NMR data and fluorescence microscopy data

For details on the used compounds and all compound numbers see 10.1002/cbic.202200363 . Technical information - NMR: 1 H and 13 C NMR spectra were recorded on a Bruker Ascend 600 MHz spectrometer at 20 °C. - Fluorescence microscopy: HT1080 cells were seeded into a 96-well plate at 3,000 cells per well and allowed to grow overnight. The medium was removed, and the cells were treated with a 200 nM solution of sulfo-cTCO-DMEDA-CA4 (compound 12) in media.

In situ click-to-release was initiated by addition of DMT (compound 7) at a final concentration of 10 µM. As controls, cells were left untreated or incubated with either the parent drug CA4 (200 nM) or 10 µM DMT (compound 7). After an incubation time of 6 h cells were stained with SiR-tubulin (a fluorogenic, cell permeable and highly specific probe for microtubules). An 11X stock solution of the probe was directly added to the growth medium to obtain a final concentration of 1 µM and incubation was carried out for 1 h.

Subsequently, the medium was removed, and cells were stained with Hoechst 33342 nuclear dye (Invitrogen, 5 µM in growth medium) for 10 minutes and washed once with PBS. Multichannel imaging of the cells was carried out in FluoroBrite DMEM medium (Gibco) on an Olympus IX82 microscope. Summary of results - NMR: The obtained data confirmed the chemical structures of all synthesized compounds.

Results of data analysis are provided in 10.1002/cbic.202200363 (freely accessible). - Fluorescence microscopy: Cell imaging via fluorescence microscopy (scale bars: 50 µm) upon staining with Hoechst 33342 (blue, nuclei) and SiR-tubulin (red, microtubules) shows comparable depletion of tubulin signals after 6 h treatment with CA4 (200 nM) or bioorthogonal activation of prodrug 12 (200 nM) by in situ reaction with DMT (compound 7).

No significant change (compared to untreated cells) was observed after treatment with DMT (compound 7) or prodrug 12.