Figure 3.

Tz acids mediate ultrafast release. (a) Black hole quencher-Tz probes for kinetics. (b) FRET assay mechanism: BHQ-Tz efficiently quenches the MayaFluor emission after click reaction with 3; fluorescence is restored upon release of the MayaFluor amine, allowing direct quantification of fast release. (c) Analytical configuration for the kinetic experiments, employing a triangular cuvette due to the very high absorbance of the BHQ-Tz at the mM concentrations required to temporally resolve click and release. (d) Fluorescence intensity vs time profiles for the reaction of 3 and 7; dual exponential fits of the click and release phases are superimposed for the four independent experiments at varying reaction pH. (e) Release rates are strikingly pH dependent, increasing ∼65-fold between pH 7.4 and pH 4 in tandem with release yields (corresponding data in gray from Figure 1e). (f) Release rates for 7–9 at pH 7.4, with respective half-lives superimposed; the PA-group accelerates release by nearly 2 orders of magnitude. Data are mean ± sd for three independent measurements. (g) Release for 8 and 9 is further accelerated by lower pH; *the reported rate for PA-directed release at pH 6 is a minimum estimate, as release is too fast to fully resolve.