NMR binding experiments were performed on BAF compounds and the dye orange G. By monitoring the aromatic regions of the 1H NMR spectra of BAFs 1, 8, and 31, these compounds were shown to bind to both Aβ16–21 and Aβ1–42 fibers more tightly than does orange G. As shown in (A and B), BAF1 binds to Aβ16–21 fibers with affinity stronger than orange G. The determination of binding parameters for Aβ16–21 fibers is detailed in Table 5 and Figure 5—figure supplements 1 and 3. In panel (A), the 1H NMR spectrum of compound BAF1 (at 100 μM) is shown as a function of increasing concentration of Aβ16–21 fibers (0–500 μM, as monomer). The insert shows the area decrease of BAF1 NMR peaks as a function of Aβ16–21 concentration, and the red curve fitting the data defines an apparent Kd of 12 ± 7 µM. In panel (B), the NMR spectrum of orange G (50 μM) is plotted against increasing concentration of Aβ16–21 fibers (0–950 μM), giving an apparent Kd of 43 ± 21 µM. In (C, D and E), BAFs 1 and 8 both bind to Aβ1–42 fibers more strongly than orange G. Notice that the molar ratio of BAFs to Aβ1–42 fibers is comparable to that used in cell toxicity assays (Figure 3). (F). The calculated binding energies of BAFs—1, 8, and 31—to Aβ1–42 fibers are compared to the decreases in NMR peak of these compounds upon their binding to full-length Aβ fibers. These three BAFs have higher affinities and a larger NMR peak reduction than orange G while the ‘knock-out’ derivative with removal of key interactions (BAF31ΔOH) discussed below has a weaker calculated affinity and a smaller NMR peak reduction than orange G. We observe good correlation between computed energies and experimental data from NMR.