Tetrazine has been widely used in bio-orthogonal click chemistry for diverse biological applications due to its short reaction time and excellent bio-orthogonality. For efficient click reaction, the stability of tetrazine in physiological conditions is one of the key issues in biological applications. However, the reactions between tetrazine and biomolecules have barely been studied. Here, we investigated nonspecific proteome labeling by tetrazine derivatives. Systematic investigation of proteome reactivities of 23 tetrazine derivatives showed their structure-dependent proteome reactivities. We further investigated the relationship between the proteome reactivity of tetrazine derivatives and selectivity of in situ tetrazine-trans-cyclooctene (Tz-TCO) click chemistry-mediated fluorescent labeling of BTK protein in live cells. Intriguingly, a tetrazine derivative SiR-Tz20 with minimal proteome reactivity showed a highly selective BTK labeling efficiency in live cells and in an ex vivo mouse model. Our results demonstrate that the proteome reactivity of tetrazine derivatives is critical for their selectivity in click reaction toward accurate fluorescent protein imaging in live cells.
pCAG-mitoYFP plasmid was kindly provided from Dr. S.-K. Kwon from Korea Institute of Science and Technology (KIST). This work was supported by Samsung Research Funding & Incubation Center of Samsung Electronics under Project Number SRFC-MA2202-07 (E.K.). This work was also supported by the Basic Science Research Program (2023R1A2C1007899 to Jongmin Park) and Priority Research Institute Program (RS-2023-00271205 to Jongmin Park) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning. This work was also supported by Korea Basic Science Institute (KBSI) National Research Facilities & Equipment Center (NFEC) grant (2019R1A6C1010006 to Jongmin Park), Global-Learning & Academic research institution for Master\u2019s\u00B7PhD students and Postdocs (G-LAMP) program (RS-2023-00301850 to Jongmin Park) from the Ministry of Education, South Korea, Korea Institute of Science and Technology (KIST) Institutional Program (Grant Nos. 2E33791, 2V10650 to Sanghee Lee), Korea US Collaboration Research Fund (KUCRF) funded by the Ministry of Science and ICT and Ministry of Health & Welfare (RS-2024-00467483 to Sanghee Lee), Creative-Pioneering Researchers Program through Seoul National University (860-20240107 to H.P.) and GRRC program of Gyeonggi province (GRRCAjou2023-B03 to E.K.).
