Monitoring the aggregation processes of amyloid-β using a spin-labeled, fluorescent nitroxyl radical

Article abstract of DOI:10.1039/c0cc05764a

Amyloid nitroxyl radical (nitroxide) ligands were used to detect amyloid-β fibrils, the main constituents of senile plaques in Alzheimer's disease, using anisotropic ESR spectra, and were found to affect the aggregation process due to the radical functionality. These compounds have great potential as novel and multifunctional probes, combining spin labels, spin probes, and fluorescence probes.

Full text of DOI:10.1039/c0cc05764a

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COMMUNICATION
Monitoring the aggregation processes of amyloid-b using a spin-labeled,
fluorescent nitroxyl radicalw
Fumiya Mito,a Toshihide Yamasaki,a Yuko Ito,a Mayumi Yamato,b Hiroyuki Mino,c
Hiromi Sadasue,a Chisato Shirahama,a Kiyoshi Sakai,b Hideo Utsumib
and Ken-ichi Yamada*a
Received 24th December 2010, Accepted 11th March 2011
DOI: 10.1039/c0cc05764a
Amyloid nitroxyl radical (nitroxide) ligands were used to detect
amyloid-b fibrils, the main constituents of senile plaques in
Alzheimer’s disease, using anisotropic ESR spectra, and were
found to affect the aggregation process due to the radical
functionality. These compounds have great potential as novel
and multifunctional probes, combining spin labels, spin probes,
and fluorescence probes.
formation10 and the interactions of Ab with aggregation
inhibitors.11 The antioxidant properties of nitroxyl radicals
also allow them to be used as spin probes for monitoring of
oxidative processes, as their reaction with ROS and redox
enzymes leads to the loss of paramagnetism.12 Furthermore,
covalent coupling of a nitroxyl radical and a fluorophore
leads to intramolecular quenching of fluorescence emission
by electron exchange interaction.13,14 If the nitroxyl radical
is lost by reaction with free radicals, fluorescence may be
recovered. These fluorophore–nitroxyl radical coupled systems
can therefore act as highly sensitive optical probes for free
radicals.15 Although the unique properties of nitroxyl radicals
have been exploited in a variety of contexts, their use to
simultaneously probe both peptide structural changes and free
radical reactions has not yet been achieved.
Alzheimer’s disease is the most common neurodegenerative
disorder and is characterized by progressive memory loss,
cognitive impairment, and changes in personality. Pathological
features include neuronal loss, neurofibrillary tangles and
senile plaques.1 The principle pathogenic factor is thought to
be oligomers of short peptides (39 to 42 amino acids long),
known as amyloid-b (Ab), which form the major components
of plaques and cause neuronal damage.2 Ab aggregation itself
may be initiated by the formation of various metastable states,
which act as nucleation points.3,4 The overall aggregation
process is characterized by an initial lag time, during which
no detectable aggregation occurs, and by a maximal aggregation
rate at which the proteins are converted into fibrils.5 Reactive
oxygen species (ROS) and free radicals formed during aggregation
appear to play a role in promoting aggregation.6–8 Improving
our understanding of Ab aggregation and the role played by
oxidative stress in this process may aid in the development of
targeted compounds designed to reduce neurotoxicity.
We recently reported a new synthetic route to 2,6-substituted
nitroxyl radicals, whose chemical properties can be tuned by
varying the substituent groups.19–21 If a fluorogenic compound
recognizing Ab fibrils were to be combined with a suitable
nitroxyl radical, it would be anticipated that the nitroxyl
radical would allow simultaneous monitoring of the molecular
mobility of the Ab proteins (by monitoring changes in the
ESR spectrum) and free radical reactions (by monitoring
recovered fluorescence). Hence, we aimed to develop a fluorescent
nitroxyl radical displaying Ab fibril recognition.
For nitroxyl radical 3, piperidin-4-one derivative
1
(Scheme 1) was first prepared by the reaction of 1,2,2,6,6-
pentamethylpiperidin-4-one and 4-bromoacetophenone following
our previously reported synthetic method for 2,6-substituted
nitroxyl radicals.19 The coupling of 1 with 6-methoxy-
benzothiazole was conducted using a direct palladium-catalyzed
arylation. Subsequent oxidation of 2 with hydrogen peroxide
in the presence of sodium tungstate afforded nitroxyl radical 3.
Nitroxyl radical 8 was synthesized by esterification of the
known methylaniline, 7.16–18 Both nitroxyl radicals were deri-
vatized as methoxyamines (R2N–O–Me) using a Fenton like
reaction22 yielding diamagnetic 4 and 9 to allow the role of the
nitroxyl radical to be probed.
Small molecules containing a nitroxyl radical (nitroxide)
with a relatively stable unpaired electron are used as reporters
in biological systems. They can act as ‘‘spin labels’’, reporting
on their microenvironment through changes in their electron
spin resonance (ESR) spectrum.9 Site-directed spin labeling of
Ab has been shown to allow time resolved monitoring of fibril
a Department of Bio-functional Science, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan.
E-mail: yamada@pch.phar.kyushu-u.ac.jp; Fax: +81-92-642-6626;
Tel: +81-92-642-6624
b Innovation Center for Medical Redox Navigation, Kyushu
University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan
c Graduate School of Science, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya, Japan
After producing nitroxyl radicals 3 and 8, their binding
affinities for Ab fibrils were evaluated. The fibrils were
prepared as described in the ESI.w Fluorescence of the
supernatant after incubation of the nitroxyl radicals with
w Electronic supplementary information (ESI) available: Experimental
procedure, spectroscopic data and Fig. S1–S3. See DOI: 10.1039/
c0cc05764a
c
5070 Chem. Commun., 2011, 47, 5070–5072
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sodium dodecyl sulfate solution was added as a denaturing
agent, the anisotropic interaction disappeared (data not
shown). The rotation correlation time tc of nitroxyl radical 3
in the presence of Ab fibrils was 1.4 ꢀ 10ꢁ8 s (estimated
by simulation) indicating that nitroxyl radical 3 binds strongly
to Ab fibrils. For nitroxyl radical 8, the broadened signal
included a sharp component, and tc could not be accurately
simulated. Complex formation with the fibrils would be
expected to cause a decrease in mobility of the radical moiety,
resulting in anisotropic peaks in the ESR spectrum. Nitroxyl
radical 3 showed a particularly notable change in the ESR
spectrum upon binding to fibrils (Fig. 1c).
We tested the effects of the nitroxyl radicals on Ab1-42
aggregation using the thioflavin T (ThT) fluorescence assay.23
No quenching of ThT fluorescence was observed in the
presence of the nitroxyl radicals, indicating that no direct
interaction occurs between the dye and the nitroxyl radicals.
Fig. 2 shows the results of ThT assays measured as a function
of time, with or without nitroxyl radicals 3 and 8. The lag time
t1 and the maximal aggregation rate ka were calculated as
reported previously.5 The lag phase t1 for nitroxyl radicals 3
and 8 was shortened significantly compared with the control
(control, 83.8 min; nitroxyl radical 3, 22.6 min; nitroxyl radical
8, 2.20 min). Nitroxyl radical 3 increased ka by approximately
Scheme
1
Synthesis of nitroxyl radicals and methoxyamine
derivatives. (a) 40-Bromoacetophenone, DMSO, NH4Cl, Triton B; (b)
6-MeO-benzothiazole, CuBr, Pd(OAc)2, P(t-Bu)3, Cs2CO3, DMF; (c)
H2O2, Na2WO4ꢂ2H2O, MeOH, H2O; (d) FeSO4ꢂ7H2O, H2O2, DMSO;
(e)–(g) ref. 16–18; (h) carboxy-PROXYL, EDC, HOBt, Et3N, THF.
fibrils and centrifugation to remove the proteins was observed
and compared with the fluorescence of the fibril-free solution,
to estimate the quantities of fibril-bound nitroxyl radicals.
Both nitroxyl radicals bound dose-dependently to Ab fibrils
(Fig. 1a). The ESR spectra of the nitroxyl radicals in aqueous
solution (Fig. 1b) showed narrow lines indicating rapid motion
of the nitroxyl radical. In the presence of fibrils, the nitroxyl
radical ESR spectra showed significant line broadening,
indicating slow motion of the nitroxyl radicals. Without
nitroxyl radicals, no ESR signal was observed. When 10%
2-fold (control, 0.024 minꢁ1; nitroxyl radical 3, 0.044 minꢁ1
;
nitroxyl radical 8, 0.021 minꢁ1). These results suggest that
both nitroxyl radicals are capable of acting as nucleation sites
decreasing the lag time, and nitroxyl radical 3 can also
influence the elongation phase during fibril formation.
Nitroxyl radical 3 promoted aggregation to form fibrils in
a concentration-dependent manner after incubation with
the Ab peptide for 24 h (Fig. 3). Conversely, nitroxyl radical
8 significantly suppressed aggregation. The methoxyamine
derivatives 4 and 9 of the nitroxyl radicals did not affect
aggregation, suggesting that the radical is responsible for
influencing aggregation (Fig. S1 in the ESIw). 4-Hydroxy-
2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol, up to 100 mM)
did not significantly affect aggregation (data not shown).
These results indicate that nitroxyl radicals 3 and 8 alter the
thermodynamic equilibrium during the aggregation process.24
Interestingly, a 4-fold increase in fluorescence intensity of
nitroxyl radical 3 after incubation with Ab1-42 for 24 h was
observed (Fig. S2 in the ESIw). This recovered fluorescence
results from the loss of the radical to form a diamagnetic
analogue. Monitoring the increase in fluorescence allows redox
Fig. 1 (a) Binding of nitroxyl radicals 3 (K) and 8 (J) to Ab1-42
fibrils. The nitroxyl radicals, at various concentrations, were incubated
in the presence or absence of fibrils (25 mg mLꢁ1) and the extent of
binding was determined as described in the ESI.w (b) ESR spectra at
room temperature in the absence of fibrils. (c) ESR spectra measured
as described in the ESIw, in the presence of fibrils (75 mg mLꢁ1).
Fig. 2 Changes in ThT fluorescence intensity (a.u.) during Ab1-42
aggregation. Ab1-42 alone (E); Ab1-42 in the presence of nitroxyl
radical 3 (K); Ab1-42 in the presence of nitroxyl radical 8 (J).
c
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the fibrils and oxidative stress on one point during aggregation
process although the utilization might be limited.
In conclusion, we have developed a new dual spin labeling
and highly sensitive fluorescent probe that can be used to
investigate changes in the redox state during protein aggrega-
tion. We anticipate that this technology could be extended for
use in understanding protein–lipid and receptor–ligand inter-
actions. The use of spin-labeled, fluorescent nitroxyl radicals
should improve our understanding of amyloid protein
dynamics, allowing therapeutic strategies to be devised for
Alzheimer’s diseases.
Fig. 3 Effects of nitroxyl radicals 3 and 8 on Ab1-42 aggregation, as
measured by ThT fluorescence intensity (%). Ab1-42 (100 mM) was
incubated for 24 h in the presence or absence of each concentration of
nitroxyl radical. *p o 0.001.
This study was partially supported by a Grant-in-Aid for
Young Scientists from the Japan Society for the Promotion of
Science, and a Development of Systems and Technology for
Advanced Measurement and Analysis grant from the Japan
Science and Technology Agency.
conditions around the fibrils to be monitored. In the absence of
fibrils, the fluorescence of compound 4 was about 20-fold higher
than that of nitroxyl radical 3 confirming that the radical is
responsible for fluorescence quenching. On the other hand, the
fluorescence intensity of nitroxyl radical 8 did not change during
the course of Ab aggregation and methoxyamine 9 did not show
significantly higher fluorescence than 8 (Fig. S3 in the ESIw).
These results suggest that the radical moiety in 3 efficiently
interacts with the proximal fluorogenic moiety, but this inter-
action is minimal in 8. Radical generation has been widely
reported during Ab aggregation processes, and the antioxidant
has been found to attenuate aggregation.25 The free radical
activities of nitroxyl radicals 3 and 8 are therefore thought
to alter the redox status by promoting or attenuating Ab
aggregation. These results show that when a radical moiety is
placed in close proximity to a fluorophore in an appropriate
probe (nitroxyl radical 3) it can be used as a fluorescence switch
in the presence of an electron exchange interaction.
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to Ab fibrils. In particular, a straightforward synthetic route to
molecules featuring both Ab recognition sites and ESR-active
nitroxyl radical moieties has been developed. These probes
afforded information regarding environmental changes during
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5072 Chem. Commun., 2011, 47, 5070–5072
This journal is The Royal Society of Chemistry 2011