Sulfonothioated meso-Methyl BODIPY Shows Enhanced Uncaging Efficiency and Releases H2Sn

meso-Methyl BODIPY photocages stand out for their absorption properties and easy chromophore derivatization. However, their low uncaging efficiencies often hinder applications requiring release of protected substrates in high amounts. In this study, we demonstrate that the sulfonothioated BODIPY group photocleaves a sulfonylthio group from the meso-methyl position with a 10-fold higher quantum yield than the most efficient leaving groups studied to date. Photocleavage, observed in solution and in cells, is accompanied by the spatiotemporally controlled photorelease of H2Sn. For this reason, sulfonothioated BODIPY may be applied in cell signaling, redox homeostasis, and metabolic regulation studies.


Experimental section
The reagents and solvents used in the study were purchased from commercial suppliers and used without purification unless stated otherwise.The solvents were removed on a rotary evaporator at 25-40 °C, and the products were dried in vacuo overnight.TLC analysis was performed on silica gel-coated aluminum plates (60F254, Merck, Darmstadt, Germany).The compounds were visualized using one of the following methods: exposure to UV light at 254 nm or 365 nm, KMnO4 spraying (1 % aqueous solution, yellow color of oxidizable compounds), and exposure to iodine vapors (yellow to brown spots, non-specific).Flash chromatography purifications were performed on silica gel (40-63 µm, Sigma-Aldrich).Gradient chromatography was performed on an ECOM flash chromatograph. 1 H and 13 C NMR spectra were measured on Bruker-400 AVANCE III HD ( 1 H at 400.13 MHz, 13 C at 100.62 MHz, 19 F at 376 MHz) in CDCl3, CD 3 OD or DMSO-d 6 solution at 300 K. H,H-COSY, H,C-HSQC, and H,C-HMBC spectra were recorded and used for the structural assignment of proton and carbon signals.HR-MS (high-resolution mass spectrometry) spectra were recorded on an FTMS mass spectrometer LTQ-orbitrap XL (Thermo Fisher Scientific) in electrospray ionization mode.HPLC-MS was performed on an HPLC-MS-2020 (Shimadzu Corporation).UV vis spectrometry was measured on an Agilent Cary 8454 UV-vis spectrometer (Agilent), and emission spectra were measured on a Duetta fluorescence spectrometer (Horiba).Mixtures of solvents are given in a v/v ratio.

5-(Dimethylamino)naphthalene-1-sulfinic acid
Dansyl chloride (400 mg, 1.5 mmol, 1 eq) was added to a solution of NaHSO3 (154 mg, 1.5 mmol, 1 eq) and K2CO3 (204 mg, 1.5 mmol, 1 eq) in water.The suspension was stirred at 50 °C for 8 h in a heating mantle and then stirred at room temperature overnight.Subsequently, the solvent was removed on a rotary evaporator, and the crude product was used in the next reaction without further purification.The purity of the target product was confirmed by HPLC-MS.
The organic layer was evaporated with SiO 2 and dry-loaded on the chromatography column.The product was purified by gradient chromatography using CHEX:DCM (0-100 % of DCM) as a mobile phase.The solvent was removed, yielding dark brown crystals (347 mg, 68 %).
The reaction mixture was left to stir for 10 minutes.The solvent was removed, and the mixture was dissolved in DCM and co-evaporated with SiO2.Then, the mixture was dry-loaded on the column, and the crude product was purified by gradient chromatography with a DCM:CHEX mobile phase (0-100 % of DCM).The target compound was obtained as an orange-red solid (40 mg, 48 %).

Fluorescence quantum yields
All compounds were dissolved in approximately 100 μL DCM, and the concentration was adjusted with MeOH to an absorbance of 0.1-0.2 at 410 nm.Then, the emission spectrum was measured (λexc = 410 nm, slits = 5 nm, spectra accumulation = 1, integration time = 1 for compound 1 or 10 s for sulfinates).The peak area was calculated from the measured spectrum.The measurement was repeated 4 times with independent samples.The same process was performed for the standard (1-OAc) with a known fluorescence quantum yield   , determining the slope of the curve of the variation of peak area as a function of absorbance at 410 nm.The quantum yield   was calculated using the following equation.

𝜙 𝑥 = 𝜙 𝑠𝑡𝑑 ( slope 𝑥 slope 𝑠𝑡𝑑 )
Due to the presence of a highly emissive impurity (9), the quantum yields were corrected using the standard addition method.The content of compound 1 was calculated based on the NMR purity.Then, compound 9 was added as a standard (0.25, 0.5, 0.75, 1.0 eq).Thanks to the known amount of impurity, the corrected peak area of the sample was calculated.Subsequently, the ratio (areaREAL/areaCORR) = 1.5 was calculated, and the quantum yields were corrected using this factor.

Figure S3
. a) Reaction scheme.UV-Vis spectra of 1 irradiated at 400 nm in degassed MeOH: b) without TX; c) with TX (1000 eq); d) with TX (100 eq).e) Time-dependent absorption of 1 at 520 nm with and without TX.

A B C D H 2 S yield determination
The procedures for methylene blue (MB) reagent mixture preparations, H2S calibration curves, and NaSH standardization were adapted from protocols previously published in the literature. 7A solution of 1 (0.5 mL, 300 μM) in a MeOH:DMSO mixture (1:0.03,v/v) was placed in an aluminumcapped vial, bubbled with argon for 10 min, and irradiated at 525 nm for 30 min, subsequently adding a solution of glutathione (corresponding concentration, 0.5 mL in PBS).The variation of the yield as a function of the incubation time is shown in Figure S4c.UV-Vis spectra were recorded after 1 h of reaction upon adding a methylene blue cocktail solution (1 mL),.Absorbance at 670 nm was used to calculate the concentration according to H2S calibration curves (Figure S4a).Methylene blue cocktail was freshly prepared before every use by mixing zinc acetate (1 % w/v), FeCl3 (30 mM in 1.2 M HClaq), and N,N-dimethyl-p-phenylene diamine (20 mM in 7.2 M HClaq) in a 1:2:2 ratio.

Sulfonothioate sensitization
A solution of compounds 3 (0.3 mM) and 7 (3 mM) in 0.5 mL MeOH (with 3 % water) was irradiated at 525 nm for 60 min in aluminum-capped vials bubbled with argon prior to irradiation.The resulting mixture was analyzed by HPLC-MS.The conversion of 3 to 8 was 20% in 60 min.

Photoreactivity and hydrolytic stability of 3
The photostability of 3 was analyzed by irradiating the compound with a 365 nm light source in a H2O:MeOH mixture (1:1, v/v).The reaction mixture was further analyzed by HPLC-MS.The hydrolytic stability of 3 was assessed at 50 °C in H2O:MeOH mixture (1:1, v/v), and the solution was further analyzed by HPLC-MS.The compound is stable for more than 24 h in solution at room temperature in the dark.

Confocal microscopy
A U-2 OS cell line (cat.no.ACC-785, DSMZ) was cultured in DMEM high glucose 4.5g/L medium (cat.no.R8758, Merck, Germany) supplemented with 10% fetal bovine serum -FBS (cat.no.F7524, Merck, Germany; inactivated at 56 °C for 30 minutes) and 1% L-glutamine (cat.no.G7513, Merck, Germany).Cells were cultured in an incubator (37°C, 5% CO2).Adherent cells were dissociated using a Trypsin-EDTA solution (cat.no.T4049-500 mL, Merck, Germany) and seeded into 96-well glass bottom plates (Cellvis, P96-1.5H-N) at a concentration of 30 000 cells per well in complete Leibowitz L-15 medium (10% FBS) 24 hours prior to the experiment.Immediately before the experiment, the medium was changed to a complete L-15 medium with 100 μM of 1 and left standing in the dark for 1 min.Subsequently, the cells were continuously irradiated with a 492-nm laser (40% power) for 3 minutes, setting the detector (GaAsP) range to 516 -543 nm.The first and last images are shown in the manuscript (Figure 2e-f).As a control, cells were imaged at the beginning and 3 minutes later, in the dark, using the same settings (Figure S34), under a confocal microscope ZEISS LSM 980 fitted with an incubator and a 20x/0.75Plan-Apochromat objective (Carl Zeiss Jena GmbH, Germany).The images were processed in Zeiss Imaging Software ZEN 3.2 (blue edition) and Fiji software (2.9.0), calculating the mean pixel value of 8-bit images (Figures S35, irradiated sample, and S36, dark control).

HPLC-MS analysis
The reaction in phosphate buffer (PBS, cat.No. D8537, Sigma-Aldrich) was initiated by dissolving 1 in a final concentration of 1 μM (from 1 mM DMSO stock solution) in 1.5 mL Eppendorf tubes.The tubes were irradiated at 525 nm or kept at room temperature in the dark for 5 minutes.The reactions were stopped immediately by adding an equal volume of 100% acetonitrile with 40 mM N-ethylmaleimide (NEM) as the derivatization agent 10 and kept in the dark on ice.All experiments were performed in triplicates.
For analysis, 2 μL of the sample was injected into an Agilent 1290 HPLC system and separated on a Luna Omega 1.6 μm Polar C18 Column (100 x 2.1 mm) using a mobile phase consisting of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B), gradient of 2 to 95% B over 6.5 minutes, and a flow rate of 0.5 mL/min.
The Sciex 6500 triple-quadrupole mass spectrometer, operating with an electrospray ionization source, was run in multiple-reaction-monitoring (MRM) mode, with the interface heated to 350 °C.The declustering potential was 20 V, the entrance potential was 10 V, and the collision energy was 28 eV.The characteristic product ions were (1) m/z 528.

Figure S1 .A solution of 1 6 Figure S2 .
Figure S1.Standard addition method for quantum yield correction

Figure S4 .
Figure S4.a) Reaction scheme of triplet quenching.UV-Vis spectra of 1 irradiated at 525 nm in degassed MeOH: b) without COT; c) with COT (2600 eq).d) Time-dependent absorption of 1 at 520 nm with and without COT.

Figure S6 .
Figure S6.A zoomed HR-MS spectrum of the crude mixture obtained after irradiating 1 in D2 18 O (273-283 m/z) at 525 nm.The number highlighted in violet corresponds to the non-isotopically labeled aldehyde, and the assigned value in yellow corresponds to non-isotopically labeled alcohol.

Figure S7 .
Figure S7.A zoomed HR-MS spectrum of the crude mixture after irradiating 1 with 525 nm light in D2 18 O (298-306 m/z).The number highlighted in violet corresponds to the non-isotopically labeled aldehyde, and the assigned value in yellow corresponds to non-isotopically labeled alcohol.

Figure S8 . 8 Figure S10 .
Figure S8.HR-MS spectrum of the crude mixture after irradiating 1 with 525 nm light in D2 18 O

Figure S33 .
Figure S33.Cell viability of 1. HeLa S3 cells were exposed to 3 different concentrations of 1 (1 μM, 10 μM, and 100 μM) and irradiated with 525 nm LED (marked as *) for 5 min.Subsequently, cells were placed in 384-well plates, and cell viability was measured after 6 (dark columns) and 24 (grey columns) hours with a CellTiter-Glo® Luminescent Cell Viability Assay.The results are expressed as percentage of relative luminescence normalized to that of the control -untreated cells (mean ± SD, n = 8).

Figure S37 .
Figure S37.Concentration of 1 (c = 1 μM) before and after irradiation in aqueous soutions determined by LCMS (left) and the corresponding increase of the concentration of 5 in the respective solution (right).Note: The conversion of 1 to 5 remained below 50%.The presence of 5 in the solution before irradiation indicates partial conversion of 1 to 5 during the sample preparation.The quantity of photoproduced 5 decreased in subsequent processes (e.g., photooxidation and bleaching) and did not correspond to the quantity of converted 1.The results are presented mean ± SD, n = 3.The Student's t test was used for statistics evaluation, two stars indicate P < 0.01, three stars indicate P < 0.001.

Figure S38 .
Figure S38.Concentration of GSSG found in the mixture of 1 (c = 1 μM) in the presence of GSH (c = 1 μM) before (left) and after irradiation (right) The results are presented mean ± SD, n = 3.The Student's t test was used for statistics evaluation, two stars indicate P < 0.01.