ABSTRACT: Despite their unique advantages, the full potential of molecular probes for fluorescent monitoring of amyloid-? (A?) aggregates has not been fully exploited. This limited utility stems from the lack of knowledge about the hydrophobic interactions between the molecules of A? probes, as well as those between the probe and the A? aggregate. Herein, we report the first mechanistic study, which firmly establishes a structure-signaling relationship of fluorescent A? probes. We synthesized a series of five fluorescent A? probes based on an archetypal donor-acceptor-donor scaffold (denoted as SN1-SN5). The arylamino donor moieties were systematically varied to identify molecular factors that could influence the interactions between molecules of each probe and that could influence their fluorescence outcomes in conditions mimicking the biological milieu. Our probes displayed different responses to aggregates of A?, A?40 and A?42, two major isoforms found in Alzheimer's disease: SN2, having pyrrolidine donors, showed noticeable ratiometric fluorescence responses (?? = 797 cm-1) to the A?40 and A?42 samples that contained oligomeric species, whereas SN4, having N-methylpiperazine donors, produced significant fluorescence turn-on signaling in response to A? aggregates, including oligomers, protofibrils, and fibrils (with turn-on ratios of 14 and 10 for A?42 and A?40, respectively). Mechanistic investigations were carried out by performing field-emission scanning electron microscopy, X-ray crystallography, UV-vis absorption spectroscopy, and steady-state and transient photoluminescence spectroscopy experiments. The studies revealed that the SN probes underwent preassembly prior to interacting with the A? species and that the preassembled structures depended profoundly on the subtle differences between the amino moieties of the different probes. Importantly, the studies demonstrated that the mode of fluorescence signaling (i.e., ratiometric response versus turn-on response) was primarily governed by stacking geometries within the probe preassemblies. Specifically, ratiometric fluorescence responses were observed for probes capable of forming J-assembly, whereas fluorescence turn-on responses were obtained for probes incapable of forming J-aggregates. This finding provides an important guideline to follow in future efforts at developing fluorescent probes for A? aggregation. We also conclude, on the basis of our study, that the rational design of such fluorescent probes should consider interactions between the probe molecules, as well as those between A? peptides and the probe molecule.
