A curcumin-based molecular probe for near-infrared fluorescence imaging of tau fibrils in Alzheimer's disease

Article abstract of DOI:10.1039/c5ob01847a

In recent years, there has been growing interest in the near-infrared (NIR) fluorescence imaging of tau fibrils for the early diagnosis of Alzheimer's disease (AD). In order to develop a curcumin-based NIR fluorescent probe for tau fibrils, structural modification of the curcumin scaffold was attempted by combining the following rationales: the curcumin derivative should preserve its binding affinity to tau fibrils, and, upon binding to tau fibrils, the probe should show favorable fluorescence properties. To meet these requirements, we designed a novel curcumin scaffold with various aromatic substituents. Among the series, the curcumin derivative 1c with a (4-dimethylamino-2,6-dimethoxy)phenyl moiety showed a significant change in its fluorescence properties (22.9-fold increase in quantum yield; K_d, 0.77 μM; λ_em, 620 nm; Φ, 0.32) after binding to tau fibrils. In addition, fluorescence imaging of tau-green fluorescent protein-transfected SHSY-5Y cells with 1c confirmed that 1c detected tau fibrils in live cells.

Full text of DOI:10.1039/c5ob01847a

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Submitted to Organic & Biomolecular Chemistry
Communication
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Curcumin-based Molecular Probe for Near-Infrared Fluorescence Imaging of Tau Fibrils in
Alzheimer’s Disease†
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Kwangꢀsu Park,1,‡ Yujin Seo,1,‡ Mi Kyoung Kim,1 Kyungdo Kim,1 Yun Kyung Kim,2,3 Hyunah
Choo,2,3* 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; 3Department of
Biological Chemistry, Korea University of Science and Technology, Youseong-gu, Daejeon 305-350,
Korea
†Electronic supplementary information (ESI) available: The synthetic procedures and characterization
of the new compounds, experimental details for the preparation of aggregated tau, fluorescenceꢀbased
tauꢀbinding assay, evaluation of the fluorescent properties of the curcumin derivatives upon binding to
the tau fibrils, and detection of the tau aggregates in transfected SHSYꢀ5Y cells.
‡These two authors contributed equally on this work.
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*Corresponding authors
Hyunah Choo, Ph.D., Eꢀmail: hchoo@kist.re.kr, Tel: +82ꢀ2ꢀ958ꢀ5157
Youhoon Chong, Ph.D., Eꢀmail: chongy@konkuk.ac.kr, Tel: +82ꢀ2ꢀ2049ꢀ6100
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ABSTRACT
In recent years, there has been growing interest in the nearꢀinfrared (NIR) fluorescence imaging of
tau fibrils for the early diagnosis of Alzheimer’s disease (AD). In order to develop a curcuminꢀbased
NIR fluorescent probe of tau fibrils, structural modification of the curcumin scaffold was attempted
by combining the following rationales: the curcumin derivative should preserve its binding affinity to
the tau fibrils, and, upon binding to the tau fibrils, the probe should show favorable fluorescent
properties. To meet these requirements, we designed a novel curcumin scaffold with various aromatic
substituents. Among the series, the curcumin derivative 1c with a (4ꢀdimethylaminoꢀ2,6ꢀ
dimethoxy)phenyl moiety showed a significant change in its fluorescent properties (fold increase in
quantum yield, 22.9; Kd, 0.77 ꢁM; λ 620 nm; Φ, 0.32) after binding to tau fibrils. In addition,
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fluorescence imaging of tauꢀgreen fluorescent proteinꢀtransfected SHSYꢀ5Y cells with 1c confirmed
that 1c selectively detected tau fibrils in live cells.
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Alzheimer’s disease (AD), which is the most common cause of dementia, is characterized by
extracellular deposits of amyloid plaques and intraneuronal deposits of neurofibrillary tangles
(NFTs).1 Although there are wellꢀdefined pathological phenotypes, there is no single test to date that
can diagnose AD. Definitive confirmation of a diagnosis of AD can be obtained only with a
postmortem histopathological examination of Aβ plaques and NFTs in a patient’s brain. Therefore,
great effort has been made to develop noninvasive methods to visualize Aβ plaques and NFTs in order
to assess disease progression and monitor the effectiveness of an antiꢀAD agent in patients with AD.
Among the diverse optical imaging techniques, fluorescence imaging at the nearꢀinfrared (NIR)
spectral region (650–900 nm) provides enormous potential as a noninvasive method for in vivo
imaging. In the NIR region, biomolecules have low absorption and autofluorescence,2 thus allowing
an optimal penetration depth and high sensitivity. Therefore, there has been an increasing demand for
new chemical entities that can be used as NIR fluorescence probes for the detection of Aβ plaques and
NFTs in AD. In particular, the NIR fluorescence labeling of NFT3 is of special interest because
accumulated evidence has suggested that the severity of dementia correlates better with the load of tau
fibrils than with Aβ.4 Nevertheless, tauꢀtargeting probes have emerged more slowly than Aβ probes,
and only a handful of chemical entities that function as molecular probes for NFT5ꢀ8 or tau
aggregates9ꢀ10 have been identified. In terms of the NIR imaging probes of tau pathology, the
examples are much more limited: BODIPYꢀbased Zn(II) complexes,5 bis(arylvinyl)pyrimidines,9 and
CyDPA2.10
Curcumin (Fig. 1) is a natural yellow pigment that is derived from rhizomes of Curcuma longa. In
addition to its wellꢀknown healthꢀpromoting benefits, curcumin has recently been recognized as an
optical probe for the in vivo visualization of both Aβ plaques and NFTs.11ꢀ12 However, the practical use
of curcumin as an NIR contrast agent is limited because of its fluorescence emission wavelength,
which is outside the NIR range (520 nm). In order to induce a redshift of the fluorescence emission,
the HOMOꢀLUMO gap needs to be narrowed, and this is generally achieved by employing a pushꢀ
pull architecture: a terminal electronꢀrich donor and electronꢀdeficient acceptor groups that are
bridged by a highly polarizable πꢀconjugated system.13 In addition to redshifts, probes with the pushꢀ
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pull architecture can be selectively turned on when they bind to the target because the nonlinear
optical properties that are associated with this architecture make the fluorescence intensity susceptible
to the environmental changes that are conferred by target binding.14 Curcumin derivatives with pushꢀ
pull structures have been devised and tested as NIR fluorescence probes for the in vivo imaging of AD
pathology, and this has culminated in the recent discovery of CRANADꢀ2 (Fig. 1)14 and its
congeners15 as NIRꢀAβ probes. The structure of CRANADꢀ2 is characterized by an aromatic N,Nꢀ
dimethylamino substituent and difluoroboronyl functionality that act as an electronꢀrich donor and an
electronꢀdeficient acceptor group, respectively. CRANADꢀ2 shows fluorescence emission in the NIR
range (805 nm), high affinity to Aβ, and a drastic change in fluorescence properties (70ꢀfold increase
in intensity, 90 nm Stokes shift) upon binding to aggregated Aβ.14 However, CRANADꢀ2 is not able
to detect tau aggregates, and, to the best of our knowledge, no curcuminꢀbased NIR probe has been
reported to reveal tau fibrils.
Therefore, we were interested in developing a novel curcuminꢀbased molecular probe that could be
utilized for the NIR imaging of tau fibrils. For this purpose, structural modification of the curcumin
scaffold was attempted by combining the following rationales: the curcumin derivative should
preserve the binding affinity to tau fibrils, and, upon binding to the tau fibrils, the probe should show
favorable fluorescence properties (emission wavelength in the NIR range and enhanced fluorescence
intensity). In order to meet these requirements, a novel curcumin scaffold was designed by structural
modification of CRANADꢀ2. (1) Through a structural comparison of CRANADꢀ2 and the recently
reported tauꢀbinding curcuminꢀsugar conjugate16 (Fig. 1), we reasoned that the lack of tauꢀbinding
capacity by CRANADꢀ2 can be attributed to the difluoroboron chelate of the 1,3ꢀdiketo functionality
of curcumin, and, thus, the 1,3ꢀdiketo functionality of the curcumin scaffold should be kept intact for
the recognition of tau fibrils. (2) In order to induce a redshift of the fluorescence emission, a N,Nꢀ
dimethylamino group, which is a wellꢀknown donor group that increases the emission wavelengths of
NIR probes,17 was positioned at the paraꢀposition of the aromatic ring of curcumin. (3) In addition, to
enhance the tau binding as well as the fluorescence properties of the curcuminꢀbased probes, the
heretofore unexplored role of the aromatic substituent was investigated by introducing various
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substituents at the ortho positions (R = Br, Cl, OMe, Me, H). Herein, we describe the preparation of a
series of novel curcumin derivatives with a (4ꢀdimethylaminoꢀ2,6ꢀdisubstituted)phenyl moiety (1, Fig.
1) and their capacity as fluorescent probes of tau fibrils.
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Fig. 1 Structures of the indicated compounds
Synthesis of the title compounds (1a–1e) was accomplished by the aldol condensation of
acetylacetone (6) with appropriately substituted benzaldehydes (5a–5e)18 (Scheme 1). Other than the
commercially available 5e, the benzaldehydes 5a–5d were prepared in 1 to 4 steps starting from 2, 3b,
3c, or 4d, respectively. Thus, the reductive removal of an amino group of 2,6ꢀdibromoꢀ4ꢀnitroaniline
(2) via diazonium salt provided the aniline 3a, which was converted into the desired benzaldehyde 5a
by Nꢀmethylation that was followed by a VilsmeierꢀHaack reaction. Commercially available anilines
(3b and 3c) or N,Nꢀdimethylaniline (4d) were used for preparation of the corresponding
benzaldehydes (5b, 5c, and 5d) by using the same reaction conditions. Generally, the curcumin
scaffold is constructed by the aldol condensation of benzaldehydes and acetylacetone in the presence
of boric oxide (B2O3), tributyl borate [(nBuO)3B], and butylamine (nBuNH2), but this reaction results
in a low chemical yield. In this study, we modified the reaction conditions by heating the reaction
mixture in a sealed tube, and the synthesis of the curcumin derivatives (1a–1e) resulted in relatively
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high yields (41–68%).
R
R
R
R
NH2
CHO
a, b
c
d
Me
Me
O2N
R
H2N
R
N
R
N
R
Me
Me
2
R = Br
3a R = Br
3b R = Cl
3c R = OMe
4a R = Br
4b R = Cl
4c R = OMe
4d R = Me
5a R = Br
5b R = Cl
5c R = OMe
5d R = Me
5e R = H
R
O
OH
R
O
O
e
Me
Me
N
R
R
N
Me
Me
6
1a R = Br
1b R = Cl
1c R = OMe
1d R = Me
1e R = H
Reagents and Conditions: (a) NaNO2, H2SO4, EtOH, 80 oC; (b) Fe, NH4Cl, H2O, acetone, 80 oC; (c) K2CO3,
MeI, CH3CN, 65 oC or CH2O, NaBH3CN, AcOH; (d) POCl3, DMF, CH2Cl2, 0 oC; (e) 5a - 5e, B2O3, (nBuO)3B,
nBuNH2, DMF
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Scheme 1. Synthesis of the curcumin derivatives (1a–1e)
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The prepared curcumin derivatives were evaluated for their fluorescent properties upon binding to
preaggregated tau. The fourꢀrepeat tau construct K18 readily aggregated in the presence of heparin,19
and formation of the tau fibrils was confirmed by a thioflavinꢀS binding assay and atomic force
microscopy (Fig. S1 ~ S3 in the Supplementary Information). In order to examine the change in the
fluorescent properties upon binding to tau fibrils, we compared the fluorescence spectra of the
curcumin derivatives before and after mixing with tau fibrils, and the fluorescent properties are
summarized in Table 1. In all of the cases, after mixing the curcumin derivatives with aggregated tau,
the fluorescence quantum yields increased 1.2ꢀ to 22.9ꢀfold. Curcumin showed a 19.1ꢀfold increase
and a Stokes shift (100 nm) in fluorescence upon binding to tau fibrils, but the maximum emission
was observed only at a short wavelength (520 nm). Among the series, the curcumin derivative 1c with
orthoꢀmethoxy substituents showed the most notable fluorescent properties (λem 620 nm; Stokes shift,
,
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120 nm; 22.9ꢀfold quantum yield increase), which indicated that it had the desired optical properties
of a useful fluorescence probe for tau fibrils. However, upon mixing with tau fibrils, other curcumin
derivatives with a bromoꢀ (1a), chloroꢀ (1b), or methylꢀ (1d) substituent showed only slightly
increased fluorescence quantum yields (1.2 ~ 1.4 fold), and these were even smaller than that of the
unsubstituted analog (1e, 12.4 fold). On the other hand, the lipophilicity of the curcumin derivatives
showed that curcumin, 1a and 1c possess optimum ClogP values (2.17 ~ 3.26) for penetration of
bloodꢀbrain barrier (BBB) but others (1b, 1d and 1e) are too lipophilic to cross BBB (5.72 ~ 7.08).
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Table 1. Changes in the fluorescence profiles of the curcumin probes (1a–1e) upon interaction with
tau fibrils
Unbound (free)b
Boundc
ε a
Fold
increaseg
e
e
d
d
QYf
(Φ)
QYf
(Φ)
Compd
ClogPh
λex
λex
(Mꢀ1cmꢀ1)
λem
λem
(nm)
420
470
450
500
(nm)
420
470
450
500
(nm)
520
(nm)
520
54980
43440
52620
52480
32820
55620
0.010
0.057
0.065
0.014
0.084
0.012
0.191
0.078
0.081
0.321
0.098
0.149
19.1
1.4
2.17
2.77
5.72
3.26
6.00
7.08
Cur
1a
580
600
620
620
620
580
600
620
620
1.2
1b
1c
22.9
1.2
500
500
500
500
1d
1e
620
12.4
b
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aMolar extinction coefficient measured in dimethylsulfoxide, Fluorescence properties of the probe
c
molecules measured in PBS without tau fibrils, Fluorescence properties of the probe molecules
measure in PBS with tau fibrils, dMaximum excitation wavelength of the probe, eMaximum emission
wavelength of probe, fQuantum yield of the probe, gFold increase = QY (bound) / QY (unbound, free),
hClogP was calculated by using ChemDraw Ultra 12.0.
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Comparison of the fluorescence spectra of curcumin and its derivatives upon binding to tau fibrils
(Fig. 2) provided a clue for understanding this interesting structureꢀproperty relationship. Compared
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to curcumin (Fig. 2a), the curcumin derivatives with small fluorescence fold increases (1a, 1b and 1d)
shared a common feature: strong native fluorescence in the absence of tau (broken lines in Fig. 2b, 2c,
and 2e). In addition, fluorescence from the curcumin derivatives 1a, 1b and 1d was not affected by
solvents while curcumin, 1c and 1e showed dramatic changes in fluorescence intensities in viscous
organic solvent (DMSO) (Fig. S4 in the Supplementary Information). The solventꢀdependent
fluorescence of curcumin, 1c and 1e is highly reminiscent of molecular rotors whose fluorescence
emission increases when the internal free rotation is hindered due to the high viscosity of their
microenvironment. In this context, the fluorescence properties of the curcumin derivatives
investigated in this study can be attributed to their conformational flexibilities. Thus, sterically
demanding substituents, such as Br, Cl, or Me, might constrain the internal rotation of the curcumin
scaffold, and, as a result, the rigid conformations of the derivatives serve to intensify the fluorescence
in the free unbound state. By the same token, the relatively low native fluorescence of the
unsubstituted 1e as well as the orthoꢀmethoxyꢀsubstituted 1c can be explained by their conformational
flexibility. In particular, the internal rotation of 1c does not seem to be hindered by the orthoꢀmethoxy
substituent due to the relatively small atomic size of the oxygen atom compared to the others (Br, Cl,
or CH3) as well as the bent conformation of the methoxy group, which might have resulted in the
relatively low native fluorescence of 1c (Fig. 2d).
On the other hand, upon mixing with the tau fibrils, all the curcumin derivatives showed significant
fluorescence. It is noteworthy that intense fluorescence was observed from 1c and 1e, which might be
attributed to rigidification of their conformation by binding to the tau fibrils. In order to quantitatively
evaluate the binding affinity of 1c to tau fibrils, an in vitro saturationꢀbinding assay was conducted.
The apparent binding constant (Kd) of 1c was determined by plotting the fluorescence maximum at
various concentrations of the probe (100, 10, 1, 0.5, 0.01, and 0.001 ꢁM) to preaggregated tau (50
ꢁM), and 1c (Kd, 0.77 ꢁM) bound to tau fibrils more favorably than thioflavinꢀS did (Kd, 1.90 ꢁM, Fig.
S1 in the Supplementary Information). Titration of 1c (50 ꢁM) with tau aggregates (Fig. S8 in the
Supplementary Information) showed that fluorescence staining of tau aggregates with 1c (lower limit
of detection = 4 ng/mL) is as effective as the immunoassay (lower limit of detection = 1 ng/mL)20 and
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sensitive enough to detect the tau protein in its physiological concentration (26 ~ 66 ng/mL)21. The
fluorescence quantum yields (Φ) of curcumin, 1c, and 1e were obtained in comparison to fluorescein
isothiocyanate (Φ, 0.52) at pH 8 (phosphateꢀbuffered saline) and were estimated to be 0.19, 0.32, and
0.16, respectively. The twoꢀfold increase in the quantum yield of 1c in comparison to curcumin or 1e
demonstrated that the methoxy substituent either strengthened the binding affinity to tau fibrils or
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selectively rigidified the curcumin scaffold in the tauꢀbound state. As was the case of curcumin11ꢀ12
,
the curcumin derivative 1c was also effective in visualization of Aβ and, upon mixing with Aβ fibrils,
it showed intense fluorescence at 610 nm (Fig. S5 in the Supplementary Information).
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Fig. 2 Fluorescence intensities of (a) curcumin and (b–f) curcumin derivatives (1a–1e) (dashed line:
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without tau aggregate, solid line: with tau aggregate) upon interaction with aggregated tau in PBS
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In order to assess whether the curcumin derivative 1c could monitor intracellular tau aggregation,
human neuroblastoma cells (SHSYꢀ5Y) were transfected with a mammalian expression vector
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expressing fullꢀlength human tau [pCMV6ꢀhtau40ꢀgreen fluorescent protein (GFP)] and then treated
with 1c (Fig. 3). With confocal microscopic observation, the intracellular expression of tauꢀGFP was
confirmed by green fluorescence (Fig. 3a). It was noteworthy that circular vacuoleꢀlike subcellular
structures were observed in the transfected SHSYꢀ5Y cells (arrows in Fig. 3a–3c), and this result was
reminiscent of the findings in a previous report by Schaeffer et al.22 that autophagic vacuoles are
colocalized with tau inclusions. The circular structures were also intensely stained by 1c (Fig. 3b), and
the merged image (Fig. 3c) clearly showed that GFP and 1c stained the same circular structures. In
contrast, the untransfected cells without tau expression did not show fluorescence even in the presence
of 1c (Fig. S7, A and B, in the Supplementary Information). Also, the confocal microscope images of
tauꢀGFPꢀtransfected SHSYꢀ5Y cells before treatment with 1c (Figure S7, C and D, in the
Supplementary Information) showed fluorescence from circular cellular compartment. In addition,
blurry fluorescence presumably from tau monomers or oligomers was also observed from the cells,
which is coincident with the colocalized images of GFPꢀtau and 1c (Fig. 3). Taken together, the
colocalization in the fluorescence microscopy images suggested that 1c specifically detected tau
aggregates in live cells.
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Fig. 3 Colocalization of tau aggregates and 1c in live cell imaging. Confocal images of tauꢀgreen
fluorescent protein (GFP)ꢀtransfected SHꢀSY5Y cells after treatment with 1c showing dual staining
for (a) GFP and (b) 1c. (c) Merged images with the sites of colocalization shown in yellow. The tau
aggregates in the vacuoleꢀlike subcellular structures are indicated by white arrows.
In summary, development of a novel curcuminꢀbased NIR contrast agent to visualize tau fibrils was
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attempted. In order to optimize the fluorescent properties of the tauꢀbinding probes, the 1,3ꢀdiketo
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functionality of curcumin was kept intact while its aromatic moiety was substituted with various
functionalities at the paraꢀ and orthoꢀ positions. The newly synthesized curcumin derivatives showed
fluorescence emissions at 580–620 nm but, presumably due to the rigid conformation, suffered from
high native fluorescence and thereby exhibited a small increase in fluorescence intensity upon binding
to tau fibrils. The only exception among the series was the curcumin derivative 1c with a methoxy
substituent, which showed a significant change in its fluorescent properties (fold increase in quantum
yield, 22.9; Kd, 0.77 ꢁM; λ 620 nm; Φ, 0.32) after binding to tau fibrils. The small atomic size of
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oxygen and the bent conformation of the methoxy group might have contributed to the selective
rigidification of the tauꢀbound conformation of 1c while leaving the free and unbound conformation
unhindered. In addition, live cell imaging of the tauꢀGFPꢀtransfected SHSYꢀ5Y cells with 1c
confirmed that 1c specifically detected tau aggregates in live cells. Taken together, the favorable
fluorescent properties upon binding to tau fibrils of 1c suggested that it might be a promising NIR
fluorescent probe for noninvasive imaging in patients with AD.
226 Acknowledgements
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This study was supported by a grant of the Korean Health Technology R&D project, Ministry of
Health & Welfare, Republic of Korea (HI14C2341) and a grant from the Priority Research Centers
Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry
of Education, Science and Technology (2009ꢀ0093824).
232 Notes and references
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1. D. J. Selkoe, Alzheimer’s disease: genes, proteins, and therapy. Physiol. Rev. 2001, 81, 741.
2. R. Weissleder, A clearer vision for in vivo imaging. Nat. Biotechnol. 2001, 19, 316.
3. N.Mohorko, G. Repovš, M. Popović, G. G. Kovacs and M. Bresjanac, Curcumin labeling of
neuronal fibrillar tau inclusions in human brain samples. J. Neuropathol. Exp. Neurol. 2010, 69,
405.
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4. C. M. Wischik, C. R. Harrington and J. M. D. Storey, Tauꢀaggregation inhibitor therapy for
Alzheimer’s disease. Biochem. Pharmacol. 2014, 88, 529, and the references cited therein.
5. A. Ojida, T. Sakamoto, M. –A. Inoue, S. –H. Fujishima, G. Lippens and I. Hamachi, Fluorescence
BODIPYꢀbased Zn(II) complex as a molecular probe for selective detection of neurofibrillary
tangles in the brains of Alzheimer’s disease patients. J. Am. Chem. Soc. 2009, 131, 6543.
6. M. Ono, S. Hayashi, K. Matsumura, H. Kimura, Y. Okamoto, M. Ihara, R. Takahashi, H. Mori and
H. Saji, Rhodanine and thiohydantoin derivatives for detecting tau pathology in Alzheimer’s brains.
ACS Chem. Neurosci. 2011, 2, 269.
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