Currently, the most common neurodegenerative disease is Alzheimer's disease (AD), which is characterized by the loss of memory, impairment in language and execution of everyday tasks, and is the main cause of dementia. Therapy at this stage is quite limited and ineffective, but earlier diagnosis could help to improve treatment outcomes. One of the indicators of AD prior to the onset of the symptoms is the presence of amyloid plaques in the cerebral cortex, and their early detection offers good potential for a more successful treatment. Fluorescent probes, which increase the emission of fluorescence when bound to such fibrils, would be of great value in the diagnostics. These probes have a characteristic structure: an electron-donor and an electron-acceptor group connected by linkers – π-systems that allow the establishment of a "push-pull" system. Compounds with high sensitivity and specificity for amyloid plaques are therefore desirable for successful diagnosis. To this end, we have characterized a new series of pyrone-based probes and tested their binding properties. We measured the absorbance and amplification of the emission signal in the presence of Aβ1–42 fibrils as well as the binding affinity of the probes. The binding of the probes to Aβ1–42 fibrils is associated with changes in the environment that result in limited intramolecular rotation, thereby increasing the fluorescence emission. Probes ALZ177, AnaBr-14a and AnaBr-16a showed the highest signal enhancement, while a much lower signal enhancement was measured for AnaBr-4b and ALZ153, which already show high responses in the buffer. The reason for this is presumably self-association due to the poor aqueous solubility. The spectral properties of the fluorescent probes were also measured after binding to fibrils of insulin. Here, the results agreed well with the measurements of binding to Aβ1–42 fibrils, indicating lower binding specificity. When we analyzed the fluorescence emission maxima in detail, we found that they were slightly different after binding to Aβ1–42 and insulin fibrils, which would allow to distinguish between these two using fluorescence microscopy. According to the measured binding affinity values, the most optimal probe is AnaBr-16a, which has a number of functional groups that can in addition to nonspecific interactions form directed hydrogen bonds and dipole-dipole interactions. In addition to the AnaBr-16a, ALZ177 is also promising for further development, since they both display high amplification of fluorescence emission intensity and have high binding affinities.
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