Detection and Imaging of Aβ1‐42 and Tau Fibrils by Redesigned Fluorescent X‐34 Analogues

Abstract

We revisited the Congo red analogue 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) to develop this highly fluorescent amyloid dye for imaging Alzheimer's disease (AD) pathology comprising Aβ and Tau fibrils. A selection of ligands with distinct optical properties were synthesized by replacing the central benzene unit of X-34, with other heterocyclic moieties. Full photophysical characterization was performed, including recording absorbance and fluorescence spectra, Stokes shift, quantum yield and fluorescence lifetimes. All ligands displayed high affinity towards recombinant amyloid fibrils of Aβ1-42 (13-300 nm K ) and Tau (16-200 nm K ) as well as selectivity towards the corresponding disease-associated protein aggregates in AD tissue. We observed that these ligands efficiently displaced X-34, but not Pittsburgh compound B (PiB) from recombinant Aβ1-42 amyloid fibrils, arguing for retained targeting of the Congo red type binding site. We foresee that the X-34 scaffold offers the possibility to develop novel high-affinity ligands for Aβ pathology found in human AD brain in a different mode compared with PiB, potentially recognizing different polymorphs of Aβ fibrils.

Intramolecular Proton and Charge Transfer of Pyrene‐based trans‐Stilbene Salicylic Acids Applied to Detection of Aggregated Proteins

“…In this regard, the pan‐amyloid targeting molecule X‐34 [2,5‐bis(4′‐hydroxy‐3′‐carboxy‐styryl) benzene], containing two salicylic acid (SA) moieties, is an important scaffold for amyloid fibril probe development . A new series of fluorescent X‐34 analogues were designed and synthesized, by replacing the central benzene unit with other heterocyclic moieties, which displayed selectivity towards the corresponding disease‐associated protein aggregates in AD tissue . SA and its derivatives are well known to exhibit excited‐state intramolecular proton transfer (ESIPT) through intramolecular hydrogen bonds (IMHBs) .…”

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“…It can thus skew the apparent fibrillization kinetics revealed by Thioflavin T, that binds to the outer surface of the amyloid assemblies, parallel to the main fibril axis. Interestingly, the Thioflavin T binding site differs from the binding site of dyes, such as Congo red and its X-34 derivative [ 41 ]. As polymorphs adopt different amyloid folds, the differences in the outer exposure of the C and N termini at each amyloid stack level undoubtedly affect dye binding, enabling or not Thioflavin T and the access of other trimethine cyanine skeleton dyes to their binding site (or other parameters).…”

Fluorescent Diagnostic Probes in Neurodegenerative Diseases

“…The derivative of Congo red azo dye is more lipophilic and has a high binding affinity to Aβ plaques as well as NFTs, but displays poor optical properties. [ 61 ] The further modified molecule with ethenyl‐linked central phenyl structure, such as probe 3 , not only stained Aβ plaques and NFTs but also crossed the BBB; this led to the development of further molecular structures with bis ‐styrylbenzenes. [ 62 ] Zhang et al were inspired to apply heterocyclic ring replacement of the bis ‐styrylbenzenes with naphthalene, thiophene, quinoxaline, and benzodithiazole for further derivation, thereby generating ligands probes 84 (NSB), 85 (TSB), 86 (QSB), and 87 (BTDSB, Figure 8E).…”

“…[ 62 ] Zhang et al were inspired to apply heterocyclic ring replacement of the bis ‐styrylbenzenes with naphthalene, thiophene, quinoxaline, and benzodithiazole for further derivation, thereby generating ligands probes 84 (NSB), 85 (TSB), 86 (QSB), and 87 (BTDSB, Figure 8E). [ 61 ] All the ligands displayed notable optical features after binding with Aβ plaques and Tau fibrils, along with different selectivity index values in the order 86 > 87 > 85 = 84 , demonstrating that probes 85 and 84 could be used to fluorescently detect the recombinant Aβ plaques and Tau fibrils. Moreover, these ligands exhibited large Stokes shifts when interacting with Aβ fibrils, in the order 87 (133 nm) > 86 (129 nm) > 85 (72 nm) > 84 (66 nm), which was attributed to the ICT effect.…”