A Difluoroboron β-Diketonate Probe Shows turn-on Near-Infrared Fluorescence Specific for Tau Fibrils

Article abstract of DOI:10.1021/acschemneuro.7b00224

Tau aggregation in neuronal cells has recently received significant attention as a robust predictor of the progression of Alzheimer's disease (AD) because of its proven correlation with the degree of cognitive impairment in AD patients. Accordingly, noninvasive imaging of tau aggregates has been highlighted as a promising diagnostic tool for AD. We have previously identified a tau-specific turn-on near-infrared fluorescent (NIRF) probe (1), and, in this study, structural modification was performed to optimize its physicochemical as well as fluorescence properties. Thus, a series of fluorescent dyes (2a-2j) composed of a variously substituted difluoroboron β-diketonate and an N,N-dimethylaniline moiety linked by a length-extendable π-bridge were prepared. Among those, isobutyl-substituted difluoroboron β-ketonate with a π-conjugated 1,4-butadienyl linker (2e) showed the most promising properties as a tau-specific NIRF probe. Compared with 1, the turn-on fluorescence of 2e was more specific to tau fibrils, and it showed 8.8- and 6.2-times higher tau-over-Aβ and tau-over-BSA specificity, respectively. Also, the fluorescence intensity of 2e upon binding to tau fibrils was substantially higher (～2.9 times) than that observed from 1. The mechanism for tau-specificity of 2e was investigated, which suggested that the molecular rotor-like property of 2e enables specific recognition of the microenvironment of tau aggregates to emit strong fluorescence. In transgenic cell lines stably expressing GFP-tagged tau proteins, 2e showed good colocalization with tau-GFP. Moreover, the fluorescence from 2e exhibited almost complete overlap with p-Tau antibody staining in the human AD brain tissue section. Collectively, these observations demonstrate the potential of 2e as a tau-specific fluorescent dye in both in vitro and ex vivo settings.

Full text of DOI:10.1021/acschemneuro.7b00224

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Letter
A Difluoroboron #–Diketonate Probe Shows “Turn–
on” Near–Infrared Fluorescence Specific for Tau Fibrils
Kwang-su Park, Mi Kyoung Kim, Yujin Seo, Taewoong Ha, Kyeongha Yoo, Seung Jae
Hyeon, Yu Jin Hwang, Junghee Lee, Hoon Ryu, Hyunah Choo, and Youhoon Chong
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00224 • Publication Date (Web): 24 Jul 2017
Downloaded from http://pubs.acs.org on July 25, 2017
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A Difluoroboron β–Diketonate Probe Shows “Turn–on” Near–Infrared Fluorescence Specific
for Tau Fibrils
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Kwang–su Park,1,† Mi Kyoung Kim,1,† Yujin Seo,1 Taewoong Ha,1 Kyeongha Yoo,1 Seung Jae Hyeon,2
Yu Jin Hwang,2 Junghee Lee,3,4 Hoon Ryu,2,3,4,* Hyunah Choo,2,5,* and Youhoon Chong1,*
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1Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University,
Hwayang–dong, Gwangjin–gu, Seoul 143–701, Korea; 2Center for Neuro–Medicine, Korea Institute
of Science and Technology, 39–1 Hawolgok–dong, Seoungbuk–gu, Seoul 136–791, Korea; 3Veteran’s
Affairs Boston Healthcare System, Boston, MA, USA; 4Boston University Alzheimer’s Disease Center
and Department of Neurology, Boston University School of Medicine, Boston, MA, USA; 5Department
of Biological Chemistry, Korea University of Science and Technology, Youseong–gu, Daejeon 305–
350, Korea
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† These two authors contributed equally on this work
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*Corresponding authors
Hoon Ryu, Ph.D.; Eꢀmail: hoonryu@bu.edu; Tel: +1ꢀ857ꢀ364ꢀ5910
Hyunah Choo, Ph.D.; E–mail: hchoo@kist.re.kr; Tel: +82–2–958–5157
Youhoon Chong, Ph.D.; E–mail: chongy@konkuk.ac.kr; Tel: +82–2–2049–6100; Fax: +82–2–454–
8217
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ABSTRACT
Tau aggregation in neuronal cells has recently received significant attention as a robust predictor of
the progression of Alzheimer’s disease (AD) because of its proven correlation with the degree of
cognitive impairment in AD patients. Accordingly, non–invasive imaging of tau aggregates has been
highlighted as a promising diagnostic tool for AD. We have previously identified a tau–specific “turn–
on” near–infrared fluorescent (NIRF) probe (1) and, in this study, structural modification was
performed to optimize its physicochemical as well as fluorescence properties. Thus, a series of
fluorescent dyes (2a–2j) composed of a variously substituted difluoroboron β–diketonate and an N,N–
dimethylaniline moiety linked by a length–extendable π–bridge were prepared. Among those,
isobutyl–substituted difluoroboron β–ketonate with a π–conjugated 1,4–butadienyl linker (2e) showed
the most promising properties as a tau–specific NIRF probe. Compared with 1, the “turn–on”
fluorescence of 2e was more specific to tau fibrils, and it showed 8.8– and 6.2–times higher tau–over–
Aβ and tau–over–BSA specificity, respectively. Also, the fluorescence intensity of 2e upon binding to
tau fibrils was substantially higher (~2.9 times) than that observed from 1. The mechanism for tauꢀ
specificity of 2e was investigated, which suggested that the molecular rotor–like property of 2e
enables specific recognition of the microenvironment of tau aggregates to emit strong fluorescence. In
transgenic cell lines stably expressing GFP–tagged tau proteins, 2e showed good co–localization with
tau–GFP. Moreover, the fluorescence from 2e exhibited almost complete overlap with p–Tau antibody
staining in the human AD brain tissue section. Collectively, these observations demonstrate the
potential of 2e as a tauꢀspecific fluorescent dye in both in vitro and ex vivo settings.
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Key words: Alzheimer’s disease, tau–specific probe, fluorescence imaging, molecular rotor
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Alzheimer’s disease (AD), the leading cause of dementia, has been highlighted due to an
enormous socioeconomic burden imposed by the ever–growing number of elderly patients
with dementia.1–2 In the absence of fundamental cure3, delaying the onset or progression of
AD has been proposed as a more practical strategy for the management of this devastating
disease4. Therefore, early diagnosis of AD has become one of the most fundamental precepts
of AD care and, for this purpose, a large body of research has focused on visualization of the
major pathological hallmarks of AD5: amyloid plaques and neurofibrillary tangles, which are
composed of misfolded amyloid–β (Aβ) and hyperphosphorylated tau proteins, respectively.
In particular, a strong correlation between the degree of cognitive impairment in AD patients
and the level of aggregated tau6–8 suggests that detection of tau aggregates would provide
potential benefit to confirmative diagnosis of AD9–11. Several molecular probes targeting tau
fibrils have thus been discovered12–22 and, among those, near–infrared (650–900 nm)
fluorescent (NIRF) probes19–22 have gained attention because, compared to visible fluorescent
dyes, NIRF dyes provide significant advantages offering higher resolution imaging, a greater
imaging depth, and a higher signal–to–background ratio.23
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Recently, we reported a smart NIRF probe (
1
, Figure 1) with “turn–on” fluorescence upon
exhibited selective fluorescence sensing
exhibited fluorescence with an
binding to the target proteins.24 More interestingly,
1
behavior for tau fibrils over Aβ: upon binding to tau fibrils,
1
effective quantum yield (Φ) of 16.5%, which is 6.6 times higher than that observed in binding
to Aβ fibrils (Φ = 2.5%).24 The “turn–on” fluorescence property as well as tau–specificity of
1
were attributed to its molecular rotor–like architecture because fluorescent molecular rotors,
characterized by a fluorophore with a rotatable donor–acceptor bond, tend to emit fluorescence
which is selectively turned on when the intramolecular rotational relaxation about the donor–
acceptor bond is constrained.25 Also, as fluorescence intensity from the molecular rotors is
subject to change depending on their microenvironment,26–27 it was conceived that the
molecular rotor property of
1 enabled its specific recognition of the tau aggregates to result in
tau–specific fluorescence behaviour.
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Nevertheless, unsatisfactory properties associated with 1 such as poor solubility and
stability in aqueous medium, weak fluorescence intensity and suboptimal tau–specificity (1.6
times over Aβ) prevented its further development. In addition, synthetic difficulties in
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preparing the 3,6–dimethoxy–
through structure–activity relationship study. Scaffold change has thus been attempted by
replacing the structural units incorporated in . The molecular rotor like architecture of is
composed of three modules: a donor ( –dimethylaniln–3,6–dimethoxy–4–yl group; dotted
circle, , Fig. 1) and an acceptor (α–cyanoester; dotted box, ) bridged by a π–linker
(hexatriene; bold lines, ). Among those, the acceptor, α–cyanoester, was supposedly
responsible for the unfavourable physicochemical as well as fluorescent properties of
N,N–dimethylanilne functionality hampered optimization of 1
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N,N
1
1
1
1
.
Therefore, alternatives for the α–cyanoester have been sought, and difluoroboron β–diketonate
which is well characterized by intense fluorescence28, solvatochromism29–30 and good aqueous
stability31 was proposed as the optimal candidate. A difluoroboronꢀbased dye, styrylꢀBODIPY,
was recently discovered to have favourable photochemical and physicochemical properties for
monitoring realꢀtime in vitro tau protein fibrillization32, which supports our reasoning that a
difluoroboron β–diketonate dye could be exploited for specific detection of tau fibrils. On the
other hand, for facile synthesis and structure–activity relationship study, the donor part of
1
–
–
was changed into an unsubstituted
N
,
N
–dimethylanilne instead of the 3,6–dimethoxy–
N
N
,
N
N
dimethylanilne functionality. The two modules, a difluoroboron β–diketonate and an
,
dimethylanilne moiety, were then combined by using a π–linker to provide a new probe
scaffold (Figure 1). The newly designed difluoroboron β–diketonate , however, resembles
2
2
CRANAD–233 (Figure 1), a well–known Aβ–specific NIRF probe. Moreover, difluoroboron
β–diketonate dyes have not been utilized for detection of tau fibrils. Notwithstanding these
issues, it is worth to note that the mechanism for Aβ–specificity of CRANAD–2 is unexplored
and, in particular, contributions of the symmetric nature as well as the length of the π–linker of
CRANAD–2 for specific staining of Aβ fibrils are elusive. In this context, a potential role of
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the π–linker and the α–cyanoacetate functionality in conferring tau–specificity to the resulting
difluoroboron β–diketonate dye was assumed, which led to the installation of a length–
extendable π–linker (n=1–2, Figure 1) as well as various alkyl substituents (R, Figure 1) to the
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newly designed probe scaffold 2.
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Figure 1. Structure of the title compound 2 in comparison with 1 and CRANAD–2. Dotted circles,
dotted boxes and bold lines denote donors, acceptors and π–bridges, respectively.
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Preparation of the title compound is outlined in Scheme 1. Conjugated aldehydes (
obtained from 4– –dimethylaminobenzaldehyde through consecutive Wittig reactions with
(1,3–dioxolan–2–ylmethyl)triphenylphosphonium
commercially available acetylacetones ( ) to provide the title compounds (2a
3–4),
N,N
bromide,
were
condensed
with
5
–
9
–
2j) (Scheme
1). This aldol–type reaction has been widely applied for the condensation of aromatic
aldehydes with acetylacetones, but in our hands, the reaction proceeded in disappointingly low
yields. After surveying a variety of reaction conditions, we found out that the reaction
proceeds in fair to moderated yields (55%–77%) within a sealed tube.34
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Scheme 1. Synthesis of the title compounds (2a–2j)
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The difluoroboron β–ketonates (2a–2j) thus obtained were dissolved in phosphate–
buffered saline (PBS) (50 ꢁM) and fluorescence from these compounds was observed before
and after mixing with pre–aggregated tau, Aβ fibrils or bovine serum albumin (BSA) (Figure
S1; Supporting Information)24. In the absence of the aggregated peptides, none of the title
probes showed fluorescence emission (brown lines, Figure S1; Supporting Information).
Interestingly, upon exposure to the target peptides, only a limited number of the probes
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exhibited substantial fluorescence intensity. In particular, the tau
fluorescence was observed from the probes with a 1,4–butadienyl π–bridge (n=1) and an
aliphatic substituent at R position (2a 2d and 2e). On the other hand, fluorescence from the
probes with a longer π–linker (n=2, 2f 2j) or with a conformationally rigid aromatic R
–specific “turn–on”
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,
–
substituent (2b and 2c) was very weak regardless of the type of the aggregated peptides. Under
the same conditions with pre–aggregated tau, no measurable fluorescence signal was detected
from CRANADꢀ2, which indicates that it is not capable of staining tau fibrils (Figure S2;
Supporting Information). Table 1 collectively shows structure–dependent fluorescence
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properties as well as tau–specificities of the title probes. Among the compounds prepared,
isobutyl–substituted difluoroboron β–ketonate with a π–conjugated 1,4–butadienyl linker (2e
)
showed the most promising properties as a tau–specific fluorescent probe; fluorescence
emission at NIR range (λem = 660 nm) with a large Stokes’ shift (110 nm), 313–fold
fluorescence “turnꢀon” response upon binding to tau–fibrils (FItau, Table 1), and significant
specificity for tau (14–fold over Aβ and 7.2–fold over BSA, Table 1).
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Table 1. Fluorescence properties of the synthesized probes (50 ꢁM) in the presence of tau, Aβ fibrils,
and BSA
FId
FIAβ
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SIe
b
c
εa
λex
λem
Cmpd
(M–1cm–1)
(nm)
(nm)
FItau
430
160
120
440
310
87
FIBSA
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28
SIAβ
3.3
7.3
3.0
5.8
14
SIBSA
3.7
5.6
3.9
7.4
7.2
13
2a
2b
2c
2d
2e
2f
35980
68330
52220
59290
53500
17270
34870
23750
46090
12840
30711
560
600
610
550
550
560
610
610
570
570
540
660
690
710
660
660
720
700
680
700
720
650
22
40
76
22
13
31
59
43
7
6.5
g
g
g
g
g
–
–
–
–
–
2g
2h
2i
g
g
g
g
g
–
–
–
–
–
100
79
43
88
67
6
5
2.3
0.9
1.6
17
16
2j
1
106
85
1.2
143
144
b
aMolar extinction coefficient (in dimethylsulfoxide). Maximum excitation wavelength of the probe
c
d
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(in PBS). Maximum emission wavelength of the probe (in PBS). Fold Increase = fluorescence
intensity of the probe bound to tau fibrils (or Aβ fibrils or BSA)/fluorescence intensity of the unbound
e
probe. Selectivity index (SI) = fluorescence intensity of the probe upon binding to tau/fluorescence
intensity of the probe upon binding to Aβ fibrils (or BSA). fNot determined due to low fluorescence.
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In addition to the fluorescence characteristics, physicochemical properties of the title
probes were also determined. The difluoroboron β–diketonate scaffold was anticipated to
increase the water solubility of the resulting probes but, due to the planar structure, most of the
title probes were shown to be only sparingly to moderately soluble in water (Table 2, Figure
S3; Supporting Information). Nevertheless, the isobutyl–substituted difluoroboron β–
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diketonates 2e showed significantly increased aqueous solubility compared to
1 (Table 2,
Figure S3; Supporting Information), which might be attributed to rotation of the methyleneꢀ
linked substituent (R = CH2CH(CH3)2) out of the difluoroboron β–diketonate plane and
thereby decrease in intermolecular forces. Bloodꢀbrain barrier (BBB) permeability is another
important property of brainꢀimaging molecular probes. Several physicochemical properties are
known to be related to the BBB permeability and, among those, logP value has a major effect
on BBB penetration: top 25 CNS drugs are known to have logP values between 2 and 5.35
Thus, the logP values of the title probes were assessed, which were shown to be correlated
with the solubility profile (Table 2): the probes with sufficient solubility (2c and 2e) showed
optimum logP values for BBB penetration while others with low solubility presented less
favourable (2b and 1) or nonꢀmeasurable (2a, 2d, 2f–2j) logP values (Table 2).
Table 2. Physicochemical properties of the synthesized probes
Cmpd
Solubility (ꢁM)a
logPb
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
1
40
40
90
40
>100 <10
<10
<10
<10
<10
40
c
c
c
c
c
c
c
–
4.68 2.43
–
2.19
–
–
–
–
–
4.88
168
169
b
c
aDetermined by UVꢀVis spectroscopy. logP = log([probe]octanol/[probe]water). Not determined due to
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low aqueous solubility.
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173
174
Taken together, the probe 2e exhibited significantly improved fluorescence properties as a
tau–selective NIRF probe compared with the mother compound 1. Neither 2e nor 1 was
fluorescent in the presence of the monomeric tau or Aβ (Figure 2), but the “turn–on”
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fluorescence of 2e was more specific to tau fibrils than
1
: it showed 8.8– and 6.2–times higher
tau–over–Aβ and tau–over–BSA specificity, respectively (Figure 2). Also, the fluorescence
intensity of 2e upon binding to tau fibrils showed substantial increase compared with that of
1
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(~2.9 times) (Figure 2).
(a)
1600
1400
1200
1000
800
600
400
200
0
Buffer only
+ Tau fibrils
+ Tau monomer
+ Aβ fibrils
+ Aβ monomer
+ BSA
550
600
650
700
750
800
850
Wavelength (nm)
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(b)
1600
1400
1200
1000
800
600
400
200
0
Buffer only
+ Tau fibrils
+ Tau monomer
+ Aβ fibrils
+ Aβ monomer
+ BSA
550
600
650
700
750
800
850
Wavelength (nm)
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Figure 2. Fluorescence spectra of 50 ꢁM of (a) 2e and (b) 1 in the absence or presence of tau, Aβ and
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BSA. Both monomeric and fibrillar forms of tau and Aβ were evaluated for fluorescence staining by
the probe molecules.
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The tau–specific fluorescence property of 2e was further investigated. First, a fluorescence titration
assay revealed that fluorescence staining with 2e can detect concentrations of tau aggregates as low as
8–16 ng/mL (Figure S2, Supporting Information), which is sufficient to detect tau protein in its
physiological concentration (26–66 ng/mL)36. For quantitative evaluation of the fluorescence
properties, various concentrations of 2e were incubated with pre–aggregated tau and, at each
concentration, the fluorescence intensity was detected. Plotting fluorescence intensities of 2e as a
function of its concentration on a semi–log scale (●, Figure 3) exhibited an apparent binding constant
(Kd) of 1.41 ꢁM, which is comparable to that of thioflavin–S (ThS, Kd = 1.90 ꢁM)25. The same
experiment was repeated using Aβ fibrils (50 ꢁM) and BSA (10 ꢁM) (▼ and ○, respectively, in Figure
3), but 2e was barely fluorescent in the presence of these polypeptides. Even at high concentrations,
fluorescence intensity of 2e did not show significant enhancement.
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Figure 3. Saturation binding curve for a fluorescence probe 2e. Specific binding of 2e to tau fibrils
β fibrils (▼) and BSA (○).
(●) was compared with its binding to A
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Fluorescence of 2e was also examined in solvents with distinct viscosities.37 A linear increase in
the fluorescence emission intensity of 2e with increasing solvent viscosity was observed (Figure 4),
which demonstrate that 2e behaves as a molecular rotor. Therefore, in the context of the environment–
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sensitivity of molecular rotors, the tau–specific fluorescence of 2e might be explained by the typical
microenvironment of tau aggregates that accommodates 2e in a conformation adequate for
fluorescence.
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Figure 4. Dependence of fluorescence of 2e on solvent viscosity. Fluorescence emission was
measured (λex = 550 nm/λem = 660 nm) in various solvents at 25 °C: (1) H2O, (2) DMSO, (3) glycerol,
(4) ethylene glycol (EG), (5) methanol, (6) EG/glycerol (1:1, v/v).
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The tau–selective “turn–on” fluorescent probe 2e was next evaluated for staining of
intracellular tau proteins. First, in vitro cytotoxic effects of different concentrations of 2e
against human neuroblastoma cells (SHSYꢀ5Y) were assessed in the absence or presence of
light to excite the fluorescent molecule38, but 2e was not cytotoxic at any tested concentration
(Figure S5, Supporting Information). Transgenic SHSY–5Y cells stably expressing GFP–
tagged tau proteins22 were then treated with 2e, and fluorescence emitted by GFP (λem = 420
nm) and 2e (λem = 660 nm) was observed through green (Figure 5A) and red (Figure 5B)
fluorescence, respectively. Notably, the merged image (Figure 5C) showed good co–
localization of tau–GFP and 2e in yellow fluorescence, which demonstrates that 2e is able to
accurately and specifically detect intracellular tau aggregates.
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Figure 5. SHSY–5Y cells expressing GFP–tagged tau proteins were stained with 2e, and
fluorescence emitted from the cells was observed by confocal microscopy. Green and red fluorescence
were observed from (A) GFP (λem = 420 nm) and (B) 2e (λem = 660 nm) respectively. A merged image
(C) shows perfect co–localization of GFP and 2e in yellow fluorescence. White arrows indicate co–
localization foci of tau–GFP with 2e.
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The selective fluorescence labelling of tau aggregates by 2e was finally assessed by ex vivo
staining of human AD brain tissue. Thus, normal (control) and AD human brain sections
(Table S1, Supporting Information) were treated with phosphorylated tau (p–Tau) antibody
(Ser202/Thr205) followed by 2e, and the staining was observed using fluorescence
microscopy (Figure 6A). While only very limited staining by 2e and p–Tau antibody was
observed in control brain sections (upper panel, Figure 6A), AD brains showed red (lower left
panel, Figure 6A) and green (lower middle panel, Figure 6A) fluorescence emitted by 2e and
p–Tau antibody, respectively. One of the most striking features of this study is the almost complete
overlap of the fluorescence from 2e with the p–Tau antibody staining, which is shown in yellow
fluorescence in the merged image (lower right panel, Figure 6A). Quantitative analysis of the
fluorescence images performed by mapping the intensity profiles of the fluorescence from 2e and p–
Tau antibody (Figure 6B) also confirmed their co–localization in the AD brain sections.
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Figure 6. (A) Confocal fluorescence images of human normal (upper panel) and AD (lower panel)
brain sections after treatment with 2e and p–Tau antibody (Ser202/Thr205). Very limited staining by
2e and p–Tau antibody was observed in control brain tissue (upper panel). AD brain sections stained
with 2e and p–Tau antibody are shown in red (lower left panel) and green fluorescence (lower middle
panel), respectively. Co–localization of 2e with p–Tau antibody is shown as yellow fluorescence in the
merged image (lower right panel). Scale bars (white): 50 ꢁm (10 ꢁm in the inset). (B) Magnified view
of fluorescence images (left panels) and the intensity profiles of 2e (red lines, right panels) and p–Tau
antibody (green lines, right panels) in the area indicated by white lines in the left panels.
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The ex vivo staining of human AD brain tissue by 2e was also performed in conjunction
with Aβ antibody (Figure 7). In normal human brain tissue, neither mouse monoclonal Aβ
antibody nor 2e emitted fluorescence due to the lack of Aβ plaque (Figure 7A, upper panel). In
contrast, brain sections from AD patients were fluorescently stained by Aβ antibody and 2e but
with different specificity and efficiency (Figure 7A, lower panel). Thus, while treatment of the
AD brain section with Aβ antibody showed both diffuse and dense core green fluorescence
(Column “Aβ”, lower panel, Figure 7A), the 2eꢀpositive signals were observed only as red
puncta with weaker fluorescence (Column “2e”, lower panel, Figure 7A). Co–localization of
red and green fluorescence was specifically detected in the punctum area (Column “Merge”,
lower panel, Figure 7A), but the puncta immunoreactivity assay (Figure 7B) showed that the
number of 2e and merge signal was significantly lower than the number of Aβ plaques in the AD
patient’s brain. In addition, the line measurement analysis demonstrated that 2e (red, Figure 7C)
is only partially localized together with an Aβ plaque (green, Figure 7C).
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Figure 7. (A) Confocal fluorescence images of human normal (upper panel) and AD (lower panel)
brain sections after treatment with 2e and mouse monoclonal anti–Aβ antibody. No fluorescence
signal by anti–Aβ antibody and 2e staining was observed in control brain tissue (upper panel). AD
brain sections stained with antiꢀAβ antibody and 2e are shown in red (lower left panel) and green
fluorescence (lower middle panel), respectively. Scale bars (white): 100 ꢁm. (B) The puncta
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immunoreactivity of Aβ plaque, 2e, and merge (co–localization of Aβ plaque and 2e) was counted in
AD. The number of 2e and merge signal was significantly lower than the number of Aβ plaque in AD.
Significantly different at **p<0.01 (Student tꢀtest). (C) Line measurement analysis showed that co–
localization of Aβ plaque (green) and 2e (red) is only partial. Scale bars (white): 100 ꢁm.
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In summary, in order to optimize the physicochemical and suboptimal fluorescence properties of the
previously reported tau–specific NIRF probe 1, scaffold change was attempted, which provided novel
fluorescent dyes (2a–2j) composed of a variously substituted difluoroboron β–diketonate and an N,N–
dimethylaniline moiety linked by a length–extendable π–bridge. The newly prepared difluoroboron β–
ketonates (2a–2j) were tested for their fluorescence properties in the absence or presence of pre–
aggregated tau, Aβ fibrils or BSA. Among those, the probes with a 1,4–butadienyl π–bridge (n=1) and
an aliphatic substituent at R position (2a, 2d and 2e) exhibited tau–specific “turn–on” fluorescence
properties in the NIR range. In particular, the isobutyl–substituted difluoroboron β–ketonate with a π–
conjugated 1,4–butadienyl linker (2e) showed the most promising properties as a tau–specific NIRF
probe. Most of all, in comparison with 1, the “turn–on” fluorescence of 2e was more specific to tau
fibrils, and it showed 8.8– and 6.2–times higher tau–over–Aβ and tau–over–BSA specificity,
respectively. Also, the fluorescence intensity of 2e upon binding to tau fibrils showed substantial
increase compared with that of 1 (~2.9 times). The molecular rotor–like property and thereby specific
recognition of the microenvironment of tau aggregates was proposed as the mechanism for tauꢀ
specificity of 2e. On the other hand, in transgenic cell lines stably expressing GFP–tagged tau proteins,
2e showed very good co–localization with tau–GFP. Also, the fluorescence from 2e was almost
completely overlapped with p–Tau antibody staining in the human AD brain tissue section.
Collectively, these observations demonstrate the potential of 2e as a tauꢀspecific fluorescent dye in
both in vitro and ex vivo settings.
METHODS
General procedure for synthesis of difluoroboron β–ketonate derivatives (2a–2j): To a
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solution of the variously substituted acetylacetones (5–9) (0.5 mmol) in DMF (1 mL) was added
BF3‧
Et2O (0.1 mL, 0.75 mmol) in a sealed tube. The resulting mixture was stirred at 65 oC for 30 min.
To this mixture, a solution of the aldehyde (3 or 4) (0.5 mmol) in DMF (1 mL) was added followed by
tributylborate (0.27 mL, 1.0 mmol) and nBuNH2 (0.01 mL, 0.1 mmol). After stirring at 90 oC for 4 h,
the reaction mixture was cooled to rt and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel.
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Synthesis of 6–((1E,3E)–4–(4–(dimethylamino)phenyl)buta–1,3–dien–1–yl)–2,2–difluoro–4–
isobutyl–2H–1,3,2–dioxaborinin–1–ium–2–uide (2e): The desired compound 2e was obtained as a
1
dark violet powder in 77% yield starting from 3; H NMR (400 MHz, CDCl3) δ 7.87 (dd, J = 14.4,
11.5 Hz, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 15.1 Hz, 1H), 6.80 (dd, J = 15.0, 11.5 Hz, 1H),
6.66 (d, J = 8.8 Hz, 2H), 6.05 (d, J = 14.6 Hz, 1H), 5.81 (s, 1H), 3.05 (s, 6H), 2.33 (d, J = 7.1 Hz, 2H),
2.17–2.22 (m, 1H), 0.99 (d, J = 6.6 Hz, 6H); 13C NMR (125 MHz, DMSO–d6) 189.9, 179.9, 152.0,
151.2, 148.6, 130.4, 123.1, 121.9, 119.1, 112.0, 100.5, 45.1, 39.6, 26.7, 22.1; 19F NMR (376 MHz,
DMSO–d6) δ –138.7; FAB–MS calcd for C19H24BF2NO2 [M]+ 347.2, found 347.4.
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ASSOCIATED CONTENT
Supporting Information
The synthetic procedures and characterization of the new compounds, experimental details for the
preparation of aggregated tau, fluorescence–based tau–binding assay, evaluation of the fluorescent
properties of the fluorescence probes upon binding to the tau fibrils, detection of the tau aggregates in
transfected SHSY–5Y cells, and fluorescence p–Tau imaging in human AD brain sections.
AUTHOR INFORMATION
Corresponding Authors
*For H.C.: phone, +82–2–958–5157; E–mail, hchoo@kist.re.kr.
*For Y.C.: phone, +82–2–2049–6100; Fax: +82–2–454–8217, E–mail, chongy@konkuk.ac.kr.
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ACKNOWLEDGMENTS
This study was supported by a grant from the Korean Health Technology R&D project,
Ministry of Health & Welfare, Republic of Korea (HI14C2341). This study was also
supported by NIH Grant (AG054156 to H.R.) and National Research Council of Science &
Technology (NST) grant (CRC–15–04–KIST) by the Korea government (MSIP). Additional
funding was provided by the Korea Institute of Science and Technology (KIST) Institutional
Program (2E26850).
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AUTHOR CONTRIBUTIONS:
K.P. and M.K.K. contributed equally to this work. H.C. and Y.C. designed the current
project. K.P., M.K.K., Y.S., T.H., and K.Y. performed synthesis of the title probes and in vitro
fluorescence assays. S.J.H., Y.J.H., J.L., and H.R. performed ex vivo staining experiments by
using human brain tissue. H.C. and Y.C. wrote the first draft of the manuscript, which all other
authors reviewed. All the authors approved publication.
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Table of Contents Graphic
1600
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Buffer only
w/ Tau
w/ BSA
w/ Abeta
600
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Wavelength (nm)
2e
P-Tau (S202/T205)
Merge
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Figure 1. Structure of the title compound 2 in comparison with 1 and CRANAD–2. Dotted circles, dotted
boxes and bold lines denote donors, acceptors and π–bridges, respectively.
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Scheme 1. Synthesis of the title compounds (2a–2j)
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Figure 2. Fluorescence spectra of 50 ꢀM of (a) 2e and (b) 1 in the absence or presence of tau, Aβ and BSA.
Both monomeric and fibrillar forms of tau and Aβ were evaluated for fluorescence staining by the probe
molecules.
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Figure 3. Saturation binding curve for a fluorescence probe 2e. Specific binding of 2e to tau fibrils (●) was
compared with its binding to Aβ fibrils (▼) and BSA (○).
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Figure 4. Dependence of fluorescence of 2e on solvent viscosity. Fluorescence emission was measured (λex
= 550 nm/λem = 660 nm) in various solvents at 25 °C: (1) H2O, (2) DMSO, (3) glycerol, (4) ethylene glycol
(EG), (5) methanol, (6) EG/glycerol (1:1, v/v).
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