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DOI: https://doi.org/10.6023/A25040102

A Catalyst-Free Example of Photomediated Biological Orthogonal Fluorescence Probes

  • Zhao Yu-Qiang ,
  • Na Di ,
  • He Xiaobo ,
  • Zhu Liping ,
  • Zhou Ying ,
  • Zeng Guang-Zhi ,
  • Fan Baomin
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  • aYunnan Key Laboratory of Chiral Functional Substance Research and Application,School of Chemistry & Environment,Yunnan Minzu University,Kunming 650504, Yunnan, China
    bCollege of Chemical Science and Technology, Yunnan University, Kunming 650091, China

Received date: 2025-04-01

  Online published: 2025-05-09

Supported by

Yunnan Key Laboratory of Chiral Functional Substance Research and Application (No.202402AN360010), Yunnan Provincial Department of Science and Technology-Basic Research-Major project (No.202401BC070018), Yunnan Provincial Science and Technology Department-Yunnan University Joint Special Project (No. 202201BF070001-001), and National Natural Science Foundation of China (Nos. 22067019, 22367023).

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Abstract

A bioorthogonal fluorescent probe, NATz, was meticulously crafted by incorporating 1,8-naphthimide onto a 1,2,4,5-tetraazine group endowed with bioorthogonal reaction activity. The spectral examination revealed the activation of fluorescence following the interaction between NATz and TCO in a water (PBS) solution. Systematic time-Dependent Density Functional Theory (TDDFT) calculations elucidate that the fluorescence quenching and activation of NATz are predominantly mediated by a Photoinduced Electron Transfer (PET) mechanism, involving excited-state charge redistribution between the tetrazine moiety and naphthalimide fluorophore. Significantly, the UV light at 365 nm substantially accelerated the reaction rate between NATz and TCO. High-resolution mass spectrometry (HRMS) confirmed that 365 nm UV irradiation exclusively promotes inverse electron-demand Diels-Alder (IEDDA) adduct formation, with no detectable photodegradation byproducts under the experimental conditions. Integrated with reaction pathway calculations, these data conclusively establish that light enhances the bioorthogonal IEDDA reaction between 1,2,4,5-tetrazine (NATz) and trans-cyclooctene (TCO) by photon energy transfer surpassing the reaction energy barrier. The MTS cytotoxicity assay revealed that NATz, even at elevated concentrations (40 μM), maintained cell proliferation inhibition rates below 30% across five cell lines (LO2, BEAS-2B, HeLa, HepG2, and A549), thereby confirming its favorable biocompatibility. Laser Scanning Confocal Microscopy (LSCM) confirms NAtz's suitability for light-activated bioimaging applications, achieving cellular-resolution visualization in single cells and intact multicellular organisms (e.g., Caenorhabditis elegans). In order to further verify the applicability of light-mediated bioorthogonal fluorescent probes, a lysosome-targeted morpholine group was introduced on the basis of NATz, and the lysosome-targeted bioorthogonal fluorescent probe Lyso-NATz was prepared. The spectral test results indicated that after the introduction of the morpholine group, this series of probes still maintained the characteristics of photo-mediated bioorthogonal imaging. Meanwhile, the cytotoxicity experiment proved that the introduction of the morpholine group did not affect its biocompatibility. Finally, Lyso-NATz was used to achieve photo-mediated bioorthogonal fluorescence imaging targeting lysosomes at the cellular level.

Key words： Photomediated; Bioorthogonal chemistry; Fluorescence probe; Tetrazine; Targeting to lysosomes

Cite this article

Zhao Yu-Qiang , Na Di , He Xiaobo , Zhu Liping , Zhou Ying , Zeng Guang-Zhi , Fan Baomin . A Catalyst-Free Example of Photomediated Biological Orthogonal Fluorescence Probes[J]. Acta Chimica Sinica, 0 : 0 . DOI: 10.6023/A25040102

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