Stabilized copper(I) oxide nanoparticles catalyze azide-alkyne click reactions in water

Article abstract of DOI:10.1002/adsc.201000206

A novel form of polyvinylpyrrolidone (PVP) coated copper(I) oxide nanoparticle (Cu₂ONP) was prepared and used to catalyze azidealkyne click reactions in water under aerobic conditions. The nanoparticles were well dispersed in aqueous solutions

Full text of DOI:10.1002/adsc.201000206

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DOI: 10.1002/adsc.201000206
Stabilized Copper(I) Oxide Nanoparticles Catalyze Azide-Alkyne
Click Reactions in Water
Zhenfang Zhang,a Changming Dong,b Cuihong Yang,a Di Hu,b Jing Long,b
Ling Wang,b He Li,b Yue Chen,b,c,* and Deling Konga,*
a
Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071, Peopleꢀs Republic of
China
Fax : (+86)-22-2349-8775; phone: (+86)-22-2350-2111; e-mail: kongdeling@nankai.edu.cn
College of Pharmacy, Nankai University, Tianjin 300071, Peopleꢀs Republic of China
b
Fax : (+86)-22-2350-7760; phone: (+86)-22-2350-8090; e-mail: yuechen@nankai.edu.cn
The State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, Peopleꢀs Republic of China
c
Received: March 16, 2010; Revised: June 6, 2010; Published online: June 30, 2010
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201000206.
Abstract: A novel form of polyvinylpyrrolidone
(PVP) coated copper(I) oxide nanoparticle (Cu2O-
NP) was prepared and used to catalyze azide-
alkyne click reactions in water under aerobic condi-
tions. The nanoparticles were well dispersed in
aqueous solutions and have a size of 20Æ10 nm, as
determined by transmission electron microscope
(TEM). Inductively coupled plasma (ICP), X-ray
powder diffraction (XRD), and X-ray photoelec-
tron spectroscopy (XPS) analyses demonstrated
that the main content of Cu2O-NP is copper(I). The
cytotoxicity of it was evaluated by an in vitro 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay and its catalytic efficiency for
azide-alkyne click reactions was studied in water
and organic solvents at physiological temperatures.
Our results indicate that Cu2O-NP is more efficient
in catalytic reactions in water for both aliphatic and
aromatic azides and alkynes and less toxic than the
commonly used CuSO4/reductant catalyst systems.
tion began with the vital discovery by the groups of
Meldal[4] and Sharpless,[5] in which copper(I) catalysis
was found to dramatically accelerate the reaction
under mild conditions. Recently, Bertozzi and co-
workers have devised a copper-free cycloaddition re-
action, which has achieved significant progress in dy-
namic in vivo imaging,[6] chemoselective ligation reac-
tions,[7] and metabolic labeling for proteomic analy-
sis,[8] but these reactions are usually slow and the re-
gioselectivity is unpredictable. Therefore, copper-cata-
lyzed processes are still being preferred for organic
synthesis. Although a variety of copper catalysts for
this important chemistry have been developed, only a
few of these systems are active in aqueous solution,
such as CuSO4/reductant or copper ion complex.[9]
Usually these systems require a high concentration of
copper ions or a copper ion complex.
Due to increasing concern about environmental
impact, tremendous efforts have been made towards
the development of green chemistry that minimizes
pollution in chemical synthesis. For this reason, not
only copper-free methodology needs to be investigat-
ed further,[10] but also a new generation of biocompat-
ible and efficient catalysts requires development.[11]
Recently, much attention has been attracted to the
use of Cu2O nanoparticles as catalyst in organic reac-
tions.[12] We envision that Cu2O nanoparticles might
have the potential to act as a new catalyst system for
Keywords: click chemistry; copper(I) oxide; 1,3-di-
polar Huisgen cycloaddition; nanoparticles; polyvi-
nylpyrrolidone
Here we report on the preparation of polyvinylpyrro- 1,3-dipolar Huisgen cycloadditions in water under
lidone (PVP) coated copper(I) oxide (Cu2O) nanopar- aerobic conditions because of their inherent proper-
ticles as an efficient catalyst for azide-alkyne click ties, for example, high surface areas, facile surface
chemistry in water at physiological temperatures. functionality and higher stability than bulk Cu2O.[13]
Azide-alkyne click chemistry has been widely used in Thus, we prepared the PVP-coated Cu2O nanoparti-
material sciences,[1] drug discovery,[2] and bioconju- cles (Cu2O-NPs) and applied these nanoparticles as
gation.[3] The enormous attention devoted to this reac- the catalyst for the azide-alkyne click chemistry in
1600
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Adv. Synth. Catal. 2010, 352, 1600 – 1604

Stabilized Copper(I) Oxide Nanoparticles Catalyze Azide-Alkyne Click Reactions
aqueous
solutions
under
aerobic
conditions
Then transmission electron microscopy (TEM) was
(Scheme 1). Our results demonstrated that the Cu2O- used to characterize the Cu2O-NPs. Figure 1, A shows
NP is more efficient in the catalytic reaction and less that the nanoparticles were well dispersed in aqueous
toxic than the commonly used CuSO4/reductant cata- solutions, they have a size of 20Æ10 nm and form
lyst systems.
small aggregates. Dynamic light scattering (DLS) re-
sults indicated that the size of these small aggregates
was less than 200 nm (50–60 and 140–170 nm,
Figure 1, B) and no larger aggregates were observed.
The Cu2O composition of the Cu2O-NPs was 59.5Æ
1% which was obtained by inductively coupled
plasma (ICP) analysis. These results imply that the
nanoparticles are coated with PVP, making the whole
composite stable in aqueous solution. To prove the
nanoparticles are Cu2O-NPs, the X-ray powder dif-
Scheme 1. The azide-alkyne click reaction with Cu2O-NPs.
In order to solve the problem that conventional fraction (XRD) method was used (Figure 1, C). Peaks
copper nanoparticles can only catalyze the click reac- at 36.568, 42.558, 62.778, and 73.938 belong to Cu2O
tion in organic solvents,[14] PVP-coated Cu2O nanopar- (111), Cu2O (200), Cu2O (220), and Cu2O (311), re-
ticles were first synthesized by adding copper acetate spectively, which clearly demonstrate that the nano-
(2.2 mmol) to the aqueous solution of 20 mmol particles are Cu2O-NPs. We also used X-ray photo-
sodium borohydride and 0.8% (w/v) polyvinylpyrroli- electron spectroscopy (XPS, Figure 1, D) to provide
done (PVP) (40 kDa) at room temperature in air. information about the composition of the nanoparti-
Here PVP was chosen as a stabilizing and dispersing cleꢀs surface. As shown by the Cu 2p3/2 peaks in
agent to make a stable Cu2O-NP catalyst system that Figure 1, D, Cu(I) has a single peak at 932.56 eV
dispersed well in aqueous solution (as shown in the (peak 1), whereas Cu(II) has one main peak at
insertion of Figure 1, A).
934.81 eV (peak 2), and shakeup satellites (peaks 3
Figure 1. (A) TEM image (insert: optical image of Cu2O-NP in aqueous solutions), (B) the granulometric distribution, (C)
XRD pattern, and (D) deconvolution of their respective Cu 2p peaks about XPS pattern.
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Zhenfang Zhang et al.
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and 4) at higher binding energies. Based on the XPS the established one reported in an earlier report.[15]
results, the percentages of Cu(I) and Cu(II) were Cu(I) is considered to be the most active species in
90% and 10%, respectively, which indicates the pres- this mechanism, forming a Cu(I) acetylide complex
ence of copper(II) oxide (CuO) in the Cu2O-NPs, by initial coordination with the alkyne followed by
probably because of oxidation in aqueous solution. addition to the azide group to give a 1,2,3-triazole.
The XRD result does not show the existence of CuO, With the assistance of sonication, significantly higher
while XPS indicates the presence of Cu2+ ions on the yields were obtained (entries 6, 8, 10, 12, and 14), and
nanoparticleꢀs surface, suggesting that CuO is present the product could be detected even when the concen-
only on the surface of the Cu2O-NPs as a thin amor- tration of Cu2O-NP was only 0.057 mol% (entry 14).
phous outer shell.
The possible mechanism for the improved yields ob-
The catalytic activity of the PVP-coated Cu2O tained through sonication is that sonication loosens
nanoparticles system was investigated in the Huisgen the PVP coating of the catalyst, allowing the alkyne
cycloaddition reaction using 6-azidohexan-1-ol (1) better access to bind to the Cu2O core. Therefore,
and hex-5-yn-1-ol (2) as model substrates (Scheme 1). sonication of the reaction containing the Cu2O-NPs
The reactions were conducted under aqueous, aerobic and the alkyne increases the reaction rate over that of
conditions. It was found that our Cu2O-NPs exhibited the unsonicated reaction or that when only the Cu2O-
higher efficiency than Cu2O powder, the widely used NPs are sonicated. Although in general click reactions
CuSO4/tris(2-carboxyethyl)phosphine (TECP), and are aided by base or high pH, the addition of the base
CuSO4/sodium ascorbate (NaAsc) catalyst systems remarkably reduced the rate and yield of the click re-
(Table 1). Over 90% of the desired 1,3-dipolar Huis- action in our catalytic system.[16]
gen cycloaddition product was formed in 24 h
The optimized reaction conditions were applied to
(entry 5) with 0.94 mol% of Cu2O-NPs, while only a variety of terminal alkynes and azides (Table 2). All
28%, 1.3%, and 0.55% of the product was detected
when using 100 mol% of bulk Cu2O, 0.94 mol% of
CuSO4/TECP and CuSO4/NaAsc, respectively.
Table 2. Examples of Cu2O-NP-catalyzed click reactions.
We also examined different catalyst loadings using
the same model compounds and reaction conditions.
We observed the formation of the product with a
yield of 21% when using 0.31 mol% of Cu2O-NPs.
Entry[a]
1
R1
R2
Product, Yield
The mechanism for the reaction should be similar to
HO
G
6a, 90%
Table 1. The reaction conditions for Cu2O-NP-catalyzed
click reactions.
2
HO
G
6b, 85%
Entry[a] Catalyst
mol% Yield [%] Yield [%]
at 24 h
at 48 h
3
4
5
HO
HO
HO
N
6c, 85%
6d, 92%
6e, 74%
1
2
3
4
Cu2O[c]
Cu2O-NP
CuSO4/NaAsc 0.94
100
9.4
28
95
1.3
0.55
92
90
39
92
21
43
4.2
33
0
N/A
N/A
2.0
0.69
96
99.7
45
99.8
28
68
7.4
42
0
13
HOCH2
CuSO4/TECP
Cu2O-NP
Cu2O-NP
Cu2O-NP
Cu2O-NP
Cu2O-NP
Cu2O-NP
Cu2O-NP
Cu2O-NP
Cu2O-NP
Cu2O-NP
0.94
0.94
0.94
0.63
0.63
0.31
0.31
0.09
0.09
0.057
0.057
5
6
7[b]
HO
HO
6f, 86%
6g, 68%
6[b]
7
HOACHTNGUETRNNU(G CH2)2
8[b]
9
8
6h, 87%
10[b]
11
12[b]
13
14[b]
9
6i, 91%
[a]
All reaction mixtures were stirred for 24 hours at 378C
under aerobic condition, and yields were obtained
through filtration. All reactions were conducted with
azide (1 mmol), alkyne (1 mmol) and Cu2O-NP
(0.94 mol%) in H2O (20 mL).
The mixture of alkyne (5 mmol) and Cu2O-NP
(0.016 mol%) in water (total volume is 1 mL) was ultra-
sonicated for 1 hour, followed by addition of azide (neat,
5 mmol). The reaction mixtures were stirred for 48 hours
at 378C under argon.
5.5
[a]
All reactions were conducted with azide (1 mmol) and of
alkyne (1 mmol) in H2O (20 mL). The reaction mixtures
were stirred at 378C under aerobic condition unless spe-
cially indicated, and yields were obtained through HPLC
analysis.
The Cu2O-NPs and the alkyne in H2O were ultrasonicat-
ed for 1 hour, followed by the addition of azide.
Cu2O is 200 mesh.
[b]
[b]
[c]
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Adv. Synth. Catal. 2010, 352, 1600 – 1604

Stabilized Copper(I) Oxide Nanoparticles Catalyze Azide-Alkyne Click Reactions
of the reactions were high yielding and were highly Experimental Section
regioselective towards the 1,4-disubstituted triazoles.
Furthermore, the corresponding 1,2,3-triazoles were Preparation of the PVP-Coated Cu2O Nanoparticles
generally isolated in excellent yields, after a simple
Copper ions were introduced in the form of copper salt, that
work-up involving filtration and solvent evaporation.
is, copper acetate. In a typical experiment, the solution of
Then the crude product was subjected to column
copper acetate (0.4 g, 2.2 mmol) in 20 mL water was added
chromatography for further purification followed by
to the mixture of sodium borohydride (0.76 g, 20 mmol) and
0.8% (w/v) PVP (40 kDa) in 200 mL water with stirring.
After about 2 to 3 h, the solution turned to a dark brown
color. Then the solution was centrifuged at 10000 rpm for
10 min and the precipitate was dried under vacuum. Treat-
ment of the black power we got with water under sonication
yields a dark brown dispersion with a minor insoluble resi-
due. The concentration is about 2 mgmLÀ1 (ca. 1.2 mgmLÀ1
for copper).
spectroscopic
characterization
using
1H NMR,
13C NMR, infrared (IR), and high resolution mass
(HR-MS) spectrometry. As shown in Table 2, these
reactions worked well for both aliphatic and aromatic
azides and alkynes. The typical turnover value for
entry 7 was 4300. The catalytic efficiency of Cu2O-
NPs was also investigated in organic solvents when
one of the reactants is lipophilic. It was found that
click reaction also worked well in acetonitrile, but in
other organic solvents such as ethanol and tetrahydro-
furan, the yield is lower than 50%.[16] The possible
reason is that our nanoparticles didn’t disperse well in
organic solvents except acetonitrile.
General Procedure for Cu2O-NPs Catalyzed
[3+2]Cycloaddition of Azides and Terminal Alkynes
A solution of azide 1 (1 mmol), alkyne 2 (1 mmol) and
Cu2O-NP dispersion in water (20 mL) was stirred at 378C
for 24 hours. The reaction mixture was filtered through a
celite pad to remove the Cu2O-NP catalyst. The solvent was
evaporated under reduced pressure. The products were gen-
erally of sufficient purity.
Beside investigating its catalytic efficiency, we also
tested the cytotoxicity of Cu2O-NPs using HepG2
cells by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assay. It was found that
more than 80% cell viability was retained in the pres-
ence of Cu2O-NP (0.94 mol%),[16] which proved its
better biocompatibility over other catalyst systems at
the same effective concentrations. Then we used ICP
to measure the concentration of copper ions within
the cells. It was found that the concentration of Cu
ions in the Cu2O-NP-treated cells is about 5.5–6.5
times higher than that in the untreated cells and
about 3 times that of CuSO4-treated cells, probably
because of the active uptake of Cu2O-NP by cells. Dy-
namic light scattering analysis found that the electron-
ic potential of Cu2O-NP dispersed in water was
+25 mV,[16] which would have helped it to penetrate
into the cells.
Cytotoxicity Assay
The cytotoxicities of Cu2O-NP and CuSO4/TECP were eval-
uated by the MTT assay. HepG2 cells were seeded into 96-
well plates at a density of 104 cells/well and incubated for
24 h. Cu2O-NP or CuSO4/NaAsc solutions were added into
the cells. After incubation for another 24 h, 25 mL of MTT
solution (5 mgmLÀ1 in PBS) were added to each well. Four
hours later, the medium was removed and the samples in
the wells were air dried. Acidic isopropyl alcohol (100 mL,
0.04M HCl in absolute isopropyl alcohol) was added to dis-
solve the formed crystals. The optical density of the solution
was measured at 570 nm using a microplate reader (Labsys-
tem, Multiskan, Ascent, Finland). The cytotoxicities of
Cu2O-NP and CuSO4/TECP were evaluated at their effec-
tive concentrations. 0.16 to 0.94 mol% is the working con-
centration of Cu2O-NP. However, for CuSO4/TECP system,
the concentration has to be over 1.25 mol% to generate a
detectable yield.
In summary, compared with CuSO4/reductant or
bulk Cu2O, our polyvinylpyrrolidone coated copper(I)
oxide nanoparticles catalyst is highly efficient for
azide-alkyne click chemistry in aqueous and aerobic
conditions. Moreover, the preliminary results demon-
strated that our Cu2O-NPs catalyst could effectively
penetrate into cells and was less toxic than the
CuSO4/reductant catalyst systems at the same effec-
Uptake of Cu2O-NP by HepG2 Cells
tive concentrations. The easily changed surface func- HepG2 cells were seeded at a density of 2ꢂ106 cells in each
10 cm cell culture dish and incubated for 24 h. Then culture
medium was removed and cells were washed twice with
warmed PBS. Following this, 10 mL of serum-free medium
or 10 mL of serum-free medium containing Cu2O-NP
(20 mgmLÀ1) were added into the dishes separately and incu-
bated with the cells at 378C. After 5 h, the medium was re-
moved and cells were washed twice with warmed PBS prior
the addition of trypsin (0.25%, 378C, 5 min.).The cell sus-
pensions were collected and freeze-dried and then digested
by the addition of 2 mL of concentrated HNO3 and 1 mL of
perchloric acid for the measurement of Cu content by ICP.
tionality of the Cu2O-NPs and the development of
PVP derivatives make it a good catalytic system for
1,3-dipolar Huisgen cycloadditions in water under
aerobic condition.
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Acknowledgements
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2010, 352, 1600 – 1604