ACS Chemical Neuroscience
Research Article
idine, respectively. Intermediates 8−11 were then subjected to
(λmax) of the free probe in solution compared to the parent
ARCAM (1) (Table 1), whereas the introduction of the
trifluoromethyl group in 3 resulted in a hypsochromic shift in
emission λmax compared to 1. These observations suggest that,
on the one hand, electron-donating groups (e.g., the methyl
group in 2) near the electron-acceptor region (e.g., the nitrile
group in 1−5) of the molecular rotor can help stabilize the
dipolar photoexcited LE state, leading to a lower energy (or
higher wavelength) of photon emission, whereas electron-
withdrawing groups (e.g., the trifluoromethyl group in 3) at
the same position lead to a destabilization of the LE state and a
higher energy (or lower wavelength) of photon emission upon
relaxation to the ground state. Electron-donating substituents
on the 2-piperidinyl position of the ARCAM scaffold (e.g., the
methyl or ethyl groups in 4 and 5), on the other hand,
apparently increased the energy of fluorescence emission of the
free probe compared to the parent ARCAM probe, albeit the
effect was relatively small. The excitation and emission λmax of
1−5 spanned ranges of 73 and 54 nm, respectively,
demonstrating that additions of small aliphatic groups on the
ARCAM scaffold can lead to very large changes in spectral
characteristics of these fluorophores and can help fine-tune the
spectroscopic properties of these probes for specialized
21
a Knoevenagel condensation with previously reported α-
cyanoamide 12 to afford fluorescent ARCAM analogues 2−5.
Because of the limitations on the commercial availability of
enantiomerically pure 2-substituted piperidines, we generated
synthesis and characterization of these probes.
With compounds 1−5 in hand, we first evaluated whether
aliphatic substituents near the rotatable single bonds a and b
on the ARCAM scaffold (Figure 1B) had an effect on the QY
We measured the absorption and emission spectra of
compounds 1−5 in an aqueous solution using a similar
protocol as previously described for an estimation of the QY of
2
4
ThT. Table 1 summarizes the estimates for QY for
compounds 1−5. These solution studies revealed that the
introduced aliphatic substituents on the ARCAM scaffold, the
QY decreased compared to the parent ARCAM (1), with the
substituents on the vinylic position causing a larger decrease in
QY than substituents on the piperidine ring (Table 1). This
trend of decreased QY for substituted ARCAM analogues was
reflected in the relative intensity of the fluorescence emission
spectra of the free probes (black lines, Figure 2) in aqueous
solution, as compounds 2 and 3 decreased in fluorescence
intensity by 2 orders of magnitude compared to ARCAM (1),
while the fluorescence intensity of free compounds 4 and 5 in
solution were lower but similar in magnitude to that of
ARCAM (1). These trends did not change when the emission
14,16,17,25
applications.
In order to examine the binding and fluorescence properties
of ARCAM and its analogues in the presence of Aβ aggregates,
we prepared a solution of aggregated Aβ(1−42) peptides using
26
21
the characterization). Binding measurements revealed that
all of the compounds 1−5 bound with similar low micromolar
substituents on the ARCAM scaffold do not significantly affect
the binding to amyloids. Interestingly, all of the probes with
aliphatic substituents show a trend of stronger binding to Aβ
aggregates, which could be due to increased hydrophobic
interactions betwen these ARCAM analogues with the binding
pockets on Aβ aggregates compared to the parent ARCAM
In order to probe whether the aliphatic substituents could
introduce any electronic effects on the spectroscopic properties
27
(1). However, we found substantial differences in the
fluorescence enhancement properties between these probes
in amyloid-containing versus amyloid-free solutions. For the
parent compound ARCAM (1), we observed a 2.1-fold
increase in the fluorescence intensity in the presence of
aggregated Aβ compared to the background fluorescence of
the probe in the absence of Aβ (Figure 2 and Table 1). For
compounds 2 and 3, containing substituents on the vinylic
group, we found a 3.5- and 5.4-fold increase in the fluorescence
intensity in the presence of aggregated Aβ compared to the
background fluorescence, respectively. ARCAM analogues 4
and 5 with substituents on the piperidine ring, however,
exhibited only a 1.9- and 1.2-fold increase in the fluorescence
intensity in the presence of aggregated Aβ compared to the
background, respectively. While the changes in the maximal
(red dotted lines)), all of the probes displayed a hypsochromic
shift in emission λmax in the presence of aggregated Aβ
compared to free probes in solution. These observations are
consistent with previous studies on ARCAM (1) and suggest
that spectroscopic measurements for all of the probes in the
presence of aggregated Aβ were dominated by their
(
profiles of compounds 1−5. We found that an introduction of
the methyl or ethyl group on the piperidine ring (as in probes
4
and 5, respectively) led to a bathochromic or red-shift in the
excitation maximum (Table 1) relative to ARCAM (1). These
observations suggest that an introduction of σ-donating groups
near the electron-donating nitrogen within the piperidine
group lowers the energy required for photoexcitation. The
introduction of substituents on the vinylic position of the
ARCAM scaffold also exhibited an effect on excitation maxima,
where a σ-donating methyl group on the vinylic position (as in
probe 2) led to a hypsochromic or blue-shift in the excitation
maximum, and a σ-withdrawing trifluoromethyl group on the
vinylic position (as in probe 3) led to a bathochromic shift in
the excitation maximum compared to the parent ARCAM (1);
the effects of substituents at the vinylic position in probes 2
and 3 are consistent with the expectation that σ-donating
groups near the electron acceptor of the fluorophore will
increase the energy required to generate the photoexcited state
and that σ-withdrawing groups will have the opposite effect.
For the effects of substituents on emission profiles, we found
that the introduction of the methyl group in 2 resulted in a
bathochromic shift in the maximum emission wavelength
14,15,17−21
fluorescence properties in the bound state.
These
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ACS Chem. Neurosci. 2021, 12, 2946−2952