Discovery of boronic acid-based fluorescent probes targeting amyloid-beta plaques in Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2016.02.042

A boronic acid-based fluorescent probe was developed for diagnosis of amyloid-β (Aβ) plaques from Alzheimer's disease (AD). Probe 4c, which included boronic acid as a functional group, exhibited a significant increase (64.37-fold, F_Aβ/F₀) in fluorescence intensity as a response to Aβ aggregates, with a blue shift (105?nm) in the maximum emission wavelength. We found that boronic acid as a functional group improved the binding affinity (K_Dvalue?=?0.79?±?0.05?μM for 4c) for Aβ aggregates and confirmed that 4c selectively stained Aβ plaques in brain sections from APP/PS1 mice. Ex vivo fluorescence imaging using mice (normal and APP/PS1) also revealed that 4c was able to penetrate the blood–brain barrier (BBB) and to stain Aβ plaques in the brain. From these results, we believe that 4c will be useful as a fluorescent probe in preclinical research related to AD. Furthermore, we believe that our results with boronic acid also provide valuable information for the development of a probe for Aβ plaques.

Full text of DOI:10.1016/j.bmcl.2016.02.042

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1784–1788
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of boronic acid-based fluorescent probes targeting
amyloid-beta plaques in Alzheimer’s disease
Seung-Jin Jung , Jun Young Lee , Tae Ho Kim , Dong-Eun Lee , Jongho Jeon a,d, Seung Dae Yang a,
a
a
c
a
a
b
a,b,
⇑
Min Goo Hur , Jung-Joon Min , Yong Dae Park
a
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea
Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Republic of Korea
Department of Chemistry and Research Institute of Natural Science, Gyeongsang National University, 501 Jinju Daero, Jinju 660-701, Republic of Korea
Department of Radiation Biotechnology and Applied Radioisotope Science, Korea University of Science and Technology, Daejeon 34113, Republic of Korea
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
A boronic acid-based fluorescent probe was developed for diagnosis of amyloid-b (Ab) plaques from
Alzheimer’s disease (AD). Probe 4c, which included boronic acid as a functional group, exhibited a signif-
Received 22 September 2015
Revised 12 February 2016
Accepted 16 February 2016
Available online 16 February 2016
icant increase (64.37-fold, FAb/F
shift (105 nm) in the maximum emission wavelength. We found that boronic acid as a functional group
improved the binding affinity (K value = 0.79 ± 0.05 M for 4c) for Ab aggregates and confirmed that 4c
0
) in fluorescence intensity as a response to Ab aggregates, with a blue
D
l
selectively stained Ab plaques in brain sections from APP/PS1 mice. Ex vivo fluorescence imaging using
mice (normal and APP/PS1) also revealed that 4c was able to penetrate the blood–brain barrier (BBB)
and to stain Ab plaques in the brain. From these results, we believe that 4c will be useful as a fluorescent
probe in preclinical research related to AD. Furthermore, we believe that our results with boronic acid
also provide valuable information for the development of a probe for Ab plaques.
Ó 2016 Published by Elsevier Ltd.
Keywords:
Alzheimer’s disease (AD)
Amyloid plaque
Boronic acid
Fluorescent probe
Alzheimer’s disease (AD) is a progressive neurodegenerative
disease characterized by cognitive decline, irreversible memory
loss, disorientation, and language impairment.1,2 One of the patho-
logical hallmarks is the presence of amyloid-b (Ab) plaques in the
brains of AD patients. Ab plaques are central in the molecular
pathogenesis and form oligomers, fibrils, and plaques. Biomarkers
to monitor Ab aggregation are important for further detailed stud-
ies of the disease mechanisms and drug development. Ab is mis-
folded into a cross b-sheet structure and thereby binds to dyes,
such as Congo red and Thioflavin T.3
Fluorescent probes have attracted interest in the labelling and
imaging of Ab plaques because these probes are relatively safe,
low cost and user friendly and have real-time and multiplexing
capabilities. Appropriate fluorescent probes for Ab plaques should
have the following properties: (a) specificity for Ab plaques, (b)
sufficient binding affinity to Ab peptides, (c) an emission wave-
length above 650 nm to minimize background fluorescence from
brain tissue, (d) appropriate lipophilicity (logP value between 1
and 3) to rapidly cross the blood–brain barrier (BBB), (e) a signifi-
cant change in fluorescence upon binding to Ab plaques, and (f)
,4
1
0
A number of imaging techniques, such as magnetic resonance
straightforward synthesis. Although fluorescent probes are unfa-
vorable for in vivo imaging on humans and animals except mice
due to their limited penetration depth, they are a useful tool for
5
6,7
imaging (MRI) , positron emission tomography (PET), and sin-
gle-photon emission computed tomography (SPECT),8 have been
developed for the specific imaging of Ab plaques. Although imaging
techniques employing these probes have produced promising
results in vitro, ex vivo, and in vivo, using these imaging methods
with probes carries the disadvantages of poor spatial and temporal
resolution, low sensitivity, exposure to radioactivity, and short-
1
1
preclinical evaluation. Therefore, fluorescent probes, such as
Congo red, NIAD-4, BODIPY 7, and CRANAD-2, have been continu-
ously developed for the detection of Ab plaques and have subse-
1
2–15
quently been reported.
Recently, we developed and reported fluorescent chalcone-
9
lived isotopes. Additionally, the handling of these probes requires
mimic probes for the detection of Ab plaques that rapidly bind to
1
6
stringent safety regulations due to their radioactivity.
Ab plaques upon intravenous injection in mice. Using chalcone-
mimic probes, we found that their clearance from the brain occurs
much more quickly than expected because of their relatively low
binding affinity to Ab plaques.
⇑
Corresponding author. Tel.: +82 63 570 3574; fax: +82 63 570 3549.
E-mail address: ydpark@kaeri.re.kr (Y.D. Park).
http://dx.doi.org/10.1016/j.bmcl.2016.02.042
960-894X/Ó 2016 Published by Elsevier Ltd.
0

S.-J. Jung et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1784–1788
1785
N
N
O
O
O
O
Br
O
O
i
ii
iii
O
O
1
2
3
4a-d
R
4
4
4
4
a, R = 4-N(CH )
3 2
b, R = 4-N(CH CH )
2
3 2
c, R = 4-B(OH)
2
d, R = 3-B(OH)
2
Scheme 1. Reaction scheme for synthesis of the optical probes. Reagents and conditions: (i) DMF (dimethylformamide), NBS (N-bromosuccinimide), room temperature, 24 h;
(
ii) DME (1,2-dimethoxyethane), 2.0 M aqueous Na
2
CO
3 3 4
, Pd(PPh ) , 4-(dimethylamino) phenylboronic acid; and (iii) DMF, NaOH, aldehyde derivatives, room temperature, 8 h.
6
5
4
3
2
00
00
00
00
00
4
a
4b
1
000
probe only
probe with Aβ aggregates
8
6
4
2
00
00
00
00
0
100
0
4
50
550
650
750
4
50
550
650
750
4
c
4d
5
4
3
2
1
000
000
000
000
000
0
2
2
1
1
500
000
500
000
5
00
0
4
50
550
650
750
450
550
650
750
Wavelength (nm)
Figure 1. Emission spectra of probes upon responses with and without Ab aggregates.
Here, we describe the discovery and further optimization of
AD.20 In this study, we report the optical and biological properties
of fluorescent probes with boronic acid for the diagnosis of Ab pla-
ques from AD.
Probe synthesis is outlined in Scheme 1. The formation of back-
bone 3 was synthesized by the Suzuki coupling reaction of 4-
(dimethylamino) phenylboronic acid with compound 2. Probes
(4a, 4b, 4c, and 4d) were successfully prepared by the condensa-
tion of 3 and aldehyde derivatives in the presence of NaOH
(53.4%, 48.4%, 37.9%, and 32.6% yields, respectively). The X-ray
crystal structure of 4d is depicted in Figure S5.
chalcone-mimic probes. In this process, we developed boronic
acid-based probes and found that boronic acid was effective at
binding to Ab plaques. In recent years, boronic acid compounds
have attracted much attention. Many of these compounds have
been used in the fields of medicinal chemistry, such as fluorescent
1
7
reporter , enzyme inhibition, cancer therapy, and antibody mimic
1
8
development. As a Lewis acid, boronic acid can form a covalent
19
bond with Lewis bases, such as alcohol, amine, thiol. We envi-
sioned that the boronic acid analogue might form stronger bonds
with amino acid residues of Ab plaques to enhance its affinity.
These compounds have also been used in research related to
First, we evaluated the fluorescent properties, including the
excitation and emission wavelengths, of the synthesized probes

1
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S.-J. Jung et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1784–1788
Table 1
Structures, fluorescence profiles, and K
D
values of probes with Ab aggregates
ex (nm)
Probe
R
k
k
em (nm)
Fold increasea (FAb/F
0
)
D
K (lM)
(mean ± SD)b
4
4
4
4
a
b
c
4-N(CH
4-N(CH
4-B(OH)
3-B(OH)
3
)
2
415
415
445
450
610
610
685
685
6.66
3.69
64.37
70.66
—
—
2
3 2
CH )
2
0.79 ± 0.05
0.90 ± 0.02
d
2
a
Fold-change values were calculated using the fluorescence emission intensities at kem of probes (10
measurements).
lM compound and 20 lM Ab42 aggregates were used for the
b
D
The K values are expressed as the means ± S.D. of values obtained from three independent replicates (N = 3).
in PBS (phosphate-buffered saline, 0.1 M, pH = 7.4). There were
large Stokes shifts of 195 nm for 4a and 4b (kmax(ex) = 415 nm,
strong binding affinities (K
0.90 ± 0.02 M, respectively) (Fig. S2), and the binding affinities
for the Ab aggregates were higher than that of a previously
D
values = 0.79 ± 0.05
l
M
and
l
kmax(em) = 610), 240 nm (kmax(ex) = 445 nm, kmax(em) = 685) for 4c,
1
6
and 235 nm (kmax(ex) = 450 nm, kmax(em) = 685 nm) for 4d (Fig. S1).
The large Stokes shifts easily separated the excitation and emission
wavelengths, which could also be applied to the fluorescent probes
with great advantage.
Next, the synthesized probes were tested with the synthetic
Ab42 aggregates for their fluorescence responses. Probes 4a and
D
reported fluorescent probe (K value = 1.59 ± 0.13 lM). All of
the values of the fluorescent properties of the probes are shown
in Table 1. These results indicated that boronic acid improved
the binding affinity for the Ab plaques and that the probes (4c
and 4d) with boronic acid specifically stained Ab plaques in the
brain. However, the position of boronic acid did not have a signif-
icant influence on the properties of the Ab aggregates. This finding
might be due to the fact that boronic acids are Lewis acids and iso-
4
b did not contain boronic acid and thus did not show changes
in fluorescent response with the Ab42 aggregates. In contrast,
probes 4c and 4d, which included boronic acid, exhibited blue
shifts of 105 nm (from 685 to 580 nm) for 4c and 75 nm (685–
steres of carboxylic acid. Boronic acid pK
a
values are ꢀ9–10; there-
22
fore, at physiological pH, boronic acids remain unionized. Thus,
the electron-deficient boronic acid moiety could increase the bind-
ing affinity by forming a hydrogen bond between its OH group and
a histidine residue of the Ab peptides.
6
10 nm) for 4d on the maximum emission wavelength, with a
remarkable increase in fluorescence intensity (64.4- and 70.6-fold,
respectively) in response to Ab42 aggregates (Fig. 1). Blue shifts in
the fluorescence emission spectra and significant increases in fluo-
rescence intensity upon binding with Ab aggregates are important
properties for good fluorescent probes for the detection of Ab
Based on the above results, we selected probes 4c and 4d with
boronic acid as fluorescent probes to stain Ab plaques, and
additional experiments were conducted using these probes. In
vitro pathological fluorescent staining of Ab plaques in slices of
brain tissue from double-transgenic mice (APP/PS1) was per-
formed to evaluate the affinity of the boronic acid probes (4c and
2
1
aggregates. Additionally, to quantitatively evaluate the binding
affinities of the two boronic acid probes for Ab aggregates, a satu-
ration binding assay was conducted. Probes 4c and 4d exhibited
(A)
4
c
Anti-Aβ
Merge
(
B)
4
d
Anti-Aβ
Merge
Figure 2. Confocal images of histological double-staining in transgenic mice (APP/PS1) brain sections. Ab plaques stained with anti-Ab (middle, red), stained with probes 4c
A) and 4d (B) (left, green), and merged with anti-Ab and probes (right, orange). The spots stained by anti-Ab and probes were cologically matched (white arrow).
(

S.-J. Jung et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1784–1788
1787
(
A)
2000 (B)
1
20
00
brain
heart
1
lung
8
6
4
2
0
0
0
0
0
1
0
000
liver
spleen
kidney
Non-injection
Normal
APP/PS1
(
C)
(D)
Normal
APP/PS1
Probe 4c
Anti-Aβ
5
0 μm
50 μm
25 μm
25 μm
Figure 3. Ex vivo fluorescent images of mouse organs. (A) Fluorescent images of organs removed from APP/PS1 and normal mice 30 min after intravenous injection of probe
TM
4
c. The fluorescent images were obtained using an orange filter at an excitation of 415 nm using software provided with the MAESTRO 2.4 fluorescence imaging system. (B)
Comparison of the fluorescence intensities in the brain from non-injection, injected-normal, and injected-APP/PS1 mice. (C) Fluorescent images of brain sections from normal
and APP/PS1 mice 30 min after intravenous injection of probe 4c (Ab plaques are indicated with white arrows). (D) Fluorescent images of Ab plaques (white arrow) bound
with probe 4c and anti-Ab. The excitation wavelengths for probe 4c and anti-Ab were 455 and 550 nm, respectively. Fluorescent images were obtained using a laser confocal
scanning microscope.
4
d). As shown in Fig. 2, specific staining of plaques was observed in
(Fig. S4) those were consistent with the in vitro data. These results
further confirmed that 4c penetrated the BBB and specifically
stained Ab plaques. The livers from APP/PS1 mice also exhibited
higher fluorescent signals than those of normal mice because the
accumulated probe 4c in the brains of APP/PS1 mice underwent
slower excretion via the hepatobiliary pathway (Fig. 3A). Addition-
ally, we checked whether 4c selectively stained the Ab plaques in
the brain sections from the APP/PS1 mice 30 min after intravenous
injection with 4c. Weak fluorescent signals were observed from the
brains of normal mice (Fig. 3C), while large amounts of Ab plaques
were confirmed in the cortex region of the brain from APP/PS1
mice (Fig. 3C); these Ab plaques were also cologically stained with
probe 4c in the brain section restrained with anti-Ab (Fig. 3D).
These results supported the specific binding of probe 4c with the
Ab plaques in the brain.
brain slices of APP/PS1 mice treated with probes 4c and 4d. The
presence and distribution of Ab plaques were confirmed by stain-
ing with anti-Ab; the plaques colocalized with the staining results
with our probes. From probes 4c and 4d, we selected 4c (due to its
better binding affinity compared to 4d) for additional in vivo
experiments using mice. Probe 4c exhibited a logP value = 2.97
and met the requirement to penetrate the BBB. It has been reported
that BBB penetration is dependent on lipophilicity, and a moderate
logP value is between 1 and 3.23
We conducted an ex vivo experiment in APP/PS1 and normal
mice using probe 4c to further confirm binding of the probe to
Ab plaques in vivo. Mice were injected with 5 mg/kg of 4c through
the tail vein, and whole organs were removed 30 min after injec-
TM
tion and scanned using a fluorescence imaging system (MAESTRO
2
.4 software). Fluorescent images of ex vivo organs from normal
In conclusion, we developed a fluorescent probe, 4c, with boro-
nic acid for the detection of Ab plaques from AD. We found that
boronic acid favorably affected the binding affinity for Ab plaques.
Probe 4c satisfied the requirements of a fluorescent probe, such as
the fluorescent properties, good binding affinity, specificity for Ab
plaques, and reasonable lipophilicity in both in vitro and in vivo
experiments. We believe that probe 4c with boronic acid will be
useful as a fluorescent probe in preclinical experiments related to
AD. Our result with boronic acid also provides valuable informa-
tion for the development of probes for Ab plaques.
mice showed that large amounts of probe 4c initially accumulated
in the brain and liver. The maximum uptake values were observed
1
0 min after injection (Fig. S3). The observed signals in both organs
then quickly decreased over the time; the fluorescent signals from
other organs displayed similar trends. The results indicated that
most of the injected probe was rapidly excreted from the body
without significant retention of the probe in the brain region. How-
ever, fluorescent signals in the brains of APP/PS1 mice were opti-
cally more abundant than those of normal mice. Additionally,
there were approximately two-fold differences in the regions of
interest (ROIs) of brains from both the APP/PS1 and the normal
mice (Fig. 3A and B). These results demonstrated show that probe
Acknowledgments
4
c showed slow clearance kinetics from the brains of APP/PS1 mice
than those of normal mice due to abundance of Ab plaques in the
brain. Additionally, the fluorescent signals from the brains of
APP/PS1 and normal mice exhibited different emission spectra
This work was supported by a National Research Foundation
(NRF) grant funded by the Korean government Ministry of Educa-
tion, Science and Technology (MEST).

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