The protonated and deprotonated states of D and A have different peak positions in both absorption and emission. The ground-state pKa values of the D and A UAAs (without Fmoc protection), investigated by UV-Vis spectroscopy using aqueous acid-base titration, were found to be 4.31 and 7.55 for D and A, respectively (Figure ). The excited-state pKa * values were determined using Förster cycle calculations and were found to be 11.12 and -0.32 for Glu-6AQ and Lys-7HC, respectively, thus creating an enormous ΔpKa * of 11.4 units between D and A that serves as the PMF for PT across the bridge. Importantly, using fluorescence measurements while exciting the D in its protonated form or the A in its deprotonated form allows an immediate understanding of whether an excited-state PT process happened by observing the emissive species of the D/A. At first, we validated using absorption that the D and the A are primarily protonated and deprotonated, respectively, in all solvents (Figure ). Switching to fluorescence measurements, we found that only in water did the D and A undergo deprotonation and protonation, respectively, while in MeOH and CHCl3, the D could not deprotonate, and the A could not protonate (Fig. and 2b for the D and A, respectively). To understand the kinetics associated with the process, we recorded the time-resolved emission spectra (TRES) that were converted into time-resolved area-normalized emission spectra (TRANES). The results revealed a sub-ns deprotonation of the D (Fig. ) and protonation of the A (Fig. ), but only in water, meaning that the D and A UAAs cannot undergo excited-state PT in both MeOH and CHCl3 (the TRES results are in Figures and for the D and A, respectively).