Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates

Article abstract of DOI:10.1039/c1ob05637a

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting (i) early non-thioflavinophilic protein assemblies of Aβ1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimer's diease brain tissue (Aβ plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob05637a

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PAPER
Synthesis of a library of oligothiophenes and their utilization as fluorescent
ligands for spectral assignment of protein aggregates†
˚
Ther e´ se Klingstedt, Andreas Aslund, Rozalyn A. Simon, Leif B. G. Johansson, Jeffrey J. Mason, Sofie Nystr o¨ m,
Per Hammarstr o¨ m and K. Peter R. Nilsson*
Received 21st April 2011, Accepted 23rd September 2011
DOI: 10.1039/c1ob05637a
Molecular probes for selective identification of protein aggregates are important to advance our
understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report
the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can
be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to
be necessary for detecting (i) early non-thioflavinophilic protein assemblies of Ab1-42 and insulin
preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two
main pathological hallmarks observed in human Alzheimer’s diease brain tissue (Ab plaques and
neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a
backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated
thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of
protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for
these diseases.
Introduction
pre-fibrillar states preceding the formation of well-defined amyloid
fibrils are likely to play a critical role in the pathogenesis of protein
aggregation diseases.
The development of molecular probes for the detection of
proteinaceous deposits is of great importance, as the formation
of extra- or intracellular protein aggregates is the common
pathological hallmark associated with many diseases, including
Alzheimer’s, Parkinson’s and Huntington’s disease, and the infec-
17–19
ThT and CR have also shown limitations for detecting disease
associated protein aggregates in tissue samples and recently
we introduced luminescent conjugated polythiophenes (LCPs)
as a novel class of fluorescent probes for the detection of
1
tious prion diseases. From an ultrastructural perspective, these
20–25
˚
26
protein aggregates.
In 2009, Aslund et al. improved the
protein deposits consist mainly of amyloid fibrils with a diameter
concept and presented defined pentameric structures termed
luminescent conjugated oligothiophenes (LCOs) that could be
utilized for in vivo imaging of protein aggregates. In comparison
with conventional amyloid ligands, LCPs and LCOs have a
conjugated thiophene backbone, which brings about a connection
2–4
of 7–10 nm and the detection of these species has for several
decades relied on small hydrophobic dyes, such as derivatives of
5–7
8–10
Congo red (CR) and Thioflavin T (ThT).
not bind to specific proteins but are rather selective towards protein
aggregates having an extensive cross b-pleated sheet conformation
and a structural regularity, the typical molecular characteristics
These ligands do
between the conformation of the thiophene backbone and its
6,11
22,27
spectral properties.
When binding to protein aggregates, the
12,13
of presumably all amyloid fibrils.
The affinity of CR with
conformation of the backbone is dictated by the protein, which
results in conformation-sensitive spectral signatures from the LCP
a resulting yellow-green birefringence is one of the amyloid
14
15,16
criteria, but the specificity of CR has been questioned.
The
28
or LCO. LCPs and LCOs have been utilized for staining of
information that can be obtained from these conventional probes is
also somewhat limited and they cannot be utilized to characterize
various conformational entities, such as pre-fibrillar species or
heterogenic populations of protein aggregates. It is evident that
significant morphological variation can exist between different
amyloid fibrils formed from the same peptide or protein and that
thioflavinophilic and congophilic protein aggregates associated
21–26
with a variety of diseases
and also for identifying disease-
associated protein aggregates that go undetected with ThT and
23,26,29–34
CR.
For instance, LCPs and LCOs have proven very useful
for identification of protein aggregates associated with distinct
26,29,31
prion strains
bodies.
and for staining of intracelllular inclusion
34
Department of Chemistry, Link o¨ ping University, SE-581 83 Link o¨ ping,
Sweden. E-mail: petni@ifm.liu.se; Tel: +46 13 28 27 87
Staining of brain sections from Alzheimer’s disease (AD)
patients with the anionic LCO p-FTAA has revealed a spectral
discrimination between the two classical pathological hallmarks
of AD, amyloid b (Ab) plaques and neurofibrillary tangles (NFTs)
†
Electronic supplementary information (ESI) available: Supplemen-
tary Fig. 1, supplementary Table 1 and NMR spectra. See DOI:
0.1039/c1ob05637a
1
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formed of hyperphosphorylated tau protein. Interestingly, this
phenomenon was lost when the carboxyl groups were removed
from the 2- or 5¢¢¢¢-positions of the p-FTAA backbone. The spec-
tral distinction was also abolished when the thiophene backbone
products 12, 13, 14, 17, 19, 21 and 22 in 80–99% yield. Lastly,
all methylesters (12, 13, 14, 17, 19, 21 and 22) were hydrolyzed in
dioxane using 1.5 equiv. of sodium hydroxide per ester and acid on
the starting material. Once precipitation started, water was added
to the reaction to dissolve the product. After completion, the
products 1, 2, 3, 6, 7, 8, 9 were lyophilized and used without further
purification. The LCOs and all precursor molecules were verified
with H and C NMR, IR, and UV/Vis absorbance (see the ESI†).
However, due to their rather extensive backbones, compounds 14,
17 and 22 probably form rather large complexes in most solvents.
Therefore, we achieved less resolved NMR spectra of these three
compounds, compared to the other molecules.
26
was extended and more negatively charged groups were added.
Therefore, we now wanted to investigate how the carboxyl groups
and various lengths of the thiophene backbone influence the
observed spectral difference between Ab plaques and NFTs.
Furthermore, we also wanted to examine if the capacity to detect
non-thioflavinophilic protein assemblies preceding the formation
1
13
35
of amyloid fibrils, as recently reported for p-FTAA, is affected by
changing the chemical composition of the LCOs. Herein we report
the synthesis of a novel set of anionic oligothiophene probes and
the utilization of this library to provide further insight of the basic
molecular requirements for achieving an optimal performance
of these molecules as ligands for spectral assignment of protein
aggregates.
Fibrillation of Ab1-42 and insulin monitored by LCOs
To verify selective binding of the LCOs to protein aggregates,
we screened all nine LCOs against ThT positive amyloid fibrils
made from recombinant Ab1-42 peptide. All of the LCOs revealed
well-resolved excitation and emission spectra with typical shifts
in wavelengths when binding to recombinant Ab fibrils (Fig. 1,
Fig. 2 and Table S1†). These significant changes in the spectral
profiles were not seen for LCOs in buffer solution or when
mixed with freshly dissolved Ab1-42. The enhanced emission
and the well-resolved spectral substructures most likely arise
Results and discussion
Synthesis of luminescent conjugated oligothiophenes
To investigate if and how the length of the thiophene backbone and
different chemical substituents affect the performance of LCOs,
we synthesized a library of nine LCOs with backbone lengths
varying from four to seven thiophenes and with end thiophenes
substituted with hydrogen or carboxyl groups at the a-position
37,38
from a conformationally restricted thiophene backbone,
due
to selective interactions with the Ab fibrils.
The pronounced shifts (around 50 nm) of the excitation spectra
towards longer wavelengths indicate that the LCOs adopt a
more extended conformation upon binding to the amyloid fibril
resulting in a larger transition dipole moment along the conjugated
molecular framework. A variety of studies suggest that other
amyloid ligands bind in grooves along the long axis of the amyloid
(
Fig. 1 and Fig. 2). The synthesis of the thiophene trimer (10)
and the three pentameric oligothiophenes, 15, p-HTAA (4) and
26,36
p-FTAA (5) have been reported previously.
The thiophene
trimer 10 is the starting point for the synthesis of tetramers q-
HTAA (2) and q-FTAA (3), pentamers p-HTAA (4) and p-FTAA
11,39–41
(
5), and heptamers h-HTAA (8) and h-FTAA (9) (Scheme 1).
fibril.
LCOs are presumably intercalating with the amyloid
42
Tetrathiophene 19 on the other hand serves as the core building
block for tetramer t-HTAA (1) and hexamers hx-HTAA (6) and
hx-FTAA (7) (Scheme 2). As mentioned above, the LCOs are
also varied by having hydrogen or carboxyl groups at the end
a-positions of the LCOs. These different variants are referred to
as “H”-variants (1, 2, 4, 6 and 8) for the hydrogen-substituted
ones and “F”-variants (3, 5, 7 and 9) for the carboxyl-substituted
ones. 10 was mono-brominated using N-bromosuccinimide (NBS)
fibrils through a similar mechanism and the constraints of the
fibril binding pocket are inducing unique excitation and emission
pathways for the LCO. Hence, the nine LCOs provide distinct op-
tical signatures upon binding to Ab1-42 fibrils (Fig. 1 and Fig. 2).
The development of tools able to identify species preceding
the final fibrillar state has lately emerged as crucial, since there
is an increasing awareness of these pre-fibrillar species playing
17–19
an important role in the pathogenesis of misfolding diseases.
◦
43
in DMF at -15 C giving product 11 in 70% yield. The two
Recently, Jovin and co-workers presented an ESIPT (excited-
state intramolecular proton transfer) probe sensitive to early and
intermediate stages of a-synuclein aggregation and the pentameric
a-positions of 10 are equal in reactivity as the reaction starts.
However, when the first a-position has reacted, the reactivity of the
second a-position drops. Hence, a yield above the statistical 50%
can be afforded. The mixture of unreacted starting material, mono-
brominated product and di-brominated product was purified by
reversed phase. Two other bromination reactions were used during
the synthesis. The bromination of thiophene acetic acid gave an
acceptable yield (60%) of 18. Position 2 is more reactive as the
intermediate carbocation in position 3 becomes stabilized by the
side chain during the reaction. The last bromination performed
was the di-bromination of oligothiophenes 15 and 19, this reaction
was rather easy as both a-positions should react, but one should
be careful not to over-brominate the compound. The coupling of
brominated oligothiophenes 11, 16, 18 and 20 with the appropriate
boronic acid or boronic ester was performed using Suzuki cross
coupling conditions with the commercially available PEPPSI-IPr
as Pd-catalyst and potassium carbonate as base. This gave the
26,35
LCO p-FTAA was recently reported
to detect early non-
thioflavinophilic species occurring during the fibrillation pathway
of several amyloidogenic proteins. Therefore, we next utilized the
library of LCOs for monitoring the kinetics of recombinant Ab1-
42 fibrillation in situ. As we were not able to identify pre-fibrillar
species with transmission electron microscopy (TEM) or p-FTAA
26
utilizing our previously reported protocol for Ab1-42 fibrillation,
we tried different fibrillation protocols to achieve reproducible
fibrillation kinetics of Ab1-42 having a prolonged lag phase
offering the possibility to observe pre-fibrillar states preceding the
formation of amyloid fibrils. The conditions identified were close
◦
to physiological (37 C, 10 mM phosphate buffer supplemented
with 140 mM NaCl and 2.7 mM KCl pH 7.4, quiescent) and
the concentration of probe was well below the one used for Ab
(0.3 mM probe and 10 mM Ab peptide, respectively). When the
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Fig. 1 Chemical structure (left panel), excitation spectrum (middle panel) and emission spectrum (right panel) of t-HTAA (a), q-HTAA (b), q-FTAA
(
c), p-HTAA (d) and p-FTAA (e). The spectral graphs display excitation/emission spectra of the different LCOs in PBS (blue dotted line), in PBS with
soluble Ab1-42 (magenta dashed line) or in PBS with fibrillar Ab1-42 (black solid line). The excitation/emission wavelengths were 430/490 nm for
t-HTAA, 430/490 nm for q-HTAA, 430/505 nm for q-FTAA, 450/490 nm for p-HTAA and 450/507 nm for p-FTAA. All probes show well-resolved
excitation and emission spectra when binding to Ab fibrils.
fluorescence intensity was plotted against time it became
evident that a LCO backbone consisting of five or more
thiophenes is a prerequisite for detecting non-thioflavinophilic
species preceding mature fibrils in the Ab fibrillation pathway
(Fig. 3). Interestingly, the main difference between the fibrillation
protocol reported herein compared to our previously reported
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Fig. 2 Chemical structure (left panel), excitation spectrum (middle panel) and emission spectrum (right panel) of hx-HTAA (a), hx-FTAA (b), h-HTAA
(
(
c) and h-FTAA (d). The spectral graphs display excitation/emission spectra of the different LCOs in PBS (blue dotted line), in PBS with soluble Ab1-42
magenta dashed line) or in PBS with fibrillar Ab1-42 (black solid line). The excitation/emission wavelengths were 480/522 nm for hx-HTAA, 480/
5
35 nm for hx-FTAA, 480/538 nm for h-HTAA and 480/548 nm for h-FTAA. All probes show well-resolved excitation and emission spectra when
binding to Ab fibrils.
2
6
protocol is the lack of agitation during fibrillation and it is
well-known that shaking can be used as a parameter to induce
rapid formation of amyloid fibrils. Hence, the LCO positive
pre-fibrillar species of Ab1-42 are most likely occurring as a
consequence of slowing down the formation of amyloid fibrils as
these species could not be observed for p-FTAA when agitation
ThT was rather expected due to the structural similarity. When
the backbone was extended by one thiophene residue, the effect
on the detection capacity was obvious. Both p-HTAA and p-
FTAA revealed an Ab1-42 fibrillation process with a growth phase
initiated around 60 min, which was 300 min earlier than ThT
(Fig. 3b). The pentameric LCOs had reached the plateau phase by
the time ThT indicated initiation of growth phase, which further
emphasized the difference. Further extension of the thiophene
backbone to yield hexamers and heptamers did not significantly
enhance nor diminish the effect. hx-HTAA, hx-FTAA, h-HTAA
and h-FTAA all bound to Ab species not detected by ThT with an
onset of fibrillation around 90 min (Fig. 3c). hx-FTAA and the two
heptamers displayed almost identical kinetic trajectories, whereas
26
was used during fibrillation.
The kinetic profile monitored by the tetrameric probes t-HTAA,
q-HTAA and q-FTAA revealed an onset of fibrillation around
3
00 min after initiation (Fig. 3a). The kinetic curves were similar
to the one obtained with ThT when it came to length of lag
phase and fibrillation onset, however, the tetramers reached the
plateau phase more rapidly than ThT. The resemblance with
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Scheme 1 The synthesis of q-HTAA (2), q-FTAA (3), h-HTAA (8) and h-FTAA (9).
hx-HTAA reached the end of the lag phase at a later time-point.
Hence, the kinetic results indicated that the length of the backbone,
not the chemical substituents, is the crucial factor for the detection
of early species preceding the formation of Ab1-42 fibrils. This
finding accentuates the fact that small variations of the LCO
structure have a strong impact on their properties for detecting
distinct protein assemblies. Hence, having access to a library of
different LCOs is highly valuable for assessing a more complete
evaluation of different protein aggregation reactions. Since the
protocol for monitoring the kinetics of Ab fibrillation was designed
with LCOs present during the reaction (in situ), transmission
electron microscopy (TEM) was applied to investigate the possible
scenario of LCOs enhancing protein aggregation. TEM images
were obtained after 180 min of reaction both with and without
LCO present and indicated no difference in the fibrillation of Ab,
hence the earlier growth phase monitored by LCOs was not caused
by the probes accelerating the kinetics (ESI, Fig. S1†). In addition,
◦
kinetic experiments under identical conditions (37 C, 10 mM
phosphate buffer supplemented with 140 mM NaCl and 2.7 mM
KCl pH 7.4, quiescent) having only the pentameric, hexameric or
heptameric LCOs present or the LCOs mixed with 10 mM bovine
serum albumin (BSA) or 100 mM bovine insulin (BI) were also
performed. To our knowledge, these proteins will not form protein
aggregates under these conditions. All LCOs, except hx-HTAA,
showed identical spectra throughout the experiments (1020 min)
compared with time-point 0 min, verifying that the characteristic
spectroscopic signature obtained from these LCOs during the
kinetic measurements of Ab1-42 fibrillation is associated with
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Scheme 2 The synthesis of t-HTAA (1), hx-HTAA (6) and hx-FTAA (7).
pre-fibrillar and fibrillar aggregated species of Ab1-42 (Fig. S2†).
For hx-HTAA we observed a slight decrease of the spectral
intensity over time when the dye was free in solution, whereas the
opposite occurred when the dye was mixed with BSA (Fig. S2i and
S2l†). However, the spectrum was unaffected over the same time
period when mixed with BI, and under none of these experiments
were we able to obtain a spectroscopic signature similar to the
one observed for hx-HTAA interacting with pre-fibrillar and
fibrillar aggregated species of Ab1-42 (Fig. S2j and S2k†). This
different behavior from hx-HTAA compared to the other LCOs
might occur due to solubility problems of hx-HTAA, as this dye
has only two negatively charged side chain functionalities on a
hexameric thiophene backbone and hx-HTAA is therefore deemed
less suitable for performing kinetic measurements than the other
LCOs.
by the extended thiophene backbone enhancing the affinity of
the LCOs towards these aggregated species. Qin et al. recently
45
reported a similar observation for a dimeric version of ThT.
However, the mechanism by which ThT and other amyloid
ligands recognize fibrillar structures is still poorly understood.
In addition to binding, ThT also needs to be sterically locked in
order to fluoresce, since free rotation around the benzylamine
46–48
and benzathiole rings quenches the excited states of ThT.
Hence, it is possible that ThT and the tetrameric LCOs are
capable of binding to the early states seen in the Ab1-42 and
insulin fibrillation, but in their “quenched” mode. Further
elongation of the fibrils and more ordered structures might cause
the immobilization of the ThT molecule that is necessary for the
dramatic increase in fluorescence seen at the later stages of the
kinetic experiment. In contrast, the pentameric, hexameric and
heptameric LCOs seem to adopt a sterically locked conformation
giving rise to a highly fluorescent mode of these LCOs when
interacting with protein assemblies preceding the formation of
amyloid fibrils. In fact, the plateau phase of pentameric and longer
LCOs coincided with the initiation of growth phase monitored by
ThT, supporting the theory that ThT needs more ordered fibrils
in order to fluoresce. The fluorescence of all investigated LCOs
decayed after the plateau phase was reached, which indicated
increased stacking of probes due to more densely organised
amyloid fibrils. Alternatively different conformations of the LCOs
when bound to the early formed assemblies compared to mature
fibrillar states could be invoked to explain these observations.
However, both the excitation and emission spectral profiles of
the LCOs bound to these species are similar, suggesting that the
LCOs bind in an analogous fashion to early formed assemblies
and mature fibrils. Nevertheless, it is well established that the LCO
It is well known that the formation of amyloid fibrils is a
rather complex event. The simultaneous occurrence of different
aggregate species in solution and involvement of heterogeneous
aggregation mechanisms render it possible that the LCOs detect
the existence of non-fibrillar aggregates occurring in competition
with the formation of amyloid fibrils during the Ab aggregation
pathway. Therefore, we utilized the pentameric, hexameric and
heptameric LCOs to monitor fibrillation kinetics of BI under
acidic conditions, as it was recently shown that insulin under
the conditions used assembled into amyloid fibrils through a
44
hexameric oligomer unit. Similar to the results from the Ab1-
2 kinetics, all LCOs detected early non-thioflavinophilic species
4
of insulin and optical density (OD) measurements verified that the
LCOs did not affect the fibrillation rate (ESI, Fig. S3 and S4†).
The detection of non-thioflavinophilic Ab and insulin species by
pentameric, hexameric or heptameric LCOs, might be explained
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26,30,32,35
under the fluorescence microscope.
Hence, the decaying
intensity effect of LCOs over time seems to occur when LCOs
are present in situ during fibrillation after the growth of more
stable and ordered aggregates in solution/suspension. The full
nature of this decrease in intensity will be further elucidated in a
future photophysical characterization of all LCOs included in the
library.
Obviously, it would be of great interest to determine the nature
of the LCO positive non-thioflavinophilic protein assemblies
preceding amyloid fibril formation, especially as LCOs and LCPs
have proven useful for detection of pathological protein aggregates
that go undetected by conventional ligands such as ThT and
23,26,29–34
CR.
However, such a study must include extensive fibrilla-
tion experiments of Ab performed under different conditions and
also include different variants of the Ab peptide as it was recently
shown that Japanese mutant Ab rapidly formed amyloid fibrils that
49
were only weakly stained by ThT. So far we can only speculate
about the nature of the LCO positive protein assemblies preceding
amyloid fibril formation. These assemblies might be fibrillar or
non-fibrillar aggregates formed on the Ab aggregation pathway or
in competition with the formation of amyloid fibrils. However, the
excitation and emission spectra from the pentameric, hexameric
and heptameric LCOs bound to the pre-fibrillar protein assemblies
or to mature amyloid fibrils are similar, suggesting that these
entities have a related structural arrangement as the LCOs seem
to interact in a similar fashion with both of these entities. Initial
experiments have also shown that p-FTAA positive Ab aggregates
preceding amyloid fibril formation are more toxic to cells than ThT
positive amyloid fibrils (manuscript in progress), suggesting that
LCOs would be highly significant as tools for identifying distinct
aggregated species occurring during the amyloid fibril formation
pathway.
Spectral signatures of LCOs binding to protein aggregates in
human AD brain tissue
We have previously reported that p-FTAA can be used to spectrally
discriminate between Ab plaques and NFTs in frozen brain
26
sections from AD patients. Since this spectral ability was believed
to depend on the length of the thiophene backbone and the
nature of the chemical substituents along the backbone, the
library of LCOs displaying variations in these properties was
applied to cryosections from human cases with AD pathology.
The histological analyses were performed with a fluorescence
microscope equipped with a Spectraview system, as this set up
offers the possibility to acquire the spectral signatures of each
LCO when binding to aggregated Ab or tau in tissue samples.
Additionally, blue (excitation: 405 or 436 nm), green (excitation:
Fig. 3 Fibrillation of recombinant Ab1-42 monitored by fluorescence
from ThT or LCOs. (a) Time plot showing the fibrillation kinetics of
Ab1-42 monitored by fluorescence from ThT (black solid line), t-HTAA
(
red dashed line), q-HTAA (green dotted line) or q-FTAA (blue dot–
dashed line). (b) Time plot of Ab1-42 fibrillation kinetics monitored by
fluorescence from ThT (black solid line), p-HTAA (red dashed line) or p-F-
TAA (green dotted line). The pentamers detect early non-thioflavinophilic
species in the fibrillation pathway and showed initiation of a growth
phase already after 60 min. (c) Time plot of Ab1-42 fibrillation kinetics
monitored by fluorescence from ThT (black solid line), hx-HTAA (red
dashed line), hx-FTAA (green dotted line), h-HTAA (blue dot-dashed line)
or h-FTAA (magenta dot-dot-dashed line). Both hexamers and heptamers
detected non-thioflavinophilic species in the fibrillation pathway and
showed initiation of the growth phase around 90 min.
4
70 or 480 nm) or red (excitation: 535 or 546 nm) long pass filters
were utilized to record a complete emission spectral profile of the
LCOs. Ab plaques and NFTs were identified due to their well-
known characteristic morphology seen with antibody staining
(ESI, Fig. S5†).
All the tetrameric LCOs (t-HTAA, q-HTAA and q-FTAA)
specifically stained both Ab plaques and NFTs in AD affected
regions. Moreover, the probes also highlighted cerebrovascular
amyloid deposits and dystrophic neurites, two other well-known
pathological markers of this disease. All tetramers displayed well-
resolved emission spectra upon binding to amyloid deposits, but
p-FTAA fluoresces very strongly when bound to mature
amyloid fibrils in tissue (and pelleted fibrils in vitro) as observed
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Fig. 4 Emission spectra and fluorescence images of p-HTAA, p-FTAA, hx-HTAA and hx-FTAA bound to pathological hallmarks in AD human brain
tissue. (a, d, j, p) The emission spectra of the indicated LCO bound to Ab plaques (green dashed line) or NFTs (red solid line) when excited at 405 nm.
The results show that carboxyl end groups are crucial for obtaining a spectral shift between the two protein entities. (b, e, q) The emission spectra of the
indicated LCO bound to Ab plaques (green dashed line) or NFTs (red solid line) when excited at 470 nm. (f, r) The emission spectra of the indicated
LCO bound to Ab plaques (green dashed line) or NFTs (red solid line) when excited at 546 nm. (k, l) The emission spectra of hx-HTAA bound to Ab
plaques (green dashed line) and NFTs (red solid line) are identical when excited at 480 nm (k) or 535 nm (l). (g, s) The emission spectra of the indicated
LCO, bound to Ab plaques (green solid line) or NFTs (red dashed line), when it has been excited at 405 nm and 546 nm and the resulting emission
spectra have been merged together. (m) The resulting emission spectra of hx-HTAA bound to Ab plaques (green dashed line) and NFTs (red solid line)
when the emission at 405 and 535 nm have been merged together. (h, n, t) Plot of the ratio of light intensity emitted at 517 and 613 nm (517/613
R
) for
p-FTAA, 521 and 608 nm (521/608 ) for hx-HTAA, 538 and 613 nm (538/613 ) for hx-FTAA bound to Ab plaques (green triangles) or NFTs (red
R
R
squares) shown with standard deviation. The intensities are obtained from the double excitation spectra. (c, i, o, u) Fluorescence images of human AD
brain sections stained with the indicated LCO. The images visualize typical pathological entities, Ab plaques (green arrow) or NFTs (red arrow), from
which the emission spectra were obtained. Scale bars represent 20 mm.
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Fig. 5 Emission spectra and fluorescence images of h-HTAA and h-FTAA bound to pathological hallmarks in AD human brain tissue. (a, g) The
resulting emission spectra of the indicated LCO bound to Ab plaques (green dashed line) and NFTs (red solid line) excited at 405 nm. (b, h) The emission
spectra of the indicated LCO bound to Ab-plaques (green dashed line) and NFTs (red solid line) when excited at 470 nm. (c, i) The emission spectra of
the indicated LCO bound to Ab plaques (green dashed line) and NFTs (red solid line) when excited at 546 nm. (d, j) The resulting emission spectra of the
indicated LCO bound to Ab plaques (green dashed line) and NFTs (red solid line) when the emission at 405 and 546 nm have been merged together. (e, k)
R R
Plots of the ratio of light intensity emitted at 545 nm and 613 nm (545/613 ) for h-HTAA or 553 nm and 613 nm (553/613 ) for h-FTAA when bound to
Ab plaques (green triangles) or NFTs (red squares) shown with standard deviation. The intensities are obtained from the double excitation spectra. (f, l)
Fluorescence images of human AD brain sections stained with the indicated LCO. The images visualize typical pathological entities, Ab plaques (green
arrow) or NFTs (red arrow), from which the emission spectra were obtained. Scale bars represent 20 mm.
there was no significant spectral distinction between Ab and tau
deposits when using two different long pass filters (excitation at
pass filters. Secondly, these LCOs also exhibited a substantial
amount of emission at longer wavelengths when a red long pass
filter was used (excitation: 535 or 546 nm) (Fig. 4 and Fig.
5). A red-shift of the emission maxima was observed as the
electronic conjugation length of the thiophene backbone was
extended and the “F” variants always showed more reddish
spectra compared to their “H” counterparts, mainly due to
extension of the conjugated thiophene backbone by carboxyl
groups. Interestingly, the difference in spectral shift was most
pronounced when comparing p-FTAA with p-HTAA, indicating
that the maximum effect of the carboxyl moieties on the electronic
system is obtained with a pentameric thiophene backbone.
When examining the spectral differences for Ab plaques and
NFTs, the pentameric, hexameric and heptameric “F” variants
showed a clear difference in the overall appearance of the emission
spectra (excitation: 405, 470 or 480 nm) for these pathological
hallmarks, whereas this spectral difference was not observed for
the “H” counterparts (Fig. 4 and Fig. 5). Binding of the “F”
4
05 or 436 nm) (ESI, Fig. S6†). The tetrameric LCOs showed
no significant emission when using a long pass filter for longer
wavelengths (excitation: 535 or 546 nm). It also became evident
that the tetrameric probes tended to photobleach, which was
indicated by the change of the normally blue-shifted emission
spectra against longer wavelengths upon prolonged exposure to
light.
26
In accordance with previous results, LCOs having a pen-
tameric thiophene backbone (p-HTAA and p-FTAA) displayed
selective binding towards protein aggregates and these patholog-
ical hallmarks were also specifically stained by LCOs having a
hexameric (hx-HTAA and hx-FTAA) (Fig. 4) or a heptameric
thiophene backbone (h-HTAA and h-FTAA) (Fig. 5). Similar
to the tetrameric LCOs, all longer LCOs showed well-resolved
emission spectra upon binding to Ab or tau deposits using blue
(
excitation: 405 nm) or green (excitation: 470 or 480 nm) long
8
364 | Org. Biomol. Chem., 2011, 9, 8356–8370
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variants to aggregated tau in NFTs and dystrophic neurites
displayed increased contribution of red-shifted wavelengths com-
pared to the spectra from Ab plaques. This was evident from
the disappearance of the most blue shifted peak and a more
pronounced shoulder at longer wavelengths (Fig. 4 and Fig. 5).
By exciting the LCOs at 405 nm and 535/546 nm and then
merging the resulting emission spectra, the spectral difference
became even more evident, since this operation highlights both
green and red contributions to the spectrum. When plotting the
ratio of the intensity of the emitted light at the blue-shifted and
red-shifted emission maxima (Fig. 4 and Fig. 5), there was a clear
separation between Ab and tau aggregates proving the discrepancy
in emission from the “F” variants upon binding to the respective
pathological entity. The merged emission spectrum of one of
the “H” variants, hx-HTAA, also revealed a distinction in color
between the protein aggregates. The spectra from tau deposits were
green-shifted compared to the spectra observed from Ab plaques,
a result that was opposite that of the “F” variants. The ratio plot
showed a rather wide distribution of the tau aggregates and a
partial overlap between the two pathological hallmarks (Fig. 4n).
When the ratio plots were compared, it became evident that the
AD. Preferably, such a ligand should have a backbone consisting
of five to seven thiophene units and carboxyl groups extending
the conjugated thiophene backbone. The addition of such LCOs
to the toolkit of fluorescent ligands will be important for studying
the underlying molecular events of protein aggregation diseases
and for obtaining sensitive diagnostic tools for these diseases.
Experimental
General methods
Organic extracts were dried over anhydrous magnesium sulfate,
◦
filtered and concentrated in vacuo at 40 C. 3-Thiopheneacetic
acid, 2-thiopheneboronic acid and PEPPSI-IPr ([1,3-bis(2,6-
diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladi-
um(II) dichloride) are commercially available from Sigma-Aldrich
Co (Sigma-Aldrich, St. Louis, MO, USA). 5-(Dihydroxyboryl)-2-
thiophenecarboxylic acid was acquired from Maybridge (May-
bridge, Cambridge, UK). UV/VIS absorbance spectra were
2
recorded with a Tecan Saphire microplate reader (Tecan,
M a¨ nnedorf, Switzerland) or a Hitachi U-1900 spectrophotometer
(
Hitachi, Tokyo, Japan). FT-IR spectra were recorded using
“
F” variants were superior LCOs for spectral distinction of the two
a Perkin Elmer Spectrum 1000 spectrometer (Perkin Elmer,
Waltham, MA, USA). NMR spectra were recorded on a Varian
pathological hallmarks seen in AD. Secondly, p-FTAA surpassed
both hx-FTAA and h-FTAA, again showing that the maximum
effect of the carboxyl extensions on the electronic system was
obtained having a pentameric thiophene backbone. Although it is
evident that the carboxyl groups are crucial to obtain a spectral
difference between Ab and tau deposits, the spectral observations
cannot explain the difference between these two protein aggregates
on a molecular basis. The variation of spectral fingerprints
reported for LCPs bound to distinct protein aggregates, such
as prion strains or heterogenic populations of Ab deposits, has
mainly been interpreted as a result of a conformational variety
of the protein aggregates, leading to separation and stacking of
1
13
instrument ( H 300 MHz, C 75.4 MHz, Varian Inc., Santa
Clara, CA, USA). Chemical shifts were assigned with the sol-
51
vent residual peak as a reference according to Gottlieb et al.
Thin layer chromatography (TLC) was carried out on Merck
precoated 60 F254 plates (Merck, Whitehouse Station, NJ, USA)
using UV light (l = 254 nm and 366 nm) and charring with
ethanol/sulfuric acid/p-anisaldehyde/acetic acid 90 : 3 : 2 : 1 for
visualization. Flash column chromatography (FC) was performed
using silica gel 60 (0.040–0.063 mm, Merck). Gradient HPLC-
MS was performed on a Gilson system (Gilson, Middleton, WY,
USA). Column: Waters X-Bridge C-18 5 m, 250 ¥ 15 mm and
Waters X-Bridge C-18 2.5 m, 150 ¥ 4.6 mm for semipreparative
and analytical runs respectively; using acetonitrile with 0.05%
formic acid and deionized water with 0.05% formic acid as mobile
22,23
polythiophene chains.
However, the distinct spectral signatures
from the LCOs reported herein were different, as the spectral
distinction between Ab and tau deposits was dependent on an
extension of the thiophene backbone with carboxyl groups rather
than on a conformational change of the thiophene backbone. The
fact that the polymeric LCP PTAA was incapable of spectrally
TM
phase. For preparative reversed phase purifications a VersaFlash
(
TM
Sigma–Aldrich) system equipped with VersaPak C18 cartridges
26
was used. MALDI-TOF MS was recorded in linear positive mode
with the analyte as matrix. Normal work up means that the
discriminating Ab and tau deposits in human tissue also indicates
that the underlying molecular mechanism of the spectral signature
from p-FTAA, hx-FTAA and h-FTAA might not be dependent on
conformational differences between Ab and tau deposits. Instead,
the red-shift in emission might arise from interplay between the
end carboxyl groups extending the LCO backbone and chemical
groups in the binding pocket of tau aggregates. Tau aggregates
organic extracts were dried on MgSO , filtered and concentrated
4
in vacuo.
General procedure A: Suzuki coupling with a thiopheneboronic
acid and a mono- or dibrominated oligothiophene
50
are known to carry a diverse array of chemical modifications,
To a solution of the brominated oligothiophene (1 mmol) in
including extensive phosphorylations, and these modifications
might affect the conjugation system causing the observed red-shift
of the emission.
toluene/methanol (1 : 1, 5 mL, degassed) K
2
CO (3 equiv./
3
bromine), the desired thiopheneboronic acid (2 equiv./bromine)
and PEPPS-IPr (0.02 equiv.) were added. The mixture was heated
◦
to 70 C for 15 min, whereupon work-up followed.
Conclusions
General procedure B: dibromination of an oligothiophene
In conclusion, we have shown that certain molecular requirements
are necessary for achieving an anionic oligothiophene-based lig-
and capable of detecting non-thioflavinophilic aggregated species
preceding amyloid fibril formation and for obtaining distinct spec-
tral signatures of the two main pathological hallmarks observed in
To a solution of the oligothiophene (1 mmol) in DMF (5 mL,
◦
-15 C) NBS (2 mmol) was added slowly, whereupon the reaction
reached room temperature. After 2 h, the reaction was diluted with
toluene, washed with HCl (1 M, aq.) twice and brine three times.
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General procedure C: Suzuki coupling with a diboronic acid or
ester with a monobrominated oligothiophene.
(dH
2
O)/nm 418. FT-IR nmax (KBr pellet): 649, 687, 786, 866, 1384,
-
1
1411, 1456, 1576, 3422 cm .
1
H NMR (DMSO-d
H); 7.24 (d, J = 3.9 Hz, 2H); 7.27 (s, 2H); 7.34 (dd, J = 1.1, 3.6
Hz, 2H); 7.39 (d, J = 3.9 Hz, 2H); 7.55 (dd, J = 1.1, 5.2 Hz, 2H).
6
) d: 3.76 (s, 4H); 7.11 (dd, J = 3.6, 5.2 Hz,
The desired boronic derivative (1 mmol), the monobrominated
2
oligothiophene (3 mmol), K
2
CO (4 mmol) and PEPPSI-IPr (0.02
3
mmol) were dissolved in degassed toluene/methanol (1 : 1, 5 mL)
13
C NMR (DMSO-d
6
) d: 35.4, 125.2, 126.1, 126.7, 128.1, 128.7,
◦
and heated to 70 C for 15 min, whereupon work-up followed.
129.2, 130.9, 133.5, 134.3, 135.3, 136.3, 136.6, 172.3.
General procedure D: hydrolysis methylesters
Synthesis of hx-FTAA (7)
To a solution of the oligothiophene (1 mmol) in dioxane (10 mL),
NaOH (1 M, 1.5 equiv./acid and ester) was added. When
General procedure A was applied to 20 and 5-(dihydroxyboryl)-
2
-thiophenecarboxylic acid. After the reaction, the mixture was
acidified with acetic acid, diluted with ethyl acetate and washed
three times with H O and subjected to normal work-up. The crude
precipitation started, H O (10 mL) was added. When only product
2
was seen on MS, the reaction was lyophilized and stored under
refrigerated, dark conditions.
2
product was recrystallized from dioxane to give 22 (82%) as a red
powder. General procedure D was applied to 22 to afford the
product hx-FTAA (7) quantitatively as a red salt. UV Abs: lmax
Synthesis of t-HTAA (1)
(
1
dH
2
O)/nm 428. FT-IR nmax (KBr pellet): 722, 783, 803, 1384,
General procedure D was applied to compound 19 to give the
-1
443, 1462, 1570, 1630, 3432 cm .
product t-HTAA (1) quantitatively. UV Abs: lmax (dH
2
O)/nm 385.
1
H NMR (D
d, J = 4.0 Hz, 2H), 7.04 (s, 2H), 7.09 (d, J = 3.9 Hz, 2H), 7.38 (d,
J = 3,9 Hz, 2H).
2
O) d: 3.68 (s, 4H); 7.00 (d, J = 4.0 Hz, 2H), 7.02
FT-IR nmax (KBr pellet): 638, 690, 786, 833, 1284, 1387, 1560, 1650,
(
-
1
3
484 cm .
1
H NMR (D
2
O) d: 3.49 (s, 4H), 6.78 (d, J = 3.7 Hz, 2H), 6.83
d, J = 4.9 Hz, 2H), 6.87 (d, J = 3.7 Hz, 2H), 7.12 (d, J = 4.9 Hz,
13
C NMR (D
2
O) d: 38.6, 124.2, 124.6, 126.4, 129.3, 131.9, 132.3,
(
1
33.9, 134.2, 134.6, 136.4, 139.3, 140.9, 169.8, 179.3.
2
H).
1
3
C NMR (D
2
O) d: 38.2, 124.4, 124.6, 126.7, 131.7, 132.0, 133.8,
Synthesis of h-HTAA (8)
134.9, 136.4, 178.0.
General procedure C was applied to 11 and thiophene-2,5-
diboronoic acid. After the reaction, the solution was decanted
and evaporated to relative dryness. The remaining portion in
the reaction vessel, containing salts, was repeatedly washed with
EtOAc (5 ¥ 20 mL) and combined with the mother liquor, which
was then washed with water (2 ¥ 50 mL), then 1 M HCl solution
Synthesis of q-HTAA (2)
General procedure D was applied to compound 12 to give the
product q-HTAA (2) quantitatively. UV Abs: lmax (dH
2
O)/nm
3
70. FT-IR nmax (KBr pellet): 641, 691, 835, 1235, 1268, 1384,
-
1
1
420, 1498, 1566, 3059, 3395 cm .
(2 ¥ 50 mL). The organic phase was dried over MgSO , filtered and
4
1
H NMR (D
2
O) d: 3.63 (s, 2H), 3.69 (s, 2H), 7.01 (dd, J = 5.0,
evaporated to dryness. The solid was slurried in ethanol, filtered
3
3
.6 Hz, 1H), 7.05 (d, J = 5.2 Hz, 1H), 7.08 (s, 3H), 7.16 (dd, J =
.6, 1.1 Hz, 1H), 7.32 (dd, J = 5.0, 1.1 Hz, 1H), 7.36 (d, J = 5.2
and dried under vacuum giving the compound 14 (99%) as an
amorphous orange/red powder. General procedure D was applied
to 14 to afford the product h-HTAA (8) quantitatively as a red salt.
UV Abs: lmax (dH O)/nm 432. FT-IR n (KBr pellet): 630, 705,
Hz, 1H).
1
3
C NMR (D O) d: 38.1, 124.1, 124.7, 125.2, 126.6, 128.0, 128.4,
2
2
max
-
1
130.8, 131.5, 131.8, 134.0, 134.6, 135.0, 135.4, 135.7, 136.7, 179.5,
788, 834, 916, 1221, 1253, 1411, 1700, 2921, 3432 cm .
1
180.0.
H NMR (D O) d: 7.07 (d, J = 5.1 Hz, 2H); 6.84 (s, 2H); 6.81
2
(
d, J = 5.2 Hz, 2H); 6.70 (m, 4H), 6.49 (s, 1H); 4.65 (s, 8H).
13
C NMR (D
2
O) d: 38.4, 38.5, 124.4, 124.9, 126.3, 126.4, 128.3,
Synthesis of q-FTAA (3)
131.3, 131.5, 132.1, 133.5, 134.2, 135.3, 135.5, 135.7, 179.3, 179.9.
General procedure D was applied to compound 13 to give the
product q-FTAA (3) quantitatively. UV Abs: lmax (dH
2
O)/nm
Synthesis of h-FTAA (9)
3
92. FT-IR nmax (KBr pellet): 636, 688, 775, 788, 866, 1394, 1448,
-
1
1568, 3416 cm .
General procedure A was used with 5-(dihydroxyboryl)-2-
thiophenecarboxylic acid and 16. After the reaction was finished,
the solution was decanted and evaporated to relative dryness.
The remaining portion in the reaction vessel, containing salts,
was repeatedly washed with EtOAc (5 ¥ 20 mL) and combined
with the mother liquor, which was then washed with water (2 ¥
50 mL), then 1 M HCl solution (2 ¥ 50 mL). The organic phase was
1
H NMR (D
2
O): d 3.68 (s, 4H), 7.01 (d, J = 5.1 Hz, 1H), 7.15
(
d, J = 3.9 Hz, 1H), 7.20 (d, J = 3.9 Hz, 1H), 7.23 (d, J = 3.9 Hz,
1
1
H), 7.23 (s, 1H), 7.36 (d, J = 5.1 Hz, 1H), 7.46 (d, J = 3.9 Hz,
H).
1
3
C NMR (D O): d 38.3, 124.3, 124.5, 126.3, 126.6, 129.1, 131.7,
2
131.9, 132.0, 132.1, 133.8, 134.2, 134.4, 135.1, 136.0, 139.5, 141.0,
1
69.8, 179.5, 180.0.
dried over MgSO
was slurried in ethanol, filtered and dried under vacuum giving
7 in 94% yield as an amorphous orange/red powder. General
procedure D was applied to 17 to afford the product h-FTAA (9)
quantitatively as a red salt. UV Abs: lmax (dH O)/nm 433. FT-IR
max (KBr pellet): 774, 1383, 1443, 1464, 1521, 1569, 3406 cm .
4
, filtered and evaporated to dryness. The solid
1
Synthesis of hx-HTAA (6)
General procedure D was applied to compound 21 to give
the product hx-HTAA (6) in quantitative yield. UV Abs: lmax
2
-
1
n
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366 | Org. Biomol. Chem., 2011, 9, 8356–8370
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1
H NMR (D
.7 Hz).
2
O) d: 3.6 (bs, 4H), 6.6–6.8 (bm, 10H), 7.3 (d, J =
Synthesis of product 16
3
1
1
3
General procedure B was applied to compound 15. The crude
product was purified on flash silica gel chromatography (hep-
tane/ethyl acetate 7 : 3) followed by purification of the desired frac-
C NMR (D2O) d: 38.8, 124.1, 125.0, 125.8, 128.4, 131.7, 131.9,
33.9, 134.0, 135.2, 136.3, 139.4, 141.0, 169.7, 179.1.
tions by reversed phase chromatography (CH
3
CN/H O gradient
2
Synthesis of product 11
60 : 40 → 80 : 20 → 100 : 0). The isolated fractions were combined
and dried under reduced pressure giving compound 16 (96%) as
an amorphous orange/red powder. UV Abs: lmax (ACN)/nm 389.
NBS (2.35 g, 13.2 mmol) was added to solution of 10 (5.36 g,
◦
1
3.7 mmol) dissolved in DMF (100 mL) at -15 C, after which
FT-IR nmax (KBr pellet): 792, 840, 965, 1013, 1172, 1197, 1254,
the stirring solution was allowed to achieve room temperature
and stir for a further hour. When TLC and MALDI-TOF
MS indicated that the reaction was complete the mixture was
diluted with toluene and washed with brine and water before
being subjected to normal work-up. The product was purified on
reversed phase (acetonitrile/water (30 : 70 0.05% formic acid)) to
afford compound 11 (70%). UV Abs: lmax (ACN)/nm 340. FT-IR
-1
1
3
1
336, 1428,1500, 1733, 2950, 3437 cm .
1
H NMR (DMSO-d
6
) d: 3.66 (s, 6H); 3.85 (s, 4H); 7.19 (d, J =
.9 Hz, 2H); 7.24 (d, J = 3.9 Hz, 2H); 7.27 (d, J = 0.8 Hz, 4H).
) d: 169.9; 137.0; 134.2; 133.4; 131.8; 131.4;
30.7; 127.9; 127.3; 124.8; 110.4; 51.5; 33.8.
13
C NMR (DMSO-d
6
Synthesis of product 18
n
max (KBr pellet): 626, 711, 764, 792, 833, 989, 1137, 1179, 1203,
-
1
1225, 1251, 1328, 1417,1434, 1732, 3446 cm .
3
-Thiopheneacetic acid (14.2 g, 100 mmol) was dissolved in
1
H NMR (CDCl
3
) d: 3.67 (s, 4H), 3.73 (s, 6H), 6.99 (s, 1H), 6.99
◦
diethyl ether (150 mL, dry) and cooled to 0 C. Thionyl chloride
(
d, J = 5.6 Hz, 1H), 7.06 (d, J = 3.5 Hz, 1H), 7.10 (d, J = 3.5 Hz,
(15.8 mL, 300 mmol) was added slowly. After 1 h, the solution was
1
H), 7.19 (d, J = 5.6 Hz, 1H)
13
evaporated, dissolved in diethyl ether (150 mL, dry) and cooled
in an ice bath. Methanol (dry, excess) was added slowly to the
solution and allowed to stir at room temperature for 1 h. The
solvents were evaporated while the product was redissolved in
C NMR (CDCl ) d: 34.1, 34.4, 51.9, 52.0, 111.2, 124.7, 127.1,
3
127.6, 130.3, 130.4, 130.8, 132.3. 132.8, 133.9, 134.2, 136.1, 170.6,
171.0.
ethyl acetate and washed with NaHCO (sat. aq.) followed by
3
water. The methyl ester of 3-thiopheneacetic acid was dissolved in
chloroform/acetic acid (4 : 1, 180 mL) and NBS (17.8 g, 100 mmol)
was added portionwise over two hours. After 18 h the solution was
Synthesis of product 12
General procedure A was used with 11 and 2-thiopheneboronic
acid. After the reaction, the product was purified by FC (toluene)
to afford compound 12 (92%) as a yellow powder. UV Abs: lmax
washed with NHCO (sat., aq.) until basic. FC (heptane/ethyl
3
acetate 9 : 1) gave compound 18 (60%) as a colorless oil. UV Abs:
(
1
ACN)/nm 368. FT-IR nmax (KBr pellet): 630, 702, 832, 1014,
lmax (ACN)/nm 236, 194. FT-IR nmax (KBr discs): 604, 636, 682,
-
1
171, 1197, 1251, 1333, 1434, 1734, 2949, 3102, 3438 cm .
701, 828, 893, 911, 971, 995, 1014, 1094, 1167, 1198, 1265, 1334,
1
-1
H NMR (CDCl
3
) d: 3.74 (s, 3H), 3.75 (s, 3H), 3.78 (s, 2H), 3.80
1375, 1413, 1435, 1541, 1738, 2841, 2951, 2999, 3108, 3460 cm .
1
(
7
7
s, 2H), 7.02 (dd, J = 5.1, 3.6 Hz, 1H), 7.06 (d, J = 5.3 Hz, 1H),
.13 (s, 1H), 7.15 (d, J = 3.7 Hz, 1H), 7.18 (d, J = 3.7 Hz, 1H),
.18 (dd, J = 3.6, 1.1 Hz, 1H), 7.23 (dd, J = 5.1, 1.1 Hz, 1H), 7.26
H NMR (D O) d: 3.63 (s, 2H); 3.71 (s, 3H); 6.92 (d, J = 5.5 Hz,
2
1H); 7.23 (d, J = 5.5 Hz, 1H).
1
3
C NMR (D O) d: 34.9, 52.2, 111.7, 125.9, 128.8, 133.6, 170.6.
2
(
d, J = 5.3 Hz, 1H).
13
C NMR (CDCl ) d: 34.8, 34.9, 52.3, 52.4, 124.2, 124.9, 125.0,
3
Synthesis of product 19
1
26.9, 127.4, 127.5, 128.0, 130.5, 130.6, 131.0, 131.8, 132.9, 135.5,
1
35.9, 136.3, 136.8, 171.2, 171.4.
General procedure C was used with 18 and 2,2¢-bithiophene-5,5¢-
diboronic acid bis(pinacol) ester. After the reaction the solution
was diluted with toluene and washed with HCl (1 M, aq.) twice
and brine 3 three times. The product was purified by FC (toluene)
Synthesis of 13
General procedure A was used with 11 and 5-(dihydroxyboryl)-
to give compound 19 (85%) as yellow oil. R : 0.42 (toluene/ethyl
acetate 18 : 1). UV Abs: lmax (ACN)/nm 371. FT-IR nmax (KBr
f
2
-thiophenecarboxylic acid. After the reaction, acetic acid and
ethyl acetate were added to the reaction mixture. The solution was
washed three times with H O and recrystallized from methanol
to afford compound 13 (87%) as a red powder. UV Abs: lmax
ACN)/nm 378. FT-IR nmax (KBr pellet): 748, 784, 803, 852, 896,
pellet): 638, 686, 716, 798, 835, 868, 925, 963, 1018, 1058, 1079,
-
1
1123, 1215, 1265, 1300, 1426, 1437, 1453, 1736, 2953, 3448 cm .
2
1
H NMR (CDCl ) d: 3.74 (s, 6H); 3.80 (s, 4H), 7.06 (d, J = 5.2
3
(
Hz, 2H); 7.10 (d, J = 3.77 Hz, 2H); 7.23 (d, J = 3.77 Hz, 2H); 7.25
9
35, 972, 1013, 1044, 1108, 1176, 1203, 1299, 1455, 1506, 1532,
(d, J = 5.2 Hz, 2H).
-
1
13
1668, 1733, 2516, 2951, 3444 cm .
C NMR (CDCl ) d: 34.7, 52.3, 124.4, 124.7, 127.6, 130.4,
3
1
H NMR (DMSO-d
6
) d: 3.65 (s, 3H), 3.67 (s, 3H), 3.84 (s, 2H),
130.5, 133.1, 134.3, 137.5, 171.4.
3
.88 (s, 3H), 7.10 (d, J = 5.1 Hz, 1H), 7.25 (d, J = 3.9 Hz, 1H), 7.30
(
d, J = 3.9 Hz, 1H), 7.39 (d, J = 3.9 Hz, 1H), 7.46 (s, 1H), 7.56 (d,
Synthesis of product 20
J = 5.1 Hz, 1H), 7.68 (d, J = 3.9 Hz, 1H).
1
3
C-NMR (DMSO-d
6
) d: 34.0, 34.1, 51.9, 52.0, 125.0, 125.5,
General procedure B was used on 19. The crude product was
purified by FC (toluene) to give compound 20 (88%). UV Abs:
1
27.3, 127.7, 129.6, 131.2, 131.3, 131.4, 132.1, 132.3, 133.3, 133.5,
133.9, 134.2, 135.5, 141.6, 162.5, 170.4, 170.7.
l
max (ACN)/nm 375. UV Abs: lmax (ACN)/nm 375. FT-IR nmax
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(
1
KBr pellet): 625, 791, 830, 1002, 1131, 1195, 1217, 1285, 1304,
Emission spectrum and excitation spectrum for each probe
in PBS were also obtained using the same settings as described
above.
-
1
334, 1425, 1455, 1505, 1734, 2954, 3439 cm .
1
H NMR (CDCl
3
) d: 3.72 (s, 4H); 3.74 (s, 6H); 7.03 (s, 2H); 7.05
(
d, J = 3.77 Hz, 2H); 7.30 (d, J = 3.77 Hz, 2H).
Amyloid fibrils of bovine insulin were prepared according to
1
3
35,44
C NMR (CDCl
3
) d: 34.6, 52.5, 111.7, 124.7, 128.2, 131.2,
a previously reported protocol.
In brief, a stock solution
-
1
1
33.2, 133.1, 137.9, 170.9.
containing 5 mg mL bovine insulin (I6634, Sigma Aldrich) in
2
M acetic acid supplemented with 500 mM NaCl was placed in
◦
a water bath kept at 50 C to induce formation of amyloid-like
insulin fibrils. Aliquots of 100 mL were withdrawn at different
time-points and mixed with 2 mL of the LCO stock solution (15
mM) or 2 mL of a ThT stock solution (15 mM). The fibrillation
was also performed having 300 nM of LCO present during the
fibrillation event. The samples were prepared in micro-titer plates
and incubated for 10 min at room temperature. The emission
spectra were recorded as described above.
Synthesis of product 21
General procedure A was used with 2-thiopheneboronic acid and
20. After the reaction was finished, chloroform was added and the
solution was washed with HCl (1 M, aq.) twice and brine three
times. The crude product was recrystallized from toluene to give
compound 21 (80%) as red crystals. UV Abs: lmax (ACN)/nm 402.
FT-IR nmax (KBr pellet): 686, 814, 842, 1020, 1139, 1229, 1272,
-
1
1
308, 1433, 1507, 1725, 2948, 3428 cm .
1
H NMR (CDCl
3
) d: 3.76 (s, 6H); 3.79 (s, 4H); 7.03 (dd, J =
LCO staining and spectral analysis
3
.63, 5.10 Hz, 2H); 7.13 (s, 2H); 7.13 (d, J = 3.79 Hz, 2H); 7.16 (d,
J = 3.79 Hz, 2H); 7.19 (dd, J = 1.15, 3.61 Hz, 2H); 7.24 (dd, J =
Cryosections (20 mm) of human brain tissue with AD pathology
were fixed with ice cold acetone for 10 min at -20 C and
◦
1
.15, 5.10 Hz, 2H).
C NMR (CDCl
1
3
3
) d: 35.0, 52.4, 124.2, 124.7, 125.0, 126.9,
then allowed to dry. The sections were incubated with PBS for
10 min and then stained for 30 min at RT with t-HTAA, q-
HTAA, q-FTAA, p-HTAA, p-FTAA, hx-HTAA, hx-FTAA, h-
HTAA or h-FTAA. All probes were diluted 1 : 500 in PBS from
a 1.5 mM stock. After rinsing with PBS three times, the sections
were mounted with Dako fluorescence mounting medium (Dako
Cytomation, Glostrup, Denmark). The medium was allowed to
solidify for three hours before the rims were sealed with nail
polish. Fluorescence and spectral images were collected with
a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Jena,
Germany) or a Leica DM6000 B fluorescence microscope (Leica,
Wetzlar, Germany) equipped with a SpectraCube module (Applied
Spectral Imaging, Migdal Ha-Emek, Israel). Bandpass filters
405/30 (LP450), 470/40 (LP515) and 546/12 (LP590) were used
for the Zeiss microscope and bandpass filters 405/40 (LP455),
436/10 (LP475), 480/20 (LP 515) and 535/50 (LP590) were used
for the Leica microscope. The emission profile of each LCO was
obtained by collecting the emission spectrum for 7–8 regions
of interest in five Ab plaques and five tau tangles, respectively,
using the above mentioned excitation wavelengths and the same
exposure time for the two protein entities. Computational analyses
were performed with the SpectraView 3.0 EXPO software (Applied
Spectral Imaging), which is a program that renders it possible not
only to obtain emission spectra for each excitation wavelength but
also to merge two different excitations into one resulting emission
spectrum. This operation was performed after exciting p-FTAA,
hx-FTAA, h-HTAA and h-FTAA at 405 and 546 nm, and hx-
HTAA at 405 and 535 nm. The same thing could not be done with
t-HTAA, q-HTAA, q-FTAA or p-HTAA since their emission at
535 or 546 nm was almost nonexistent. The double-excitation
emission spectra were used to make ratio plots for each LCO and
determine their capability to spectrally separate Ab plaques and
tau tangles.
1
27.6, 128.0, 131.1, 131.9, 134.2, 136.3, 136.8, 137.5 171.2.
Detection of pre-fibrillar and fibrillar Ab and insulin species
Recombinant Ab1-42 (rPeptide, Athens, GA, USA, lyophilized
from hexafluoroisopropanol and dissolved in 2 mM NaOH) was
diluted in 10 mM phosphate buffer supplemented with 140 mM
NaCl and 2.7 mM KCl (PBS pH 7.4) to a final concentration
of 10 mM and added to a 96-well microtiter plate (Corning
Incorporated, Corning, NY, USA). Stock solutions of LCOs (1.5
mM) and ThT (2 mM) were diluted in distilled water to 15 mM and
added to the wells to a final concentration of 0.3 mM. The samples
◦
2
were incubated at 37 C in a Tecan Saphire microplate reader
(
Tecan, M a¨ nnerdorf, Switzerland) and the emission spectrum of
each probe was collected every 30 min. The emission spectra for
t-HTAA, q-HTAA and q-FTAA were collected between 470–
6
for p-HTAA and p-FTAA were collected between 480–650 nm
with excitation wavelength 450 nm. The emission spectra for hx-
HTAA, hx-FTAA, h-HTAA and h-FTAA were collected between
40 nm with excitation wavelength 430 nm. The emission spectra
5
00–670 nm with excitation wavelength 480 nm. ThT was excited
at 430 nm (emission between 470–640 nm) when used as a reference
for the tetramers and it was excited at 450 nm (emission between
480–650 nm) when used as a reference for the pentamers, hexamers
and heptamers. All samples were done in triplicate. Experiments
replacing the Ab1-42 peptide with 10 mM bovine serum albumin
(
MP Biomedicals, Solon, USA) or 100 mM bovine insulin (I6634,
Sigma Aldrich) were also performed in a similar fashion.
Excitation spectra for the LCOs were obtained at time-point
min (right after the addition of soluble Ab1-42) and for probes
0
binding to Ab1-42 fibrils (at the plateau phase). The excitation
spectra for t-HTAA (emission at 490 nm), q-HTAA (emission at
4
90 nm), q-FTAA (emission at 505 nm) and p-HTAA (emission
at 490 nm) were collected between 270–470 nm. The excitation
spectra for p-FTAA (emission at 507 nm), hx-HTAA (emission at
Immunostaining with antibodies
522 nm), hx-FTAA (emission at 535 nm), h-HTAA (emission at
538 nm) and h-FTAA (emission at 548 nm) were collected between
300–500, 310–510, 325–525, 335–535 nm, respectively.
Cryosections of human Alzheimer’s diseased brain were fixed with
4% PFA for 15 min, washed in PBS and then incubated in blocking
buffer (PBS with 0.1% triton x-100 and 2% BSA) for 30 min. Ab
8
368 | Org. Biomol. Chem., 2011, 9, 8356–8370
This journal is © The Royal Society of Chemistry 2011

View Article Online
plaques and NFTs were visualized by using monoclonal mouse
anti-Ab antibody (1 : 200, Mabtech, Nacka Strand, Sweden)
diluted in PBS and monoclonal mouse anti-phosphorylated tau
antibody AT8 (1 : 100, Pierce Endogen, Rockford, IL, USA) di-
luted in blocking buffer. Both antibodies were incubated overnight
at 4 C. The sections were washed with PBS (Mabtech) or blocking
buffer (AT8) and then incubated with secondary Alexa Fluor 594-
conjugated goat anti-mouse antibody (1 : 500 (Mabtech) or 1 : 400
14 J. D. Sipe and A. S. Cohen, J. Struct. Biol., 2000, 130, 88–
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8.
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001, 276, 22715–22721.
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R. Melki, Biochemistry, 2004, 43, 5022–5032.
17 J. Hardy and D. J. Selkoe, Science, 2002, 297, 353–356.
◦
1
8 D. M. Walsh, I. Klyubin, J. V. Fadeeva, W. K. Cullen, R. Anwyl,
M. S. Wolfe, M. J. Rowan and D. J. Selkoe, Nature, 2002, 416, 535–
539.
1
9 J. L. Tomic, A. Pensalfini, E. Head and C. G. Glabe, Neurobiol. Dis.,
(
AT8), Molecular Probes, Eugene, OR, USA) for 30 min at RT.
2
009, 35, 352–358.
After washing, the sections were mounted with Dako mounting
medium (Dako) for fluorescence and analysed by using a Zeiss
Axioplan epifluorescence microscope (Carl Zeiss).
2
0 K. P. R. Nilsson, A. Herland, P. Hammarstr o¨ m and O. Ingan a¨ s,
Biochemistry, 2005, 44, 3718–3724.
21 K. P. R. Nilsson, P. Hammarstr o¨ m, F. Ahlgren, A. Herland, E. A.
Schnell, M. Lindgren, G. T. Westermark and O. Ingan a¨ s, Chem-
BioChem, 2006, 7, 1096–1104.
Abbreviations
2
2 K. P. R. Nilsson, A. A˚ slund, I. Berg, S. Nystr o¨ m, P. Konradsson, A.
Herland, O. Ingan a¨ s, F. Stabo-Eeg, M. Lindgren, G. T. Westermark, L.
Lannfelt, L. N. Nilsson and P. Hammarstr o¨ m, ACS Chem. Biol., 2007,
2, 553–560.
CR
Congo red
Thioflavin T
Luminescent conjugated polythiophenes
Luminescent conjugated oligothiophenes
Alzheimer’s disease
ThT
LCPs
LCOs
AD
Ab
NFTs
23 C. J. Sigurdson, K. P. R. Nilsson, S. Hornemann, G. Manco, M.
Polymenidou, P. Schwarz, M. Leclerc, P. Hammarstr o¨ m, K. Wuthrich
and A. Aguzzi, Nat. Methods, 2007, 4, 1023–1030.
2
4 O. Philipson, P. Hammarstr o¨ m, K. P. R Nilsson, E. Portelius, T.
Olofsson, M. Ingelsson, B. T. Hyman, K. Blennow, L. Lannfelt, H.
Kalimo and L. N. G. Nilsson, Neurobiol. Aging, 2009, 30, 1393–
amyloid b
neurofibrillary tangles;
1
405.
2
2
5 K. P. R. Nilsson, K. Ikenberg, A. A˚ slund, S. Fransson, P. Konradsson,
Acknowledgements
C. Rocken, H. Moch and A. Aguzzi, Am. J. Pathol., 2010, 176, 563–
5
74.
6 A. A˚ slund, C. J. Sigurdson, T. Klingstedt, S. Grathwohl, T. Bolmont,
Our work is supported by the Swedish Foundation for Strategic
Research (K.P.R.N, P.H. T.K.), the Knut and Alice Wallenberg
foundation (K.P.R.N, P.H.), and European Union FP7 HEALTH
D. L. Dickstein, E. Glimsdal, S. Prokop, M. Lindgren, P. Konradsson,
D. M. Holtzman, P. R. Hof, F. L. Heppner, S. Gandy, M. Jucker, A.
Aguzzi, P. Hammarstr o¨ m and K. P. R. Nilsson, ACS Chem. Biol., 2009,
˚
4, 673–684.
(
Project LUPAS, P.H., K.P.R.N., A.A, J.M., S.N.). K.P.R.N is
financed by an ERC Starting Independent Researcher Grant
Project: MUMID) from the European Research Council and P.H.
2
2
2
7 A. A˚ slund, K. P. R. Nilsson and P. Konradsson, J. Chem. Biol., 2009,
2
, 161–175.
(
8 T. Klingstedt and K. P. R. Nilsson, Biochim. Biophys. Acta, Gen. Subj.,
2011, 1810, 286–296.
is a Swedish Royal Academy of Science Research Fellow sponsored
by a grant from the Knut and Alice Wallenberg Foundation. We
would also like to thank Prof. David M. Holztman, Prof. Nigel
Cairns and the Knight Alzheimer’s Disease Research Center (NIH
grants P50AG05681 and 5PO1-AG03991) Washington University
9 C. J Sigurdson, K. P. R. Nilsson, S. Hornemann, M. Heikenwalder,
G. Manco, P. Schwarz, D. Ott, T. R u¨ licke, P. P. Liberski, C. Julius, J.
Falsig, L. Stitz, K. W u¨ thrich and A. Aguzzi, Proc. Natl. Acad. Sci. U.
S. A., 2009, 106, 304–309.
30 I. Berg, K. P. R. Nilsson, S. Thor and P. Hammarstr o¨ m, Nat. Protoc.,
010, 5, 935–944.
2
(
St. Louis, MO, USA) for supplying us with human brain tissue
3
1 K. P. R. Nilsson, S. Joshi-Barr, O. Winson and C. J. Sigurdson, J.
Neurosci., 2010, 30, 12094–12102.
32 A. Lord, O. Philipson, T. Klingstedt, G. Westermark, P. Hammarstr o¨ m,
and Mikael Lindgren for illuminating discussions on optical
spectroscopy. A generous gift from Astrid and Georg Olsson is
also gratefully acknowledged. T.K., R.A.S, L.B.G.J are enrolled
in the doctoral program Forum Scientum.
K. P. R. Nilsson and L. N. Nilsson, Am. J. Pathol., 2011, 178, 2286–
2
298.
3
3
3 S. W. Schultz, K. P. R. Nilsson and G. T. Westermark, PLoS One, 2011,
6
, e20221.
4 V. Mahajan, T. Klingstedt, R. Simon, K. P. R Nilsson, A. Thueringer,
K. Kashofer, J. Haybaeck, H. Denk, P. M. Abuja and K. Zatloukal,
Gastroenterology, 2011, 141, 1080–1090.
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