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

研究论文

无需催化剂的光介导生物正交荧光探针实例

  • 赵玉强 ,
  • 那迪 ,
  • 和晓波 ,
  • 朱立平 ,
  • 周莹 ,
  • 曾广智 ,
  • 樊保敏
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  • a云南民族大学 化学与环境学院、云南省手性功能物质研究与利用重点实验室 昆明 650091
    b云南大学 化学科学与工程学院 昆明 650091

收稿日期: 2025-04-01

  网络出版日期: 2025-05-09

基金资助

云南省手性功能物质研究与利用重点实验室(202402AN360010)、云南省科技厅-基础研究专项-重大项目（202401BC070018）、云南省科技厅-云南大学联合特殊项目资助（202201BF070001-001）和国家自然科学基金（22067019, 22367023）资助

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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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摘要

将具有生物正交反应活性的1,2,4,5-四氮嗪基团引入到1, 8-萘酰亚胺荧光团上设计合成了光介导生物正交荧光探针NATz。光谱测试显示,NATz与TCO在水（PBS）溶液中反应后荧光被激活,理论计算清晰解释了NATz的猝灭机制和荧光激活机制。最重要的是,在不加光催化剂的条件下,365 nm紫外光显著提高了NATz与TCO的反应速率。高分辨质谱和反应路径计算表明,光对生物正交反应的促进效应是通过克服NATz与TCO之间的环加成反应能垒实现的。细胞毒性测试证明了NATz具有良好的生物相容性,在此基础上实现了细胞和多细胞生物（秀丽隐杆线虫）水平上的光介导生物正交成像。最后,基于NATz的结构,引入吗啉基因设计并合成了具有溶酶体靶向能力的生物正交荧光探针Lyso-NATz。一系列实验证明了Lyso-NATz在保持光介导生物正交荧光成像特性的同时,实现了对溶酶体的靶向作用。

关键词： 光介导; 生物正交; 荧光探针; 四氮嗪; 溶酶体靶向

本文引用格式

赵玉强 , 那迪 , 和晓波 , 朱立平 , 周莹 , 曾广智 , 樊保敏 . 无需催化剂的光介导生物正交荧光探针实例[J]. 化学学报, 0 : 0 . DOI: 10.6023/A25040102

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

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