A structurally compact aqueous soluble oxypicolinium photocage with high photosensitivity†

Photocages are protecting groups that can be unmasked with light irradiation and provide spatiotemporal control over the activity of a biomolecule. Ideally, photocages have high quantum yields for deprotection and good water solubility. Small photocages are preferred in applications where minimal biological perturbations of the photocage are desired or where large photocage structures stymie needed binding or activity, such as in the incorporation of unnatural photocaged amino acids into proteins. Here, we report N-methyl-3-oxypicolinium ester, by molecular weight the smallest photocage reported to date. The photocage is smaller (by MW) than even the parent nitrobenzyl photocage, but has improved properties, including being more water soluble as a result of its zwitterionic character, a higher quantum yield of release of 0.32 at pH 7 for release of the mediocre leaving group AcOH, and a superior chromophore within a single benzene ring (λ_max = 320 nm, λ_em = 402 nm) that matches the absorbance wavelength of the larger 7-hydroxy coumarin photocage. The zwitterionic character aids in water solubility, red-shifts the chromophore absorption and emission by raising the HOMO and lowering the LUMO, and improves the quantum yield of release. Cell studies show that the photocage crosses the HEK293 cell membrane and shows no observable toxicity (trypan blue exclusion assay, 25 μM), while mechanistic studies indicate a singlet photoheterolysis mechanism that is supported by ultrafast transient absorption spectroscopy, oxygen sensitivity studies, computational investigations, and photoproduct analysis.