We then investigated the self-immolation chemistry of SIE2 to confirm its molecular releasing capability (Figure ). We incubated a 2 mM SIE2 solution at room temperature overnight (90% pH 6.5 phosphate buffer in D₂O and 10% DMSO-d6) and purified the reaction mixture using silica gel flash chromatography (Figure ). The hypothesized cyclization product and the released coumarin cargo were successfully recovered in high yields, with their identities confirmed by NMR and mass spectrometry (see SI for details).