Design, Synthesis, and Photochemical Properties of Gene-directed Caged RyR Probes for Photopharmacological Studies

Ryanodine receptor (RyR) is a crucial intracellular Ca²⁺ channel involved in various physiological processes associated with several diseases. This paper presents the synthesis and evaluation of novel photoactivatable caged compounds for the RyR. We used (6-bromo-7-hydroxycoumarn-4-yl)methyl (Bhc) as the photolabile protecting group to develop caged versions of 4-CmC, a broad-spectrum RyR agonist, and FLA365, an antagonist. The synthesized compounds, Bhcmoc-4-CmC and Bhcmoc-FLA365, exhibited photoreactivity that enabled spatiotemporal control over the RyR activity. Bhcmoc-4-CmC presents a quantum yield of 25 % and a photolysis efficiency of 1,075 M⁻¹ cm⁻¹ under 405 nm light, triggering Ca²⁺ release from the endoplasmic reticulum. Conversely, Bhcmoc-FLA365 presents a reduced quantum yield of 0.61 %, which can be attributed to the quenching effects of its tertiary amine structure. We also applied gene-directed caging, synthesizing β-galactosidase (β-Gal) activatable Gal-Bhcmoc-4-CmC and Gal-Bhcmoc-FLA365, and porcine liver esterase (PLE)-activatable CM-Bhcmoc-FLA365, to target these probes to the specific cell types. These caged compounds contribute significantly to photopharmacology, providing tools for the precise analysis and manipulation of the RyR functions. Future research objectives involve further optimization and analysis of the photochemical mechanisms of these caged compounds to enhance their applications in biological and medical research.