Imidazolium salts: A mild reducing and antioxidative reagent

Article abstract of DOI:10.1021/ja8037883

The concept of directly using imidazolium salts (IMSs) as mild reducing and antioxidative reagents was proposed and investigated. A simple and robust protocol for the synthesis of stable, ultrafine gold nanoparticles has been established using IMSs under mild conditions. IMSs showed remarkably lower toxicity but greater antioxidative power than N-acetyl-l-cystein and (-)-epigallocatechin gallate on HSC-T6 cells. These studies demonstrate that the simple and inexpensive IMSs represent a new type of antioxidant with potential biomedical applications Copyright

Full text of DOI:10.1021/ja8037883

Published on Web 08/30/2008
Imidazolium Salts: A Mild Reducing and Antioxidative Reagent
Lan Zhao, Chunyan Zhang, Lang Zhuo, Yugen Zhang,* and Jackie Y. Ying*
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669
Received May 29, 2008; E-mail: jyying@ibn.a-star.edu.sg; ygzhang@ibn.a-star.edu.sg
Imidazolium salts (IMSs) are well-known as room-temperature
ionic liquids (RTILs) that can be used as electrolytes or green
solvents because of their low vapor pressure and wide chemical
stability.1 IMSs are also known as precursors for stable carbenes
or bisimidazolidine with many applications in organic synthesis.2,3
Most of the N-heterocyclic carbenes (NHCs) are sensitive to
moisture.4 However, a natural analogue of this group, thiamine or
vitamin B, plays a very important biological role in the aqueous
environment.5 Although the physical behavior of IMSs in aqueous
media has been widely studied, their chemical properties and
applications have seldom been mentioned.6 Amyes and co-workers
have reported the carbon acid pKa of a series of imidazolium cations
in aqueous solutions.7 The equilibrium in eq 1 is shown to always
exist. Therefore, bisimidazolidine (3) is known as a superelectron-
donor (S.E.D.) reagent and has been used as a reducing agent in
some reactions.8 These studies prompted us to investigate the redox
properties of IMSs.
Figure 1. IMS attenuated cellular ROS level. HSC-T6 cells were incubated
with 4 of different concentrations, NAC (1 mM) and EGCG (25 µM). Data
were presented as relative value after normalization against vehicle control,
and as mean and standard error of mean (SEM), N ) 6. *c P < 0.005; *d
P < 0.0005.
stress.15 There have also been attempts to investigate the biological
effects of some NHC-metal complexes in a cell-free system.16
To examine if the metal-free IMSs possessed beneficial biological
properties, herein we investigated the antioxidative capacity of IMSs
by accessing the cellular oxidative stress level in cultured hepatic
stellate cells (HSC-T6) treated with 4. Cultured HSCs are known
to be routinely activated when cultured on the plastic surface of
the culture flask and are therefore used as an in Vitro model of
liver fibrosis. Oxidative stress resulting from the metabolic genera-
tion of reactive oxygen species (ROS) is believed to be implicated
in HSC activation and liver fibrogenesis and many other diseases,
such as cancer, autoimmune disorders, neurodegeneration, and
aging.17 Cells treated with 4 (10, 50, 100, and 300 µM) for 48 h in
full serum medium were assayed for ROS. Cells treated with
N-acetyl-L-cystein (NAC) (1 mM) and (-)-epigallocatechin gallate
(EGCG) (25 µM) separately were included as references.18 ROS
level was significantly attenuated under the treatment of 4. As
shown in Figure 1, ROS was dose-dependently suppressed (25%
for 50 µM (P < 0.005) and 34% for 300 µM (P < 0.0005)). In
comparison, EGCG (25 µM) was able to attenuate 14% ROS (P <
0.005), while NAC (1 mM) did not show any apparent inhibition
of ROS level under the same scheme of treatment. In addition, 4
exhibited a much lower cytotoxicity (IC50 ) 1.7 mM) as compared
to the natural antioxidant EGCG (IC50 ) 30 µM) assayed on the
same HSC-T6 cells (Figure 2). A full characterization of 4 and
additional IMSs in cultured HSCs will be presented in detail
elsewhere.19b
This study demonstrated IMSs as a promising antioxidant that
could effectively attenuate ROS in a dose-dependent manner. Other
assays (glutathione peroxidase (GPx), catalase (CAT), and super-
oxide dismutase (SOD)) were also conducted, and similar effects
were achieved with IMSs.19 The antioxidative power of IMSs was
proposed to be through the direct neutralization of hydrogen
peroxide or other radical species by carbene or bisimidazolidine
(see eq 1). However, the interactions between carbene and radicals
were not well-known.13 To support this hypothesis, two model
reactions between stable carbenes and radical oxidants were
conducted (see Scheme 1). Stable carbene mesitylimidazolinylidene
First, IMS (1,3-dibenzylimidazolium bromide (4)) was employed
in the synthesis of gold particles. It was found that ultrafine gold
nanoparticles could be very efficiently derived in IMS/thiol solution
in seconds at room temperature. The small gold nanoparticles were
well dispersed in the solvent to form a clear solution that was stable
for at least 6 months at 4 °C. Transmission electron microscopy
images showed that uniform gold nanoparticles (1-2 nm in size)
were produced. Similar products were obtained by using different
IMSs, solvents, and thiol/Au ratios.9 Compared to the commonly
used borane or borohydride reduction processes,10 no strong
reducing reagent was added in this case. The ultrafine gold
nanoparticles were obtained under a very mild reaction condition
with remarkable efficiency. This new method could be easily scaled
up for industrial applications.11,12
Further studies showed that there were large amounts of
1-benzylimidazole, benzyl chloride (bromide), and disulfide present
in the reaction system. Combined with other observations (see
Supporting Information (SI)), it was proposed that the carbene
derivative7 of 4 coordinated with and reduced the gold cations,
followed by decomposition to benzyl chloride (bromide) through
a radical pathway (SI, Scheme S1). The thiol further reduced gold
complex to Au(0) and released benzylimidazole, which played a
very important role as a ligand generated in situ in coordination
with and protection of Au(0) to form stable, ultrafine nanoparticles.
This radical intermediate pathway was also commonly accepted in
the carbene or bisimidazolidine reduction processes.13,8
On the basis of the understanding of IMS-promoted gold
reduction, it was proposed that stable IMS could also be a potential
candidate as a radical scavenger antioxidant in a cellular system.14
Thiamine deficiency has been known to be related to oxidative
9
12586 J. AM. CHEM. SOC. 2008, 130, 12586–12587
10.1021/ja8037883 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
chemical and biological aspects of IMSs’ actions in vitro and in
vivo are under way.
Acknowledgment. This work was supported by the Institute
of Bioengineering and Nanotechnology (Biomedical Research
Council, Agency for Science, Technology and Research, Singapore).
Supporting Information Available: Synthesis of gold nanoparticles,
antioxidative properties of IMSs, toxicity of 4 on HSC-T6 cells, and
reaction between stable carbenes with disulfide and peroxide. This
material is available free of charge via the Internet at http://pubs.acs.org.
Figure 2. Cytotoxicity of 4. HSC-T6 cells were seeded at a density of
5000 cells/well in 96-well plate, and adapted for 18-24 h in Dulbecco
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum
(FBS), before the addition of compounds of various concentrations (0-400
µM) in DMEM containing 10% FBS for 48 h. They were then assayed for
proliferation. Signal was normalized against vehicle treatment (0 µM
compound). Data were obtained from four independent experiments, and
presented as mean and SEM. *a P < 0.01; *b P < 0.005; *c P < 0.0005.
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