, PortugalA Black Hole Quencher-3 (BHQ-3) derivative was synthesized through an azo-coupling reaction between Methylene Violet 3RAX and a tertiary aniline functionalized with a pendant primary amine, allowing subsequent peptide conjugation. The synthesized compounds were characterized using NMR, UV–vis absorption, fluorescence spectroscopy, and mass spectrometry. The spectral properties of a Cy5/BHQ-3 amine pair were investigated through titration experiments in PBS (pH 7.4). The results confirmed Förster Resonance Energy Transfer (FRET), along with additional dynamic quenching, as evidenced by the Stern–Volmer analysis. The Stern–Volmer constant (<i>K<sub>SV</sub></i>) was determined to be 1.40 × 10<sup>5</sup> M<sup>−1</sup>. These findings confirm the potential of this system for use in molecular probes and bioimaging applications.https://www.mdpi.com/2673-4583/16/1/48azo dyesBHQ-3FRETproteasesquenching |
| spellingShingle | Cátia D. F. Martins Maria Manuela M. Raposo Susana P. G. Costa Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity Assays Chemistry Proceedings azo dyes BHQ-3 FRET proteases quenching |
| title | Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity Assays |
| title_full | Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity Assays |
| title_fullStr | Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity Assays |
| title_full_unstemmed | Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity Assays |
| title_short | Straightforward Synthesis of BHQ-3 Amine: An Azo Dark-Quencher for FRET-Based Protease Activity Assays |
| title_sort | straightforward synthesis of bhq 3 amine an azo dark quencher for fret based protease activity assays |
| topic | azo dyes BHQ-3 FRET proteases quenching |
| url | https://www.mdpi.com/2673-4583/16/1/48 |
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