Pro-fluorescent ethynylthiophene-based <i>o</i> -nitrobenzyl photolabile protecting group for hydroxamic acid synthesis

Photolabile protecting groups (PPGs) that enable real-time monitoring of uncaging processes are highly sought after for tracking product release during and after photolysis. Few PPGs facilitate direct detection of uncaging events through a fluorescence signal, with <i>o</i>-nitrobenzyl (<i>o</i>-NB) PPG derivatives being the only known examples exhibiting pro-fluorescent properties. In this study, we broaden the scope of accessible pro-fluorescent <i>o</i>-NB PPGs for direct monitoring of product release by reporting two new pro-fluorescent, ethynylthiophene-based, and visible light-absorbing <i>o</i>-NB PPGs, referred to as NPETs 1 and 2. UV-Vis spectroscopy confirmed the complete cleavage of hydroxamic acid (HA) derivatives from NPETs 1 and 2, as evidenced by a blue shift and reduced absorbance intensity. This step likely proceeds through an <i>aci</i>-nitro intermediate, supported by both spectroscopic and computational examinations. We also assessed the released HA products by monitoring the corresponding increase in fluorescence intensity, which corresponds to the co-generated nitrosoketone by-product. The 4-fold and 3-fold increase in fluorescent intensity for NPETs 1 and 2, respectively, was easily observable with the naked eye. Time-course ¹H NMR experiments revealed that NPET 2 exhibits greater stability than NPET 1, showing only minor degradation after 30 days at ambient conditions. (TD)-DFT calculations revealed that the nitrosoketone by-product emission occurs from the S₂ singlet excited state, violating Kasha's rule. This study highlights the efficacy of pro-fluorescent, ethynylthiophene-based <i>o</i>-NB PPGs in facilitating precise photoreactions under mild acidic conditions. Their pro-fluorescence response and minimal degradation under ambient conditions indicate their potential for application in releasing synthetically difficult-to-synthesize functional groups.