Synthesis and Characterization of Thiophene‐based Donor–Acceptor–Donor Heptameric Ligands for Spectral Assignment of Polymorphic Amyloid‐β Deposits

Abstract Protein deposits are associated with many devastating diseases and fluorescent ligands able to visualize these pathological entities are essential. Here, we report the synthesis of thiophene‐based donor–acceptor–donor heptameric ligands that can be utilized for spectral assignment of distinct amyloid‐β (Aβ) aggregates, one of the pathological hallmarks in Alzheimer's disease. The ability of the ligands to selectively distinguish Aβ deposits was abolished when the chemical composition of the ligands was altered. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species consisting of the same peptide or protein. In addition, such ligands might aid in interpreting the potential role of polymorphic Aβ deposits in the pathogenesis of Alzheimer's disease.

[13][14] Lately, an inter-subjectv ariability of Ab deposits has also been found in familiala nd sporadic AD. [19][20][21] Likewise, distinct age dependent Ab deposits were observed in transgenic mousem odels, suggesting that different aggregate specieso fA b are presentd uring different stageso ft he pathologicalp rocess. [22,23] 26][27][28] Notably,s eedingw ith Ab aggregates extracted from two AD patients with distinctc linical history and pathology resultedi n fibrilsw ith two different structures, implying ac orrelation betweena ggregate structure and disease progression. [28]Thus,l igandst hat can differentiate different types of Ab aggregates are essential as such molecular agents will aid in accurate clinical diagnostics of AD, as well as assist in deciphering the impact of polymorphicA b deposits in the pathogenesis of AD.
1][32][33] Owing to their electronically delocalized conjugated thiophene backbones, LCOs exhibit intrinsic conformational dependent fluorescencec haracteristics that can be recorded by different modes of detection. [33]Lately, at hiophene-based pentameric ligand, HS-169( Figure1A), with donor-acceptor-donor (D-A-D) type electronic structure was also presented. [34]The D-A-D photophysical characteristics were obtainedb yr eplacing the central thiophene moiety with 2,1,3-benzothiadiazole (BTD), rendering ap entameric ligand wheree lectron rich bithiophene units act as donors and the electron-withdrawing BTD as acceptor.H S-169 selectively identified Ab pathology in humanA Db rain tissue sections and the ligandd isplayed similarn ear-infrar ed emission bound to Ab core plaques or cerebral b-amyloid angiopathy (CAA). [34]Furthermore, HS-169 enabled optical assignmentofspecific carbohydrates,c ellulose or starch,i np lant tissue and this spectral distinction could not be obtained by the corresponding oligothiophene derivative. [35]Thus, the D-A-D electronic structure of HS-169 was crucial for distinguishing specific carbohydrates.Herein,w ep resent the synthesis and characterizationo f anionic heptameric oligothiophened erivativesh aving thiophene or BTD as the central heterocyclic moiety (Figure 1, Scheme 1).The ligands were utilized for spectral assignment of recombinant Ab fibrils and Ab deposits in brain sections from transgenic mice with AD-like pathology.T he ligandss electively identified Ab aggregates and subtle changes in the chemical composition of the ligandsw ere shown to eliminate their capacity for spectrals eparation of specific Ab deposits.Thus, these findings might aid in the chemical design of ligandsr ecognizing different aggregated proteinaceous species consisting of ad istinct protein.

Synthesis and optical characterizationo fthe ligands
To achieveavariety of anionic heptameric oligothiophene derivatives having thiophene or BTD as the central heterocyclic moiety,w es tartedw ith ap reviously reported monobrominated thiopheneb uildingb lock [30] and boronic acid pinacole sters of thiophene and BTD (Scheme 1).By applying palladium-mediated Suzuki-Miyaura cross-coupling reactions and bromination with N-bromosuccinimide (NBS) in DMF/chloroform, two distinct dibrominated trimer building blocks were achieved in affordable yields.From these trimeric building blocks, four different heptameric ligands, h-FTAA, LL-1, LL-2 and LL-3, were generated by applying sequential symmetric addition of different thiopheneo rb ithiophene units through Suzuki-Miyaura cross-coupling reactions and regioselective brominations followed by removal of protecting groupst oh ave anionic side chain functionalities (Scheme 1a nd Figure 1).LL-1 (Figure 1C)r esembles the previously synthesized heptameric oligothiophene, h-FTAA (Figure 1B), with aseven units long backbone and four carboxylate side chain functionalities at distinct positions, as well as ac entral BTD moiety instead of thiophene.Similar to h-FTAA, thiophenes constitute the entire backbone of LL-2 (Figure 1D), though, this ligand comprises six carboxylate side chain functionalities which render ah ighern egative net charge compared to both h-FTAA and LL-1.The ligand denoted LL-3 (Figure 1E)i st he corresponding BTD analogue to LL-2 andc ontainss ix anionic substituents, as well as ac entral BTD moiety.I na ddition to the heptameric ligandsd escribed above,t he previously reportedp entameric thiophene-based ligand with ac entral BTD moiety,H S-169 (Figure 1A), [34] was included in the study.
For LL-3, the spectrald ifferenceb etween free ligand or the ligand bound to recombinant amyloid-like Ab 1-42 fibrils was even more evident.In PBS, LL-3 showeda ne mission maximum around5 00 nm, whereas bound to Ab 1-42 fibrils, the ligand exhibitedared-shifted spectrumw ith an emission maximum around6 80 nm (Figure 2B).This phenomenonw as also observed in excitation mode, since the spectrum for LL-3 bound to fibrils showeda na dditional peak around5 10 nm compared to the spectra for the ligand in PBS (Figure 2B).The excitation-and emission spectrum for LL-3 in PBS resemble the fluorescencec haracteristics observed from pure oligothiophenes, [23,[29][30][31] suggesting that the intramolecular charge transfer transition to the BTD motif is restricted for the free ligand in PBS.On the other hand, this intramolecular charge transfer probably occurs when LL-3 binds to Ab 1-42 fibrils, since the optical characteristics resemble the excitation-and emissions pectrum for LL-1 (Figure 2A), as well as HS-169 boundt oA b 1-42 fibrils. [34]Hence,L L-3 is most likely adopting as trikingly different conformation bound to Ab 1-42 fibrils compared to unbound ligand in PBS.Further photophysical studies and theoretical calculations are necessary to resolve this matter in more detail.O verall, we concludet hat both ligands provided distinct optical signatures upon binding to Ab 1-42 fibrils, verifying that these ligandsc ould be utilized for fluorescent assignment of recombinant amyloid-like fibrils.

Opticalcharacterization of LL-1 and LL-3 bound to Ab deposits in brain tissue sections from transgenic mice
34] Therefore, the ligands weren ext evaluated towards brain tissue sections from APP23 transgenic mice with AD-like pathology.
All the heptameric ligands, as well as HS-169, showed selective binding to Ab core plaques and CAA (Figure 3a nd Supporting Information, Figure S2 and S3).As previously reported, [30] h-FTAA displayed well-resolved emission spectra with characteristic double peaks upon binding to both Ab assemblies and the novel heptameric oligothiophene, LL-2, showed similar spectralc haracteristics with slightly less defined double peaks (Supporting Information, Figure S2).HS-169 displayed similar emission spectra as reported for the ligand bound to Ab deposits in human brain tissue sections with AD pathology. [34]When bound to Ab core plaques, an emission profile with am aximum around 675 nm was obtained, whereas the emission spectrum was slightly red-shifted from the ligand bound to CAA (SI, Figure S2).
When stained in combination with an antibody (4G8) towards Ab pathology,t he novel heptameric D-A-D ligands showedg ood correlation with the antibodys taining (Supporting Information, Figure S3).Thus, LL-1 and LL-3 selectively identifiedi mmunopositive aggregated Ab species.Similar to the observation on recombinantA b 1-42 amyloid-like fibrils, upon excitation at4 05 nm or 535 nm, LL-1 showedabroad fluorescences pectrum with an emission maximum close to 700 nm when bound to Ab deposits in brain tissue sections (Figure 3A and 3B).Moreover,t he spectra from CAA were slightly blue-shifted comparedt ot he emissions pectrum obtained from Ab core plaques.In contrast, LL-3 showedt wo distinct emission profiles bound to CAA or Ab core plaques (Figure 3C,D).Upon excitation at 405 nm, LL-3 bound to Ab core plaquesd isplayed an emissionp rofile with as trong emission peak around6 75 nm andaweakere mission peak around 500 nm, whereas as trikingly different emission spectrum (l max around5 00 nm) was observed for LL-3 bound to CAA (Figure 3C).Thus, the spectral signatures from LL-3 could be utilized to distinguish these two aggregated Ab species.Analogous to the observation for LL-3 free in PBS or bound to recombinant Ab 1-42 amyloid-like fibrils (Figure 2B), the ligand is presumably binding in differentm odes to CAA or Ab core plaques, which renders distinct photophysical properties and specific emission profiles.
From ac hemical perspective,t hese experimentss upported that thiophene-based heptameric ligands having the central thiophene unit replaced with aB TD moietyc ould selectively detect CAA or Ab core plaques in brain tissue sections from APP23 mice.Secondly, LL-3 gave ab etter spectral separation of thesea ggregated Ab speciest han LL-1, suggesting that the amount of carboxylic acid side chain functionalities, as well as their spacingalong the conjugated backbone are crucial chemical determinantsf or achieving superior ligands for assigning distinct Ab species.These chemical determinantsh ave also been essential for obtainingi mproved tetrameric oligothiophenesf or spectrals eparation of age-related Ab and tau aggregates, [23] as well as for achieving pentameric oligothiophenese xhibiting ag reater therapeutic effect in prion infected mice. [39]Furthermore,w hen using ac ombination of at etrameric LCO and h-FTAA( Figure 1B), the spectra from the cores of Ab plaques differed significantly among familiala nd sporadic AD subtypes. [19]However, previously reported LCOs have not been able to distinguish CAA and Ab core plaquesa se fficiently as LL-3.Therefore, it would be of great interest to evaluate the novel D-A-D heptameric ligands, independently or in combination with other LCOs, towards brain tissue samples from differentc ases with familial or sporadic AD and such studies are ongoing.

Synthesis and characterization of an additional thiophenebased D-A-Dh eptameric ligand
To elucidate the importance of the amount of carboxylic acid side chain functionalities, as well as their spacinga longt he conjugated backbone, we synthesized an additional thiophene-based D-A-D heptameric ligand,d enoted LL-4 (Scheme 2a nd Figure 4A).LL-4 was synthesized in as imilar fashion as the other ligandss tarting with methyle ster protected 2-bromothiophene acetic acid. [30]By applying ap alladiummediated Suzuki-Miyaura cross-coupling reaction followed by bromination with NBS in sequential steps with am onoborylated bithiophene derivative [23,34] andd iborylated BTD, LL-4 was achieved in affordable yield (Scheme 2).Similar to LL-3, LL-4 has six carboxylate side-chain functionalities.However,t he positions of the acetic acetate side chains are alteredo nt he thiophene rings adjacent to the central BTD moiety.T hus, LL-4 is an isomer to LL-3 having the acetate side chainso ft he central trimerict hiophene-BTD-thiopheneu nit tail-to-tail instead of head-to-head.
When diluted in PBS, LL-4 showed ad istinctive absorption spectrum with two absorption maxima at 400 and 545 nm (Figure 4B).As mentioned earlier,t hese absorption bands likely arise from the p-p*t ransition and charge-transfer transition, respectively,a nd the latter band was much more pronounced for LL-4 compared to LL-1 and LL-3 (Figure 1C,E).Instead, the spectrum resembled the optical signature from HS-169 (Figure 1A).Hence, the charge-transfer transition seems to be more favorable for ligands having the acetate sidec hains of the central trimeric thiophene-BTD-thiophene unit tail-to-tail (HS-169a nd LL-4) instead of head-to-head (LL-1 and LL-3).
LL-4 was next evaluated towards brain tissue sections from APP23 transgenic mice with AD-like pathology.T he ligand revealed selectiveb inding to both Ab core plaques and CAA (Figure 4C and 4D).The emission spectra from LL-4 were similar for both aggregated Ab speciesw ithe missionm axima around7 30 nm.Thus, in contrast to LL-3, LL-4 lacked the ability to distinguish Ab core plaques from CAA, suggesting that a distinct periodicity of carboxylic groups along the heptameric backbonei sn ecessary to achievealigand that can differentiate these aggregated Ab entities.Overall,t he characterization experiments confirmed that the two isomers, LL-3 and LL-4, displayed different photophysical characteristics, both in solution and bound to Ab aggregates.

pH-dependent optical characteristics of LL-1, LL-3 and LL-4
To clarify the observed emissionc haracteristics of the thiophene-based D-A-D heptameric ligands when interacting with aggregated Ab morphotypes, we next explored solvent induced photophysical properties of the ligands.As it has previously been reported that changes in pH can mimict he optical behavior of anionic LCOs boundt od ifferent Ab and tau morphotypes, [23] absorption-and emission spectra were recorded for LL-1, LL-3 and LL-4 in buffer solution with pH 3o rp H7 (Figure 5).For LL-1, decreasing pH inducedaslight increase of the low energy absorption band around 490 nm, suggesting that the charge-transfer transition seems to be more favorable at acidic pH (Figure 5A).In addition, at pH 3, as trong increase of the emission intensity around 700 nm was observed.Hence, by changing the pH and thereby the chargeo ft he carboxyl groups,the emission profile observed for LL-1 bound to Ab aggregates could be largely mimicked.
LL-3 showed even more striking pH-dependent photophysical transitions than LL-1.Upon protonation of the carboxylates, the absorption spectrum was red-shifted and the low energy absorption band at longer wavelengths becamem ore pronounced (Figure 5B).Excitation of LL-3 at aw avelength correspondingt ot he absorption maximaw ith highest energy,r esulted in two completely differente mission profiles (Figure 5B).At pH 7, LL-3 displayed ane mission spectrum with a maximum intensity around 510 nm, whereas at pH 3, dominant emissiona round 710 nm was detected.Thus, the two different emissionp rofiles displayed from LL-3 boundt oA b core plaques or CAA in tissue sections (Figure 3C)c ould be recreated in solution.T he LL-3 emission characteristics associated with CAA in APP23 transgenic mice could be obtained upon deprotonation of the carboxyl groups.Likewise, the spectral features obtained in acid conditions, when the anionic side chainsw ere protonated, resembled the emission spectra acquired from LL-3 bound to Ab core plaques.
The third ligand,L L-4, revealed similarp H-dependent photophysical transitions as LL-1 (Figure 5C).The absorption spectrum at pH 3o r7 were comparable, but under acidic conditions LL-4 displayed as imilar emission profile as obtained for the ligand bound to Ab aggregatesw ith an enhanced emis-  sion around 750 nm.Overall,t he photophysical characterization of the ligandsa td ifferent pH verified that similar spectral transitions to the ones observed for the ligandsb ound to Ab deposits could be induced by alteringt he charge of the anionic side chain functionalities along the conjugated backbone.Moreover,t hese experimentsa lso confirmed that minor alterations of the amount and periodicity of anionic groups along the heptameric backboneh ighly influenced the pH-dependent opticalc haracteristics of the ligands.Thus, similarc hemical determinants that seemed important for achieving superior ligands for assigning distinct Ab species, could be correlated to distinct pH-dependent photophysical transitions.Additional advanced photophysical studies and theoretical calculations will be required to clarify theseissues in more detail.

Conclusions
In conclusion, thiophene-based D-A-D heptameric ligands were identified as optical ligands for spectral assignment of Ab aggregates.The spectral signature from one ligand could also be utilized to distinguish different Ab morphotypes and the superior functionality of this ligand compared to structurally related compounds could be assigned to ad istinct periodicity and number of carboxys ubstituents along the conjugated backbone.We foreseethat our findings will aid in the chemical design of ligandst hat can be utilized for exploring different aggregated morphotypes composed of Ab,a sw ell as other polymorphic protein aggregates that are observed in neurodegenerativeprotein aggregation disorders.

Experimental Section
Full experimental details including additional characterization data and NMR spectra of new compounds, as well as supporting figures are given in the Supporting Information.

Figure 3 .
Figure 3. Optical characterization of LL-1 and LL-3 bound to Ab deposits in brain tissuesection from APP23t ransgenic mice:A,B) Images of LL-1 labelled CAA (left) and Ab core plaques (middle), as well as emission spectra (right) for LL-1 bound to the different Ab deposits uponexcitationat4 05 nm (A) or 535 nm (B).C,D) Images of LL-3 labelled CAA (left) and Ab core plaques (middle), as well as emission spectra(right) for LL-3 bound to the different Ab deposits upon excitationat4 05 nm (C) or 535 nm (D).Spectra were collected from 10 individual deposits.Scale bar represents 50 mm.

Figure 4 .
Figure 4.Chemical structure and optical characterizationo fL L-4:A)Chemical structure of LL-4.B) Absorption spectrumo f3 0mm LL-4 in PBS pH 7.4.C,D) Images of LL-4 labelled CAA (left) and Ab core plaques (middle), as well as emission spectra (right) for LL-4 bound to the different Ab deposits upon excitation at 405 nm (C) or 535 nm (D).Spectra were collected from 10 individual deposits.Scale bar represents 50 mm.