We recently described the development and application of a new bioorthogonal conjugation, the triazinium ligation. To explore the wider application of this reaction, in this work, we introduce a general method for synthesizing C3-substituted triazinium salts based on the Liebeskind-Srogl cross-coupling reaction and catalytic thioether reduction. These methods enabled the synthesis of triazinium derivatives for investigating the effect of different substituents on the ligation kinetics and stability of the compounds under biologically relevant conditions. Finally, we demonstrate that the combination of a coumarin fluorophore attached to position C3 with a C5-(4-methoxyphenyl) substituent yields a fluorogenic triazinium probe suitable for no-wash, live-cell labeling. The developed methodology represents a promising synthetic approach to the late-stage modification of triazinium salts, potentially widening their applications in bioorthogonal reactions.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám 2 16000 Prague Czech Republic

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Oliveira B. L.; Guo Z.; Bernardes G. J. L. Inverse electron demand Diels–Alder reactions in chemical biology. Chem. Soc. Rev. 2017, 46 (16), 4895–4950. 10.1039/C7CS00184C. PubMed DOI

Battigelli A.; Almeida B.; Shukla A. Recent Advances in Bioorthogonal Click Chemistry for Biomedical Applications. Bioconjugate Chem. 2022, 33 (2), 263–271. 10.1021/acs.bioconjchem.1c00564. PubMed DOI

Yang J.; Zhu B.; Ran C. The Application of Bio-orthogonality for In Vivo Animal Imaging. Chem. Biomed. Eng. 2023, 1 (5), 434–447. 10.1021/cbmi.3c00033. PubMed DOI PMC

Liong M.; Fernandez-Suarez M.; Issadore D.; Min C.; Tassa C.; Reiner T.; Fortune S. M.; Toner M.; Lee H.; Weissleder R. Specific Pathogen Detection Using Bioorthogonal Chemistry and Diagnostic Magnetic Resonance. Bioconjugate Chem. 2011, 22 (12), 2390–2394. 10.1021/bc200490r. PubMed DOI PMC

Spitzberg J. D.; Ferguson S.; Yang K. S.; Peterson H. M.; Carlson J. C. T.; Weissleder R. Multiplexed analysis of EV reveals specific biomarker composition with diagnostic impact. Nat. Commun. 2023, 14 (1), 1239.10.1038/s41467-023-36932-z. PubMed DOI PMC

Ko J.; Wilkovitsch M.; Oh J.; Kohler R. H.; Bolli E.; Pittet M. J.; Vinegoni C.; Sykes D. B.; Mikula H.; Weissleder R.; Carlson J. C. T. Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes. Nat. Biotechnol. 2022, 40 (11), 1654–1662. 10.1038/s41587-022-01339-6. PubMed DOI PMC

Cañeque T.; Müller S.; Rodriguez R. Visualizing biologically active small molecules in cells using click chemistry. Nat. Rev. Chem. 2018, 2, 202–215. 10.1038/s41570-018-0030-x. DOI

Nguyen S. S.; Prescher J. A. Developing bioorthogonal probes to span a spectrum of reactivities. Nat. Rev. Chem. 2020, 4 (9), 476–489. 10.1038/s41570-020-0205-0. PubMed DOI PMC

Kozma E.; Bojtar M.; Kele P. Bioorthogonally Assisted Phototherapy: Recent Advances and Prospects. Angew. Chem., Int. Ed. 2023, 135, e20230319810.1002/ange.202303198. PubMed DOI

Bauer D.; Cornejo M. A.; Hoang T. T.; Lewis J. S.; Zeglis B. M. Click Chemistry and Radiochemistry: An Update. Bioconjugate Chem. 2023, 34 (11), 1925–1950. 10.1021/acs.bioconjchem.3c00286. PubMed DOI PMC

Zhong X.; Yan J.; Ding X.; Su C.; Xu Y.; Yang M. Recent Advances in Bioorthogonal Click Chemistry for Enhanced PET and SPECT Radiochemistry. Bioconjugate Chem. 2023, 34 (3), 457–476. 10.1021/acs.bioconjchem.2c00583. PubMed DOI

Devaraj N. K. The Future of Bioorthogonal Chemistry. ACS Cent. Sci. 2018, 4 (8), 952–959. 10.1021/acscentsci.8b00251. PubMed DOI PMC

Blackman M. L.; Royzen M.; Fox J. M. Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels–Alder Reactivity. J. Am. Chem. Soc. 2008, 130 (41), 13518–13519. 10.1021/ja8053805. PubMed DOI PMC

Zhao G. X.; Li Z. T.; Zhang R. S.; Zhou L. M.; Zhao H. B.; Jiang H. F. Tetrazine bioorthogonal chemistry derived in vivo imaging. Front. Mol. Biosci. 2022, 9, 105582310.3389/fmolb.2022.1055823. PubMed DOI PMC

McFarland J. M.; Alečković M.; Coricor G.; Srinivasan S.; Tso M.; Lee J.; Nguyen T.-H.; Mejía Oneto J. M. Click Chemistry Selectively Activates an Auristatin Protodrug with either Intratumoral or Systemic Tumor-Targeting Agents. ACS Cent. Sci. 2023, 9 (7), 1400–1408. 10.1021/acscentsci.3c00365. PubMed DOI PMC

Mitry M. M. A.; Greco F.; Osborn H. M. I. In Vivo Applications of Bioorthogonal Reactions: Chemistry and Targeting Mechanisms. Chem. - Eur. J. 2023, 29 (20), e20220394210.1002/chem.202203942. PubMed DOI

Peplow M. ‘Clicked’ drugs: researchers prove the remarkable chemistry in humans. Nat. Biotechnol. 2023, 41 (7), 883–885. 10.1038/s41587-023-01860-2. PubMed DOI

Row R. D.; Prescher J. A. Constructing New Bioorthogonal Reagents and Reactions. Acc. Chem. Res. 2018, 51 (5), 1073–1081. 10.1021/acs.accounts.7b00606. PubMed DOI PMC

Knall A. C.; Slugovc C. Inverse electron demand Diels-Alder (iEDDA)-initiated conjugation: a (high) potential click chemistry scheme. Chem. Soc. Rev. 2013, 42 (12), 5131–5142. 10.1039/c3cs60049a. PubMed DOI

Novák Z.; Kotschy A. First cross-coupling reactions on tetrazines. Org. Lett. 2003, 5 (19), 3495–3497. 10.1021/ol035312w. PubMed DOI

Sun H.; Xue Q.; Zhang C.; Wu H.; Feng P. Derivatization based on tetrazine scaffolds: synthesis of tetrazine derivatives and their biomedical applications. Org. Chem. Front. 2022, 9 (2), 481–498. 10.1039/D1QO01324F. DOI

Bender A. M.; Chopko T. C.; Bridges T. M.; Lindsley C. W. Preparation of Unsymmetrical 1,2,4,5-Tetrazines via a Mild Suzuki Cross-Coupling Reaction. Org. Lett. 2017, 19 (20), 5693–5696. 10.1021/acs.orglett.7b02868. PubMed DOI

Ros E.; Prades A.; Forson D.; Smyth J.; Verdaguer X.; Pouplana L. R. d.; Riera A. Synthesis of 3-alkyl-6-methyl-1,2,4,5-tetrazines via a Sonogashira-type cross-coupling reaction. Chem. Commun. 2020, 56 (75), 11086–11089. 10.1039/D0CC03482G. PubMed DOI

Wu H.; Yang J.; Šečkutė J.; Devaraj N. K. In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live-Cell Imaging Probes. Angew. Chem., Int. Ed. 2014, 53 (23), 5805–5809. 10.1002/anie.201400135. PubMed DOI PMC

Lambert W. D.; Fang Y.; Mahapatra S.; Huang Z.; am Ende C. W.; Fox J. M. Installation of Minimal Tetrazines through Silver-Mediated Liebeskind–Srogl Coupling with Arylboronic Acids. J. Am. Chem. Soc. 2019, 141 (43), 17068–17074. 10.1021/jacs.9b08677. PubMed DOI PMC

Xie Y.; Fang Y.; Huang Z.; Tallon A. M.; am Ende C. W.; Fox J. M. Divergent Synthesis of Monosubstituted and Unsymmetrical 3,6-Disubstituted Tetrazines from Carboxylic Ester Precursors. Angew. Chem., Int. Ed. 2020, 59 (39), 16967–16973. 10.1002/anie.202005569. PubMed DOI PMC

Slachtova V.; Bellova S.; La-Venia A.; Galeta J.; Dracinsky M.; Chalupsky K.; Dvorakova A.; Mertlikova-Kaiserova H.; Rukovansky P.; Dzijak R.; Vrabel M. Triazinium Ligation: Bioorthogonal Reaction of N1-Alkyl 1,2,4-Triazinium Salts. Angew. Chem., Int. Ed. 2023, 62 (36), e202306828.10.1002/anie.202306828. PubMed DOI

An initial version of this work was deposited in chemRxiv on October 27, 2023 10.26434/chemrxiv-2023-lpf8m. DOI

Galeta J.; Šlachtová V.; Dračínský M.; Vrabel M. Regio- and Diastereoselective 1,3-Dipolar Cycloadditions of 1,2,4-Triazin-1-ium Ylides: a Straightforward Synthetic Route to Polysubstituted Pyrrolo[2,1-f][1,2,4]triazines. ACS Omega 2022, 7 (24), 21233–21238. 10.1021/acsomega.2c02276. PubMed DOI PMC

Markovic T.; Rocke B. N.; Blakemore D. C.; Mascitti V.; Willis M. C. Pyridine sulfinates as general nucleophilic coupling partners in palladium-catalyzed cross-coupling reactions with aryl halides. Chem. Sci. 2017, 8 (6), 4437–4442. 10.1039/C7SC00675F. PubMed DOI PMC

Svatunek D.; Wilkovitsch M.; Hartmann L.; Houk K. N.; Mikula H. Uncovering the Key Role of Distortion in Bioorthogonal Tetrazine Tools That Defy the Reactivity/Stability Trade-Off. J. Am. Chem. Soc. 2022, 144 (18), 8171–8177. 10.1021/jacs.2c01056. PubMed DOI PMC

Devaraj N. K.; Weissleder R. Biomedical Applications of Tetrazine Cycloadditions. Acc. Chem. Res. 2011, 44 (9), 816–827. 10.1021/ar200037t. PubMed DOI PMC

Modak A.; Maiti D. Metal catalyzed defunctionalization reactions. Org. Biomol. Chem. 2016, 14 (1), 21–35. 10.1039/C5OB01949D. PubMed DOI

Wu Z.-C.; Boger D. L. Synthesis, Characterization, and Cycloaddition Reactivity of a Monocyclic Aromatic 1,2,3,5-Tetrazine. J. Am. Chem. Soc. 2019, 141 (41), 16388–16397. 10.1021/jacs.9b07744. PubMed DOI PMC

Jemas A.; Xie Y. X.; Pigga J. E.; Caplan J. L.; am Ende C. W.; Fox J. M. Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally Controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells Using Long Wavelength Light. J. Am. Chem. Soc. 2022, 144 (4), 1647–1662. 10.1021/jacs.1c10390. PubMed DOI PMC

Karver M. R.; Weissleder R.; Hilderbrand S. A. Synthesis and Evaluation of a Series of 1,2,4,5-Tetrazines for Bioorthogonal Conjugation. Bioconjugate Chem. 2011, 22 (11), 2263–2270. 10.1021/bc200295y. PubMed DOI PMC

Meimetis L. G.; Carlson J. C. T.; Giedt R. J.; Kohler R. H.; Weissleder R. Ultrafluorogenic Coumarin-Tetrazine Probes for Real-Time Biological Imaging. Angew. Chem., Int. Ed. 2014, 53 (29), 7531–7534. 10.1002/anie.201403890. PubMed DOI PMC

Galeta J.; Dzijak R.; Obořil J.; Dračínský M.; Vrabel M. A Systematic Study of Coumarin–Tetrazine Light-Up Probes for Bioorthogonal Fluorescence Imaging. Chem. - Eur. J. 2020, 26 (44), 9945–9953. 10.1002/chem.202001290. PubMed DOI PMC

Carlson J. C. T.; Meimetis L. G.; Hilderbrand S. A.; Weissleder R. BODIPY-Tetrazine Derivatives as Superbright Bioorthogonal Turn-on Probes. Angew. Chem., Int. Ed. 2013, 52 (27), 6917–6920. 10.1002/anie.201301100. PubMed DOI PMC

Siegl S. J.; Galeta J.; Dzijak R.; Dračínský M.; Vrabel M. Bioorthogonal Fluorescence Turn-On Labeling Based on Bicyclononyne–Tetrazine Cycloaddition Reactions that Form Pyridazine Products. ChemPlusChem. 2019, 84 (5), 493–497. 10.1002/cplu.201900176. PubMed DOI PMC