Tetrazine‐Triggered Bioorthogonal Cleavage of trans‐Cyclooctene‐Caged Phenols Using a Minimal Self‐Immolative Linker Strategy

Abstract Bond‐cleavage reactions triggered by bioorthogonal tetrazine ligation have emerged as strategies to chemically control the function of (bio)molecules and achieve activation of prodrugs in living systems. While most of these approaches make use of caged amines, current methods for the release of phenols are limited by unfavorable reaction kinetics or insufficient stability of the Tz‐responsive reactants. To address this issue, we have implemented a self‐immolative linker that enables the connection of cleavable trans‐cyclooctenes (TCO) and phenols via carbamate linkages. Based on detailed investigation of the reaction mechanism with several Tz, revealing up to 96 % elimination after 2 hours, we have developed a TCO‐caged prodrug with 750‐fold reduced cytotoxicity compared to the parent drug and achieved in situ activation upon Tz/TCO click‐to‐release.

HRMS analysis of aqueous or acetonitrile solutions of the compounds (sample concentration: 10 ppm) was carried out on an Agilent 6545 Q-TOF mass spectrometer equipped with an Agilent Dual AJS ESI source. The mass spectrometer was connected to a liquid chromatography system comprised of an Agilent G7167B multi sampler, an Agilent G7120A binary pump with degasser and an Agilent G7116B oven (Agilent Technologies, Palo Alto, CA, USA). A SecurityGuard Cartridge (Phenomenex) was used as a stationary phase. Data evaluation was performed using Agilent MassHunter Workstation Qualitative Analysis 10.0. Identification was based on peaks obtained from extracted ion chromatograms (extraction width ± 20 ppm).

Instrument and solvents
Reaction monitoring of release experiments was performed on a Nexera X2® UHPLC system (Shimadzu®) with a temperature-controlled autosampler at 37 °C. For acidic HPLC conditions, the aqueous solvent was prepared by addition of 2.5 mL of neat formic acid to 2.5 L of HPLC-water to yield a final concentration of 0.1% formic acid. For buffered HPLC conditions, the aqueous solvent was prepared by addition of 625 μL of 10 M ammonium formate (BioUltra, Sigma-Aldrich) to 2.5 L of HPLCgrade water followed by adjusting the pH to 8.4 by addition of 25% aqueous ammonia (for HPLC, LiChropur, Merck). Since its pH declines over time, this volatile buffer was freshly prepared each day. HPLC-grade acetonitrile was used without any additives.

Release kinetics measurements
rTCO-DMEDA-Tyr-BODIPY (6): The stock solution of 6 was diluted with PBS to a concentration of 100 µM in an HPLC vial (2.5 µL 20 mM TCO stock, 497.5 µL PBS). Tetrazine stock solutions were diluted with PBS to a concentration of 200 µM in an HPLC vial (2.5 µL Tz stock, 122.5 µL PBS), and the click-torelease reaction was initiated by addition of the diluted TCO solution (125 µL) to obtain starting concentrations of 50 µM TCO and 100 µM Tz. The samples were immediately incubated at 37 °C in the autosampler and subjected to serial HPLC analysis. All measurements were conducted in triplicates.
sulfo-cTCO-DMEDA-CA4 (12): The stock solution of 12 was diluted with DMSO (30 µL TCO stock, 30 µL DMSO) to give a 10 mM stock solution. Tz stock solution (10 µL) was added to PBS (985 µL, containing 8.6% DMSO), and the click-to-release reaction was initiated by addition of the stock solution of 12 (5.24 µL) to obtain starting concentrations of 50 µM TCO and 100 µM Tz (in 10% DMSO/PBS). The samples were immediately incubated at 37 °C in the autosampler and subjected to serial HPLC analysis in intervals of 30 min. All measurements were conducted in triplicates.

Determination of exact TCO stock concentrations
The exact TCO stock concentrations were determined by absorbance titration (535 nm) with a freshly prepared stock solution of 3,6-bis(2-pyridyl)tetrazine 2Pyr2 (Sigma Aldrich) in DMSO using a Thermo Fisher Scientific NanoDrop One C Microvolume UV-Vis Spectrophotometer in cuvette mode at 25 °C. The TCO stock solution (20 mM) was diluted with DMSO to reach a concentration of 1 mM, and then spiked with an excess of 2Pyr2 stock solution (20.4 mM). Upon IEDDA reaction, the remaining tetrazine absorbance at 535 nm was measured. This procedure was repeated twice (standard addition) to determine the exact TCO stock concentration.
Analytical HPLC analysis rTCO-DMEDA-Tyr-BODIPY (6): PDA data was collected for all samples and used to identify all signals showing a characteristic BODIPY absorption. Relative quantification of reactants, intermediates and products was done using extracted chromatograms (wavelength: 500 nm).
sulfo-cTCO-DMEDA-CA4 (12): PDA data was collected for all samples. Relative quantification of intermediates and products was done using extracted chromatograms (wavelength: 254 nm). In addition, released CA4 was quantified via external calibration.

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External CA4 calibration A CA4 stock solution (20 mM) was prepared in DMSO and diluted with PBS (containing 10% DMSO) to reach a concentration of 100 µm. CA4 standard solutions (1 µM -75 µM) were prepared by serial dilution into PBS (containing 10% DMSO). All measurements were conducted in triplicates.

Click Kinetics
A stock solution of rTCO-PEG4 [3] in DMSO was prepared at an approximate concentration of 20 mM. The exact concentration was determined by absorbance titration with DMT (7) (extinction coefficient 510 M -1 cm -1 at 520 nm), quantifying the decrease in tetrazine absorbance upon reaction with TCO. The initial DMSO stock was diluted with PBS to prepare solutions for stopped-flow analysis at a final TCO concentration of 1 mM. 20 mM stock solutions of DMT (7), PA2 (8), and PymK (9) in DMSO were prepared. Serial dilution into PBS gave solutions for stopped-flow analysis at a Tz concentration of 100 µM.
Stopped-flow measurements were performed using an SX20-LED stopped-flow spectrophotometer (Applied Photophysics) equipped with a 535nm LED (optical pathlength 10 mm, full width half-maximum 34 nm) to monitor the characteristic tetrazine visible light absorbance (520-540 nm). The reagent syringes were loaded with tetrazine and TCO solutions and the instrument was primed. Measurements were done in sextuplicate for each Tz. Reactions were conducted at 37 °C and recorded automatically at the time of acquisition. Data sets were analyzed by fitting an exponential decay using Prism 6 (GraphPad) to calculate the observed pseudo-first-order rate constants that were converted into second-order rate constants (Table S1) by dividing through the TCO concentration.

Cell Viability Assays
HT1080 human fibrosarcoma cells (ATCC) were cultivated in EMEM (Minimum Essential Medium Eagle, with Earleʹs salts, L-glutamine and sodium bicarbonate; Sigma Aldrich) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (100X, Sigma-Aldrich) at 37 °C and 5% CO2. HT1080 cells were seeded into 96-well plates (triplicates for each group) at 10.000 cells per well and allowed to grow overnight.
Cell viability was assessed by replacing the medium with 100 μL of PrestoBlue solution (Invitrogen, 1:9 in growth medium) followed by incubation for 30 minutes at 37 °C. Read-out of the fluorescence signal was carried out using a PerkinElmer EnSpire Multimode Plate Reader and data processing was done in GraphPad Prism.
Following the same procedure, cells were treated with DMT (7), PymK (9), or 1,3dimethylimidazolidin-2-one (= byproduct of the self-immolation process) with concentrations of up to 10 µM, revealing no significant effect on cell viability.

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6 Cell Imaging 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 (12) in media. In situ click-to-release was initiated by addition of DMT (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 (7). After an incubation time of 6 h cells were stained with SiR-tubulin (a fluorogenic, cell permeable and highly specific probe for microtubules). [6] 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 (Fig. S1). Figure S1. Cell imaging via fluorescence microscopy (scale bars: 50 µm) upon staining with Hoechst 33342 (blue, nuclei) and SiR-tubulin [6] (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 (7). No significant change (compared to untreated cells) was observed after treatment with DMT (7) or prodrug 12. Prodrug 12 was incubated in (i) PBS and (ii) cell growth medium (DMEM + 10% fetal bovine serum) at 37 °C at a concentration of 100 µM (1 µL 10 mM stock in DMSO + 99 µL PBS/medium). Samples in PBS were analyzed by serial HPLC measurements (n=3) for 120 h. No degradation of the DMEDA bis(carbamate)-linkage and <5% isomerization of the cTCO linker was observed.
Aliquots (50 µL) of samples in cell growth medium were diluted with ice cold MeCN (200 µL) followed by centrifugation at 14,000 rpm for 8 min at 4 °C. A sample of CA4 (100 µM in cell growth medium) was prepared following the same procedure as a control. HPLC analysis of the supernatants (n=3) revealed 33.0 ± 0.2% trans-to-cis isomerization of the cTCO linker after 72 h and 43.6 ± 1.0% after 120 h (as verified by addition of 2Pyr2), but no release of CA4, confirming integrity of the DMEDA bis(carbamate)-linkage (Fig. S2). cis-12 S18 8 NMR Spectra, Chromatograms and MS Data S1, 1 H NMR S1, 13 C NMR