Synthesis of Thiophene-Based Optical Ligands That Selectively Detect Tau Pathology in Alzheimer's Disease

Article abstract of DOI:10.1002/chem.201703846

The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the development of molecular ligands able to detect these pathological hallmarks is essential. Here, the synthesis of thiophene based optical ligands

Full text of DOI:10.1002/chem.201703846

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Accepted Article
Title: Synthesis of thiophene based optical ligands that selectively
detect tau pathology in Alzheimer´s disease
Authors: Peter Nilsson, Hamid Shirani, Hanna Appelqvist, Marcus
Bäck, Therese Klingstedt, and Nigel Cairns
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Synthesis of thiophene based optical ligands that selectively
detect tau pathology in Alzheimer´s disease
Hamid Shirani,[a] Hanna Appelqvist, [a] Marcus Bäck,[a] Therése Klingstedt,[a] Nigel J. Cairns,[b] and K.
Peter R. Nilsson*[a]]
Abstract: The accumulation of protein aggregates is associated with
many devastating neurodegenerative diseases and the development
of molecular ligands able to detect these pathological hallmarks is
essential. Here, we report the synthesis of thiophene based optical
ligands, denoted bi-thiophene-vinyl-benzothiazoles (bTVBTs) that
can be utilized for selective assignment of tau aggregates in brain
tissue with Alzheimer´s disease (AD) pathology. The ability of the
ligands to selectively distinguish tau deposits from the other AD
associated pathological hallmark, senile plaques consisting of
aggregated amyloid-β (Aβ) peptide, were reduced when the chemical
composition of the ligands were altered, verifying that specific
molecular interactions between the ligands and the aggregates are
necessary for the selective detection of tau deposits. Our findings
provide the structural and functional basis for the development of new
fluorescent ligands that can distinguish between aggregated
proteinaceous species consisting of different proteins. In addition, the
bTVBT scaffold might be utilized to create powerful practical research
tools for studying the underlying molecular events of tau aggregation
and for creating novel agents for clinical imaging of tau pathology in
AD.
disease-associated protein aggregates consisting of a distinct
protein.
In AD, the two major proteinaceous deposits are extracellular
senile plaques consisting of aggregated amyloid-β (Aβ) peptide
and intraneuronal neurofibrillary tangles (NFTs) composed of
aggregated tau.[1,9] Thus, the development of ligands specifically
targeting Aβ or tau deposits are essential for clinical diagnosis of
AD, as well as for evaluating the contribution of these respective
aggregated species to the complex molecular pathology in AD
brain. Molecular scaffolds enabling visualization of Aβ deposits in
humans with AD by positron emission tomography (PET) imaging
have been presented.[10-12] More recently, some molecular
scaffolds targeting the other pathological hallmark, tau deposits,
in AD have also been recognized.[13-18] However, different
morphotypes of Aβ and tau aggregates have been reported[19-23]
and the existence of distinct aggregate morphotypes has been
suggested to explain the heterogeneous phenotype reported for
several neurodegenerative protein aggregation diseases. Hence,
a variety of ligands might be necessary to achieve an accurate
assessment of the diversity of pathological protein deposits
present in AD.
Luminescent conjugated oligothiophenes (LCOs) have been
utilized for fluorescence imaging of protein aggregates and
compared to conventional ligands, LCOs have been shown to
detect a wider range of disease-associated protein aggregates.[24-
Introduction
Protein deposits are the pathological hallmarks of a wide range of
neurodegenerative diseases,[1] including Alzheimer´s disease
(AD), and small hydrophobic ligands that are selective for protein
aggregates having an extensive cross β-pleated sheet
conformation and sufficient structural regularity have been
developed.[2-7] The most common ligands are derivatives of
Congo Red or Thioflavins and in addition to these conventional
ligands, a variety of other molecular scaffolds has also been
reported.[8] However, most of these ligands detect disease-
associated protein aggregates in general and the next scientific
challenge is to develop small molecular ligands that target specific
26]
In addition, LCOs having distinct chemical compositions can
also be utilized for spectral assessment of different protein
aggregates, such as Aβ or tau deposits in AD.[27,28] Lately, a
thiophene based tetrameric ligand, q-FTAA-CN (Figure 1A) with
a striking higher affinity for Aβ deposits than aggregated species
composed of tau was also identified.[29]
Herein, we explored if a thiophene-based ligand with a high
selectively toward tau pathology in AD could be synthesized. As
a template, a previously reported tau specific ligand, PBB3
(Figure 1B)[17] was selected and two classes of thiophene-based
ligands,
bi-thiophene-vinyl-benzothiazoles/benzoimidazoles
(bTVBTs/bTVBIs) and tert-thiophene-benzothiazoles (tTBTs),
were generated (Figure 1C). The ligands were applied on human
brain tissue sections with AD pathology and bTVBTs were
identified as a novel class of optical ligands that selectively
identified tau pathology in AD. Furthermore, subtle changes in the
chemical composition of the bTVBTs were shown to reduce or
eliminate their capacity for selective identification of tau deposits.
Thus, these findings might aid in the chemical design of ligands
recognizing aggregated proteinaceous species consisting of a
distinct protein, as well as in the development of novel agents for
clinical imaging of tau pathology in AD.
[a]
[b]
Dr. H. Shirani, Dr. H. Appelqvist, Dr. M. Bäck, Dr. T. Klingstedt,
Prof. K.P.R. Nilsson
Division of Chemistry, Department of Physics, Chemistry and
Biology, Linköping University, SE-581 83 Linköping, Sweden
E-mail: petni@ifm.liu.se
Prof. N.J. Cairns
Department of Pathology and Immunology, Washington University
School of Medicine, St. Louis, Missouri, U.S.A.
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. Chemical structure of fluorescent ligands for protein aggregates. (A) The chemical structure of q-FTAA-CN, a high-affinity ligand for Aβ deposits in
human AD brain. (B) The chemical structure of PBB3, a high-affinity ligand for tau deposits in human AD brain. (C) Chemical structure of novel bi-thiophene-vinyl-
benzothiazoles/benzoimidazoles (bTVBTs/bTVBIs). (D) Chemical structure of novel tert-thiophene-benzothiazoles (tTBTs)
bi-thiophene building blocks (Scheme 1b). These synthetic routes
were utilized to synthesize five different bTVBT variants denoted
bTVBT1, bTVBT2, bTVBT3, bTVBT4 and bTVBT5, respectively
(Figure 1C). In addition, a compound, bTVBI1, having a
Results and Discussion
benzimidazole moiety, 1-ethyl-1H-benzimidazole, instead of a
benzothiazole/benzothiazolium attached to the bi-thiophene-vinyl
moiety was synthesized in an analogous fashion as for the
benzothiazoliums variants (Scheme 1c).
Synthesis of bTVBTs, bTVBIs and tTBTs
As PBB3 (Figure 1B) was reported to display a selectivity towards
tau aggregates in human AD brain tissue sections,[17] this
molecular scaffold was selected as a template to synthesize two
classes of novel thiophene based compounds. Firstly, the
pyridinyl-butadienyl-motif was replaced by a bi-thiophene-vinyl
moiety rendering a set of compounds denoted bi-thiophene-vinyl-
The second class of compounds (Figure 1D), denoted tert-
thiophene- benzothiazoles (tTBTs) was achieved by replacing the
pyridinyl-butadienyl-motif of PBB3 with different tert-thiophene
moieties and this class of compounds was synthesized as
outlined in scheme 2. Different brominated bi-thiophenes were
attached to compound 7 by the same Suzuki coupling reaction
used for the synthesis of bTVBT1. Overall, the synthetic schemes
described above were utilized to generate a total of nine different
thiophene based fluorescent ligands with different chemical
composition (Figure 1).
benzothiazoles/benzothiazoliums
Compound bTVBT1 was achieved through a condensation
reaction of 2-methylbenzothiazole and 5-bromo-2-
(bTVBTs)
(Figure
1C).
thiophenecarboxaldehyde followed by a Suzuki coupling to attach
the second thiophene moiety (Scheme 1a), whereas the
benzothiazoliums variants were assembled by a condensation
reaction of 3-ethyl-2-methylbenzothiazolium iodide and different
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Scheme 1. Synthesis of bTVBTs and bTVBI1. Reagents and conditions: (a) (i) DMSO, KOH (2M), RT; (ii) 1,4-dioxane/MeOH, PEPPSI-IPr, K2CO3 80 °C, 20 min.
(b) (i) Pyridine, MeOH or MeOH/THF mixture, reflux. (c) (i) DMF, TMSCl, 110 °C, 2 h.
Scheme 2. Synthesis of tTBTs. Reagents and conditions: (i) 1,4-dioxane/MeOH, PEPPSI-IPr, K2CO3, 80 °C, 20 min.; (ii) NaOH (1M), 1,4-dioxane, 70 °C, 5 h.
Histological staining of AD brain tissue sections with
bTVBTs, bTVBIs and tTBTs
brain,[30,31) it is essential to verify ligands that selectively detect tau
pathology in AD towards human brain sections with AD pathology.
Therefore, 100 nM of the ligands were applied for histological
staining of human brain tissue sections with AD pathology (Figure
2 and 3). When stained in combination with an antibody (4G8) for
Aβ pathology, bTVBT1, bTVBT2, bTVBT3, bTVBT4 and bTVBT5
As previously studies have shown that tau ligands display
differential binding towards recombinant tau fibrils, tau deposits in
transgenic mouse models or aggregated tau in human AD
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Figure 2. Fluorescence images of brain tissue sections with AD pathology stained with bTVBTs, bTVBIs and tTBTs. Frozen brain sections were fixed with
ethanol and double stained with an antibody towards Aβ (4G8, red) and 100 nM of the respective ligand (green). Scale bar represent 50 µm and white arrow-heads
indicate auto-fluorescence from lipofuscin observed in the blue- and the green channel. bTVBT1, bTVBT2, bTVBT3, bTVBT4 and bTVBT5 showed no correlation
with the antibody staining (red arrows) and the staining pattern observed for these ligands (green arrows) resembled NFTs, dystrophic neurites and neuropil threads.
bTVBT1 showed overlap with the 4G8 staining (white arrows) as well as some minor staining from dystrophic neurites. tTBT2 displayed overlap with the 4G8 staining
(white arrows) as well as staining from tau-like pathology (green arrows). tTBT1 and tTBT3 did not display any substantial staining of any of the pathological
aggregated species in AD brain.
showed no correlation with the antibody staining (Figure 2 and
Supporting Information (SI), Figure S1). Instead, the staining
pattern observed from the bTVBTs resembled classical AD tau
pathology with NFTs, dystrophic neurites and neuropil threads.
Thus, the bTVBT ligands appeared to stain aggregated species
composed of tau, whereas staining of Aβ pathology was lacking.
The bTVBTs selectivity towards aggregated tau species was also
verified with co-staining experiments using the tau-specific
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Figure 3. Fluorescence images of brain tissue sections with AD pathology co-stained with bTVBTs and the hyperphosphorylated tau specific antibody
AT100. Frozen brain sections were fixed with ethanol and double stained with an antibody towards tau (AT100, red) and 100 nM of the respective ligand (green).
All the bTVBTs displayed overlap with the AT100 staining (white arrows), verifying that the ligands stained tau pathology in brain tissue sections with AD pathology.
Scale bar represent 10 µm.
antibody AT-100 (Figure 3). Clearly, all the bTVBTs labelled both
immuno-positive NFTs, dystrophic neurites and neuropil threads.
However, certain parts of individual NFTs or dystrophic neurites
were intensively labelled with AT100 and displayed weak bTVBT
fluorescence, whereas other parts of the same NFTs or dystrophic
neurites showed the opposite pattern, namely strong bTVBT
fluorescence and weak AT100 labeling. This discrepancy in
fluorescence intensity is most likely a consequence of the different
binding modes for AT100 and the bTVBTs. AT100 is recognizing
an epitope (paired helical filaments phosphorylated at Thr 212
and Ser 214),[32] whereas the bTVBTs are most likely interacting
with the repetitive β-sheet structure composed of identical
protofilaments comprising residues 306-378 of the tau protein.[33]
These different motifs are most likely displayed in a different
fashion along the tau aggregates. In contrast to the tTVBTs, the
benzimidazole variant, bTVBI1, displayed staining towards
immuno-positive Aβ aggregates, as well as some weak staining
of tau-like pathology (Figure 2 and SI, Figure S1). Hence, by
rather minor chemical alteration could have an impact on the
ligand/protein aggregate interaction.
Replacing the vinyl moiety in the bTVBTs with an additional
thiophene unit also had a major influence on the ligands ability to
detect Aβ and tau aggregates. When using 100 nM of the
uncharged tTBTs, tTBT1 and tTBT3, staining of both immuno
positive Aβ aggregates and tau-like pathology were lacking
(Figure 2, SI Figure S2). In contrast, the negatively charged tTBT2,
displayed staining of both Aβ and tau aggregates (Figure 2, SI
Figure S1 and S2). Thus, similar to what has been reported earlier
for oligothiophenes,[24-29] anionic carboxyl groups along the tTBT
backbone might be an essential chemical determinant for
achieving high-affinity optical ligands towards protein aggregates.
However, none of the tTBTs displayed selective staining towards
tau pathology, signifying that the bTVBT molecular scaffold was
superior for achieving optical ligands that selectively detect tau
deposits in brain sections with AD pathology.
replacing the benzothiazole/benzothiazolium moiety with
a
Optical characterization of bTVBTs bound to NFTs
benzimidazole motif, the specificity towards tau pathology was
abolished. Interestingly, an earlier study of PBB-like compounds
showed that a benzimidazole derivative displayed different
emission spectra when bound to Aβ or tau deposits, whereas the
spectral difference was abolished when replacing the
benzimidazole moiety with a benzothiazole,[34] verifying that this
In order to elucidate the tau selective staining of the bTVBTs in
more detail, we next evaluated the photo-physical properties of
the bTVBTs bound to tau deposits in comparison to the ligands
dissolved in DMSO and further diluted in DMSO or PBS.
Representative spectra from bTVBTs stained NFTs are shown in
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Figure 4. Excitation and emission characteristics of the bTVBTs bound to NFTs or diluted in DMSO and PBS. (A) Fluorescence images of bTVBTs stained
NFTs. The staining was performed using 100 nM bTVBT in PBS pH 7.4. Scale bar represents 10 µm. (B) Excitation- (blue) and emission (red) spectra from the
bTVBTs bound to NFTs. (C) Excitation- (blue) and emission (red) spectra from bTVBT dissolved in DMSO and further diluted in DMSO (dotted line) or in PBS pH
7.4 (solid line) to a final concentration of 300 nM ligand.
figure 4. All the bTVBTs displayed an emission spectrum with a
maximum around 600 to 630 nm and an excitation spectrum with
a maximum around 525 to 550 nm (Figure 4B and SI, Table S1),
suggesting that all bTVBTs binds to tau deposits in a similar
fashion. Furthermore, when analyzing the excitation- and
emission profiles of the ligands in DMSO or PBS, the photo-
physical behavior was strikingly different (Figure 4C and SI, Table
S1). bTVBT1 exhibited a solvatochromatic behavior rendering a
blue-shifted excitation spectrum, as well as a red-shifted emission
spectrum, when switching solvent from DMSO to PBS, whereas
the other bTVBTs showed similar spectra in both solvents.
Notably, all the bTVBTs displayed a pronounced red-shift of the
excitation maxima when bound to NFTs compared to the maxima
obtained in the different solvents. Thus, upon interaction with the
NFT, the bTVBTs adopt a distinct conformation that induces a
specific optical signature from the ligands. Similar to what has
been suggested for other amyloid ligands, such as thioflavin T
(ThT),[35-41] the molecular form of the bTVBTs that gives rise to the
red-shifted excitation spectra upon binding to NFTs might be
either monomeric or dimeric. For ThT, the monomer hypothesis is
based on ThT behaving as a molecular rotor,[35-38] while the dimer
hypothesis is based on ThT forming an excimer.[39-41] To achieve
more molecular details regarding the conformation of the bTVBTs
bound to NFTs, further photo-physical studies, as well as
theoretical modeling are necessary and such experiments are
ongoing. Overall, we conclude that upon interaction with NFTs, all
the bTVBTs display distinct excitation- and emission spectra
compared to when the ligands are dissolved in DMSO or PBS.
Comparison of the bTVBTs with q-FTAA-CN and PBB3
To further investigate the binding mode of the bTVBTs to tau
deposits, tissue sections with AD pathology were simultaneously
stained with 100 nM bTVBT2 and 100 nM of q-FTAA-CN (Figure
1). As previously reported,[29] at this concentration (100 nM), q-
FTAA-CN selectively labelled Aβ deposits. In addition, tau
pathology could be identified by the spectral signature of bTVBT2
(Figure 5A-D). Thus, by applying two thiophene-based ligands, q-
FTAA-CN and bTVBT2, specific optical assignment of the two
pathological hallmarks of AD could be achieved.
Next we performed a similar staining experiment using 100 nM
bTVBT and 100 nM of PBB3. However, as the previously
reported[17] in vitro fluorescence staining of AD brain sections with
PBB3 was performed under different conditions (10-3 % ligands
dissolved in 50% ethanol for 1 hour at room temperature) than for
the bTVBTs, staining experiments with only 100 nM PBB3 in PBS
pH 7.4 was first conducted. With this staining procedure, PBB3
evidently labelled both Aβ and tau deposits, as characteristic
fluorescence was observed from both these aggregated species
(SI, Figure S3). Due to these spectral properties of PBB3,
bTVBT3 was selected as the thiophene-based ligand for being
simultaneously used with PBB3. As shown in figure 5E, when
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Figure 5. Fluorescence images of brain tissue sections with AD pathology simultaneously stained with bTVBTs and q-FTAA-CN or PBB3. (A-C)
Fluorescence spectral images of frozen brain sections with AD-pathology simultaneously stained with 100 nM q-FTAA-CN and 100 nM bTVBT2. q-FTAA-CN
fluorescence (blue) was observed from Aβ deposits (white arrow-heads) whereas tau aggregates (white arrows) displayed bTVBT2 fluorescence (yellow-red). (D)
Characteristic emission spectra (blue line) from q-FTAA-CN labelled Aβ deposits and bTVBT2 stained tau aggregates (yellow-red line). (E) Fluorescence images of
frozen brain sections with AD-pathology simultaneously stained with 100 nM PBB3 and 100 nM bTVBT3. PBB3 fluorescence (blue) was observed from Aβ deposits
(white arrow-heads) whereas tau aggregates (white arrows) displayed bTVBT3 fluorescence (red). (F) Fluorescence images of frozen brain sections with AD-
pathology pre-incubated with 1 µM PBB3 prior to staining with 10 nM bTVBT3. PBB3 fluorescence (blue/green) was observed from both Aβ deposits (white arrow-
heads) and tau aggregates (white arrows) whereas no bTVBT3 fluorescence was observed in the red channel. (G) Fluorescence images of frozen brain sections
with AD-pathology stained with 10 nM bTVBT3. bTVBT3 fluorescence (red) was observed from the tau aggregates (white arrows). (H) Fluorescence images of
frozen brain sections with AD-pathology pre-incubated with 1 µM q-FTAA-CN prior to staining with 10 nM bTVBT3. q-FTAA-CN fluorescence (blue/green) was
observed from both Aβ deposits (white arrow-heads) and tau aggregates whereas bTVBT3 fluorescence (red) was observed from the tau aggregates (white arrows).
Scale bar represent 50 µm (A) or 20 µm (B, C, E-H) and green arrow-heads indicate auto-fluorescence from lipofuscin observed in the blue- and the green channel.
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using this combination of dyes, 100 nM PBB3 and 100 nM
bTVBT3, Aβ deposits were visualized by the fluorescence from
PBB3, whereas tau deposits, NFTs, dystrophic neurites and
neuropil threads displayed characteristic emission from bTVTB3.
Thus, under these staining conditions, bTVTB3 showed a higher
selectivity and most likely a higher affinity for tau deposits than
PBB3, and seemingly these ligands also compete for the same
binding site to the tau deposits. To verify the latter, tissue sections
were first incubated in PBS or PBS with 1 µM of PBB3 for 1 hour
prior to staining with 10 nM of bTVBT3. Strikingly, in the section
pre-incubated with PBB3, both Aβ and tau deposits exhibit strong
PBB3 fluorescence, whereas no observable bTVBT3
fluorescence was obtained from the tau pathology (Figure 5F). In
contrast, for the section pre-incubated in PBS, both NFTs,
dystrophic neurites and neuropil threads displayed strong
bTVBT3 fluorescence (Figure 5G). Hence, the tau staining of
bTVBT3 was abolished by pre-incubating the sections with a 100-
fold excess of PBB3, verifying that these two ligands share a
similar mode of binding to tau aggregates.
As it was previously shown that q-FTAA-CN also stained NFTs
when using micro-molar concentrations of ligand for histological
staining,[29] similar competition experiments as described above
were also performed using q-FTAA-CN and bTVBT3. At this
higher concentration, 1 µM, q-FTAA-CN labelled both Aβ deposits
and NFTs (Figure 5H). In addition, even though the section was
pre-incubated with 1 µM q-FTAA-CN, the characteristic bTVBT3
fluorescence from the NFTs could easily be observed from the q-
FTAA-CN positive NFTs as well as from q-FTAA-CN negative
dystrophic neurites (Figure 5H). Hence, q-FTAA-CN and bTVBT3
most likely have alternative modes of binding to tau aggregates.
Previous studies have suggested that anionic oligothiophenes,
including q-FTAA-CN, have a similar mode of binding to protein
aggregates as Congo red and related analogues.[29, 42] Anionic
Experimental Section
Full experimental details including additional characterization
data and NMR spectra of new compounds, as well as supporting
figures and tables are given in the Supporting Information. Frozen
brain tissue from a clinically and neuropathologically well-
characterized case of AD was obtained from the Knight
Alzheimer’s Disease Research Center, Washington University
School of Medicine, St. Louis, Missouri, USA. Tissue was
removed according to Washington University Local Ethics
Committee guidelines and informed consent for brain donation
was obtained from the next-of-kin. Frozen tissue from human AD
brain was also purchased from Tissue Solutions Ltd, Glasgow,
Scotland. Tissue Solutions Ltd confirmed that these human tissue
samples have been collected with ethics committee approval and
with permission to use these sections for research, and that all
samples have been collected from donors following written
consent.
Acknowledgements
Our work is supported by the Swedish Foundation for Strategic
Research, the Erling Persson Foundation and the Swedish
Research Council. We would also like to thank Prof. David M.
Holztman and the Knight Alzheimer's Disease Research Center
(NIH grants P50AG05681 and 5PO1-AG03991) Washington
University, St. Louis, Missouri, USA), as well as CBD solutions for
supplying us with human brain tissue.
Keywords: fluorescent ligands • protein aggregates • tau •
amyloid-β • Alzheimer´s disease
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10.1002/chem.201703846
Chemistry - A European Journal
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A specific pathological partner: bi-
thiophene-vinyl-benzothiazoles
(bTVBTs) are superior thiophene-
based optical ligands for selective
detection of tau pathology in
Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
Alzheimer´s disease (AD). Alternating
distinct chemical motifs of the bTVBT
scaffold greatly reduced the ability of
the dye to distinguish Aβ and tau
deposits, the two pathological
hallmarks in AD.
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