Rational Design of in Vivo Tau Tangle-Selective Near-Infrared Fluorophores: Expanding the BODIPY Universe

Article abstract of DOI:10.1021/jacs.7b05878

The elucidation of the cause of Alzheimer's disease remains one of the greatest questions in neurodegenerative research. The lack of highly reliable low-cost sensors to study the structural changes in key proteins during the progression of the disease is a contributing factor to this lack of insight. In the current work, we describe the rational design and synthesis of two fluorescent BODIPY-based probes, named Tau 1 and Tau 2. The probes were evaluated on the molecular surface formed by a fibril of the PHF6 (³⁰⁶VQIVYK³¹¹) tau fragment using molecular docking studies to provide a potential molecular model to rationalize the selectivity of the new probes as compared to a homologous Aβ-selective probe. The probes were synthesized in a few steps from commercially available starting products and could thus prove to be highly cost-effective. We demonstrated the excellent photophysical properties of the dyes, such as a large Stokes shift and emission in the near-infrared window of the electromagnetic spectrum. The probes demonstrated a high selectivity for self-assembled microtubule-associated protein tau (Tau protein), in both solution and cell-based experiments. Moreover, the administration to an acute murine model of tauopathy clearly revealed the staining of self-assembled hyperphosphorylated tau protein in pathologically relevant hippocampal brain regions. Tau 1 demonstrated efficient blood-brain barrier penetrability and demonstrated a clear selectivity for tau tangles over Aβ plaques, as well as the capacity for in vivo imaging in a transgenic mouse model. The current work could open up avenues for the cost-effective monitoring of the tau protein aggregation state in animal models as well as tissue staining. Furthermore, these fluorophores could serve as the basis for the development of clinically relevant sensors, for example based on PET imaging.

Full text of DOI:10.1021/jacs.7b05878

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Article
Rational Design of In Vivo Tau Tangle-Selective Near
Infrared Fluorophores: Expanding the BODIPY Universe
Peter Verwilst, Hye-Ri Kim, Jinho Seo, Nak-Won Sohn, Seung-Yun Cha, Yeongmin Kim,
Sungho Maeng, Jung-Won Shin, Jong Hwan Kwak, Chulhun Kang, and Jong Seung Kim
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05878 • Publication Date (Web): 31 Aug 2017
Downloaded from http://pubs.acs.org on August 31, 2017
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Rational Design of In Vivo Tau Tangle-Selective Near Infrared
Fluorophores: Expanding the BODIPY Universe
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Peter Verwilst,†‡ Hye-Ri Kim,§‡ Jinho Seo,† Nak-Won Sohn,§ Seung-Yun Cha,§ Yeongmin Kim,§
Sungho Maeng,§ Jung-Won Shin,§* Jong Hwan Kwak,∆* Chulhun Kang,§* Jong Seung Kim†*
† Department of Chemistry, Korea University, Seoul 02841, Korea
§ School of East–West Medical Science, Kyung Hee University, Yongin 17104, Korea
∆ School of Pharmacy, Sungkyunkwan University, Suwon 16419, Korea
ABSTRACT: The elucidation of the cause of Alzheimer’s disease remains one of the greatest questions in neurodegenera-
tive research. The lack of highly reliable low-cost sensors to study the structural changes in key proteins during the pro-
gression of the disease is a contributing factor to this lack of insight. In the current work, we describe the rational design
and synthesis of two fluorescent BODIPY based probes, named Tau 1 and Tau 2. The probes were evaluated on the mo-
lecular surface formed by a fibril of the PHF6 (306VQIVYK311) tau fragment using molecular docking studies to provide a
potential molecular model to rationalize the selectivity of the new probes as compared to a homologous Aβ selective
probe. The probes were synthesized in a few steps from commercially available starting products and could thus prove to
be highly cost effective. We demonstrated the excellent photophysical properties of the dyes, such as a large Stokes’ shift
and emission in the near infrared (NIR) window of the electromagnetic spectrum. The probes demonstrated a high selec-
tivity for self-assembled microtubule associated protein tau (MAPT, Tau protein), both in solution and cell-based experi-
ments. Moreover, the administration to an acute murine model of tauopathy clearly revealed the staining of self-
assembled hyperphosphorylated tau protein in pathologically relevant hippocampal brain regions. Tau 1 demonstrated
efficient BBB penetrability, and demonstrated a clear selectivity for tau tangles over Aβ plaques, as well as the capacity for
in vivo imaging in a transgenic mouse model. The current work could open up avenues for the cost-effective monitoring
of the tau protein aggregation state in animal models as well as tissue staining. Furthermore, these fluorophores could
serve as the basis for the development of clinically relevant sensors, for example based on PET imaging.
neurons, through predominantly electrostatic interac-
tions, promotes microtubule growth and stability.3,5-7
Extensive post-translational modifications, such as phos-
phorylation of serine and threonine residues, methyla-
tion, acylation, glycation and truncation have been ob-
served.8-10 Whereas the dynamic phosphorylation and
dephosphorylation of tau proteins is an important feature
in healthy individuals, the disruption of this delicate bal-
ance in favor of hyperphosphorylated tau proteins triggers
a pathological response. As a result of diminished electro-
static interactions, tau proteins disassemble from micro-
tubules and are missorted to the somatodendritic com-
partment, where they self-aggregate, resulting in intracel-
lular accumulation of NFTs.11,12
■ INTRODUCTION
Intraneuronal neurofibrilary tangles (NFT), consist of
paired helical filaments, comprising the hyperphosphory-
lated microtubule associated protein tau (MAPT, tau
protein)1 and, together with extracellular deposits of β
amyloid (Aβ) protein, are classical hallmark features ob-
served in the brains of Alzheimer’s patients.2 Furthermore
NFTs are a prevalent feature of other neurodegenerative
diseases, such as Down syndrome, amyotrophic lateral
sclerosis (ALS), Pick’s disease, Parkinson’s disease and
some forms of familial dementia, collectively known as
tauopathies.3 Importantly, the NFT burden has a strong
correlation to the disease progression and severity,4 un-
like Aβ in AD. Considering the potential applicability in
other tauopathies, the development of tau based imaging
probes is of paramount importance.
The structure of self-aggregated tau proteins is hypothe-
sized to consist of a core of parallel beta sheets, surround-
ed by a fussy coat of less structured polyelectrolyte brush-
es.13 However, no definite structure based on NMR or X-
ray crystallography is available, making the discovery of
tau tangle specific beta sheet binders a laborious task.
In humans, tau proteins exist in six isoforms, ranging
from 352 to 441 amino acids in length, with no clearly
defined secondary structure.3,5 The association of these
proteins with microtubules in the axonal compartment of
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Scheme 1. Synthetic approach towards BAP-1 and Tau 1-2. The structural modifications relative to BAP-1 are marked in
blue. The N − BODIPY C8 (meso) distance, as determined from the optimized structure by DFT is shown in magenta (See
SI for DFT calculation details).
Hence, no clear pharmacophore model has been identi-
fied thus far.14 In recent years two hypotheses guiding the
design of NFT binders have emerged. Firstly, it was dis-
covered from a series of benzothiazole-containing donor-
acceptor dyes that a distance of 13 to 19 Å between the
donor and acceptor parts benefits NFT selectivity, where-
as shorter distances favor Aβ plaques.15 Secondly, amongst
similar compounds, those containing fused ring systems
frequently have an increased selectivity for tau tangles
over Aβ fibrils.14
vents (Table S1). As can clearly be seen from Figures S7-
S9, BAP-1, Tau1 and Tau2 exhibit very similar behavior,
showing increased Stokes’ shifts and decreased fluores-
cence quantum yield in relation to the solvent polarity (as
quantified by the solvent dielectric constant). None of the
dyes showed a dependence on the solvent viscosity. Par-
ticularly the Stokes’ shift’s dependence on the dielectric
constant, as well as their relatively large value in relation
to the very small Stokes’ shifts commonly observed in
BODIPY-type dyes (in the range of 10 nm), indicates the
presence of intramolecular charge transfer (ICT) upon
excitation.
Hitherto only very few NIR (near infrared) emissive fluo-
rescent probes for NFTs have been discovered, either
recognizing specific phosphorylation patterns,16 or beta
sheet binders exhibiting a certain degree of selectivity.15,17
In the current work, we discuss two rationally designed
BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s- indacene)
based probes, aptly named Tau 1 and Tau 2, resulting
from the elongation of conjugation and annulation of a
known Aβ plaque binding dye, known as BAP-118 (Scheme
1), in order to investigate the scope and the potential
translation of the above mentioned design hypotheses to
this structurally unrelated class of dyes. The BODIPY
fluorophore was chosen owing to its excellent photophys-
ical properties such as high molecular extinction coeffi-
cients, quantum yields and photostability, combined with
attractive, often orthogonal chemical modifications.19,20
The dependency of the peak wavelength of absorbance
and emission of the fluorophores were studied in relation
to the solvent polarity, using a scale for the polarity that
treats the polarizability (induced dipole) and the dipolari-
■ RESULTS AND DISCUSSION
Synthesis. BAP-1 and Tau 1-3 were synthesized from the
common precursor BODIPY 1 via Knoevenagel condensa-
tions with their corresponding aldehydes (2-4) (Scheme
1
1). The characterization of all new final products by H
and 13C NMR, as well as ESI-MS can be found in the SI
(Figures S1-S6).
Figure 1. Calculated frontier orbitals of Tau 1 and Tau 2 by
density functional theory using B3LYP at the 6-311++G** level
of theory. (a-b) HOMO and LUMO orbital of Tau 1. (c-d)
HOMO and LUMO orbital of Tau 2. Electron densities are
displayed at the 0.03 e∙bohr-3 isodensity surface.
Fluorescence and theoretical calculations. The pho-
tophysical characteristics of the dyes were determined in
relation to the environmental properties of various sol-
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nucleation event as a potential therapeutic for tauopa-
ty (permanent dipole) independently, as described by
Catalán.21 The maximum absorbance wavelength correlat-
ed well with the solvent polarizability in all fluorophores
(Figure S10), as a result of the solvent electronic stabiliza-
tion of the Franck-Codon state, whereas the maximum
emission wavelength corresponded well with the solvent
dipolarity, via the solvent cage rearrangement after the
geometrical relaxation of the excited state molecule in
Tau 1-2 (Figure S11). Taken together these solvatochromic
effects clearly point to an excited state that has a signifi-
cantly higher dipole moment than the ground state,
which is a key characteristic of fluorophores exhibiting
ICT in the excited state.
thies.24-26 In particular, the interaction between adjacent
stacks of parallel layers of 306V and 308I have been identi-
fied as a target to prevent the initiation of protein nuclea-
tion.25 The crystal structures of this hexapeptide consist of
a steric zipper architecture, and co-crystallization with
various compounds reveals a propensity to generate tun-
nels along the fibril axis, either naturally or induced by
co-crystallization with small organic ligands.27-30 A signifi-
cant degree of polymorphism exists, with crystal struc-
tures demonstrating 0 (PDB: 2ON9),27 1 (PDB: 3OVL)28 or
2 (4NP8 and 5K7N)29,30 tunnels along the fibril axis. The
structure of tunnels induced by co-crystallization with
cross-beta sheet binders DDNP and curcumin (1 tunnel)
are also highly conserved in the structures exhibiting two
tunnels.
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The energy levels of the dyes’ molecular orbitals in vacu-
um were calculated by B3LYP DFT calculations at the 6-
311++G** level of theory (Figure 1 and Table S2). The elec-
tron distributions in the frontier orbitals revealed an elec-
tron redistribution from the aniline or aminonaphthalene
pendants in the highest occupied orbital (HOMO) to the
BODIPY core in the lowest unoccupied orbital (LUMO)
(Table S2), thus confirming the involvement of an ICT
process as a rationale for the observed large Stokes’ shifts.
Firstly, the interactions of BAP-1 and Tau 1-2 were stud-
ied on a 6-mer fibril built from the axis elongation of a
single VQIVYK peptide extracted from a recent 1.1Å reso-
lution crystal structure (PDB: 5K7N).30 The docking stud-
ies revealed a predominant interaction with the surface
formed by the QVK along the fibril axis, with the affinity
in the order of BAP-1 < Tau 1 < Tau2 (data not shown).
Importantly, the probes did not show a strong affinity for
the opposite surface of the protein, including the 306V and
308I believed to be important for tau aggregation inhibi-
tion.
The spectra of BAP-1 and Tau 1-2 in acidified chloroform
resulted in significantly hypsochromic shifts in both the
absorption and emission spectra (Figure S12), as com-
pared with those in non-acidified chloroform (Figure S13),
with associated Stokes’ shifts of classical BODIPY dyes
(around 6 nm). These results further demonstrate the
involvement of ICT, as the protonation of the pendant
aniline moieties disrupts the charge transfer process.
Subsequently, a 6-mer model of the most conserved
channel, formed by four adjacent beta sheet structures
was constructed (Figure S15). The tunnel exhibits a com-
bination of hydrophobic surfaces, as well as some hydro-
philic surfaces by in-plane and stacked hydrogen bond-
ing. The interactions of BAP-1 and Tau 1-2 in the tunnel
cavity were assessed, alongside the structures of the two
previously described NIR emissive cross beta sheet-
binding tau probes 3h17 and PBB515 (Table S3). Fascinat-
ingly, Tau 1-2 as well as 3h and PBB5 demonstrated a
tight fit in the tunnel as shown in Figure 2 and S16-S17,
respectively, with BAP-1 showing a significantly lower
calculated binding affinity (Table S3), which can be ra-
tionalized by a smaller surface area and thus a smaller
amount of surface interactions.
Unlike many other probes detecting cross beta sheet
protein morphologies, BAP-1, as well as Tau 1-2, did not
show a fluorescence quantum yield dependence on the
solvent viscosity and did thus not function as a molecular
rotor, despite their structural resemblance. The selective
recognition of tau protein cross beta sheets, combined
with the relatively apolar nature of the beta sheet struc-
tures of self-assembled tau protein, should thus ensure
increased fluorescence upon binding, whereas the ICT-
based large Stokes’ shifts are greatly beneficial to imaging
applications, preventing the re-absorbance of emitted
light often encountered in dyes with small Stokes’ shifts,
thus resulting in better signal over noise ratios.
In order to test the generality of the docking study, the
calculations were repeated with 12 lead compounds in the
design of tau selective PET probes, identified from vast
libraries of compounds (Table S3, Figures S19-S31). With
the exception of two compounds (F-ATPZ-38 and Aste-
mizole; see Table S3, entries 1 and 3 respectively), all
probes demonstrated calculated affinities in the same
range or higher than Tau 1-2, 3h and PBB5. As judged
from their structures, neither F-ATPZ-38 nor Astemizole
are likely to act as cross-beta sheet binders, but are rather
expected to interact with a different topology on the hy-
perphosphorylated Tau protein aggregates.
Finally, the photostability of the dyes was determined
versus Rose Bengal in DMF. As can be seen in Figure S14,
all dyes exhibited a similar resistance to photobleaching
under the employed conditions with a stability of 2.43 ±
0.04, 2.42 ± 0.07 and 2.59 ± 0.06 -fold the stability of Rose
Bengal for BAP-1, Tau 1 and Tau 2, respectively.
Molecular docking studies. The interactions of BAP-1
and Tau 1-2 with a crystal structure of the PHF6 fragment
(
306VQIVYK311) of the R3 microtubule binding region of tau
protein were evaluated using molecular docking studies.
The PFH6 sequence has been demonstrated to play a
pivotal role in the propensity of tau protein to form self-
assembled structures and was identified as responsible for
the nucleation of the tau protein,22,23 and has as such been
studied extensively in the quest for ligands to prevent the
In view of these results, we postulate that this tunnel
architecture along the fibril axis may be a major binding
site of cross-beta sheet binding probes with hyperphos-
phorylated tau protein.
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Figure 2. Molecular docking of Tau 1 (A-C) and Tau 2 (D-F). A and D) Top view of the molecule docked in the tunnel formed by
4 adjacent stacked beta sheets, protein molecular surface depicted. B and E) zoomed top view of the tunnel, side chains compris-
ing the tunnel are depicted as sticks, others as lines. C and F) Side view of the molecule in the in the protein tunnel, side chains
comprising the tunnel are depicted as sticks, others as lines. The yellow arrow indicates the viewing direction for panels C and F.
The results of this theoretical study need to be inter-
preted with some level of caution however, as the com-
plex structure of self-assembled tau protein precludes the
definite pinpointing of cavities formed by self-aggregating
PHF6 protein as a potential, or even predominant, bind-
ing location.
demonstrated a strong fluorescent response to both of tau
and Aβ amyloid aggregation assays (Figure S32).
In order to determine the influence of the dyes on the
aggregation behavior of tau derived peptides, an aggrega-
tion inhibiting assay was performed on the AcPHF6 pep-
tide. The assays showed that the aggregation of the pep-
tide, as recorded by the time dependent fluorescence of
ThT, was not affected by the presence of BAP-1 or Tau 1
at the concentrations used in the current work (Figure
S33), as the time to reach maximum fluorescence was not
significantly different. The maximum fluorescence inten-
sity of ThT was reduced upon the addition of the dyes,
suggesting a competition for binding sites on the peptide
aggregates with BAP-1 and Tau 1. The influence of Tau 2
was not determined as the solubility in the high buffer
strength medium was suboptimal for this dye.
Nevertheless these results in the protein cavities could
potentially provide a molecular insight and rationale for
the improved binding affinities observed in longer probes
with tau tangles vs. Aβ plaques,15 and could prove useful
for the design of novel (fluorescent) probes for tau aggre-
gates.
Tau 1 and Tau 2 selectively interact with Tau protein
aggregates in solution. The emission spectra of a sam-
ple of Tau 1-2, alongside BAP-1 and the universal beta
sheet fluorophore thioflavin T (ThT)31 as the controls,
were monitored over time to analyze the probes’ selectivi-
ty for tau protein aggregates over beta amyloid aggregates
(Figures 3 and S32). As evident from Figure 3A-B, tau
protein aggregation was induced by incubation with the
polyanionic heparin,32 and a clear time-dependent fluo-
rescent enhancement could be observed in the presence
of both Tau 1 and Tau 2. The maximum fluorescence
increase (6.4-fold and 9.3-fold for Tau 1 and Tau 2, respec-
tively) was reached after 48 hour heparin incubation. How-
ever, no significant fluorescence could be observed in the
presence of heparin alone (Figure 3A-B). By contrast, Tau
1 and Tau 2 both show only trivial responses to the pres-
ence of beta amyloid solutions (Figure 3C-D). These data
suggest that Tau 1 and Tau 2 are highly selective to Tau
protein aggregates over Aβ aggregates. ThT and BAP-1
The fluorescence of BAP-1 and Tau 1 in the NIR region
did also increase under these conditions, with similar
fluorescence rise times, providing extra evidence for a
binding interaction with the AcPHF6 peptide (Figure
S34), thus validating the results of the docking studies.
Cytotoxicity of Tau 1 and Tau 2. Prior to assessing the
specificity of the probes in a cellular environment, the
cytotoxicity of the probe was assessed using an MTT assay
in a number of brain-derived cell lines: SKNMC (Human
neuroblastoma), U87 (Human glioblastoma), SH-SY5Y
(Human neuroblastoma), C6 (Rat brain glioma), N2A
(Mouse brain neuroblastoma) as well as a human primary
brain neuron culture. No significant toxicity was observed
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Figure 3. Time dependent fluorescence enhancement of Tau 1-2 (10 µM) in the presence of protein aggregates. (a) Emission
spectra of Tau 1 in the presence of tau protein (10 µM) and heparin (2.5 µM), excited at 590 nm, (b) Emission spectra of Tau 1 in
the presence of tau protein (10 µM) and heparin (2.5 µM), excited at 580 nm, (C-D) Emission spectra of Tau 1-2 in the presence
of Aβ fibrils (50 µM).
at concentrations as high as 10 µM in any of these cells
after 48 hours of incubation (Figure S35).
To characterize the fluorophores’ response to Aβ, a pos-
sible cause of interference in the selective visualization of
tau aggregates in brain tissues, their fluorescent responses
were investigated in SH-SY5Y cells pretreated with freshly
dissolved Aβ1-42 peptide (50 µg/mL) for 48 hours,35,36 the
formation of intracellular amyloid fibrils was confirmed
by Western Blot analysis of cell lysates (Figure S37).
Tau 1 and Tau 2 selectively visualize okadaic acid-
induced tau hyperphosphorylation in cells. Human
neuroblastoma SH-SY5 cells treated with okadaic acid
(OA),33 a potent inhibitor of a broad range of ser-
ine/threonine phosphatases, is a well-known cell culture
model for in vitro tau hyperphosphorylation, and the tau
phosphorylation pattern is similar to that obtained from
human AD brain tissue.34 As shown in Figure S36, the
addition of 50 nM OA to SH-SY5 cells clearly resulted in a
time-dependent increase tau protein expression and its
hyperphosphorylation.
As can be seen in Figure 4C-D, Tau 1 and Tau 2 show
little fluorescence enhancement in the Aβ-treated cells,
compared to untreated cells, whereas BAP-1 and ThT did
reveal a small but significant increase in the fluorescence
intensity (1.57 ± 0.42 and 2.35 ± 0.61-fold, respectively).
These results clearly demonstrate that Tau 1 and Tau 2
are capable of detecting hyperphosphorylated tau protein
aggregates even in cellular environments without any
significant interference by Aβ aggregates.
Having confirmed the hyperphosphorylation in the cell
model, Tau 1-2, BAP1 or ThT (500 nM each) were added
to SH-SY5 cells incubated with 100 nM OA for 6 hours,
and the fluorescent images were quantitatively analyzed.
As seen in Figure 4A and B, all probes demonstrated an
increase in the fluorescence intensity as compared to cells
not pre-incubated with OA. Whereas an increase in fluo-
rescence of 4.41 ± 1.53 and 2.92 ± 1.10 fold was observed for
Tau 1 and Tau 2, respectively, BAP1 and ThT showed a
less pronounced effect with fluorescence enhancements
(1.71 ± 0.35 and2.17 ± 0.45 fold, respectively). Thus, these
results clearly indicate Tau 1 and Tau 2’s preference for
tau protein aggregates.
Tau 1 visualizes tau hyperphosphorylation in an
acute murine model for tauopathies in aged mice.
Encouraged by the previous results of Tau 1’s selectivity
to hyperphosphorylated tau aggregates in cells, its appli-
cation to an in vivo animal model of tauopathy was at-
tempted. For a relevant animal model, aged mice (23
months) were subjected to a single injection of 10 ng of
OA to the lateral amygdala in brain to induce tau hyper-
phosphorylation and its aggregation in the region of the
hippocampus.37 The CA1 and CA3 regions are particularly
interesting since these regions are among the early
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Figure 4. (a) Fluorescence images of SH-SY5Y cells incubated with Tau 1, Tau 2, BAP-1 and ThT (500 nM) for 20 min at 37°C.
Top: Untreated cells, Bottom: pre-incubated with okadaic acid (100 nM) for 6hr. ThT was excited at 458 nm and the emission
was recorded using a 475-525 nm band pass filter. Tau 1-2 and BAP-1 were excited at 633 nm and the fluorescence was collected
at wavelengths higher than 650 nm. (b) Florescence intensity per cell relative to untreated cells (n=5), as determined using im-
age J.38 Error bars designate standard deviation. (c) Fluorescence images of SH-SY5Y cells using amyloid beta peptide with Tau 1,
Tau 2, BAP-1 and ThT. After treatment of amyloid beta peptide (50 μg/mL) for 48 hr, the probes (1 µM) were incubation for 15
min at 37°C (bottom). Untreated cells were incubated with the probes for 15 min without peptide (top). ThT was excited at 458
nm and the emission was recorded using a 475-525 nm band pass filter, Tau 1-2 and BAP-1 were excited at 633 nm and the fluo-
rescence was collected at wavelengths higher than 650 nm. (d) Fluorescence intensity per cell relative to untreated cells (n=5), as
determined using image J.38 Error bars designate standard deviation.
regions to be affected by tau protein hyperphosphoryla-
tion and intraneuronal neurofibrilary tangles deposition
in the course of disease progression in models37 and hu-
man patients.39 Indeed, ex vivo analysis of the hippocam-
pal regions of the mouse brain, resulted in distinct stain-
ing of bands in the CA1 and CA3 region of the brain, cor-
responding to the pyramidal neuronal somatodendritic
compartments (Figure S38) where, on the brain slice from
mice without OA, Tau 1 showed virtually no fluorescence
under the same imaging settings.
In order to pursue whether the tau-positive regions in
the brain slice are the locations for the hyperphospory-
lated tau aggregates or not, a confocal microscopy study
of the emission from Tau 1 and an antibody against hy-
Figure 5. Confocal imaging of the CA1 (Top) and CA3 (Bot-
tom) hippocampal regions of OA treated mice. Dual staining
with the hyperphosphorylated tau protein antibody (AT-180)
and Tau 1 reveals a very high degree of overlap. The fluores-
cence image of Tau 1 was collected using excitation at 633
obtained using excitation at 488 nm with a 505-550 nm
bandpass filter.
perphosphorylated tau protein was conducted. The imag-
es in Figure 5 show that, in CA1 and CA3 hippocampal
regions of the OA-treated mice, Tau 1 demonstrates in-
tense fluorescence spots and, as anticipated the cell bod-
ies in these regions are clearly overlapped with the fluo-
rescence of the antibody. At the scale of an individual
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Figure 6. Confocal imaging of a single hippocampal neuron
of an OA treated mouse. Dual staining with the hyperphos-
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phorylated tau protein antibody AT-180 and Tau 1 demon-
strates significant amounts of tau missorting to the soma-
todendritic compartment (*) of the neuron, as well as spo-
radic hyperphosphorylated tau deposits along the axon (ar-
row). The fluorescence image of Tau 1 was collected using
excitation at 633 nm with a 650 nm longpass filter and that of
AT180 was obtained using excitation at 488 nm with a 505-
550 nm bandpass filter.
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Figure 7. Merged confocal imaging of hippocampal regions
of 3xTg mice. Left: Dual staining with the hyper-
phosphorylated tau protein antibody AT180 and Tau 1; Right:
Dual staining with the amyloid beta antibody 6e10 and Tau 1.
The fluorescent image of Tau 1 was obtained using excitation
at 633 nm with a 650 nm longpass filter and AT180 and 6e10
were obtained using excitation at 488 nm with a 505-550 nm
bandpass filter. Cell nuclei were stained with DAPI. See Fig-
ures S42 and S43 for images of the individual channels.
neuron, some revealed a flame-like feature as shown in
Figure 6. This feature can be interpreted as tau protein
aggregates sorted to the somatodendritic compartment of
the neuron as well as along the axon, to a lesser extent.
Although hyper-phosphorylated tau aggregates were
mainly found in axonal stem of neurons in human pa-
tients, its somatic localization was often observed in ani-
mal models of tau hyperphosphorylation (Figure 6).12
Nonetheless, the results in Figures 5 and 6 demonstrate
Tau 1’s capability as selective fluorescent sensor, respon-
sive to hyperphosphorylated tau aggregates in brain tis-
sues. Co-localization experiments with a fluorescent anti-
body for Aβ1-42 showed only faint, mostly non-specific
fluorescence, thus confirming the fluorescence originat-
ing for Tau 1 in OA-treated mice did not result from Aβ1-42
staining (Figure S39).
precursor protein (APP) and the human P301L mutation
of the 4R2N (Tau441) isoform of the human tau protein, as
well as a M146V mutated version of PSEN1, involved in
the processing of APP to Aβ1-42 peptides. The combined
effect of these three mutations ensures the presence of
both tau tangles and beta amyloid plaques by 12 and 6
months of age, respectively.
Two different methods were used to ascertain the pa-
thology selectivity of Tau 1. Firstly, using hippocampal
brain sections of 15-month-old mice, we identified a clear
co-localization of Tau 1 with the hysphorylated tau pro-
tein specific antibody AT180 upon tissue staining (Figure
S42). The pyramidal neurons of the hippocampus demon-
strated fluorescence originating from the somatodendritic
compartments, both in the AT180 and Tau 1 fluorescent
windows, mirroring the findings of the primary literature
of the 3xTg model.43
The Tau 1 fluorescence dependence on the tau protein
burden in the OA-treated mouse model was further vali-
dated, as 10-week old OA-treated mice demonstrated only
very faint fluorescence of Tau 1 (Figure S40), in accord-
ance with the reported age-dependent reduction of the
PP2A (protein phosphatase 2A) expression levels,40 with
PP2A being the most important phosphatase maintaining
tau protein in its non-phosphorylated form,41 as well as
being a prime target for OA inhibition.42 Thus the effect
of OA treatment on tau hyperphosphorylation would be
expected to be more severe in aged mice.
Beta amyloid plaques in these brain sections were identi-
fied with the pan-Aβ fluorescent antibody 6e10 and co-
staining with Tau 1 demonstrated a very weak signal from
the plaque’s core only (Figure S43). Comparing the co-
localization in tau tangle rich sections and the Aβ plaques
demonstrates the high selectivity of Tau 1 for tau pathol-
ogy over Aβ aggregates (Figure 7), confirming the results
obtained in the solution and cell based assays (Figures 3
and 4).
Blood-brain barrier penetrability of Tau 1. In order to
assess the BBB penetrability of Tau 1 in mice, the dye was
injected via the tail vein and the animals were sacrificed
after one hour. The brain was cryosectioned and imaging
of Tau 1 demonstrated fluorescence originating from the
pyramidal neurons in the hippocampus (Figure S41).
Whereas a mouse injected with the vehicle only failed to
show any fluorescence. These results clearly indicate the
potential of Tau 1 to cross the blood-brain barrier.
Secondly, a 3xTg mouse was intracranially injected with
Tau 1, to assess the in vivo distribution of the dye. After
the mouse was sacrificed the hippocampal regions of the
brain were co-stained with antibodies for hyperphosphor-
ylated tau protein (AT180), Aβ plaques (sig 39200) and
APP (mab348). As can be seen from Figures 8 and S44,
Tau 1 clearly co-localizes with tau pathology but not with
Aβ pathology under these conditions as well.
Tau 1 shows selectivity of Tau aggregates over Aβ
plaques in a transgenic mouse model of AD. Tau tan-
gle selectivity was assessed using the 3xTg mouse model
of AD.43 This triple transgenic model expresses both the
Swedish (KM670/671NL) mutation of the human amyloid
In vivo imaging of Tau 1 in a transgenic mouse mod-
el of tauopathy. As NIR emissive dyes allow for deep
tissue penetration, we finally determined the in vivo imag-
ing of Tau 1 fluorescence in the Tau P301L mouse model
as well as a wild-type mouse model. The P301L mouse
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Figure 9. In vivo imaging of brains of Tau P301L mice or
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wild-type mice, 30 minutes subsequent to tail vein injection
of Tau 1 or vehicle. The fluorescence images were collected
using excitation at 670 nm and the emission was monitored
at 699 nm.
fragment of the R3 repeat of tau protein provide a poten-
tial binding mode of the dyes on the self-assembled tau
protein, and could provide extra guidance in the design of
novel tau binding molecules for imaging purposes. The
probe is accessible via a short synthetic route and was
found to exhibit excellent photophysical properties such
as a large Stokes’ shift as well as low cytotoxicity allowing
in vitro and ex vivo imaging of hyperphosphorylated tau
protein filaments with minimal background noise. The
probes were clearly able to efficiently penetrate living
cells to visualize the intracellular tau protein deposits.
Tau 1 also demonstrated efficient BBB penetration and
displayed a high selectivity for tau tangles over Aβ depos-
its in transgenic mice models exhibiting both types of
protein aggregates. Finally, Tau 1 demonstrated the
recognition of tau tangles in a transgenic mouse model in
vivo, thus clearly showing the great potential of this dye
for the discrimination of tau tangles in living organisms.
Compared to a commercially available phosphorylated
tau antibody, the probe performed exceptionally well,
demonstrating a near perfect spatial overlap. Given the
high monetary and time cost of synthesizing fluorescently
labeled antibodies, the current fluorophore could repre-
sent a significant advantage over these antibodies for
histological experiments. Furthermore, due to the effi-
cient 18F/19F exchange at the boron center of BODIPY
dyes, the current dye could also find applications as a
dual NIR fluorescent and positron emission tomography
imaging agent.
Figure 8. Confocal imaging of hippocampal regions of 3xTg
mice intracranially injected with Tau 1. First row: Control.
Second row: dual staining with the hyperphosphorylated tau
protein antibody (AT180) and Tau 1 reveals a very high de-
gree of overlap. Third row: dual staining with an Aβ antibody
(sig 39200) and Tau 1 reveals virtually no overlap. Bottom
row: dual staining with an APP antibody (mab348) and Tau 1
reveals virtually no overlap. The fluorescence image of Tau 1
was collected using excitation at 633 nm with a 650 nm
longpass filter and that of the antibodies was obtained using
excitation at 488 nm with a 505-550 nm bandpass filter.
model specifically overexpresses the 4R2N (Tau441) iso-
form of the human tau protein, with the aggregation-
prone P301L mutation.44 As can be seen in Figure 9, 30
minutes subsequent to tail vein injection, a significantly
increased fluorescence intensity could be observed in the
transgenic mouse model, as compared to the wild type of
negative control samples, clearly demonstrating the abil-
ity of Tau 1 to report the presence of tau tangles in live
mice.
■ EXPERIMENTAL SECTION
Materials. All reactants and solvents were obtained from
commercial suppliers (Sigma-Aldrich, Alfa, Samchun) and
were used without further purification. THF was distilled
over Na/benzophenone. Aβ1-42 and AcPHF6 peptides were
purchased from GenicBio Limited (Shanghai, China) and
non-tagged 4R2N Tau441 was obtained through the MRC PPU
Reagents and Services facility (MRC PPU, College of Life
Sciences, University of Dundee, Scotland, mrcppurea-
gents.dundee.ac.uk). NMR spectra were obtained on a 300 or
400 MHz Varian NMR spectrometer. UV/Vis spectra were
recorded on a Scinco S-3100 spectrometer and fluorescence
spectra were obtained using a Shimadzu RF-5301PC spectro-
fluorophotometer. Mass spectroscopy (ESI-MS) was per-
■ CONCLUSIONS
Despite the lack of a clear pharmacophore, we hereby
demonstrate the feasibility of a straightforward structural
modification affording highly sensitive and selective
probes for the molecular surface formed by self-
assembled microtubule associated tau protein. As such,
Tau 1 and Tau 2 clearly adhere to a hypothesis related to
the push-pull chromophore’s conjugation distance on
protein aggregate specificity, as formulated for an entirely
different class of fluorophores. The incorporation of an
extra lipophilic surface via ring fusion was not found to
play a significant role. The docking studies on the PHF6
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Journal of the American Chemical Society
formed on a Shimadzu LCMS-2020 mass spectrometer sys-
longpass (>650 nm) emission filter. Other information is
1
tem.
available in the Figure captions.
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Spectroscopy in the presence of proteins. For spectro-
scopic measurements in the presence of proteins stock solu-
tions of ThT, Tau 1, Tau 2, and BAP-1 were prepared in
DMSO. Both excitation and emission slit widths were 10 nm
unless noted otherwise.
Tau aggregation in vitro. Protein aggregation was per-
formed in a 25 mM Tris buffer at pH 7, supplemented with 50
mM NaCl and 1 mM DTT Buffer, at 37 °C for 2hr to reduce
any covalent tau dimers to monomers. The aggregation reac-
tion was induced by heparin (2.5 μM).
6
Quantum yields and determination of emission and
fluorescence wavelength maxima. Data were recorded in
HPLC grade solvents, with the absorbance lower than 0.1 at
wavelengths longer or equal to the excitation wavelength to
prevent the inner filter effect. The slit settings were 3 nm for
both the excitation and the emission slits. The quantum
yields of BAP-1 and Tau 1-2 were recorded versus Rhodamine
B in 95% EtOH (ΦFl = 0.70),45 using correction factors as
previously described.46
Photostability. Solutions of Rose Bengal, BAP-1 and Tau 1-
2 with an absorbance of 1.0 at the peak absorbance wave-
length were prepared in DMF. The solutions were irradiated
with the focused light of a 3300K halogen lamp using a Zeiss
KL1500 LCD apparatus and the absorbance was determined
in 5 minute intervals for 40 minutes. The photo-bleaching
slopes of the BODIPY dyes were corrected for the spectral
radiance of the lamp at the peak absorption wavelength
relative to the spectral radiance of the lamp at the peak ab-
sorption wavelength of Rose Bengal.
Beta Amyloid aggregation. Beta Amyloid peptide (1-42) in
the lyophilized form was dissolved at a concentration of 1.0
mg/ml in 100% HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) and
incubated at RT for 1 h. The solution was sonicated for 10 min
and the solvent was removed by speed vac. The peptide was
resuspended in DMSO as stock solution. Aliquots of 0.5 mM
of this stock solution were diluted to 50 μM with 10 mM
phosphate buffer (pH 7.4), containing 50 mM NaCl, 1.6 mM
KCl, 2 mM MgCl2 and 3.5 mM CaCl2, prior to each experi-
ment.
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AcPHF6 aggregation inhibition assay. The assay was car-
ried out analogous to literature procedures for 96 well
plates25,61 in a 10 mm fluorescent cuvette. Data are represent-
ed as the average of three measurements. Stock solutions
used were as follows: (A) PBS (50 mM, pH 7.4), (B) 100 µM
BAP-1 or Tau 1in water with 0.5% DMSO, (C) 100 µM ThT in
50 mM PBS (50 mM, pH 7.4), (D) 1 mM AcPHF6 in pure H2O.
Each sample contained 1400 µL of solution A, 200 µL of solu-
tion B, 200 µL of solution C and 200 µL of solution D for a
total solution of 2000 µL, controls were carried out as well.
Solution D was added just prior to the start of the experi-
ment. The fluorescence was measured for 30 minutes with
data points each 0.16s with maximal stirring. Excitation and
emission setting were 440/480 and 600/660 with slit width
3/3 and 10/10 to monitor the fluorescence of ThT or the NIR
dyes, respectively. The data were corrected for the peptide
precipitating from the solution, using an exponential decay
correction after the initial rise in fluorescence intensity.
MTT assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was used to quantify cell
viability. SH-SY5Y, SKNMC, U87, C6 and N2A cells 2×105/mL
as well as human primary brain neurons (cat. 8800-10f, Cell
Applications, San Diego, CA, USA) were treated with various
concentrations of Tau 1 and Tau 2 in a 96-well plate for 48 h
at 37°C. Then, an MTT solution of 5 mg/mL in serum free
media was added to each well and cells were incubated for 1
h. Resulting formazan crystals were solubilized upon the
addition of DMSO and the relative concentrations of forma-
zan were determined by absorbance spectroscopy using a
multi-well plate reader at 570 nm (n=3).
DFT Calculations. Theoretical calculations using the densi-
ty functional theory were performed using the B3LYP47-50
functional at the 6-311++G** level of theory.51-53 Calculations
were carried out with the commercial Gaussian G09W soft-
ware package.54 Molecular orbitals were visualized using the
Gabedit 2.4.8 software package.55 Results are summarized in
Table S1.
Docking studies. The optimized structures of Tau 1, Tau 2,
BAP-1, 3h and PBB5 as well as the lead compounds in Table
S2 were (re)calculated using the 6-31G* level of theory and
were used as the input for the ligands, with the central boron
atom of the BODIPY dyes replaced by a carbon, as parame-
ters for the boron atom are not included in the docking soft-
ware, as previously described for other BODIPY based
probes.56 For the docking studies on the isolated 6-mer
PHF6, the molecular structure was constructed using the
Avogadro 1.2.0 software package,57 based on a X-ray crystal
structure (PDB ID: 5K7N),30 and the tunnel formed by 4
adjacent beta sheets forming a steric zipper along the fibril
axis were constructed as a 5/6-mer from the same crystal
structure similarly. The input files for the calculations were
generated with the AutoDockTools 1.5.6 software package58
and docking calculations were carried out using AutoDock
Vina.59 Visualization of the calculation results was performed
using the Python Molecule Viewer 1.5.6 software package.60
Cell culture and fluorescent imaging. SH-SY5Y cells (hu-
man neuroblastoma) were grown in Dulbecco’s Modified
Eagle’s Medium (DMEM), with 10% FBS (Gipco), penicillin
(100 units/mL), and streptomycin (100 µg/mL). One day
before imaging, the cells were placed on glass-bottomed
dishes (SPL) which were incubated in a humidified atmos-
phere containing 5% (v/v) CO2 at 37 °C. Cell images were
obtained using a confocal microscope (Zeiss model LSM 510).
All fluorescence images were obtained using an excitation
wavelength of 458 nm (ThT), 633 nm (Tau 1, Tau 2 and BAP-
1) and a band pass emission filter at 475-525 nm (ThT), and a
Acute animal model. 23 month-old or 10-week-old female
wild-type C57BL/6 mice were anaesthetized with isoflurane
vaporized in oxygen and 130 nL of 100 μM OA (Sigma Al-
drich) solubilized in DMSO or DMSO alone was injected
unilaterally to the lateral amygdala (n=3). At 24 h after the
injection, 130 nL of 100 μM probe was applied at the injection
site. Longitudinal sections containing the hippocampal CA1
and CA3 regions of interest were subjected to immuno-
histochemical labeling for the tau phospho-epitope AT180
(pTh231: Thermo Fisher). Prior to processing by paraffin
embedding, brains were dissected into forebrain, hindbrain
and cerebellum, as previously described. 34,62
BBB penetration. ICR mice (male, 12-week-old) were used
for the determination of the BBB permeability by using a
modification of the in vivo mouse brain perfusion
technique.63-65 A solution of Tau 1 (500 μM) in 20% DMSO
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and 80% propylene glycol (100 μL) of was injected
anti-actin antibodies at 4°C. And the bands were visualized
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intravenously via the tail vein. After 1h, the mice were
sacrificed, and the brains were removed from the skull and
dissected on ice. The frozen brain was sliced into serial
sections of 20 μm thickness. The fluorescence images of Tau
1 were obtained using excitation at 633 nm with a 650 nm
by super signal west femto (Thermo Fisher). Intracellular
aggregation of Aβ1-42: cell extracts of cells incubated with
50µg/mL Aβ1-42 for 48 h, as well as a control, were run on a
Tris-glycine gel (4-20%) as previously described in the litera-
ture.35
Synthesis of 1. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene
(1) was obtained using a reported procedure in 85% yield.
Analytical data were in accordance with reported data in the
literature.66
longpass filter using
(LSM700, Carl Zeiss).
a
confocal microscopy apparatus
Determination of tau tangle selectivity of Tau 1. Tissue
staining: 15-month-old female 3xTg mice43 were sacrificed,
and the brain was immediately removed and fixed for 24h in
4% formaldehyde. The brain was processed for paraffin
embedding and sectioned at a thickness of 5 μm. The serial
sections were dual stained with the hyper-phosphorylated
tau protein antibody (AT180, Thermo Fisher) and Tau 1, and
the amyloid beta antibody (6e10, Biolegend) and Tau 1,
respectively. The fluorescence image of Tau 1 was collected
using excitation at 633 nm with a 650 nm longpass filter with
a confocal microscope (LSM700, Carl Zeiss). The images of
AT-180 and 6e10 were obtained using excitation at 488 nm
with a 505-550 nm bandpass filter. Intracranial injection of
Tau 1: analogous to the acute mouse model in a 17-month-
old female 3xTg mice.43 Longitudinal sections were stained
with AT180 (Thermo Fisher), sig 39200 (BioLegend) mab348
(EMD Millipore). The fluorescence image of Tau 1 was col-
lected using excitation at 633 nm with a 650 nm longpass
filter and that of AT180 was obtained using excitation at 488
nm with a 505-550 nm bandpass filter.
9
Synthesis of 4. 6-(Dimethylamino)-2-naphthaldehyde (4)
was synthesized by a Bucherer-reaction according to a modi-
fied literature procedure.67 In a 48 mL heavy walled glass
pressure vessel (CAUTION: explosion risk, use protective
equipment) 1.4 g (7.36 mmol) sodium metabisulfite and 800
mg (3.58 mmol) 6-bromo-2-naphthol was suspended in 8 mL
water. To this stirred suspension, 2 mL 40 wt% aqueous
dimethylamine (39 mmol) was added, and the reaction vessel
was sealed and heated at 120°C for 60h. After the pressure
vessel cooled down to room temperature, the reaction mix-
ture was filtered. The solids were washed with water (3 × 20
mL) and the solids were dissolved in DCM. The organic layer
was washed with water, and dried over Na2SO4. Column
chromatography (silica, EtOAc/Hexane 8/1) yielded 505 mg
(2.02 mmol, 56%) (6-bromo-2-naphthyl)dimethylamine as a
white solid. Analytical data were in accordance with reported
data in the literature.67 To a solution of 900 mg (3.60 mmol)
6-bromo-2-naphthyl)amine in 9 mL anhydrous THF, 2.7 mL
(a 1.6M solution) of n-BuLi in hexanes (4.32 mmol) were
added at -78°C under inert atmosphere. The solution was
stirred for 30 min, after which 2.7 mL anhydrous DMF was
added dropwise. The solution was allowed to attain 0°C and
is stirred at that temperature for another 2h, after which 15
mL of a saturated NH4Cl solution in water was added. The
resulting mixture was extracted with Et2O (3 × 50 mL) and
the organic layers were combined and dried over Na2SO4.
Column chromatography (silica, EtOAc/Hexane 1/6) yielded
511 mg (2.56 mmol, 71%) of 6-(dimethylamino)-2-
naphthaldehyde (4). Analytical data were in accordance with
those reported in the literature.67
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In vivo imaging. Tau P301L mice (female, 12-month-old)44 or
ICR mice (female, 12-week-old) were injected with a solution
of Tau 1 (1 mM) in 30% DMSO/30% EtOH (50 μL) or 30%
DMSO/30% EtOH (50 μL) alone via the tail vein. The mice
were anesthetized with Zoletil 50 (Virbac, New Zealand) and
in vivo fluorescence images of Tau 1 and the negative control
were collected using excitation at 670 nm and emission at
699 nm on an Optix MX3 Optical Molecular Imaging System
(ART Advanced Research Technologies Inc., Canada).
Stereotaxic surgery. For histological sampling, mice were
anaesthetized with a lethal dose of pentobarbitone followed
by transcardial perfusion with 30 ml PBS, and subsequently
30 ml 4 % paraformaldehyde. Post-fixing of the brains, re-
moved from the skull, was carried out overnight at 4 °C.
Immunocytochemistry. Serial sections were collected on
gelatin-coated glass slides and air-dried in the dark. Without
staining, glass coverslips were applied atop a fade retardant
solution and mounted with cover slips for confocal observa-
tion. The brain sections were observed using a Carl Zeiss
LSM 510 META laser-scanning microscope. The brain sec-
tions were fixed with 100% ethanol for 20 min and immuno-
stained against the phospho-tau selective marker AT 180 or
the Aβ1-42 selective marker ab10148 (Abcam).
Western Blot. Tau protein phosphorylation after okadaic
acid treatment: The SH-SY5Y cells were washed with PBS and
subsequently homogenized through sonication in 50 mM
Tris–HCl (pH 7.4), containing 150 mM NaCl, 0.5% Triton X-
100, 1 mM EDTA, a protease inhibitor cocktail, and a phos-
phatase inhibitor cocktail (Sigma-Aldrich) for 30 min at 0°C.
The homogenate was then centrifuged at 10,000 rpm for 15
min, resulting in a clear supernatant that was transferred
into a new tube. After SDS-PAGE, the proteins were trans-
ferred onto a polyvinylidene difluoride membrane (Bio-Rad).
The membrane was treated with in 3% BSA for overnight
incubated with anti-tau (sc390476), anti-p-tau (sc101815), or
Synthesis of BAP-1. BAP-1 was synthesized via
a
Knoevenagel condensation following a literature procedure
in a 50% yield.18 Analytical data were in accordance with
those reported in the literature.18
Synthesis of Tau 1. A solution of 100 mg (0.45 mmol) 1, 84
mg (0.45 mmol) 4-(dimethylamino)cinnamaldehyde (3), 0.35
mL acetic acid and 0.35 mL piperidine in 10 mL toluene was
heated under dean-stark conditions for 2h. The reaction was
allowed to cool to room temperature and 50 mL of a saturat-
ed aqueous NH4Cl solution was added. The mixture was
extracted by EtOAc (3 × 50 mL) and the organic layers were
combined and dried over Na2SO4. Column chromatography
→
(silica, EtOAc/Hexane 1/20 1/5) resulted in 22 mg (0.06
mmol, 13%) Tau 1. 1H NMR (300 MHz, CDCl3): δ 7.61 (s, 1H),
7.39 (d, J = 8.5 Hz, 2H), 7.29 − 7.16 (m, 1H), 7.13 − 6.99 (m,
2H), 6.99 − 6.78 (m, 3H), 6.75 − 6.62 (m, 3H), 6.45 − 6.36 (m,
1H), 3.03 (s, 6H), 2.28 (s, 3H) ppm. 13C NMR (100 MHz,
CDCl3): δ 159.82, 151.22, 144.43, 142.92, 140.62, 138.75, 137.34,
133.12, 129.17, 124.74, 124.69, 124.41, 121.17, 119.73, 117.87, 115.85,
+
112.33, 40.44, 11.68 ppm. MS(ESI): C22H22BF2N3 [M]• , m/z
Calculated: 377.19, Found: 377.35.
Synthesis of Tau 2. A solution of 120 mg (0.55 mmol) 1, 164
mg (0.83 mmol) 4, 0.35 mL acetic acid and 0.35 mL piperi-
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(6) Morris, M.; Maeda, S.; Vossel, K.; Mucke, L. Neuron 2011,
dine in 10 mL toluene was heated under dean-stark condi-
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4
5
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70, 410-426.
tions for 2h. The reaction was allowed to cool to room tem-
perature and 50 mL saturated aqueous NH4Cl solution was
added. The mixture was extracted by EtOAc (3 × 50 mL), the
organic layers were combined and dried over Na2SO4. Col-
(7) del Carmen Cárdenas-Aguayo, M.; Gómez-Virgilio, L.;
DeRosa, S.; Meraz-Ríos, M. A. ACS Chem. Neurosci. 2014, 5, 1178-
1191.
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→
umn chromatography (silica, EtOAc/Hexane 1/15
1/10)
1
resulted in 70 mg (0.17 mmol, 32%) Tau 2. H NMR (300
MHz, CDCl3): δ 7.81 (s, 1H), 7.74 − 7.59 (m, 5H), 7.54 − 7.45
(m, 1H), 7.13 (dd, J = 9.2 Hz, J = 2.4 Hz, 1H) 7.10 (s, 1H), 6.90 −
6.84 (m, 2H), 6.79 (s, 1H), 6.47 − 6.41 (m, 1H), 3.10 (s, 6H), 2.31
(s, 3H) ppm. 13C NMR (100 MHz, CDCl3): δ 159.98, 149.73,
144.71, 141.76, 138.46, 137.78, 136.37, 133.12, 130.01, 130.00,
129.70, 127.05, 126.38, 124.93, 124.66,121.97, 117.65, 116.47,
116.45, 116.07, 106.17, 40.76, 11.72 ppm. MS(ESI): C24H22BF2N3
[M+H]+, m/z Calculated: 402.19, Found: 402.35; [2M+H]+ m/z
Calculated: 803.38, Found: 803.50.
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■ ASSOCIATED CONTENT
1
13
Supporting Information. H and C NMR and ESI-MS data
of new compounds, absorption and emission spectra, DFT
calculation details, supporting solution experiments, probe
toxicity, Western blotting of OA treated cells and supporting
tissue staining results. This material is available free of
charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
* shinarago@khu.ac.kr (J-WS)
* jhkwak@skku.edu (JHK)
* kangch@khu.ac.kr (CK)
* jongskim@korea.ac.kr (JSK)
Author Contributions
‡ These authors contributed equally.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by the Ministry of Science, ICT &
Future Planning (MSIP) of National Research Foundation of
Korea (No. 2009-0081566, JSK), the Korea Research Fellow-
ship Program funded by the Ministry of Science, ICT and
Future Planning through the National Research Foundation
of Korea (2016H1D3A1938052, PV), a Korean Health Technol-
ogy R&D project (HI16C1010, CK), funded by the Korean
Ministry of Health &Welfare, and a healthy aging project
grant (20151022, J-WS) from the Korea Health Industry De-
velopment Institute, Republic of Korea.
(28) Landau, M.; Sawaya, M. R., Faull, K. F.; Laganowsky, A.;
Jiang, L.; Sievers, S. A.; Liu, J.; Barrio, J. R.; Eisenberg, D. Plos Biol.
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Scheme 1. Synthetic approach towards BAP-1 and Tau 1-2. The structural modifications relative to BAP-1
are marked in blue. The N - BODIPY C8 (meso) distance, as determined from the optimized structure by DFT
is shown in magenta (See SI for DFT calculation details).
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Figure 1. Calculated frontier orbitals of Tau 1 and Tau 2 by density functional theory using B3LYP at the 6ꢀ
311++G** level of theory. (aꢀb) HOMO and LUMO orbital of Tau 1. (cꢀd) HOMO and LUMO orbital of Tau 2.
Electron densities are displayed at the 0.03 e∙bohrꢀ3 isodensity surface.
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Figure 2. Molecular docking of Tau 1 (A-C) and Tau 2 (D-F). A and D) Top view of the molecule docked in
the tunnel formed by 4 adjacent stacked beta sheets, protein molecular surface depicted. B and E) zoomed
top view of the tunnel, side chains comprising the tunnel are depicted as sticks, others as lines. C and F)
Side view of the molecule in the in the protein tunnel, side chains comprising the tunnel are depicted as
sticks, others as lines. The yellow arrow indicates the viewing direction for panels C and F
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Figure 3. Time dependent fluorescence enhancement of Tau 1ꢀ2 (10 µM) in the presence of protein
aggregates. (a) Emission spectra of Tau 1 in the presence of tau protein (10 µM) and heparin (2.5 µM),
excited at 590 nm, (b) Emission spectra of Tau 1 in the presence of tau protein (10 µM) and heparin (2.5
µM), excited at 580 nm, (CꢀD) Emission spectra of Tau 1ꢀ2 in the presꢀence of Aβ fibrils (50 µM).
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Figure 4. (a) Fluorescence images of SHꢀSY5Y cells incubated with Tau 1, Tau 2, BAPꢀ1 and ThT (500 nM)
for 20 min at 37°C. Top: Untreated cells, Bottom: preꢀincubated with okadaic acid (100 nM) for 6hr. ThT
was excited at 458 nm and the emission was recorded using a 475ꢀ525 nm band pass filter. Tau 1ꢀ2 and
BAPꢀ1 were excited at 633 nm and the fluorescence was collected at wavelengths higher than 650 nm. (b)
Florescence intensity per cell relative to untreated cells (n=5), as determined using image J.38 Error bars
designate standard deviation. (c) Fluorescence images of SHꢀSY5Y cells using amyloid beta peptide with Tau
1, Tau 2, BAPꢀ1 and ThT. After treatment of amyloid beta peptide (50 ꢁg/mL) for 48 hr, the probes (1 µM)
were incubation for 15 min at 37°C (bottom). Untreated cells were incubated with the probes for 15 min
without peptide (top). ThT was excited at 458 nm and the emission was recorded using a 475ꢀ525 nm band
pass filter, Tau 1ꢀ2 and BAPꢀ1 were excited at 633 nm and the fluorescence was collected at wavelengths
higher than 650 nm. (d) Fluorescence intensity per cell relative to untreated cells (n=5), as determined
using image J.38 Error bars designate standard deviation.
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Figure 5. Confocal imaging of the CA1 (Top) and CA3 (Bottom) hippocampal regions of OA treated mice.
Dual staining with the hyperphosphorylated tau protein antibody (AT-180) and Tau 1 reveals a very high
degree of overlap. The fluorescence image of Tau 1 was collected using excitation at 633 obtained using
excitation at 488 nm with a 505-550 nm bandpass filter.
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Figure 6. Confocal imaging of a single hippocampal neuron of an OA treated mouse. Dual staining with the
hyperphosphorylated tau protein antibody AT-180 and Tau 1 demonstrates significant amounts of tau
missorting to the somatodendritic compartment (*) of the neuron, as well as sporadic hyperphosphorylated
tau deposits along the axon (arrow). The fluorescence image of Tau 1 was collected using excitation at 633
nm with a 650 nm longpass filter and that of AT180 was obtained using excitation at 488 nm with a 505-550
nm bandpass filter.
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Figure 7. Merged confocal imaging of hippocampal regions of 3xTg mice. Left: Dual staining with the
hyperphosphorylated tau protein antibody AT180 and Tau 1; Right: Dual staining with the amyloid beta
antibody 6e10 and Tau 1. The fluorescent image of Tau 1 was obtained using excitation at 633 nm with a
650 nm longpass filter and AT180 and 6e10 were obtained using excitation at 488 nm with a 505-550 nm
bandpass filter. Cell nuclei were stained with DAPI. See Figures S42 and S43 for images of the individual
channels.
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Journal of the American Chemical Society
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Figure 8. Confocal imaging of hippocampal regions of 3xTg mice intracranially injected with Tau 1. First row:
Control. Second row: dual staining with the hyperphosphorylated tau protein antibody (AT180) and Tau 1
reveals a very high de-gree of overlap. Third row: dual staining with an Aβ anti-body (sig 39200) and Tau 1
reveals virtually no overlap. Bottom row: dual staining with an APP antibody (mab348) and Tau 1 reveals
virtually no overlap. The fluorescence image of Tau 1 was collected using excitation at 633 nm with a 650
nm longpass filter and that of the antibodies was ob-tained using excitation at 488 nm with a 505-550 nm
band-pass filter.
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Journal of the American Chemical Society
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Figure 9. In vivo imaging of brains of Tau P301L mice or wild-type mice, 30 minutes subsequent to tail vein
injection of Tau 1 or vehicle. The fluorescence images were collected using excitation at 670 nm and the
emission was monitored at 699 nm.
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