Photo- and Ca2+- induced acceleration of uncaging process from hemiacetal intermediate formed in the photolysis of an o-nitrobenzyl type of photoremovable protecting group (photocage)

• Nuclear magnetic resonance (NMR) and mass spectroscopy (MS) clarify the full picture assignment of a long-lived hemiacetal intermediate, confirming that hydrolysis was the rate-determining step for product release in the photoreaction of o NB PPGs. • The decomposition of the hemiacetal was accelerated by photolysis at 365 nm light, acid, base, and Ca 2+ . • Low-temperature infrared analysis under Ar-matrix isolation conditions at 4 K confirmed the photo-induced transformation of the hemiacetal. • Low-temperature spectroscopy under the glassy matrix conditions revealed the existence of a short-lived aci -nitro intermediate at 405 nm at 95 K. • Transient absorption spectroscopy provided rate constants for the aci -nitro decay process in the microsecond time scale = 8.3 × 104 s −1 (τ = 12 µs, 43 %) and = 2.0 × 104 s −1 (τ = 51 µs, 57 %). The activation energy for the decay process was determined to be 11.5 kcal mol −1 , which was consistent with the computed cyclization reaction of the aci -nitro compound at the B3LYP/6-31G(d) in CH3CN (SMD). A long-lived hemiacetal intermediate was assigned by 1 H and 13 C nuclear magnetic resonance and mass spectroscopy. The uncaging mechanism of ortho -nitrobenzyl ( o NB) photolabile protecting groups (PPGs) has been extensively studied for the protection and deprotection of biologically active substances. Herein, nuclear magnetic resonance and mass spectroscopy studies on the photolysis of α-methyl-(6-nitropiperonyloxymethyl) alcohol derivatives reveal a full-picture assignment of a long-lived hemiacetal intermediate, confirming that hydrolysis was the rate-determining step for product release in the photoreaction of o NB PPGs. The decomposition of the hemiacetal was accelerated by photolysis at 365 nm light, acid, base, and Ca 2+ . Low-temperature infrared analysis under Ar-matrix isolation conditions at 4 K confirmed the photo-induced transformation of the hemiacetal. Additionally, low-temperature spectroscopy under the glassy matrix conditions revealed the existence of a short-lived aci -nitro intermediate at 405 nm at 95 K. Transient absorption spectroscopy provided rate constants for the aci -nitro decay process in the microsecond time scale k = 8.3 × 10 4 s −1 (τ = 12 µs, 43 %) and k = 2.0 × 10 4 s −1 (τ = 51 µs, 57 %). The activation energy for the cyclization process was determined to be 11.5 kcal mol −1 , which was confirmed by quantum chemical calculations. These findings enhance our understanding of the photochemical processes for potential applications of the o NB protecting group in caged compounds.