As we carry out our chemically fueled reaction cycle at pH 6.0, it is reasonable to assume that most of the precursor of Fmoc-GGD in the solution or in the vesicles is completely deprotonated. In the case of Fmoc-AVD, the pKa in the fibers is close to experimental conditions, which implies that, on average, less than half of the carboxylates were deprotonated in the fibers. Consequently, the driving force of disassembly, i.e., the accumulation of negative charges on the precursor, is far less prevalent on Fmoc-AVD than Fmoc-GGD. To further corroborate that hypothesis, we monitored the chemically fueled fibers by confocal microscopy in combination with Hoechst dye 33258. That dye is cationic and stains anionic assemblies like proteins and peptides, enabling us to monitor the accumulation of negative charges (Fig. ). We found that Hoechst 33258 colocalized with the vesicles formed by Fmoc-GGD throughout the reaction cycle (Fig. ). In contrast, the fibers formed by Fmoc-AVD hardly incorporated some Hoechst 33258 dye at the beginning of the reaction cycle, when deactivation is at its highest. After 10 min, the Hoechst 33258 signal was very faint or not observable, from which we conclude that the fibers are hardly anionic, which corroborates our earlier findings (Fig. ). Finally, we performed the same experiments described above, but at various pH values between pH 6.0 and 6.5. 1 H-NMR showed a clear trend that less precursor got trapped with increasing pH, further corroborating that the pH of the surrounding media is not sufficient to deprotonate the kinetically trapped precursor (Fig. , Fig. ). We assume that working above pH 6.5 would further avoid the kinetic trapping precursor. However, at pH 7 or higher, we observed the formation of significant amounts of the side product N-acylisourea and much less anhydride.