Data for Publication: Intramolecular control of click-to-release chemistry unlocks fast and quantitative bioorthogonal bond cleavage in living systems

Context and methodology

This dataset was generated within a research project investigating intramolecular control of tetrazine-triggered click-to-release chemistry through the development of iTCO, a trans-cyclooctene linker equipped with a tautomerization-directing hydroxyl group. The corresponding publication describes the scientific background, molecular design, mechanistic investigations, and biological applications in detail, while the Supplementary Information provides extended experimental, analytical, and computational methods.

The purpose of this dataset is to document the quantitative experimental data underlying the figures reported in the associated manuscript and Supplementary Information. The data support the development and characterization of iTCO, including its click-to-release efficiency and kinetics with structurally diverse tetrazines, the mechanistic analysis of post-click tautomerization and elimination pathways, the influence of pH and biological media on reaction performance, and the stability of iTCO conjugates. The dataset further supports experiments demonstrating bioorthogonal activation of iTCO-caged prodrugs in solution and in living cells, as well as real-time click-triggered cleavage of an antibody-conjugated iTCO probe from circulating red blood cells in vivo.

The data were generated using established experimental techniques, including high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), UV-Vis and fluorescence spectroscopy, NMR spectroscopy, cell-based viability assays, and fluorescence microscopy. Computational studies using density functional theory (DFT) and time-dependent DFT were additionally used to investigate the post-click reaction mechanism and photophysical properties of relevant intermediates and products. The optimized molecular geometries obtained from these calculations are provided as XYZ files. Raw NMR data supporting the structural characterization and mechanistic investigations are likewise included. Detailed experimental procedures, reaction conditions, analytical methods, and computational protocols are described in the accompanying manuscript and Supplementary Information.

Technical details

The dataset consists of a structured Excel workbook containing quantitative data underlying the experimental figures presented in the associated publication and Supplementary Information, together with raw NMR spectroscopy data and calculated molecular geometries from the computational studies.

The Excel workbook contains individual worksheets corresponding to specific figures, enabling direct mapping between the deposited data and the graphical representations in the publication.

Raw NMR spectroscopy data are provided for the structural characterization of synthesized compounds and for NMR-based mechanistic experiments reported in the manuscript and Supplementary Information. The data are provided in their original instrument format and allow independent processing and analysis using standard NMR software such as Mnova or TopSpin. Assignment of the NMR datasets to specific compounds and experiments is described by the corresponding file and folder names and in the associated Supplementary Information.

Calculated molecular geometries from the DFT and time-dependent DFT studies are provided as XYZ files. These files contain the optimized Cartesian coordinates of the molecular structures investigated computationally and can be opened, visualized, and further processed using commonly available molecular visualization and computational chemistry software. Assignment of the calculated structures to the corresponding intermediates, products, and computational analyses is provided through the file names and the associated Supplementary Information.

The Excel workbook can be accessed using standard spreadsheet software such as Microsoft Excel or equivalent open-source applications.

All additional information required for interpretation of the deposited data, including molecular structures and compound numbering, experimental procedures and conditions, analytical methods, kinetic analyses, computational methods, and details of the cellular and in vivo experiments, is provided in the associated manuscript and Supplementary Information.

Further details

The data are suitable for reuse in comparative studies, methodological benchmarking, meta-analyses, or educational contexts, provided that appropriate attribution to the original publication is given.