Theoretical Design and Synthesis of Caged Compounds Using X‐Ray‐Triggered Azo Bond Cleavage

Abstract Caged compounds are frequently used in life science research. However, the light used to activate them is commonly absorbed and scattered by biological materials, limiting their use to basic research in cells or small animals. In contrast, hard X‐rays exhibit high bio‐permeability due to the difficulty of interacting with biological molecules. With the main goal of developing X‐ray activatable caged compounds, azo compounds are designed and synthesized with a positive charge and long π‐conjugated system to increase the reaction efficiency with hydrated electrons. The azo bonds in the designed compounds are selectively cleaved by X‐ray, and the fluorescent substance Diethyl Rhodamine is released. Based on the results of experiments and quantum chemical calculations, azo bond cleavage is assumed to occur via a two‐step process: a two‐electron reduction of the azo bond followed by N─N bond cleavage. Cellular experiments also demonstrate that the azo bonds can be cleaved intracellularly. Thus, caged compounds that can be activated by an azo bond cleavage reaction promoted by X‐ray are successfully generated.

Chemical shifts were referred to tetramethyl silane or heavy solvent peak as standard and expressed in ppm values.
In the cell study, intracellular fluorescence was measured using a CytoFlex (Beckman Coulter, Pasadena, CA, USA) with a set excitation wavelength of 488 nm and fluorescence wavelength of 564-606 nm.

Quantum chemical calculations
Density functional theory (DFT) calculations were carried out with a Gaussian16 program, [2] using the long-range corrected ωB97XD functional (including Grimme's dispersion correction) and cc-pVDZ basis set.The solvent effect of water was taken into account by using the integral equation formalism variant of the polarizable continuum model (IEFPCM).For each AZO-Rhodamine derivative, equilibrium structures were systematically searched by the single-component artificial force induced reaction (SC-AFIR) method implemented in GRRM17 software, [3] and then the lowest energy structure was employed for the vertical electron affinity (VEA) calculation.The VEA is calculated as the energy gap between non-reduced (NR) and one-electron-reduced (OER) states at equilibrium structure of the NR form (see Figure 3A in the main text).

Dose dependency for Diethyl Rhodamine released from AZO-Rhodamine2
A 5 µM AZO-Rhodamine2 solution in 1 mM phosphate buffer (pH 7.4) containing 40% MeOH was prepared in a vial.The solution was bubbled with argon gas through the septum cap of a sealed vial and irradiated with X-rays at 4, 10, 20, and 40 Gy using CLINAC (irradiation dose rate: 4.7 Gy/min, tube voltage: 6 MeV).Subsequently, fluorescence spectra of these solutions were measured (excitation wavelength: 480 nm).

Cell study
Human leukemia cell lines MOLT-4 were purchased from RIKEN Cell Bank (Tsukuba, Japan).MOLT-4 cells were cultured in an RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and maintained in a humidified atmosphere containing 5% CO2 at 37 °C.The cells were sub-cultured when the cell concentration reached 1.0 × 10 4 cells/mL.
After incubating the cells with the dye solution for 80 min, the medium was removed by centrifugation at 400 g for 5 min, and the cells were resuspended in 30 mL of phosphate-buffered saline (final cell concentration: 1.0 × 10 6 cells/mL).The cell suspensions were transferred to sealed vials and bubbled with nitrogen gas for 10 min to remove oxygen, and further irradiated with X-rays at various doses (0, 2.5, 5, 10, and 25 Gy) setting the dose rate at 4.37 Gy/min.Low-energy X-rays were filtered out using a 0.3 mm copper plate.
Each irradiated sample was transferred to a measuring tube and analyzed using CytoFlex.

Diethyl Rhodamine
2-(4-Dietylamino-2-hydroxylbenzoyl) benzoic acid (2.19 g, 7.00 mmol), 3-aminophenol (763 mg, 7.00 mmol) were added to methane sulfonic acid (10 mL) in a round bottle flask and reacted at 90 °C for 7 h.The reaction solution was then dissolved in RO water and salted out using potassium chloride.The salted product was recrystallized using 100 mL of toluene to obtain red crystals of the target compound (982 mg).The yield was 29%.No further purification was performed.

AZO-Rhodamine1
Diethyl Rhodamine methane sulfonic acid (100 mg, 0.207 mmol) was dissolved in RO water (8 mL), acetonitrile (8 mL), and 1 M hydrochloric acid (4 mL) in the round bottle flask.After cooling, sodium nitrite (28.6 mg, 0.414 mmol) was added and the reaction mixture was stirred at 0 °C for 15 min.Then, phenol (39 mg, 0.415 mmol) was dissolved in MeOH (2 mL), and this MeOH solution was added dropwise to the previous mixture and allowed to react over 15 min.Next, 0.1 M aq.ammonia was added until pH 6-7 was reached.After removal of all the volatiles under reduced pressure, the residue was purified by column chromatography using silica gel

Figure S10 .
Figure S10.Raw data of the LC-MS chart of AZO-Rhodamine1 after irradiation.

Figure S11 .
Figure S11.Raw data of the LC-MS chart of AZO-Rhodamine2 after irradiation.

Figure S12 .
Figure S12.Raw data of the LC-MS chart of AZO-Rhodamine3 after irradiation.