Caged compounds are molecules protected by photolabile protecting groups, which show biological activity with light irradiation. Caged compounds have also been used for imaging, for example, caged capsaicin and caged histamine have contributed to Ca²⁺ imaging because of their ability to control Ca²⁺ concentration by light irradiation. ^[1,2] Although they are utilized as useful chemical tools for analyzing biological process and functions, the light to activate caged compounds is commonly absorbed or scattered by biological molecules, making them difficult to use in deep tissues of animals. As a stimulus for caged compounds to control in deep tissues, X-rays could be suitable because it exhibits high bio-permeability due to the weak absorption from biological materials. Since X-rays can generate small number of radical species by ionizing water molecule, if compounds can react with these radical species faster than endogenous molecules, such compounds were expected to be used as X-ray activatable caged compounds even in living organisms. Among the radical species generated by X-rays, hydroxyl radicals and hydrated electrons (e^–_aq) account for a large proportion. Previous reports suggested that 1-phenylazo-2-naphthol is decomposed to aniline by e^–_aq with kilo-order Gy γ-ray irradiation.^[3] In this work, we theoretically designed novel X-ray activatable caged compounds possessing azo bond which can be cleaved more efficiently and selectively than 1-phenylazo-2-naphthol and showed that it was cleaved even in cultured cells. 
　　Since e^–_aq possesses a monovalent negative charge and high reducing potential, compounds with positive charge and long π-conjugated system could be highly reactive with e^–_aq due to electrostatic interactions and their lower lowest unoccupied molecular orbital (LUMO) energy. Thus, we designed AZO-Rhodamine1, 2 and 3, which features a positive charge and long π-conjugated system to an azo group (Figure. A) . These compounds were synthesized by azo coupling between Diethyl Rhodamine and phenol, resorcinol or m-hydroxybenzoic acid, respectively.
Firstly, it was examined whether azo bond can be cleaved by X-ray irradiation to release Diethyl Rhodamine. AZO-Rhodamine1–3 (final 5 µM) were dissolved in PBS containing 40% methanol, and X-rays were irradiated to produce 5 µM of e^–_aq. The irradiated solutions were analyzed by HPLC. As a result, decrease of AZO-Rhodamine1–3 and increase of Diethyl Rhodamine were observed after irradiation. In contrast, AZO-Rhodamine2, which has two hydroxy groups at benzene moiety, released the most amount of Diethyl Rhodamine (Figure. B). Based on these results, the reaction mechanism of azo bond cleavage by X-rays could be assumed as follow; (1) the azo bond undergoes a two-electron reduction with e^–_aq to form a hydrazine intermediate, (2) the N─N bond of the hydrazine intermediate is cleaved by an electron flow from hydroxyl groups.  This plausible mechanism will aid in the design of molecules that will release various drugs by X-ray irradiation in the future. 
　　Finaly, it was investigated whether azo bond could be cleaved intracellularly. After MOLT-4 cells were incubated in medium containing 5 µM AZO-Rhodamine2, the cells were irradiated with 2.5–25 Gy X-rays. The fluorescence intensity of Diethyl Rhodamine from the cells was measured by flow cytometry.  As a result, an increase in fluorescence intensity was observed even in the case of 2.5 Gy, and the fluorescence was increased in a dose-dependent manner up to an irradiation dose of 25 Gy (Figure. C). This result suggested that azo bonds were cleaved by X-ray irradiation even under intracellular conditions.　

Image/Figure:

Click to view full size

Image/Figure Caption:

Figure. A shows chemical structures of AZO-Rhodamine1–3, and reaction scheme caused by X-ray irradiation.  Figure. B shows fluorescence of released Diethyl Rhodamine by X-ray irradiation from AZO-Rhodamine2 in cuvette (λex = 308 nm), and Figure. C shows fluorescence intensity of cells emitted by Diethyl Rhodamine inside the cells after X-ray irradiation. 

Author