Design, synthesis and evaluation of curcumin-based fluorescent probes to detect Aβ fibrils

Article abstract of DOI:10.1016/j.bmcl.2018.10.002

Amyloid β fibrillation is an early event in Alzheimer's disease, so its detection is important to understand its roles in Alzheimer's disease. Curcumin, which has poor water solubility, has been reported to have many pharmacological activities including potent anti-amyloid β fibril activity in Alzheimer's disease. In this study, we found that curcumin analogues with the fluorescence property instead of non-inhibition of amyloid β fibrils. The development of new curcumin analogue, Me-CUR (9), as fluorescent switchable probe to detect amyloid β fibrils is described. Me-CUR (9) shows excellent fluorescence, especially higher than ThT (4), in the presence of amyloid β fibrils. These results suggest that Me-CUR (9) can become a useful in vitro amyloid fluorescence sensor for diagnosis of Alzheimer's disease.

Full text of DOI:10.1016/j.bmcl.2018.10.002

Accepted Manuscript
Design, synthesis and evaluation of curcumin-based fluorescent probes to detect
Aβ fibrils
Taki Sato, Mayumi Hotsumi, Koki Makabe, Hiroyuki Konno
PII:
S0960-894X(18)30782-0
https://doi.org/10.1016/j.bmcl.2018.10.002
BMCL 26061
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
13 July 2018
20 September 2018
1 October 2018
Please cite this article as: Sato, T., Hotsumi, M., Makabe, K., Konno, H., Design, synthesis and evaluation of
curcumin-based fluorescent probes to detect Aβ fibrils, Bioorganic & Medicinal Chemistry Letters (2018), doi:
https://doi.org/10.1016/j.bmcl.2018.10.002
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Design, synthesis and evaluation of curcumin-based fluorescent probes to detect Aβ
fibrils
Taki Sato, Mayumi Hotsumi, Koki Makabe, Hiroyuki Konno*
Department of Biological Engineering, Graduate School of Science and Technology,
Yamagata University, Yonezawa, Yamagata 992-8510, Japan.
Key words: Alzheimer’s disease, Amyloid , Curcumin, Inhibitor, Probe
Author information
*Corresponding author
Tel & FAX +81-238-26-3131
E-mail konno@yz.yamagata-u.ac.jp
ABSTRACT: Amyloid  fibrillation is an early event in Alzheimer’s disease, so its
detection is important to understand its roles in Alzheimer’s disease. Curcumin, which
has poor water solubility, has been reported to have many pharmacological activities
including potent anti-amyloid  fibril activity in Alzheimer’s disease. In this study, we
found that curcumin analogues with the fluorescence property instead of non-inhibition
of amyloid  fibrils. The development of new curcumin analogue, Me-CUR (9), as
fluorescent switchable probe to detect amyloid  fibrils is described. Me-CUR (9)
shows excellent fluorescence, especially higher than ThT (4), in the presence of amyloid
 fibrils. These results suggest that Me-CUR (9) can become a useful in vitro amyloid
fluorescence sensor for diagnosis of Alzheimer’s disease.

Amyloid fibrils are insoluble aggregates with fibrillary morphology and a β-sheet
rich structure that can cause several diseases known as amyloidosis due to their
misfolding1-3. Among the different amyloid-like peptides, those called amyloid β (Aβ)
are very important because of their prominent role in Alzheimer’s Disease (AD) 4,5. AD
is a progressive neurodegenerative disorder characterized by cognitive decline,
irreversible memory loss, disorientation and language impairment6,7. One of the
pathological hallmarks is the aggregation of Aβ peptide and its accumulation in the
human brain has an important role in the etiology of AD8. Therefore, Aβfibrils could be
a biomarker for therapy or diagnosis for AD. Although there are several isoforms of Aβ
species ranging in length from 39 to 43 residues, Aβ40 and Aβ42 are the most abundant
(9:1 = Aβ40:Aβ42) and Aβ42 is more aggregation-prone9. Therefore, inhibiting or
disrupting the Aβ42 aggregation process represents a promising therapeutic strategy for
the prevention and treatment of AD.
Curcumin (CUR (1)), is a yellow pigment exacted from Curcuma longa
(turmeric), which is commonly used as a food colorant, spice, and cosmetic, as well as a
traditional medicine in Asian countries10,11. In the last few decades, CUR (1) has become
a research focus due to its numerous beneficial biological and pharmacological
activities such as antioxidant, anti-tumor, anti-inflammatory and other desirable
medicinal benefits12-17. CUR (1) is one of the PAINS (pan assay interference
compounds) and IMPS (invalid metabolic compounds), and more than 1,000
curcuminoids have been developed to improve their activities to date18-20. Especially, the
effect of anti-Aβfibrils as highly beneficial in the treatment of AD has been reported by
the Ono group 21. CUR (1) has a characteristic structure in which two phenolic groups
are linked by an olefin chain containing a β-diketone moiety and the high crystallinity
reduces the solubility to solvents markedly22,23. Since the insolubility of CUR (1) in
water may restrict its pharmacodynamics, we have successfully synthesized a
water-soluble CUR analogue (Ws-CUR (2)), with potent inhibitory activity against Aβ
fibrils. Ws-CUR (2) shows 31 μM of water-solubility and has intensive inhibitory
activity of Aβfibrils24.
To date, the most widely applied Aβimaging probes have used positron emission
tomography (PET) and single photon emission computed tomography (SPECT) using
Congo Red (CR, (3)) and Thioflavin T (ThT (4))25,26. CUR (1) has also been developed
as a PET imaging dye or Near-infrared (NIR) dye; however, these imaging modalities

are hindered by their intrinsic high cost and time-consuming nature and result in patient
exposure to radiation27-29. Therefore, labeling technology has been increasingly popular
for imaging of Aβfibrils as a potential alternative to other techniques30,31. Among these
molecular probes, ThT (4) is the most widely used dye for monitoring amyloid fibrils
both in vitro and in vivo32. Although the ThT (4) binding model is largely unknown, the
increasing emission intensity of ThT (4) upon binding to amyloid fibrils is due to the
inhibition of the rotation around a single C-C bond in ThT (4)33. Therefore, there is
disadvantage in the point of low material selectivity. Moreover, CRANAD-2 (5) for NIR
19
probe and Shiga-Y5 (6), F-MR diagnostic imaging probe, have the CUR-structure, so
these results suggest that it is possible they can detect Aβ fibrils as a fluorescent
molecular rotor to CUR (1)34, which has five single C-C bonds. The property of CUR
(1) is expected high selectivity and potent detection capability. In addition, most CUR
(1) and its analogues were symmetry structures in the previous work by many
researchers18-20. Development of Ws-CUR (2) by our group indicated that asymmetry
CUR structures exploit novel ability. Although a variety of organic dyes were reported,
a few compounds have been applied as biomarker of Aβ fibrils. It is anticipated that
CUR (1) and its analogues rarely show aggregation-induced emission (AIE) and can be
reversibly showed AIE efficacy depending on the aggregation state of Aβfibrils. Herein,
we report the design, synthesis and evaluation of CUR analogues as new fluorescence
probes (Figure 1).
CR (3)
λem = 432 nm
Ws-CUR (2)
CUR (1)
ThT (4)
λex = 550 nm
λem = 445 nm
Shiga-Y5 (6)
λex = 490 nm
CRANAD-2 (5)
Figure 1. Structure of CUR (1), Ws-CUR (2), CR (3), ThT (4), CRANAD-2 (5) and Shiga-Y5 (6)
In our previous work, we performed a structure activity relationship study (SAR)
to develop an Aβaggregation inhibitor. As depicted in Figure 2, inhibitory activity of

Me-CUR (7) against Aβfibrils decreased markedly compared with those of Ws-CUR (2).
In addition, ThT-induced fluorescence intensity of Me-CUR (7) with Aβ fibrils was
more intense than that in the absence of CUR derivatives as a positive control (Figure
2).
Figure 2. Time-dependent fibril formation of Aβ42 was
monitored by ThT (4), Me-CUR (7) and Ws-CUR (2)
(dye 5 μM, Aβ42 25 μM).
As for this phenomenon, we assume that Me-CUR (7) has no inhibition activity
for Aβ fibrils and may not induce their collapse. To investigate this phenomenon, we
designed CUR analogues, which converted the hydroxy group into methoxy group of
phenyl rings to develop more effective Me-CURs as fluorescence probes for Aβfibrils.
Moreover, Ws-CUR (2), which has an ortho hydroxy group, showed potent inhibition
activity even though Ws-CUR (2) has one less hydroxy group than CUR (1). Therefore,
we designed CUR analogues with two or three methoxy groups on their phenolic rings.
Especially, the necessity for the methoxy group in the ortho-position of Me-CURs was
verified to detect Aβ fibrils. Me-CURs (7) - (18) with four methoxy groups on their
phenolic rings and a similar structure to CUR, were also investigated (Table 1). To
prepare the designed compounds (7)
-
(18), aldol condensation of an
acetylacetone-boron complex and benzaldehyde derivatives in the presence of piperidine
or n-buthylamine is a key step (See supporting information SI-Scheme 1). Symmetrical
derivatives were synthesized directly and asymmetric derivatives were synthesized twice
by repeating the operation.

Ar2
Table 1. Molecular design of Me-CURs.
Ar1
a: The number of methoxy groups.
b)
a)
Figure 3. (a) Fluorescence quantities of Me-CURs (7) - (18) for Aβ fibrils (Incubate time, 10h,
ThT fluorescence, 100) (b) Time-dependent fibril formation of A42 was monitored by ThT
compared with 9 (dye 5 M, A42 25 M).
Evaluation of synthesized molecules, Me-CURs (7) - (18), as fluorescence probes
for Aβfibrils was performed by an incubation assay with aggregated Aβ42 peptide. ThT
(4), the control probe, is a benzothiazole dye that exhibits enhanced fluorescence upon

binding to amyloid fibrils. As shown in Figure 3a, fluorescence intensities of Me-CURs
(9), (15) and (16) with the Aβfibrils were stronger than those of ThT (4). In the spectra
of the Aβ42-ThT assay system in Figure 3b, 9 showed constant weak autofluorescence.
In contrast, 9 behaved as selective light-up fluorescent probe upon binding to the Aβ
fibrils, as fluorescence intensity was increasing. In other words, 9 can be selectively and
reversibly turned on and off depending on the aggregation state of Aβfibrils and act as
an Aβfibrils-responding On-Off switchable fluorescent probe (Figure 3).
To investigate the spacer unit of the CUR framework, ‘‘Downsizing’’ derivatives
of Me-CUR (9), which have a short olefin linker and reduced number of ketones, were
designed. Basically, the C7-diketone olefin linker of CUR framework was replaced with
C5-diketone, C5-monoketone and C3-monoketone olefin linkers to afford downsized
analogues (19), (20) and (21). These downsized analogues (19), (20) and (21) were
synthesized as shown in SI-Scheme 2 (See supporting information SI-Scheme 2). A
List-Barbas aldol reaction was employed to give C5-diketone derivatives (19) and
C5-monoketone derivatives (20) as a key step. The downsizing analogue (20) was
prepared by a base-catalyzed aldol condensation reaction starting from o-anis aldehyde.
C3-monoketone derivatives (21) were synthesized by coupling of o-anis aldehyde and
o-methoxy acetophenone in the presence of sodium methoxide. As shown in Figure 4,
these short-linker Me-CURs (19), (20) and (21) showed weaker or undetectable
fluorescence. The results suggested that the methoxy groups at the 3,4-positions of
Figure 4. “Downsizing” analogues of 9; C5-diketone type derivative
(19); C5-monoketone type derivative (20); C3-monoketone type
derivative (21)

phenolic rings and olefin chain having both 7 carbon atoms and the β-diketone motif as
CUR are needed to recognize the Aβfibrils (Figure 4).
We also investigated the photophysical behavior of 9, 15 and 16. There was a
significant association between the mechanism to stain Aβ fibrils and the molecular
planarity as ThT (4) behaves like a “molecular rotor”. In detail, a low energy barrier
allows the benzenamine and benzothiazole rings of ThT (4) to rotate freely about their
shared C-C bond in solution. Amyloid fibrils are likely to present a ThT-binding site that
sterically “locks” the bund dye, thus leading to enhancement of ThT fluorescence.
Me-CURs also have the methoxy groups on phenyl rings, which are linked to the olefin
chain via two single bonds. Thus, they feature high rotational flexibility and possesses
two different twisted intramolecular change transfer (TICT) channels within the whole
molecular structure. These intramolecular rotations lead to the non radiative
deactivation of the fluorescent excited state, which could be another cause for the weak
auto fluorescence of Me-CURs. To test this assumption, we investigated the effect of
solvent viscosity on Me-CUR emission. The intramolecular rotation process is reported
to be influenced by the viscosity of the medium; in a high viscosity medium, the
intramolecular rotation was slowed down and hence enhanced the emission of
Me-CURs (9), (15) and (16). Thus, we examined the viscosity effect on the emission
behavior of Me-CURs (9), (15) and (16) which detected Aβfibrils more sensitively than
ThT (4) in glycerol. As shown in Figure. 5b, the fluorescence intensity of Me-CURs (9),
(15) and (16) were dramatically enhanced with a solvent viscosity which is typically
observed for molecular rotors like ThT (4). These results support the fact that Me-CURs
act as the molecular rotor and could turn on/off their emission when free rotation of
phenyl rings to the olefin linker is restricted (Figure 5b). Based on this hypothesis, we
calculated the molecular planarity of Me-CURs (9), (15) and (16) when the two
carbon-carbon bonds sharing both sites of aromatic rings and the olefin linker of
Me-CURs (9), (15) and (16) defined as their rotor (switch). As we expected, Me-CUR
(9) had a high molecular planarity (Φ= 8.8) compared with 15 (Φ= 79.3) and 16 (Φ=
81.7) (Figure 5a). Thus, the high molecular planarity of Me-CUR (9) is an important
factor in the robust intense fluorescence when interacting with Aβ fibrils. Moreover,
Me-CUR (9) showed sufficient stokes shift to use as a fluorescence probe (Figure 5c).

The photoproperties of Me-CURs (9), (15) and (16) are shown in Figure 5c. To obtain
more emission properties of Me-CUR (9), a concentration titration test was done. The
fluorescence emission maxima of Me-CUR (9) shifted from 520 nm to 496 nm in the
presence of Aβfibrils from 0.5 μM to 25 μM as shown in Figure 5d. The concentration
dependent-fluorescence spectral changes of Me-CUR (9) showed that the fluorescence
intensity gradually increased along with increasing concentrations of Aβ fibrils,
accompanied by a slight blue shift in λ , while it did not change markedly in the
max
solution without Aβ fibrils (Figure 5). There is sigmoidal curve between fluorescence
intensity shift (nm) and concentration of Aβ fibrils (M) and consequently, Aβ
concentration at a range of 5-10 M prompts to a blue shift in λ (See supporting
ma
information SI-Figure 2). As further investigation, the detection limit between Aβfibrils
and Me-CUR (9) was estimated on fluorescence intensity. To detect 25 M Aβfibrils,
Me-CUR (9) from 0.2 M to 500 M were attempted in standard condition. More than
10 M Me-CUR (9) gave saturated data (>60000 a.u.) and 0.2 M Me-CUR (9) shows
no detection with 25 M Aβfibrils. On the other hand, we confirmed that less than 6 nM
Aβfibrils was hardly performed fluorescent detection using 10 M Me-CUR (9) and,
therefore, it needs to exist more than 60 nM Aβfibrils to exhibit the ability of Me-CUR
(9) (See supporting information SI-Figure 3).

b)
a)
9
d)
c)
Figure 5. (a) The most stable conformation of Me-CUR (9). (b) Viscosity-dependent emission
behavior of Me-CUR (9), (15) and (16) were observed compared with ThT. (c) The
absorption and emission spectra of Me-CUR (9). (d) Concentration dependent-fluorescence
titration. Aβ42 0.5 - 25 μM, Me-CUR (9), 5 μM.
To observe the morphology of Aβfibrils, the effect on amyloid formation was also
investigated by TEM (Figure. 6a-f). Aβ42 formed amyloid fibrils of 30-120 nm diameter
and 300 nm-2 μm long owing to aggregation properties (Figure 6a). The addition of ThT
(4) to Aβfibrils maintained the fibril formation as a positive control (Figure 6b). The
addition of Me-CUR (9) kept the formation of fibrils from transforming and fibril
formations of 300-500 nm in length were still observed, which corresponded to the
fluorescence intensity (Figure 6c). In addition, the other Me-CUR analogues, which
have short olefin linkers, C5-diketone type derivative (19), C5-monoketone type
derivative (20), C3-monoketone type derivative (21) were also analyzed. The results
indicated that the addition of the C5-diketone type derivative (19) led to some shorter
fibril formation (~200 nm) (Figure 6d), and the addition of the C5-monoketone type
derivative (20) and C3-monoketone (21) derivative changed the morphology of Aβ
fibrils (Figure 6e,f). To visualize the interaction of ThT (4) or Me-CURs (9), (19)-(21)

with Aβfibrils, we observed fibril formation by fluorescence microscopy (Figure 6g-l).
Aβ fibrils showed no fluorescence with an excitation filter (B) of 460-495 nm as a
negative control (Figure 6g). The fluorescence of ThT (4) was clearly evident on the
amyloid aggregate and it showed as a green light (Figure 6h). We found that our
promising probe, Me-CUR (9), stained the fibrous aggregates of Aβfibrils with a yellow
light (Figure 6i). Predictably, the C5-diketone type derivative (19) undetected the
aggregates, and only confirmed their autofluorescence. Neither the C5-monoketone type
derivative (20) nor the C3-monoketone type derivative (21) also showed any fluorescent
activity to bind to the aggregates (Figure 6j-l).
Figure 6. TEM images of the Aβ fibrils (a-e). (a) Aβ fibrils. (b) the addition of ThT. (c) the addition
of 9. (d) the addition of 19. (e) the addition of 20. (f) the addition of 21. Fluorescence microscopy
analyses of the Aβ fibrils (g-l). (g) Aβ fibrils. (h) stained with ThT. (i) stained with 9. (j) stained with
19. (k) stained with 20. (l)stained with 21.
To predict insight into the possible interaction mechanism for Aβfibrils binding,
we performed molecular docking analysis. The binding mode of Me-CUR (9) or ThT
(4) was achieved with Aβ fibrils using GOLD software on a 12-fold Aβ11-42 fibril
structure (PDB ID: 2MXU). As shown in Figure 7, Me-CUR (9) and ThT (4) located in
the inner site of Aβ fibrils (Figure 7a,b). The binding mode between Me-CUR (9) or
ThT (4) and the Aβ fibrils was mainly supported by the hydrophobic interaction. In
detail, several significant binding interactions were observed within the docked complex,
as depicted in Figure 7c,d. Our docking studies in silico further supported the fact that
the localization site of Me-CUR (9) and ThT (4) are similar and are sustained by
hydrophobic interaction and hydrogen bonding networks (Figure 7).

b)
d)
a)
a)
c)
c)
Figure 7. (a) Binding pose of ThT (4) in the Aβ fibrils; (b) Binding pose of 9 in the Aβ
fibrils; (c) Detailed view of the docked ThT structure and the corresponding interacting
amino-acid moieties within the binding site of Aβ fibrils; (d) Detailed view of the docked
9 structure and the corresponding interacting amino-acid moieties within the binding site
of Aβ fibrils.
In summary, a new series of novel curcumin analogues, an Me-CUR library
containing short linker moiety or various substituents, was successfully synthesized and
evaluated as new imaging agents for Aβ fibrils. The Me-CURs, (9), (15), (16), which
have a C7-diketone moiety and phenolic rings containing methoxy groups at
3,4-positions, showed higher fluorescence than ThT (4) as the most extensive amyloid
sensor. Detailed spectroscopic studies indicated that Me-CUR (9) has high molecular
planarity and thus it could strongly bind with the Aβfibrils resulting in a large increase
in its emission intensity with a large spectral shift. Me-CUR (9) is a fluorescent
switchable probe to detect amyloid  fibrils with high sensitivity. At this time, Me-CUR
(9) can be employed as the detecting agent of Aβfibrils in vitro experiment instead of

ThT (4). These results suggest that Me-CUR (9) can become a useful fluorescence
probe for diagnosis of AD. Our research using cell lines and a serum is in progress.
Supporting information
Additional experimental procedures, and analytical data for synthetic compounds. This
material is available free of charge via the Internet at xxxxxxx.
Acknowledgement
This work was supported in part by a Grant-in-Aid from the Japan Society for the
Promotion of Science, KAKENHI (25450145) to H.K.
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Design, synthesis and evaluation of curcumin-based fluorescent probes to detect Aβ
fibrils
Taki Sato, Mayumi Hotsumi, Koki Makabe, Hiroyuki Konno*
Graduate School of Science and Technology, Yamagata University
ON
Aβ fibrils
OFF
=
Me-CUR
twisted-structure
planner-structu

Design, synthesis and evaluation of curcumin-based fluorescent probes to detect Aβ
fibrils
Taki Sato, Mayumi Hotsumi, Koki Makabe, Hiroyuki Konno*
Department of Biological Engineering, Graduate School of Science and Technology,
Yamagata University, Yonezawa, Yamagata 992-8510, Japan.
Highlights:
Me-CUR is a fluorescent switchable probe to detect amyloid  fibrils with high
sensitivity
Me-CUR can be employed as the detecting agent of Aβ fibrils in vitro experiment
instead of ThT
Me-CUR has high molecular planarity and thus it could strongly bind with the A
fibrils