To evaluate the efficacy of the amplification strategy, we first studied the SH-triggered release of TPPTS from compound 1. Nucleophilic substitution with S-terminal nucleophiles on 1 result in the release of TPPTS and the formation of 2 (Figure ). We exposed 2.0 mM of 1 to substoichiometric amounts of 4 ranging from 0.1 to 0.8 mM and monitored the release of TPPTS by UV-vis absorbance at 260 nm (Figure ). When 40% (0.8 mM) thiol was used (vs. compound 1), we observed complete release of TPPTS within ~20 hours (Figure ). As expected, the release was slower when less thiol was added to the system. However, the release of TPPTS could stoichiometrically be correlated to the amount of SH input. Indeed, we found a linear correlation (R 2 = 0.99) between thiol input and released TPPTS (Figure ). This shows that the system displays a high sensitivity towards S-terminal nucleophiles, without the interference of Michael addition as side reactions. As control we tested compound 1 without 4 in phosphate buffer (0.1 M, pH = 7.6) by monitoring it over 24 hours with 1 H NMR spectroscopy (Supplementary Figure ). Over this time, we found that 1 is stable in the absence of thiol, and we did not observe release of TPPTS via hydrolysis. Background interference such as hydrolysis or side reactions without trigger have been a consistent issue among self-propagating amplification reactions. TPPTS alone in aqueous media showed negligible oxidation, forming less than 5% OTPPTS during the 24 hours experimental period under air (Supplementary Figure ).