Rational design of amyloid binding agents based on the molecular rotor motif

Article abstract of DOI:10.1002/cmdc.200900440

New fluorescent probes, designed based on the molecular rotor motif, displayed good affinity for aggregated b-amyloid peptides. The physical and fluorescence properties of these probes can be fine tuned by modifying their chemical structures. These findin

Full text of DOI:10.1002/cmdc.200900440

MED
DOI: 10.1002/cmdc.200900440
Rational Design of Amyloid Binding Agents Based on the Molecular Rotor
Motif
Jeyanthy Sutharsan,[a] Marianna Dakanali,[a] Christina C. Capule,[a] Mark A. Haidekker,[b] Jerry Yang,*[a] and
Emmanuel A. Theodorakis*[a]
Alzheimer’s disease (AD) is characterized by a progressive loss
of cognitive function and constitutes the most common and
fatal neurodegenerative disorder.[1] Genetic and clinical evi-
dence supports the hypothesis that accumulation of amyloid
deposits in the brain plays an important role in the pathology
of the disease. This event is associated with perturbations of
biological functions in the surrounding tissue leading to neuro-
nal cell death, thus contributing to the disease process. The
deposits are comprised primarily of amyloid (Ab) peptides, a
39–43 amino acid sequence that self aggregates into a fibrillar
b-pleated sheet motif. While the exact three-dimensional struc-
ture of the aggregated Ab peptides is not known, a model
structure that sustains the property of aggregation has been
proposed.[2] This creates opportunities for in vivo imaging of
amyloid deposits that can not only help evaluate the time
Figure 1. Structures of selected amyloid imaging reagents.
course and evolution of the disease, but can also allow the
timely monitoring of therapeutic treatments.[3]
Historically, Congo Red (CR) and Thioflavin T (ThT) have pro-
vided the starting point for the visualization of amyloid
plaques and are still commonly employed in post mortem his-
tological analyses (Figure 1).[4] However, due to their charge
these probes are unsuitable for in vivo applications.[5] To ad-
dress this issue, several laboratories developed probes with
noncharged, lipophilic (logP=0.1–3.5) and low-molecular
weight chemical structures (MW<650) that facilitate crossing
of the blood–brain barrier.[6] Further functionalization of these
compounds with radionuclides led to a new generation of
in vivo diagnostic reagents (Figure 1) that target plaques and
related structures for imaging with positron emission tomogra-
phy (PET) and single-photon emission computed tomography
(SPECT).[7] Despite these advances, there is a pressing need for
the design and development of new amyloid-targeting mole-
cules with improved physical, chemical and biological charac-
teristics.[8] At present, identification of new amyloid sensing
molecules is based mainly on modification of existing dyes[9]
and/or screening of libraries of dyes.[10]
donor unit in conjugation with an electron acceptor (d-p-A
motif). This motif is a typical feature in molecular rotors, a
family of fluorescent probes known to form twisted intramo-
lecular charge-transfer (TICT) complexes in the excited state
producing a fluorescence quantum yield that is dependent on
the surrounding environment.[11] Following photoexcitation,
this motif has the unique ability to relax either via fluorescence
emission or via an internal nonradiative molecular rotation.
This internal rotation occurs around the s-bonds that connect
the electron-rich p-system with the donor and acceptor
groups, and can be modified by altering the chemical structure
and microenvironment of the probe.[12] Hindrance of the inter-
nal molecular rotation of the probe by increasing the sur-
rounding media rigidity, or by reducing the available free
volume needed for relaxation, leads to a decrease in the non-
radiative decay rate and consequently an increase in fluores-
cence. In contrast, relaxation proceeds mainly via nonradiative
pathways in environments of low viscosity or of high free
volume. Due to these properties, molecular rotors have been
used to study polarity, free volume and viscosity changes in
solvents and organized assemblies,[13] such as liposomes,[14]
cells[15] and polymers.[16]
Examination of the chemical structures shown in Figure 1 re-
veals that the majority of these probes contain an electron-
[a] J. Sutharsan, Dr. M. Dakanali, C. C. Capule, Prof. Dr. J. Yang,
Prof. Dr. E. A. Theodorakis
Intrigued by the above observations, we asked whether we
could design amyloid-binding agents based on the molecular
rotor motif. We envisioned that p-conjugation of a dialkyl
amino group, as the electron donor (D), with a 2-cyano acry-
late unit, as the electron acceptor (A), would produce Ab-bind-
ing molecules with inherent fluorescence properties.[17] Inter-
estingly, the fluorescence properties of such a motif could be
fine-tuned by modifying the electronic density and extent of
conjugation between the donor and acceptor units. The solu-
bility of these amyloid-binding agents in aqueous media can
Department of Chemistry and Biochemistry, University of California
San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358 (USA)
Fax: (+1)858-822-0386 (ET)
Fax: (+1)858-534-4554 (JY)
E-mail: jerryyang@ucsd.edu
etheodor@ucsd.edu
[b] Prof. Dr. M. A. Haidekker
Faculty of Engineering, University of Georgia, Athens, GA 30602 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900440.
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ChemMedChem 2010, 5, 56 – 60

be achieved by the introduction of water solubilizing groups
(WSG), such as esters of triethylene glycol monomethyl ether
(TEGME) or of glycerol. The design concept is shown in
Figure 2.
Scheme 2. Reagents and conditions: a) 8.0 equiv piperidine in benzene/
HMPA: 1/1, 08C, 8.0 equiv nBuLi, 08C, 15 min, then 1.0 equiv 9, 258C, 12 h,
35%; b) 1.0 equiv 10, 1.1 equiv 7, 0.1 equiv piperidine, THF, 508C, 21 h, 82%.
Figure 2. Design of amyloid-binding agents based on the structure of a
molecular rotor (d-p-A motif).
Key to the synthesis of all probes was a Knoevenagel con-
densation of the appropriate aldehyde, for example, 6, with
the appropriate malonic acid derivative, for example, 7,
(Scheme 1). This reaction was catalyzed by piperidine and was
Scheme 3. Reagents and conditions: a) 1.0 equiv 6a, 1.1 equiv 12, 0.1 equiv
piperidine, THF, 508C, 21 h, 91%; b) 1.5 mmol 13, 0.10 g DOWEX-H+, THF/
CH3OH (1:1), 258C, 20 h, 75%.
ing aldehyde 18 with cyano ester 7 (Scheme 4, 42% combined
yield).
Scheme 1. Reagents and conditions: a) 1.0 equiv 6, 1.1 equiv 7, 0.1 equiv
piperidine, THF, 508C, 21 h.
completed within 21 h in refluxing THF.[18] After a standard
chromatographic purification on silica gel, the desired product
8 was isolated in excellent yields (Table 1).
Table 1. Structures and yields of probes 8a–8d.
Compd
R1
R2
Yield (%)
8a
8b
8c
8d
Me
Me
Et
H
OMe
H
98
98
90
78
Scheme 4. Reagents and conditions: a) 1.0 equiv 15, 15 equiv triethyl phos-
phite, 908C, 19 h, 98%; b) 1.0 equiv 16, 1.0 equiv NaOMe, 1.0 equiv 6a,
excess DMF, 258C, 24 h, 74%; c) 1.0 equiv 17, 1.0 equiv nBuLi, 1.33 equiv
DMF, THF, À788C, 60%; d) 1.0 equiv 18, 1.1 equiv 7, 0.1 equiv piperidine,
THF, 508C, 21 h, 97%.
nBu
H
Naphthalene-based probe 11 was synthesized by treatment
of commercially available methoxy naphthaldehyde 9 with
lithiated piperidine[19] and Knoevenagel condensation of the re-
sulting aldehyde 10 with cyano ester 7 (Scheme 2, 29% com-
bined yield).
An initial study to determine whether a probe can associate
with aggregated Ab is to compare its fluorescence spectra
before and after mixing with the Ab aggregates.[9,10] Typically, a
fluorescent amyloid-binding agent displays a significant fluo-
rescence intensity increase after binding to Ab aggregates as
compared to its native fluorescence in solution.[22] Along these
lines, we measured the fluorescent properties of each probe at
4 mm before and after mixing with preaggregated Ab(1–42)
peptides (5 mm, aggregated in PBS buffer for 3 days at 258C).
In all cases, a 1.3- to 9.4-fold fluorescence intensity increase
was observed in the presence of aggregated Ab, indicating
that these compounds bind to the peptide (Table 2). In most
Compound 14 was prepared by condensation of aldehyde
6a with a-cyano ester 12, followed by an acid-catalyzed de-
protection of the acetonide unit (Scheme 3, 68% combined
yield).[20]
Stilbene-based probe 19 was synthesized in four steps that
included: a) conversion of benzyl bromide 15 to phosphonate
16;[21]; b) Horner–Emmons olefination of 16 with aldehyde 6a
to form 17; c) lithiation of bromide 17 and formylation to pro-
duce aldehyde 18; d) Knoevenagel condensation of the result-
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5.0 mm pre-aggregated Ab(1–42)
peptide. The Kd can be measured
from the double reciprocal of
Table 2. Fluorescence profile, Kd, IC50 and related values for the interaction of the synthesized probes with
aggregated Ab(1–42) peptides.
[b]
[b]
Compd Excitation Maximum[a] [nm] Emission Maximum[a] [nm]
Fold
Kd
R2 Imax
IC50
LogP
the fluorescence maximum (Fmax
)
before
after
before
after
increase [mm]
[%] [mm]
and the concentration of the
probe.[22] All Kd values were mea-
sured between 1.4 and 5.3 mm
(Table 2). It is remarkable that,
despite the structural differen-
ces, these probes display similar
Kd values suggesting that they
bind in a similar fashion to ag-
gregated Ab. Moreover, these
values are similar to the reported
Kd values for ThT (2 mm).[22,26] The
8a
8b
8c
8d
11
439
442
445
432
445
437
312
435
444
442
440
440
434
319
476
478
478
466
462
476
658
470
469
470
468
538
467
638
1.8
1.3
4.2
9.4
9.3
2.2
2.3
2.6 0.93 81 129.0 1.74
5.3 0.99 92
4.8 0.96 98
4.4 0.95 91
2.5 0.98 58
3.3 0.99 79
1.4 0.98 40
1.2 1.54
11.4 2.49
90.6 4.62
74.3 3.81
82.1 1.07
33.6 4.30
14
19
[a] Before or after binding. [b] Maximum percent inhibition (Imax) and IC50 values were determined by ELISA
assay.
cases a modest blue shift (6–20 nm) was observed upon bind-
ing. Only in the case of the naphthalene-based probe 11 was a
significant red shift of 76 nm observed upon binding to preag-
gregated Ab (Figure 3c and d). Interestingly, this binding was
accompanied with a 9.3-fold intensity increase. A similar inten-
sity increase has been observed with FDDNP[23] and may be ex-
plained by the ability of the naphthalene motif to create exci-
mers upon binding to its target.[24] Probes 8a and 8b exhibited
similar fluorescence characteristics suggesting that addition of
a methoxy group on the phenyl group does not alter the bind-
ing properties of the probe. On the other hand, it is worth
noting that increasing the size of the alkyl groups of the nitro-
gen leads to a significant increase in the fluorescence intensity
after binding (Table 2, 8a, 8c, 8d). This is likely a result of the
decreased rotational freedom of the molecules upon binding
to the aggregated forms of Ab peptide.[25] Interestingly, no in-
crease of fluorescence intensity was observed upon mixing of
these probes with monomeric Ab peptide (see Supporting In-
formation). This supports the notion that these probes bind se-
lectively to aggregated forms of Ab. The fluorescence profile of
8d (excitation and emission) is shown in Figure 3a and b.
We also measured the apparent binding constants (Kd) of
the probes (in concentrations of 10, 5, 2.5 and 1.25 mm) to
double reciprocal plot of fluorescence intensity versus concen-
tration of probes 8d and 11 are shown in Figure 4. The Kd cor-
responds to the À1/(x-intercept) of the linear regression.[22]
The association of the synthesized compounds with aggre-
gated Ab peptides was tested using a semi-quantitative ELISA-
based assay developed by Yang and co-workers.[27] The assay is
based on screening for molecules that inhibit the interaction
of the aggregated Ab peptide with a monoclonal anti-Ab IgG
raised against residues 1–17 of Ab. Table 2 shows the concen-
trations of the probes corresponding to 50% inhibition (IC50)
of the IgG-Ab interactions as well as the maximal percentage
of IgG inhibited from binding to the aggregated peptide. All
probes exhibited IC50 values at micromolar levels, the lowest
value being measured for compound 8b (IC50 =1.2 mm). The
maximum inhibition (Imax), a measure of the extent of surface
coating of the aggregated peptide by the probes,[27] was deter-
mined to be between 40–98% (Table 2). Comparison of these
data indicates that the surface coating increases by decreasing
the size of the probe or the extent of the p system. Specifically,
while the maximum inhibition is between 81–98% for the
phenyl compounds, it decreases to 58% for the longer naph-
thalene compound 11 and to 40% for the more conjugated
stilbene
19.
Representative
graphs for 8d and 11 are shown
in Figure 5.
The logP values for all the
compounds were calculated to
be between 1.07 and 4.62
(Table 2)[28] indicating that most
of these probes meet the solu-
bility criteria and should be able
to cross the blood–brain barri-
er.[27,29] Finally, all compounds
showed little or no cytotoxicity
against human neuroblastoma
cells at concentrations up to
100 mm (see Supporting Informa-
tion). These properties represent
significant advantages for further
in vivo evaluation.
In conclusion, inspired by the
structures of the currently used
Figure 3. Fluorescence excitation (a, c) and emission spectra (b, d) of probes 8d (a, b) and 11 (c, d) in aqueous
PBS solution (c) and in the presence of aggregated Ab peptide (b).
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ChemMedChem 2010, 5, 56 – 60

reduces the free volume around the rotor resulting in an in-
creased fluorescence emission.[30] A similar effect has been re-
ported for the binding of molecular rotors to actin, albumin
and other proteins.[31] We have demonstrated that these mole-
cules can be readily synthesized and have no significant cyto-
toxicity. In addition, we have shown that both the physical
properties and fluorescence profile of these probes can be fine
tuned by modifying their chemical structure. Notably, substitu-
ent changes in the electron donor group can affect the intensi-
ty of fluorescence emission, while changes in the p-system can
affect the emission wavelength. These effects can be imple-
mented for the construction of multicolored dyes and can lead
to potential applications for in vitro and in vivo imaging.[32] In-
terestingly, a recent report describes the identification of
CRANAD-2,[33] a small molecule containing two electron-donat-
ing groups connected simultaneously via p-conjugation to a
single difluoroboronate acceptor. This probe has a high affinity
for Ab aggregates (Kd =38.0 nm) and suitable near-infrared
fluorescence properties for in vivo imaging, further validating
our proposed concept of exploring the molecular rotor motif
for the development of new amyloid-imaging probes. These
findings demonstrate that the d-p-A motif of molecular rotors,
presented in Figure 2, is a privileged scaffold and represents
an important first step for the rational design of new diagnos-
tic tools for Alzheimer’s disease and related amyloid-based
neurodegenerative disorders.
Figure 4. Determination of the apparent binding constant (Kd) of probes 8d
2
2
&
^
(
; R =0.95) and 11 ( ; R =0.98) to preaggregated Ab peptide.
Acknowledgements
Financial support by the NSF (CMMI-0652476) and the NIH (grant
1R21RR025358) are gratefully acknowledged. C.C.C. and J.Y. ac-
knowledge support from the Wallace H. Coulter Foundation, the
Alzheimer’s Disease Research Center (NIH 3P50 AG005131), and
the Alzheimer’s Association (NIRG 08-91651).
Keywords: amyloid peptides · fluorescent probes · imaging
agents · molecular rotors
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Received: October 21, 2009
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