Self-triggered click reaction in an Alzheimer's disease model:: In situ bifunctional drug synthesis catalyzed by neurotoxic copper accumulated in amyloid-β plaques

Article abstract of DOI:10.1039/c9sc04387j

Cu is one of the essential elements for life. Its dyshomeostasis has been demonstrated to be closely related to neurodegenerative disorders, such as Alzheimer's disease (AD), which is characterized by amyloid-β (Aβ) aggregation and Cu accumulation. It is

Full text of DOI:10.1039/c9sc04387j

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Self-triggered click reaction in an Alzheimer's
disease model: in situ bifunctional drug synthesis
catalyzed by neurotoxic copper accumulated in
amyloid-b plaques†
Cite this: Chem. Sci., 2019, 10, 10343
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
e
a
Zhi Du,ab Dongqin Yu,ac Xiubo Du,d Peter Scott,
Jinsong Ren
a
*
and Xiaogang Qu
Cu is one of the essential elements for life. Its dyshomeostasis has been demonstrated to be closely related
to neurodegenerative disorders, such as Alzheimer's disease (AD), which is characterized by amyloid-b (Ab)
aggregation and Cu accumulation. It is a great challenge as to how to take advantage of neurotoxic Cu to
fight disease and make it helpful. Herein, we report that the accumulated Cu in Ab plaques can effectively
catalyze an azide–alkyne bioorthogonal cycloaddition reaction for fluorophore activation and drug
synthesis in living cells, a transgenic AD model of Caenorhabditis elegans CL2006, and brain slices of
triple transgenic AD mice. More importantly, the in situ synthesized bifunctional drug 6 can disassemble
Ab–Cu aggregates by extracting Cu and photo-oxygenating Ab synergistically, suppressing Ab-mediated
paralysis and diminishing the locomotion defects of the AD model CL2006 strain. Our results
demonstrate that taking the accumulated Cu ions in the Ab plaque for an in situ click reaction can
achieve both a self-triggered and self-regulated drug synthesis for AD therapy. To the best of our
knowledge, a click reaction catalyzed by local Cu in a physiological environment has not been reported.
This work may open up a new avenue for in situ multifunctional drug synthesis by using endogenous
neurotoxic metal ions for the treatment of neurodegenerative diseases.
Received 31st August 2019
Accepted 14th September 2019
DOI: 10.1039/c9sc04387j
rsc.li/chemical-science
harness toxic Cu under pathological conditions for defense
against disease rather than just to chelate Cu.4–6
Introduction
Due to its high efficiency, fast kinetics, and mild reaction
conditions, the Cu(I)-catalyzed azide–alkyne cycloaddition
(CuAAC) reaction has received special attention in many
biochemical applications such as bioconjugation,7–11 biosens-
ing,12–14 and drug synthesis.15,16 Many kinds of Cu sources are
efficient for CuAAC bioorthogonal reactions. Besides typical
Cu(I) complexes and the reduction of Cu(II) complexes,17 various
Cu-containing nanoparticles (NPs) are increasingly used for
CuAAC reactions, including Cu, Cu2O and CuO NPs,18,19 Cu-
immobilized inorganic and polymer NPs,20–22 as well as Cu-
doped semiconductor NPs.23,24
Cu is indispensable to life and plays an important role in energy
generation, signal transduction, cellular respiration and anti-
oxidant defense.1 The uctuation of Cu is associated with a wide
range of diseases, including genetic diseases like Menkes and
Wilson's diseases; neurodegenerative diseases such as Alz-
heimer's, Parkinson's, Huntington's, and prion diseases;
metabolic disorders and even cancer.1,2 For diseases related to
Cu accumulation, the most common treatment strategy is the
direct use of Cu-chelators. However, systemic administration of
chelating agents may cause adverse side effects because of
indiscriminate metal chelation.3 It is a promising strategy to
A major drawback of the CuAAC reaction in biological
systems is Cu-triggered reactive oxygen species (ROS) from
Fenton's reaction.25 An additional but usually neglected side
effect is that the addition of exogenous Cu-catalysts might
disturb Cu homeostasis in physiological milieu. In terms of
intracellular chemistry, a click reaction using endogenous Cu as
the catalyst holds a special attraction.
Alzheimer's disease (AD), affecting about y million people
worldwide, is suggested to be strongly correlated to amyloid-
b (Ab) aggregation and metal dysregulation.26–28 The typical
concentration of Cu in Ab plaques reaches up to 0.4 mM, while
aLaboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource
Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun, Jilin 130022, China. E-mail: xqu@ciac.ac.cn
bUniversity of Chinese Academy of Sciences, Beijing 100039, China
cUniversity of Science and Technology of China, Hefei, Anhui 230029, China
dCollege of Life Sciences and Oceanography, Shenzhen Key Laboratory of Microbial
Genetic Engineering, Shenzhen University, Shenzhen, 518060, China
eDepartment of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL,
UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9sc04387j
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the amount of Cu in a normal brain is 0.2–1.7 mM.29,30 Inspired is delocalized to the triazole and the uorescence is sharply
by an aspartate-coordinated Cu catalyst that can accelerate the enhanced.
click reaction at low parts per million levels,31 herein we have
For catalysis of the click reaction in vitro, Ab40–Cu aggre-
found that Ab–Cu aggregates can catalyze the CuAAC reaction in gates (10 mM), ascorbic acid (AA, 50 mM) as a reducing agent,
cells, an AD model of Caenorhabditis elegans (C. elegans) and the and compounds 1 (50 mM) and 2 (50 mM) were mixed at room
brain slices of triple transgenic AD (3 ꢀ Tg-AD) mice. To the temperature. As expected, Ab40–Cu aggregates efficiently cata-
best of our knowledge, a click reaction catalyzed by local Cu in lyzed the uorogenic click reaction with a 51-fold uorescence
the physiological environment has not been reported. Our work increase (Fig. 1b). Calculated from the uorescence intensity,
will promote the application of bioorthogonal chemistry in the conversion efficiency was about 96% (Fig. 1c). Moreover, the
living systems, and may open up a new avenue for in situ CuAAC reaction for different ratios of Ab40 to Cu was explored.
multifunctional drug synthesis using endogenous neurotoxic As shown in Fig. S2,† the conversion efficiency was almost the
metal ions for the treatment of neurodegenerative diseases.
same for different ratios of Ab40 to Cu. The result veried that
the Ab40–Cu species, rather than free Cu, is responsible for the
CuAAC reaction. This conclusion was further supported by the
data shown in Fig. S3.† Ab42–Cu aggregates were also prepared
and characterized by AFM (Fig. S4†). As shown in Fig. S5,†
Ab42–Cu aggregates can efficiently catalyze the CuAAC reaction.
Inspired by the above results, the performance of Ab40–Cu
for catalyzing the uorogenic click reaction in living cells was
explored. PC12 cells were treated with Ab40–Cu aggregates (10
mM) for 12 h before compound 1 (50 mM), 2 (50 mM), and AA (50
mM) were incubated with the cells for a further 12 h. Flow
cytometry (Fig. 1d and e) in conjunction with the uorescence
images (Fig. S6†) validated that Ab40–Cu could activate the
prouorophore. Mass spectrometry (MS, Fig. S7†) further veri-
ed the feasibility of Ab40–Cu aggregates as a Cu catalyst for the
CuAAC reaction in living cells. It has been reported that large Ab
aggregates are able to enter cells via macropinocytosis.34–36
Therefore, we conducted an immunouorescent staining
experiment to investigate whether Ab40–Cu aggregates can
enter cells. As shown in Fig. S8,† PC12 cells incubated with Ab
aggregates show noteworthy reactivity to Ab bril antibody
mOC78, which means that Ab–Cu aggregates can attach to the
cytomembrane or enter cells.
Results and discussion
Ab40–Cu aggregates were prepared by incubating Ab40 (100 mM)
with equimolar CuCl2 at 37 ꢁC for 8 h and then characterized by
atomic force microscopy (AFM, Fig. S1†). A dialysis experiment
demonstrated that 97% of Cu was bound to Ab40, analyzed by
inductively coupled plasma mass spectrometry (ICP-MS, Table
S1†). Ab–Cu aggregates were used as Cu-accumulated Ab plaque
mimics to catalyze the click reaction, and conversion of non-
uorescent coumarin 1 into uorescent 3 was chosen as the
model reaction (Fig. 1a). The azido group of 1 is capable of
quenching the uorescence via internal charge transfer
(ICT).32,33 Upon reaction with alkyne 2, the lone pair of the azide
We further assessed the feasibility of an Ab–Cu-catalyzed
click reaction in a transgenic AD model of C. elegans CL2006,
which expresses Ab1–42 in muscle cells. Firstly, CL2006 worms
were cultured in nematode growth medium (NGM) containing
1 mM CuCl2 for 24 h to form Cu-accumulated Ab plaques.37 The
Cu-treated worms were then transferred to the NGM containing
compound 1 (1 mM), 2 (1 mM), and AA (1 mM). As shown in
Fig. S9 and S10,† a click reaction was promoted in a Cu-treated
AD model of CL2006 worms aer 12 h.
Organotypic brain slice cultures largely preserve brain
architecture and possess major disease features, providing
a practical platform for direct medication and real-time moni-
toring therapeutic effects.38,39 Therefore, this technology is
widely used for studying neurodegenerative diseases. In addi-
tion, it has been observed that copper levels are elevated in
senile plaques, the hippocampus, and cortex of AD model
mice.40–42 Herein, we also investigated whether the click reaction
Fig. 1 Ab40–Cu-catalyzed fluorogenic click reaction. (a) Scheme of
the click reaction to synthesize fluorescent probe 3. (b) Fluorescence
spectra at different times of the reaction medium in HEPES buffer (pH can be catalyzed by Cu-accumulated plaques in the brain slices
of triple transgenic AD (3 ꢀ Tg-AD) model mice.43 Aer incu-
7.4). (c) Normalized fluorescence intensity at 475 nm versus the
reaction time (lex ¼ 405 nm). The fluorescence intensity of 3 (50 mM)
bation for 12 h, the uorescence of the brain slices was much
was defined as 100%. (d) Flow cytometry detection of synthesized 3 in
more intense for 3 ꢀ Tg-AD mice than for normal mice, which
PC12 cells. (e) Mean fluorescence analysis of (d). PC12 cells incubated
demonstrated that the local Cu in brain slices from 3 ꢀ Tg-AD
with 3 (50 mM) were used as the control. Each experiment was
repeated three times. Error bars indicate ꢂ standard deviation (s.d.).
mice can catalyze the CuAAC click reaction (Fig. S11 and S12†).
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Repeated systemic administration is oen hindered by
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absent target specicity and unpredictable toxicity in non-
targeted tissue.44,45 Drugs may also suffer from rapid deactiva-
tion, poor pharmacokinetics, and low tissue absorption.46 In situ
drug synthesis within living cells or organisms through targeted
bioorthogonal catalysis may overcome the above mentioned
drawbacks because it can greatly improve drug concentration at
desired sites and reduce off-target effects.47–52 We speculated
that in situ drug synthesis through CuAAC reaction would
probably mitigate the cytotoxicity caused by the Ab–Cu aggre-
gates. Recent studies have indicated that the aggregation
properties and cytotoxicity of Ab are closely related to the
hydrophobicity of the protein.53,54 Photo-oxygenating Ab is
capable of disintegrating Ab aggregates through increasing the
protein polarity, which is an attractive strategy for AD
therapy.55–58 However, in the presence of Cu, just oxygenating Ab
may not entirely block its brillation because Cu can still
promote protein aggregation by coordinating to Ab. Consid-
ering that Cu and Ab are two critical pathogenic factors of
AD,59,60 we envisioned that in situ synthesized bifunctional drug
agent with Ab-oxygenating and Cu-chelating properties could
effectively disassemble Ab–Cu aggregates in vivo.
To this end, compound 6 was rationally designed based on
the following considerations: (1) thioavin T (ThT) and its
analogues can selectively photo-oxygenate amyloid aggre-
gates.61,62 Without binding to b-sheet aggregates, photo-excited
ThT undergoes twisted intramolecular charge transfer and
transforms light energy to heat; (2) propargylglycine 5 has oen
been used for synthesizing transition metal chelators through
a “click-to-chelate” approach.63,64 Therefore, the bifunctional
compound 6 was constructed by integrating an Ab-specic
photosensitizer with a Cu prochelator (Fig. 2a).
Spectral titration6 and isothermal titration calorimetry (ITC)
experiments65–67 were carried out to measure the binding stoi-
chiometry (n) and binding affinity of compound 6 with Cu. Aer
adding CuCl2, the absorbance of compound 6 at 321 nm was
slightly decreased (Fig. S13†). A breakpoint was observed at
a ratio of 0.5 : 1, illustrating the binding stoichiometry of Cu
and 6 was 1 : 2. For ITC assays, a weaker competitor glycine was
added to prevent the precipitation of Cu. As shown in Fig. 2b
and c, without considering the competing chelation of glycine,66
apparent binding constants (Ka) of Cu to Ab40 and compound 6
were 9.64 ꢀ 105 Mꢃ1 and 7.21 ꢀ 106 Mꢃ1, respectively. The
affinity of compound 6 to Cu was appropriate to disrupt Ab–Cu
interaction (Table S1†), without affecting the normal function of
the metal enzymes.67 The binding affinity between compound 5
and Cu was too low to detect by ITC, showing that prop-
argylglycine and glycine have equivalent chelating ability to Cu.
Fig. S14† indicates that chelating Cu slightly decreases the
uorescence of compound 6.
Fig. 2 Cu-chelating properties of 6. (a) Scheme of the synthesis of the
bifunctional compound 6. (b and c), ITC data of CuCl2 binding to Ab40
and compound 6, respectively. The bottom panels show the fitting
results. Details are described in the Experimental section.
produced more 1O2 in a glycerol/HEPES mixed solution
(Fig. S15†).55,61 Ab40 (10 mM) and compound 6 (20 mM) were
irradiated with UV light (1 W cmꢃ2) for 1 h and then photo-
oxygenated Ab was detected by MS. Fig. S16† illustrates that
both compounds 4 and 6 can effectively oxygenate Ab40.
Ab aggregation kinetics were measured with Nile Red (NR),
a widely used dye for studying bril formation.69 As shown in
Fig. S17,† the effect of ThT analogues 4 and 6 on NR uores-
cence is negligible. Fig. S18† demonstrates that 4 and 6 can
inhibit Ab40 aggregation under light illumination. 8-
Anilinonaphthalene-1-sulfonate (ANS), the uorescence of
which is strongly enhanced in the presence of the hydrophobic
protein, was also applied to monitor Ab brillation (Fig. S19†).
ANS uorescence was greatly enhanced for native Ab40 aer
incubation for 7 days, in contrast to the slight uorescence
change for photo-oxygenated Ab40. This implies that photo-
oxygenation decreased Ab40 brillation and, possibly, toxicity,
through increasing protein polarity.56
The inhibition effect of compound 6 on Cu-accelerated Ab40
aggregation was then evaluated. Aer UV light (1 W cmꢃ2
)
irradiation for 1 h, Ab40 monomer (10 mM) and Cu (10 mM) were
incubated with 2 equiv. of 4 or 6 for 24 h. Then, NR assays,
circular dichroism (CD), and AFM were conducted to monitor
Ab40 aggregation. As the incubation period progressed, Cu
induced Ab40 to form b-sheet-rich brils, while compound 6
blocked this process (Fig. 3). However, due to the lack of a Cu-
chelating moiety, compound 4 could not inhibit Cu-induced
Ab40 aggregation.
We then assessed the ability of compound 6 to photo-
oxygenate Ab and inhibit its aggregation. 1,3-Diphenyliso-
benzofuran (DPBF)68 was used to detect singlet oxygen (1O2)
generated by photo-excited compound 6. Glycerol, which can
increase the solution viscosity, was used to restrict the bond
rotation of the ThT analogue 6. In contrast to 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES) buffer, compound 6
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photo-activated 6 was able to reduce Cu–Ab aggregates in brain
slices. The immunouorescence study further conrmed the
effectiveness of clearing Ab plaques using photo-excited
compound 6 (Fig. S26†).39
We next investigated whether Ab40–Cu aggregates could act
as a catalyst for the in situ synthesis of the bifunctional drug 6 in
cells. It has been proven that silica nanoparticles can penetrate
the blood–brain barrier (BBB) by inducing tight junction loss
and cytoskeleton arrangement.70,71 Moreover, certain types of
silica-based nanoparticles have been approved by the Food and
Drug Administration (FDA) for clinical use.72 Herein, H2O2-
responsive mesoporous silica nanoparticles (MSN) were used as
a controlled-release nanocarrier to deliver the prodrugs to
desired sites.73,74 Firstly, phenylboronic acid (BA) was modied
on MSN and then MSN–BA was loaded with the prodrugs (4, 5,
and AA). Immunoglobulin G (IgG) acted as a capping agent and
the formed boronates were sensitive to H2O2.75 The successful
fabrication of MSN–IgG was validated by transmission electron
microscopy (TEM, Fig. S27†), nitrogen adsorption–desorption
isotherms (Fig. S28, Table S3†), Fourier transform infrared
spectroscopy (FTIR, Fig. S29†), and thermogravimetric analysis
(Fig. S30†). As shown in Fig. S31,† MSN–IgG efficiently released
the loaded prodrug 4 when treated with 0.5 mM H2O2. It has
been proved that H2O2 level is remarkedly elevated in AD
because of Ab–Cu76–78 and H2O2 levels in partial area of cells can
be 1 mM.79 Table S4† shows that MSN can enter the cells.
To evaluate the therapeutic potential of in situ synthesized
drug 6 in cells, PC12 cells were incubated with Ab40–Cu
aggregates and prodrug-loaded MSN–IgG. As shown in Fig. 4a,
Fig. 3 Inhibition of Cu-induced Ab40 aggregation. (a) Scheme of the
interruption of Cu-induced Ab40 fibrillation with compound 6 under
light illumination. (b) Fibrillation kinetics of Ab–Cu alone or in the
presence of compounds 4 and 6, monitored by NR. Each experiment
was repeated three times. Error bars indicate ꢂ s.d. (c) CD spectra of
Ab40–Cu alone or in the presence of compounds 4 and 6. (d)
Secondary structure analysis of Ab40–Cu alone or in the presence of
compounds 4 and 6. (e) AFM images of Ab40–Cu alone or in the
presence of compounds 4 and 6. Scale bars: 500 nm.
Furthermore, photo-activated compound
6 could also
dissociate Ab40–Cu aggregates. As shown in Fig. S20,† Ab40–Cu
aggregates further assembled into long brils aer incubation
for 24 h. However, NR uorescence gradually decreased for
Ab40–Cu aggregates when incubated with compound 6, indi-
cating that the Ab40–Cu aggregates were dissociated over the
course of time in the presence of compound 6. AFM and ANS
assays further supported that compound 6 dissociates Ab40–Cu
aggregates (Fig. S20†). Compound 6 also inhibits and disas-
sembles Cu-induced Ab42 aggregates (Fig. S21 and S22†).
Given the above results, we assessed the neuroprotective
effect of compound 6 on the cytotoxicity triggered by Ab40–Cu
aggregates. Immunouorescence analysis showed the co-
localization of compound 6 and anti-Ab antibodies in PC12
cells (Fig. S23†). This implied that photo-activated compound 6
reduces non-specic oxygenation to lipid, DNA or other proteins
in the cells.61 Methyl thiazolyl tetrazolium (MTT) assays revealed
that 6 protects against Ab–Cu-mediated cellular toxicity under
light illumination (Fig. S24†).
Fig. 4 In situ synthesis of bifunctional compound 6 for mitigating
Ab40–Cu-induced cytotoxicity. (a) Overview of the in situ synthesis of
compound 6 for disassembling Ab40–Cu aggregates. (b) Microscope
images of the normal PC12 cells and Ab40–Cu-treated PC12 cells in
the presence or absence of prodrug-loaded MSN. Scale bars: 20 mm.
(c) MTT assays. Each experiment was repeated three times. Error bars
indicate ꢂ s.d. PC12 cells were incubated with Ab40–Cu aggregates
(10 mM) and prodrug-loaded MSN–IgG (0.1 mg mLꢃ1) for 24 h and then
irradiated with UV light (1 W cmꢃ2) for 10 min in 20 min intervals 6
times. Cells were incubated for 24 h after the light irradiation and then
MTT assays were carried out.
We further evaluated the effectiveness of disassembling Ab–
Cu aggregates by 6 in brain slices of 3 ꢀ Tg-AD mice. Ab plaques
were labeled with NR to monitor the therapeutic effects. As
shown in Fig. S25,† many Ab aggregates could be observed in
the hippocampus and cortex. For brain slices treated with
compound 6 and UV light, the uorescence of NR became weak
aer incubating for 12 h (Fig. S25, Table S2†), indicating that
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prodrug-loaded MSN–IgG was responsive to H2O2 induced by Ab. Subsequently, it was conrmed that MSN can access worm
Ab40–Cu aggregates,75 and the prodrugs (4, 5, and AA) were tissue (Table S5†). As shown in Fig. 5, Ab–Cu aggregates induce
released in the cells. Then Ab40–Cu aggregates catalyzed the AD model CL2006 strain paralysis, reducing the lifespan, and
click reaction and compound 6 was in situ synthesized, as behavioral defects,37,83 while the in situ synthesized drug 6
veried by MS (Fig. S32†). Moreover, the yield of compound 6 decreases Ab deposits, rescues Ab-associated paralysis and
increased upon an increase in the concentration of Ab–Cu improves the motility of the CL2006 strain.
(Fig. S33†). In this regard, in situ drug synthesis without the use
of the exogenous Cu-catalyst could be self-triggered and self-
regulated by the level of Ab–Cu aggregates in cells.
Conclusion
As shown in Fig. 4b and c, Ab40–Cu aggregates destroyed the
cell morphology and caused cell death. In contrast, the in situ
synthetic drug 6 restored PC12 cells to their normal morphol-
ogies and improved the cell viability from 37% to 82%.
However, compound 4 did not display similar neuroprotective
activity. Collectively, our results show that compound 6 miti-
gates the cytotoxicity induced by Ab–Cu aggregates as a conse-
quence of chelating Cu and photo-oxygenating Ab.
The AD model C. elegans has been widely used to enhance
the understanding of the molecular pathogenesis and identify
promising therapeutic strategies.80,81 It has been reported that
ThT rescues Ab-mediated paralysis and increases the longevity
of transgenic worms,82 and Cu-chelator clioquinol (CQ)
ameliorates Ab–Cu toxicity to C. elegans.83 As shown in Fig. S34,†
photo-excited compound 6 can prolong the lifespan of C. ele-
gans CL2006. We then explored whether the in situ synthesis of
bifunctional compound 6 can suppress worm paralysis trig-
gered by Ab–Cu aggregates. CL2006 worms were cultured in
NGM containing 1 mM CuCl2 for 24 h and then Cu-treated
worms were transferred to NGM containing prodrug-loaded
MSN–IgG (1 mg mLꢃ1) for a further 24 h.
In summary, the CuAAC click reaction has been realized by
endogenous Cu in vivo, instead of using exogenous Cu-catalysts.
We have demonstrated that Ab–Cu aggregates can catalyze
a click reaction in cells, an AD model of C. elegans and brain
slices of 3 ꢀ Tg-AD mice. The reaction is self-triggered and
regulated by the level of Ab–Cu aggregates. Harnessing the
synergy of photo-oxidizing Ab and chelating Cu, the in situ
synthesized drug 6 can dissociate Ab–Cu aggregates, reduce Ab
burden, suppress Ab-mediated paralysis and diminish the
locomotion defects of the AD model CL2006 strain. Our work
provides new insights into in situ drug synthesis against AD
using bioorthogonal chemistry.
Experimental section
Synthesis of compound 4
A mixture of 2-(4-aminophenyl)-6-methyl-1,3-benzothiazole (1
mmol) and NaNO2 (1.2 mmol) in 5 M HCl (10 mL) was stirred at
4
ꢁC for 30 min. Then, a NaN3 (4 mmol) solution was added
dropwise and the mixture was stirred for 2 h at 4 ꢁC. Aer
adjusting the pH to 7, the resulting yellow precipitate was
ltered off and washed with water, affording compound 4
As shown in Fig. S35 and S36,† compound 6 was synthesized
in Cu-treated CL2006 worms and 6 was able to co-localize with
1
(189 mg, 71%). H NMR (600 MHz, CDCl3), d (ppm): 8.08–8.03
(m, 2H), 7.93 (d, J ¼ 8.3 Hz, 1H), 7.68 (s, 1H), 7.33–7.28 (m, 1H),
7.14–7.09 (m, 2H), 2.50 (s, 3H). MS (ESI) m/z: [M + H]+ calcd for
C
14H10N4S, 267.3; found, 267.2.
Synthesis of compound 6
Compound 4 (1 mmol), 5 (1.1 mmol), CuSO4 (0.1 mmol) and AA
(0.2 mmol) were added to a solution of H2O and ethanol (1 : 1,
ꢁ
20 mL) and stirred in the dark at 37 C for 24 h. Then, ethyl-
enediaminetetraacetic acid disodium salt (EDTA, 0.3 mmol) was
added and the mixture was stirred for 1 h to chelate Cu. The
ethanol was then removed under reduced pressure and the pH
of the solution was adjusted to 7. The resulting yellow precipi-
tate was ltered off and washed with saturated salt water,
affording compound 6 as a yellow solid (345 mg, 91%). 1H NMR
(600 MHz, D2O/DCl), d (ppm): 7.15 (s, 1H), 6.34 (d, J ¼ 8.3 Hz,
2H), 6.19 (d, J ¼ 8.3 Hz, 2H), 5.98 (d, J ¼ 9.1 Hz, 2H), 5.60 (d, J ¼
8.5 Hz, 1H), 2.86 (t, J ¼ 6.4 Hz, 1H), 1.91 (qd, J ¼ 15.9 Hz, 2H),
0.46 (s, 3H). MS (ESI) m/z: [M ꢃ H]ꢃ calcd for C19H17N5O2S,
378.4; found, 378.4.
Fig. 5 The in situ synthesis of compound 6 for suppressing the toxicity
induced by Ab–Cu fibrils in worms. (a) Representative ThT-staining
images of N2 and Cu-treated CL2006 in the presence or absence of
prodrug-loaded MSN on the 6th day. Scale bars: 40 mm. (b) Survival
curves. (c) Quantification of the worm movement in M9 buffer (turns
per minute). Each experiment was repeated three times. Error bars Peptide preparation
indicate ꢂ s.d. Cu-treated worms were transferred to NGM containing
Ab1–40 and Ab1–42 were purchased from ChinaPeptides Co.,
prodrug-loaded MSN–IgG (1 mg mLꢃ1) for 24 h before worms were
irradiated with UV light (1 W cmꢃ2) for 10 min in 20 min intervals 6
times. A wild-type N2 strain was used as the control.
Ltd. The peptides were dissolved in hexauoroisopropanol
ꢁ
(HFIP) and stored at ꢃ20 C as the stock solution. Before use,
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the solvent HFIP was evaporated under a gentle stream of
nitrogen and Ab was re-dissolved in HEPES buffer (20 mM, pH
7.4). For preparing Ab40–Cu aggregates, 100 mM Ab40 was
incubated with an equimolar amount of CuCl2 at 37 C for 8 h
and then diluted to the corresponding concentration before
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (21431007, 21533008, 21820102009, 21871249,
91856205) and the Frontier Science Key Program of CAS (QYZDJ-
SSW-SLH052) and 20190701028GH from Jilin Province.
ꢁ
use. For preparing Ab42–Cu aggregates, 100 mM of Ab42 was
ꢁ
incubated with an equimolar amount of CuCl2 at 37 C for 4 h
and then diluted to the corresponding concentration before
use. The Ab–Cu species were dialyzed to remove any free Cu
before the click reaction.
References
1 J. A. Cotruvo Jr, A. T. Aron, K. M. Ramos-Torres and
C. J. Chang, Chem. Soc. Rev., 2015, 44, 4400–4414.
2 Y. Liu, Q. Su, M. Chen, Y. Dong, Y. Shi, W. Feng, Z. Y. Wu and
F. Li, Adv. Mater., 2016, 28, 6625–6630.
ITC measurements
ITC experiments were carried out on a NANO ITC System (TA
Instruments Inc., USA) in HEPES buffer (20 mM, pH 7.4) with
stirring at 300 rpm at 25 ꢁC. Ab or compound 6 was placed in the
cell and the CuCl2 solution was loaded via a syringe. To prevent
the precipitation of Cu2+, 4 equiv. of glycine were added to the
CuCl2 solution. All solutions were degassed for 20 min before
titration. The apparent binding constant (Ka), binding enthalpy
(DH), and binding stoichiometry (n) were determined by tting
ITC data following information from the literature without
considering the competing effects of glycine with Cu2+.66,67
When Cu2+–glycine was taken into consideration, the Ka values
of Cu to Ab and compound 6 were 1.63 ꢀ 109 Mꢃ1 and 1.04 ꢀ
1010 Mꢃ1, respectively.65 For binding between Ab and Cu2+, [Ab]
¼ 20 mM, [Cu2+] ¼ 200 mM. For binding between 6 and Cu2+, [6]
¼ 40 mM, [Cu2+] ¼ 200 mM.
3 Q. Wang and K. J. Franz, Acc. Chem. Res., 2016, 49, 2468–
2477.
4 W. Wu, L. Yu, Q. Jiang, M. Huo, H. Lin, L. Wang, Y. Chen and
J. Shi, J. Am. Chem. Soc., 2019, 141, 11531–11539.
5 S. Bakthavatsalam, M. L. Sleeper, A. Dharani, D. J. George,
T. Zhang and K. J. Franz, Angew. Chem., Int. Ed., 2018, 57,
12780–12784.
6 J. Geng, M. Li, L. Wu, J. Ren and X. Qu, J. Med. Chem., 2012,
55, 9146–9155.
7 J. F. Reuther, J. L. Dees, I. V. Kolesnichenko, E. T. Hernandez,
D. V. Ukraintsev, R. Guduru, M. Whiteley and E. V. Anslyn,
Nat. Chem., 2018, 10, 45–50.
8 Y. Sun, S. Hong, R. Xie, R. Huang, R. Lei, B. Cheng, D. E. Sun,
Y. Du, C. M. Nycholat, J. C. Paulson and X. Chen, J. Am. Chem.
Soc., 2018, 140, 3592–3602.
9 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009,
48, 6974–6998.
CD measurements
CD spectra were collected at 37 ꢁC at a speed of 5 nm minꢃ1 over
the wavelength range from 200 to 250 nm. The secondary
structure was analyzed using the BeStSel program (http://
bestsel.elte.hu/).84
10 H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003, 8,
1128–1137.
11 J. Li and P. R. Chen, Nat. Chem. Biol., 2016, 12, 129–137.
12 Y. Zeng, J. Q. Ren, A. G. Shen and J. M. Hu, J. Am. Chem. Soc.,
2018, 140, 10649–10652.
13 G. Liu, G. Shi, H. Sheng, Y. Jiang, H. Liang and S. Liu, Angew.
Chem., Int. Ed., 2017, 56, 8686–8691.
14 W. Zheng, H. Li, W. Chen, J. Zhang, N. Wang, X. Guo and
X. Jiang, Small, 2018, 14, e1703857.
C. elegans experiments
Transfected C. elegans strain CL2006 constitutively expresses
human Ab1–42 in muscle tissues and thus displays an age-
related progressive defect of muscle-specic motility. CL2006
and wild-type (N2) strain were fed with E. coli (OP50) and
cultured at 20 ꢁC in NGM. For worm paralysis assay, N2 and
CL2006 worms were cultured at 20 ꢁC on new NGM plates
(about 50 worms per plate) aer egg synchronization. Worms
were classied as paralyzed if they failed to respond to a touch-
provoked movement. For ThT-staining experiments, worms
were xed with 4% paraformaldehyde for 24 h at 4 ꢁC and
rendered permeable in permeabilization buffer (1% Triton X-
100, 5% b-mercaptoethanol) for a further 24 h at 37 C. Aer
washing with PBS, the worms were immersed in ThT solution (2
mM) for 5 min. Aer washing with PBS, the worms were
mounted on slides and observed using
microscope.
15 D. S. Tyler, J. Vappiani, T. Caneque, E. Y. N. Lam, A. Ward,
O. Gilan, Y. C. Chan, A. Hienzsch, A. Rutkowska,
T. Werner, A. J. Wagner, D. Lugo, R. Gregory, C. Ramirez
Molina, N. Garton, C. R. Wellaway, S. Jackson,
L. MacPherson, M. Figueiredo, S. Stolzenburg, C. C. Bell,
C. House, S. J. Dawson, E. D. Hawkins, G. Drewes,
R. K. Prinjha, R. Rodriguez, P. Grandi and M. A. Dawson,
Science, 2017, 356, 1397–1401.
16 I. Glassford, C. N. Teijaro, S. S. Daher, A. Weil, M. C. Small,
S. K. Redhu, D. J. Colussi, M. A. Jacobson, W. E. Childers,
B. Buttaro, A. W. Nicholson, A. D. MacKerell Jr,
B. S. Cooperman and R. B. Andrade, J. Am. Chem. Soc.,
2016, 138, 3136–3144.
ꢁ
a uorescence
17 B. J. Adzima, Y. Tao, C. J. Kloxin, C. A. DeForest, K. S. Anseth
and C. N. Bowman, Nat. Chem., 2011, 3, 256–259.
18 M. B. Gawande, A. Goswami, F. X. Felpin, T. Asefa, X. Huang,
R. Silva, X. Zou, R. Zboril and R. S. Varma, Chem. Rev., 2016,
116, 3722–3811.
Conflicts of interest
There are no conicts to declare.
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¨
19 F. Alonso, Y. Moglie and G. Radivoy, Acc. Chem. Res., 2015, 44 M. Hoop, A. S. Ribeiro, D. Rosch, P. Weinand, N. Mendes,
48, 2516–2528.
F. Mushtaq, X.-Z. Chen, Y. Shen, C. F. Pujante,
´ ˆ
J. Puigmartı-Luis, J. Paredes, B. J. Nelson, A. P. Pego and
20 A. W. Cook, Z. R. Jones, G. Wu, S. L. Scott and T. W. Hayton,
J. Am. Chem. Soc., 2018, 140, 394–400.
´
S. Pane, Adv. Funct. Mater., 2018, 28, 1705920.
21 J. Chen, J. Wang, Y. Bai, K. Li, E. S. Garcia, A. L. Ferguson and 45 Y. Zheng, X. Ji, B. Yu, K. Ji, D. Gallo, E. Csizmadia, M. Zhu,
S. C. Zimmerman, J. Am. Chem. Soc., 2018, 140, 13695–13702.
22 C. Deraedt, N. Pinaud and D. Astruc, J. Am. Chem. Soc., 2014,
136, 12092–12098.
M. R. Choudhury, L. K. C. De La Cruz, V. Chittavong,
Z. Pan, Z. Yuan, L. E. Otterbein and B. Wang, Nat. Chem.,
2018, 10, 787–794.
23 B. Wang, J. Durantini, J. Nie, A. E. Lanterna and J. C. Scaiano, 46 Y. Li, Q. Zou, C. Yuan, S. Li, R. Xing and X. Yan, Angew.
J. Am. Chem. Soc., 2016, 138, 13127–13130. Chem., Int. Ed., 2018, 57, 17084–17088.
24 J. C. Bear, N. Hollingsworth, P. D. McNaughter, A. G. Mayes, 47 F. Wang, Y. Zhang, Z. Liu, Z. Du, L. Zhang, J. Ren and X. Qu,
M. B. Ward, T. Nann, G. Hogarth and I. P. Parkin, Angew.
Chem., Int. Ed., 2014, 53, 1598–1601.
25 J. Clavadetscher, S. Hoffmann, A. Lilienkampf, L. Mackay,
Angew. Chem., Int. Ed., 2019, 58, 6987–6992.
48 Y. Bai, J. Chen and S. C. Zimmerman, Chem. Soc. Rev., 2018,
47, 1811–1821.
R. M. Yusop, S. A. Rider, J. J. Mullins and M. Bradley, 49 A. M. Perez-Lopez, B. Rubio-Ruiz, V. Sebastian, L. Hamilton,
Angew. Chem., Int. Ed., 2016, 55, 15662–15666.
26 P. C. Ke, M. A. Sani, F. Ding, A. Kakinen, I. Javed,
F. Separovic, T. P. Davis and R. Mezzenga, Chem. Soc. Rev.,
2017, 46, 6492–6531.
C. Adam, T. L. Bray, S. Irusta, P. M. Brennan, G. C. Lloyd-
Jones, D. Sieger, J. Santamaria and A. Unciti-Broceta,
Angew. Chem., Int. Ed., 2017, 56, 12548–12552.
50 T. Volker and E. Meggers, Curr. Opin. Chem. Biol., 2015, 25,
48–54.
27 I. W. Hamley, Chem. Rev., 2012, 112, 5147–5192.
28 A. Nakamura, N. Kaneko, V. L. Villemagne, T. Kato, 51 A. Unciti-Broceta, E. M. Johansson, R. M. Yusop,
J. Doecke, V. Dore, C. Fowler, Q. X. Li, R. Martins, C. Rowe,
T. Tomita, K. Matsuzaki, K. Ishii, K. Ishii, Y. Arahata,
R. M. Sanchez-Martin and M. Bradley, Nat. Protoc., 2012, 7,
1207–1218.
´
S. Iwamoto, K. Ito, K. Tanaka, C. L. Masters and 52 X. Zhang, R. Huang, S. Gopalakrishnan, R. Cao-Milan and
K. Yanagisawa, Nature, 2018, 554, 249–254.
29 C. Hureau and P. Faller, Biochimie, 2009, 91, 1212–1217.
30 K. P. Kepp, Chem. Rev., 2012, 112, 5193–5239.
V. M. Rotello, Trends in Chemistry, 2019, 1, 90–98.
53 R. Krishnan, J. L. Goodman, S. Mukhopadhyay,
C. D. Pacheco, E. A. Lemke, A. A. Deniz and S. Lindquist,
Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 11172–11177.
31 Y. Bai, X. Feng, H. Xing, Y. Xu, B. K. Kim, N. Baig, T. Zhou,
A. A. Gewirth, Y. Lu, E. Oldeld and S. C. Zimmerman, J. 54 S. Campioni, B. Mannini, M. Zampagni, A. Pensalni,
Am. Chem. Soc., 2016, 138, 11077–11080.
32 K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and
Q. Wang, Org. Lett., 2004, 6, 4603–4606.
C. Parrini, E. Evangelisti, A. Relini, M. Stefani,
C. M. Dobson, C. Cecchi and F. Chiti, Nat. Chem. Biol.,
2010, 6, 140–147.
33 C. Le Droumaguet, C. Wang and Q. Wang, Chem. Soc. Rev., 55 J. Ni, A. Taniguchi, S. Ozawa, Y. Hori, Y. Kuninobu, T. Saito,
2010, 39, 1233–1239.
34 R. Zeineddine and J. J. Yerbury, Frontiers in Physiology, 2015,
6, 277–284.
35 Y. Li, D. Cheng, R. Cheng, X. Zhu, T. Wan, J. Liu and
R. Zhang, PLoS One, 2014, 9, e99939.
36 J. J. Yerbury, Prion, 2016, 10, 119–126.
T. C. Saido, T. Tomita, Y. Sohma and M. Kanai, Chem, 2018,
4, 807–820.
56 Z. Du, N. Gao, X. Wang, J. Ren and X. Qu, Small, 2018, 14,
e1801852.
57 A. Aliyan, B. Kirby, C. Pennington and A. A. Marti, J. Am.
Chem. Soc., 2016, 138, 8686–8689.
37 Y. Luo, J. Zhang, N. Liu, Y. Luo and B. Zhao, Sci. China: Life 58 B. I. Lee, S. Lee, Y. S. Suh, J. S. Lee, A. K. Kim, O. Y. Kwon,
Sci., 2011, 54, 527–534.
K. Yu and C. B. Park, Angew. Chem., Int. Ed., 2015, 54,
11472–11476.
59 M. G. Savelieff, G. Nam, J. Kang, H. J. Lee, M. Lee and
M. H. Lim, Chem. Rev., 2018, 119, 1221–1322.
38 J. A. Varela, J. P. Dupuis, L. Etchepare, A. Espana, L. Cognet
and L. Groc, Nat. Commun., 2016, 7, 10947–10956.
39 H. Sun, J. Liu, S. Li, L. Zhou, J. Wang, L. Liu, F. Lv, Q. Gu,
B. Hu, Y. Ma and S. Wang, Angew. Chem., Int. Ed., 2019, 58, 60 M. R. Jones, E. Mathieu, C. Dyrager, S. Faissner,
5988–5993.
Z. Vaillancourt, K. J. Korshavn, M. H. Lim,
A. Ramamoorthy, V. Wee Yong, S. Tsutsui, P. K. Stys and
T. Storr, Chem. Sci., 2017, 8, 5636–5643.
40 W. Liu, H. Dong, L. Zhang and Y. Tian, Angew. Chem., Int.
Ed., 2017, 56, 16328–16332.
41 Y. Luo, L. Zhang, W. Liu, Y. Yu and Y. Tian, Angew. Chem., 61 A. Taniguchi, Y. Shimizu, K. Oisaki, Y. Sohma and M. Kanai,
Int. Ed., 2015, 54, 14053–14056. Nat. Chem., 2016, 8, 974–982.
42 M. W. Bourassa, A. C. Leskovjan, R. V. Tappero, 62 D. Ozawa, H. Yagi, T. Ban, A. Kameda, T. Kawakami, H. Naiki
E. R. Farquhar, C. A. Colton, W. E. Van Nostrand and
L. M. Miller, Biomed. Spectrosc. Imaging, 2013, 2, 129–139.
43 G. L. Song, C. Chen, Q. Y. Wu, Z. H. Zhang, R. Zheng,
and Y. Goto, J. Biol. Chem., 2009, 284, 1009–1017.
63 J. Notni and H. J. Wester, Chem.–Eur. J., 2016, 22, 11500–
11508.
Y. Chen, S. Z. Jia and J. Z. Ni, Metallomics, 2018, 10, 1107– 64 N. Zabarska, A. Stumper and S. Rau, Dalton Trans., 2016, 45,
1115.
2338–2351.
This journal is © The Royal Society of Chemistry 2019
Chem. Sci., 2019, 10, 10343–10350 | 10349

View Article Online
Chemical Science
Edge Article
65 L. Q. Hatcher, L. Hong, W. D. Bush, T. Carducci and 76 G. F. da Silva and L. J. Ming, Angew. Chem., Int. Ed., 2005, 44,
J. D. Simon, J. Phys. Chem. B, 2008, 112, 8160–8164. 5501–5504.
66 X. Hu, Q. Zhang, W. Wang, Z. Yuan, X. Zhu, B. Chen and 77 G. F. da Silva and L. J. Ming, Angew. Chem., Int. Ed., 2007, 46,
X. Chen, ACS Chem. Neurosci., 2016, 7, 1255–1263. 3337–3341.
67 H. Zhang, C. Zhang, X. Y. Dong, J. Zheng and Y. Sun, J. Mol. 78 W. M. Tay, G. F. da Silva and L. J. Ming, Inorg. Chem., 2013,
Recognit., 2018, 31, e2697. 52, 679–690.
68 Z. Wang, Y. Zhang, E. Ju, Z. Liu, F. Cao, Z. Chen, J. Ren and 79 K. E. Broaders, S. Grandhe and J. M. Frechet, J. Am. Chem.
X. Qu, Nat. Commun., 2018, 9, 3334–3347. Soc., 2011, 133, 756–758.
69 R. Mishra, D. Sjolander and P. Hammarstrom, Mol. BioSyst., 80 T. M. Dawson, T. E. Golde and C. Lagier-Tourenne, Nat.
2011, 7, 1232–1240. Neurosci., 2018, 21, 1370–1379.
70 D. Ye, S. Anguissola, T. O'Neill and K. A. Dawson, Nanoscale, 81 Y. Guan, Z. Du, N. Gao, Y. Cao, X. Wang, P. Scott, H. Song,
2015, 7, 10050–10058.
J. Ren and X. Qu, Sci. Adv., 2018, 4, eaao6718.
82 S. Alavez, M. C. Vantipalli, D. J. Zucker, I. M. Klang and
G. J. Lithgow, Nature, 2011, 472, 226–229.
83 K. E. Matlack, D. F. Tardiff, P. Narayan, S. Hamamichi,
K. A. Caldwell, G. A. Caldwell and S. Lindquist, Proc. Natl.
Acad. Sci. U. S. A., 2014, 111, 4013–4018.
84 A. Micsonai, F. Wien, L. Kernya, Y. H. Lee, Y. Goto,
M. Refregiers and J. Kardos, Proc. Natl. Acad. Sci. U. S. A.,
2015, 112, E3095–E3103.
71 X. Liu, B. Sui and J. Sun, Biomaterials, 2017, 121, 64–82.
72 Y. Min, J. M. Caster, M. J. Eblan and A. Z. Wang, Chem. Rev.,
2015, 115, 11147–11190.
73 C. Tapeinos and A. Pandit, Adv. Mater., 2016, 28, 5553–5585.
74 J. Geng, M. Li, L. Wu, C. Chen and X. Qu, Adv. Healthcare
Mater., 2012, 1, 332–336.
75 P. Shi, M. Li, J. Ren and X. Qu, Adv. Funct. Mater., 2013, 23,
5412–5419.
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