A Palette of Fluorescent Thiophene-Based Ligands for the Identification of Protein Aggregates

By replacing the central thiophene unit of an anionic pentameric oligothiophene with other heterocyclic moities, a palette of pentameric thiophene-based ligands with distinct fluorescent properties were synthesized. All ligands displayed superior selectivity towards recombinant amyloid fibrils as well as disease-associated protein aggregates in tissue sections.

Small fluorescent ligandsa re essential for visualizing protein aggregates, the common pathological hallmark of many neurodegeneratived iseases such as Alzheimer's and Parkinson's disease (AD and PD). [1,2] In this regard, av ariety of molecular scaffolds targeting the regular cross b-pleated sheet conformation of the protein aggregates have been developed. [3][4][5][6][7][8] Lately,l uminescent conjugated poly-and oligothiophenes (LCPs and LCOs) have also been employed as novel tools for fluorescence imaging of protein aggregates andi nc omparison to conventional ligands, LCOs have been shown to detect aw ider range of disease-associatedp rotein aggregates. [9][10][11][12][13] Due to their electronically delocalized conjugated thiophene backbones, LCOs exhibit specific intrinsic fluorescencec haracteristics and offer the possibilityt ou se av ariety of imaging techniques, as well as different modeso fd etection, such as full excitation/emission spectra, and fluorescenced ecay time. [14] However,t he fluorescent characteristics of LCOs are to somee xtent restricted and it is of great interest to develop thiophene-based ligands coveringawider range of emission in the visual spectrum. Such fluorescent ligandsw ill be essential in order to design multiplex detection methodologies involving ac ombination of LCOs as well as fluorophore-labelled antibodies towards distinct proteins.
Herein, we present the synthesis and characterizationo f ap alette of anionic pentameric oligothiophene derivatives and evaluated them as fluorescent ligands for protein aggregates. To achieve ap alette of ligands, we started with ap reviously reportedm ono-borylated di-thiophene building block [12] (Scheme 1). By applying general synthetic routes, including Suzuki cross-coupling of this building block with di-brominated phenylene, selenophene, quinoxaline, or benzodithiazole, followed by de-protection of the anionic side chains, four pentameric thiohene-based ligands, HS-163, HS-165, HS-167, and HS-169 with distinct central heterocyclic moieties were synthesized. In addition, the previously reported pentameric oligothiophene, HS-84 (Scheme1), [13] was included in the study.
When diluted in phosphate-buffered saline (PBS, pH 7.4) all the novel ligandsd isplayed different absorption characteristics compared to HS-84 ( Figure 1A). HS-163 showedablueshift of the absorption maximum, whereas the absorption maximum of HS-165 was slightly redshifted. The slight redshift for HS-165 might originate from an increased quinoidc haracter of the central selenophene giving the inter-ring CÀCb ond more double-bond properties and an enhanced efficiency of conjugation through the backbone. [15][16][17] In asimilar fashion,the central phenylm oiety will disrupt the planarity and the conjugation length, leading to ab lueshifted absorption maximum for HS-163 compared to HS-84. [18][19][20] HS-167 and HS-169, which have donor-acceptor-donor (D-A-D)-type electronic structure, displayed two absorption maxima (Figure1A). This approach, utilizing nitrogen-containing heterocycles as effective electron acceptors and thiophenes as donors, has been reported previously. [21,22] The two absorbance bands, ah igh energy band around3 60 nm, followed by al ow energy band at 460 (HS-167) or 515 nm (HS-169), likely arise from the p-p*t ransition and charge-transfert ransition, respectively.
Calculationso fU Va bsorption spectra were performed on model systemsw ith the ÀCH2ÀCOOH groups on the two thiopheness urrounding the central unit being replaced by methyl groups ( Figure S1 in the SupportingI nformation). This chromophore modification is denoted with primes in the name labels, so it is to be understood that labels with inter-ringt orsional angles very close to zero. This finding is partly attributed to favorable sulfur-nitrogen non-bonded interactions between sulfur atoms belonging to thiophenes and the nitrogen atoms of the quinoxalineo rb enzothiadiazole moieties. These interactions explain the small values for dihedral angles F2a nd F3, butf ail to rationalize the equallys mall values for dihedrala ngles F1a nd F4. Calculated UV spectra were in excellent agreementw ith the experimental counterparts and the main absorption bands for the series of molecules are summarized in Ta ble S1 in the SupportingI nformation. For HS'-84, HS'-165, and HS'-163, the sole absorption band found in this region is attributed to an electronic transition between the highest occupied (HOMO) to the lowestu noccupied molecular orbital( LUMO). For HS'-167 and HS'-169, the respective main peak is also attributedt oaHOMO-LUMO transition, but they are strongly redshifted with respect to the peak for HS'-84 by 46 and 86 nm, respectively.Astrong correlation between redshift and planarity in anionic oligothiophene derivatives has been reported and it was demonstrated that shifts in transition wavelengths due to planarity alonec an easily reacho ver 100 nm. [23] For HS'-167 and HS'-169, we also observe the appearance of as econd intense band at 344 and 354 nm, respectively.T hese bands are attributed to combinations of HOMO-LUMO + 2and HOMO-1-LUMO + 1t ransitions.
In agreement with the absorbance data, the corresponding fluorescences pectra showed similars pectrals hifts with emission maximar angingf rom 480 nm for HS-163 up to 705 nm for HS-169( Figure 1C). The relativelyl arge Stokes shifts displayed for HS-167 and HS-169 (Table S1 in the SupportingI nformation)a re common for p-conjugated donor-acceptor compounds anda re most likely attributed to ad egree of charge-transfer character. [24,25] In comparison with HS-84, the selenophene-containing ligand showed ad ecrease in the fluorescence intensity.T his phenomenon has been reported previously in as tudy with thiophenea nd selenophene co-polymers and might be dependento nt he lowering of the band gap and thereby increasing the probability of non-radiativee mission. [26] Ad ecreased intensity of emission was also observed for HS-167 and HS-169, the ligands showingt he most redshifted fluorescence. As imilart rend, ad ecrease quantum yield as the fluorescence redshifts, was recently reported for af amily of thiophene-based conjugatedo ligomers. [21] In order to elucidate selective binding of the ligandst op rotein aggregates,a ll five ligandsw ere tested towards amyloidlike fibrils made from recombinant Ab 1-42 peptide. All of the ligandsr evealed distinct excitation and emission characteristics when bound to recombinant Ab fibrils (Figure 2). When mixed with the amyloid-like fibrils, HS-84 (Figure 2A www.chemeurj.org emission spectra with enhanced intensity and the characteristic double peaks reported for anionic oligothiophenes bound to recombinant amyloid-like fibrils. [10][11][12][13] HS-167 and HS-169 also display redshifted excitation maximaa nd blueshifted emission maximaw hen bound to Ab 1-42 amyloid-like fibrils (Table S2 in the Supporting Information). In addition, am ore pronounced enhancement of the emission intensity was observed from these D-A-D compounds upon interaction with the fibrils (Figure 2D,E). Thus, similart ot he mostc ommonly used amyloidspecific dye, thioflavin T( ThT), [3,27] HS-167 and 169 indicated as trong increase in fluorescenceu pon bindingt oa myloid fibrils. Overall, the five ligands provided distincto pticals ignatures upon binding to Ab 1-42 fibrils, verifying thata ll of the ligands could be utilized for fluorescent assignmento fr ecombinant amyloid-like fibrils.
Previous studies have shown that pentameric oligothiophenesi dentify ab roader subset of disease-associated protein aggregates than conventionala myloid ligands. [10][11][12] For instance,t wo of the major pathological hallmarks of AD, Ab deposits and tau neurofibrillaryt angles (NFTs), have been selec-tively identified by oligothiophenes in brain tissue sections. [10][11][12] Therefore, the anionic pentameric oligothiophene derivatives were next evaluated as fluorescentl igands in brain tissue sections with AD pathology.A ll of the ligands showed specific bindingt oe xtracellular Ab deposits in the brain parenchyma( A b core plaques) and in the vasculature (cerebral bamyloid angiopathy,C AA) (Figure 3). In addition, intracellular NFTsw ere also stained by all of the ligands (Figure 3). Similar to the observation on recombinantA b 1-42 amyloid fibrils, HS-84, HS-163, and HS-165d isplayed well-resolved emission spectra with characteristic double peaks upon binding to assemblies of Ab and tau, whereas HS-167 and HS-169 showed ab road fluorescences pectrum with redshiftede mission maximac omparedt ot he other ligands ( Figure 3a nd Table S2 in the Supporting Information). Thus, from ab iological perspective, the three common pathological hallmarks, Ab core  www.chemeurj.org plaques, CAA, and NFTs, could easily be identified due to bright fluorescencea sw ell as distinct spectrals ignatures from all of the ligands. From ac hemical perspective,t hesee xperiments also verifiedt hat the central thiophene motif could be replaced with other heterocyclicm oieties withoutr educing the ligand'ss electivity towards disease-associated protein aggregates in ac omplex environment such as tissuesections.
In conclusion, we have demonstrated that ag eneral synthetic route where the central thiophene unit is replaced with other heterocyclic motifs can be used for synthesizingapalette of ligandst hat can be utilized for fluorescent assessment of protein aggregates.W ef oresee that these novel thiophenebased pentameric ligands will expand the tool box of fluorescent ligands for identifying av ariety of disease-associatedp rotein aggregates,t he common pathological hallmarks of several neurodegenerativediseases. Keywords: Alzheimer's disease · fluorescent probes · luminescence · oligothiophenes · microscopy