A dual colorimetric-ratiometric fluorescent probe NAP-3 for selective detection and imaging of endogenous labile iron(III) pools in C. elegans

Article abstract of DOI:10.1039/c4cc09798j

The discovery of an iron(III)-selective ratiometric fluorescent probe to detect and visualize endogenous labile iron pools in living organisms at the molecular level has been long awaited. Herein we report the first dual colorimetric and ratiometric fluor

Full text of DOI:10.1039/c4cc09798j

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: A. Goel, S. Umar,
P. Nag, A. Sharma, L. Kumar, S. Shamsuzzama, Z. Hossain, J. R. Gayen and A. Nazir, Chem. Commun.,
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

Page 1 of 4
ChemComm
View Article Online
DOI: 10.1039/C4CC09798J
ChemComm
RSCPublishing
COMMUNICATION
A Dual Colorimetric-Ratiometric Fluorescent Probe
NAP-3 for Selective Detection and Imaging of
Endogenous Labile Iron (III) Pools in C. elegans
Cite this: DOI: 10.1039/x0xx00000x
Atul Goel,*,a Shahida Umar,a Pankaj Nag,a Ashutosh Sharma,a Lalit Kumar,b
Shamsuzzama,b Zakir Hossain,c Jiaur R. Gayen,c Aamir Nazirb
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
The discovery of an iron (III)-selective ratiometric fluorescent
probe to detect and visualize endogenous labile iron pools in
living organisms at molecular-level has been long awaited.
Herein we report the first dual colorimetric and ratiometric
fluorescent probe Naphtho[2,1-
for selective and ‘direct’ visualization of labile iron (III) pools
in a multicellular organism Caenorhabditis elegans
b][1,10]phenanthroline NAP-3
Fig. 1 Designing of Naphtho[2,1-b][1,10]phenanthrolines (NAPs).
.
have distinct advantage over single emission intensityꢀbased probes
because of their selfꢀcalibration/builtꢀin environmental correction via
two selected emission wavelengths.12
Iron is an essential redoxꢀactive element in all living organisms
controlling indispensable physiological and pathophysiological
functions.1 The bulk of the iron present in biological systems is tightly
bound to various proteins.2 Only a fraction of iron is free and exists as
cytosolic labile iron pool (LIP) consisting of both the ionic forms (Fe2+
and Fe3+) in dynamic equilibrium.3 If the cellular homeostasis of labile
iron gets disturbed, it can trigger uncontrolled Fenton reaction4 leading
to peroxidative tissue damage and thus causing various diseases, such
as Alzheimer’s disease (AD), Parkinson’s disease (PD), and different
types of cancers.5 The complexity of redox chemistry of labile iron and
its role in biological processes remain a challenge for chemists and
biologists due to the dearth of ironꢀselective, sensitive and cellꢀ
permeable ratiometric fluorescent probes.6 Although fluorogenic iron
probes like calcein, fluoresceinꢀdeferoxamine (FLꢀDFO), Phen green
SK and Phen green FL have been explored for cell imaging
applications, they provide information through fluorescence quenching
readouts, and exhibit poor selectivity between the two spin states of
iron and nonspecific turnꢀoff response with other interfering transition
metal ions.6,7 Few examples of Fe (II)8 and Fe (III)9 selective
fluorescent sensors and chemodosimeters are reported in the literature
with or without cell imaging studies. Remarkably, Tang et al. in 2011
reported the first ratiometric fluorescent probe for the selective
detection of both exogenous and endogenous labile Fe (II) in live
human liver HL7702 cells.10 Recently, Guo et al in 2012 reported
rhodamine based turn on fluorescent probe ‘RPE’ for imaging labile Fe
(III) pools in live neuronal cells.11 To the best of our knowledge, none
has reported Fe (III)ꢀselective ratiometric probe for in vivo
visualization of LIP in a multicellular organism. From the perspective
of accurate and quantitative analysis, ratiometric sensors and reporters
Commercially available Phen green probes possess [1,10]ꢀ
phenanthroline (PHT) scaffold, which is an entropically balanced ironꢀ
metal chelating ligand but exhibits weak fluorescence due to low lying
π–π* and n–π* singlet excited states.13 Though enormous efforts have
been made in the past to improve its emission intensity by introducing
πꢀconjugated systems mainly at the 2,9ꢀ 3,8ꢀ and 4,7ꢀpositions,
researchers still rely on fluorescent tags. Henceforth PHTꢀbased
commercial iron probes Phen green SK/FL or siderophores
(deferoxamine) are appended with fluorescein label, yet they suffer
with nagging problems of quenching readouts and/or poor selectivity.3,6
Addressing these issues and considering the necessity for selective
ratiometric fluorescent probes for iron, we conceptually designed a
novel14 naphtho[2,1ꢀ
b][1,10]ꢀphenanthroline (NAP) framework
(surprisingly yet unknown in the literature) equipped with wellꢀ
conjugated donorꢀacceptor (DꢀA) and chromophoric πꢀgroups to induce
effective charge transfer during preferential binding of metal to the
phenanthroline receptor (Fig. 1).
Our research group is engaged in targetꢀbased design and synthesis
of new fluorescent dyes for their application in material15 and biological
sciences.16 We have previously shown that controlled tuning of πꢀ
conjugation and appropriate positioning of DꢀA moieties dramatically
modulate the optical properties of aromatic and heterocyclic
compounds.15 The synthetic methodology adopted for the synthesis of
designed DꢀA NAPs is outlined in Scheme 1. The key intermediates
2
H
ꢀpyranꢀ2ꢀones 3a-d were prepared from easily accessible precursor
αꢀoxoꢀketeneꢀS,Sꢀacetal (
1
) and substituted acetophenones (2a-d).15
This was followed by the substitution of the methylsulfanyl group in
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 1

ChemComm
Page 2 of 4
VJieowuArrtnicalelONnlainme e
COMMUNICATION
DOI: 10.1039/C4CC09798J
Fig. 3 (a) Computed molecular orbital energy diagrams of NAP-3 as obtained
from TDDFT calculations. (b) Selectivity studies of NAP-3 (2.5 × 10-5 M) in the
absence or presence of a series of metal ions (5 × 10-4 M) after 1 h upon addition
of metal ion at λex= 365 nm.
offering NAP-3 as
a
Fe3+ꢀselective ‘nakedꢀeye’ chemosensor.
Scheme 1 Synthesis of Naphtho[2,1-b][1,10]phenanthrolines (NAPs).
Correspondingly in the emission spectrum, the peak at 544 nm
decreased and a new peak appeared at 605 nm with the increasing
concentration of Fe3+. The ratiometric response of fluorescence
intensities (I605/I544) enhanced from 0.48 to 4.1 with a final enhancement
factor over 8.5 fold (Fig. S8, ESI†). The titration studies with NAP-3
revealed a good linearity between the fluorescence intensity ratio and
3a-d by a piperidine donor group to furnish DꢀA appended 6ꢀarylꢀ2ꢀ
oxoꢀ4ꢀpiperidinꢀ1ꢀylꢀ2
The chelating precursor 10,11ꢀdihydrobenzo[
8(9 )ꢀone ( ) was prepared via threeꢀcomponent condensation of 8ꢀ
aminoquinoline with formaldehyde and 1,3ꢀcyclohexaneꢀdione in good
yield (Scheme 1). Further, Michael addition of the conjugate base of
to the lactones 4a at position 6 followed by intramolecular cyclization
afforded 11ꢀarylꢀ9ꢀ(piperidinꢀ1ꢀyl)ꢀ12,13ꢀdihydronaphtho[2,1ꢀ ][1,10]ꢀ
phenanthrolineꢀ8ꢀcarbonitriles 9a . To extend πꢀconjugation, the
compounds 9a-d were aromatized with DDQ in dioxane at reflux
conditions to get the desired NAPs 10a-d
H
ꢀpyranꢀ3ꢀcarbonitriles (4a-d) in good yields.15
b][1,10]phenanthrolinꢀ
H
8
Fe3+ concentration with a detection limit of approximately 9.1×10ꢀ9
M
8
(Fig. S9, ESI†) that allowed the quantification of Fe3+ by a ratiometric
fluorescence method17. Further Job’s plot18 established a 2:1 binding
stoichiometry of NAP-3 with Fe3+ (Fig. S10, ESI†), which was
supported by mass spectrometry (Fig. S11, ESI†). The binding constant
for NAP-3 was determined as 9.36 × 1010 Mꢀ2 by nonꢀlinear regression
analysis19 (Fig. S12, ESI†). The fluorescence property of the probe
NAP-3 at different pHs (1.8–10.7) revealed no change in fluorescence
intensity within pH 5.5–8.3 but a gradual decrease in the intensity was
observed beyond this range (Fig S13, ESI†).
ꢀd
b
ꢀd
.
The absorption and emission characteristics of the designed NAPs
9a-d and 10a-d were investigated in aqueous medium (10ꢀ5 M, triple
distilled water TDW/DMSO, 9:1, v/v) at pH 7.0ꢀ7.4 (Figs. S1 and S2,
Table S1, ESI†). The ironꢀsensing studies of NAPs in the presence of
perchlorate salts of Fe2+ and Fe3+ revealed that only Fe3+ exhibited a
visible color change (colorimetric response, Fig. 2a, Figs. S3ꢀS6, ESI†)
and a ratiometric fluorescence response in 10a-d (Figs. S3ꢀS5, ESI†)
It is interesting to note that donorꢀacceptor functionalized
naphtho[2,1ꢀb
][1,10]ꢀphenanthroline NAP-3 selectively binds to Fe3+
but not to Fe2+ (Figs. S6ꢀS7, ESI†). This is contrary to the nature of
1,10ꢀphenanthroline, which selectively binds to Fe2+ (Figs. S14ꢀS15,
with 10c
point at 590 nm (Fig. 2b, Fig S7, ESI†). The aqueous solution of NAP-
exhibited an absorption maximum at 300 nm (πꢀπ* transition) with a
(NAP-3) being the best having a wellꢀdefined isoemission
ESI†). To understand the electronic behaviour of NAP-3
, the
geometries were optimized at DFT/B3LYP level using a 6ꢀ31G(d,p)
basis set and timeꢀdependent density functional theory (TDDFT)
calculations were performed using a B3LYP/6ꢀ311++G(d,p) method.20
The energies of the HOMO and LUMO levels, HOMOꢀLUMO gap,
3
low intensity band at 368 nm (nꢀπ* transition), and a greenꢀyellow
fluorescence band with a maximum at 544 nm (Fig. 2). Upon addition
of increasing concentrations of Fe3+ ions, we observed emergence of a
new peak with maximum at 580 nm in the absorption spectrum
changing the solution color from light yellow to reddish pink thereby
main orbital transition, and oscillator strength
f are listed in Table S2
(ESI†). Fig. 3a shows the theoretically computed molecular orbitals in
the ground states for the probe NAP-3. TDDFT results revealed two
prominent electronic transitions observed at 305 nm (HOMOꢀ2 to
LUMO+1) and 383 nm (HOMO to LUMO). The electron density of the
HOMO is delocalized over the piperidine donor moiety and the
neighbouring naphthophenanthroline ring, while the LUMO is
delocalized on the naphthophenanthroline moiety and the nitrile
acceptor group (Fig. 3a). The electronic transition at higher wavelength
(HOMO to LUMO) corresponds to an intramolecular chargeꢀtransfer
(ICT) band. The presence of conjugated donorꢀacceptor moieties
enhanced the charge density over phenanthroline ring of NAP-3
facilitating the formation of ligand to metal charge transfer (LMCT)
complex with Fe3+. In contrast, 1,10ꢀphenanthroline forms metal to
Fig. 2 (a) Absorption and (b) Ratiometric fluorescence spectra of 10c
after 1 h upon addition of increasing concentrations of Fe3+ (0–6.8 × 10-4 M).
NAP-3] = 2.5 × 10-5 M, λex= 365 nm. During the titration studies, pH of the
(NAP-3)
[
solutions were maintained to 7.0-7.4 using 0.01M NaOH solution.
ligand
charge
transfer
(MLCT)
complex
with
Fe2+.21
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 3 of 4
COMMUNICATION
ChemComm
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C4CC09798J
cell imaging (Fig. S23, ESI†). Therefore, the in vitro cell studies
demonstrated that NAP-3 is a cellꢀpermeable fluorescent probe to
detect elevated levels of Fe3+ via exogenous supplementation and may
be used for in vivo imaging of labile iron.
The soil nematode Caenorhabditis elegans is a model of choice for
studying regulatory mechanisms associated with iron metabolism due to
homology with human gene sequences.22 Electron paramagnetic
resonance (EPR) spectroscopy has been used as a tool to ‘indirectly’
measure free Fe3+ levels in vivo in C. elegans 23
In an attempt to
.
visualize LIP ‘directly’ in an organism, we examined in vivo iron
binding specificity of NAP-3 in wild type (N2) C. elegans by confocal
fluorescence imaging experiments. We observed that treatment of
worms with 3 ꢁM of NAP-3 for 24 h exhibited staining in the green
channel, while staining was weak in the red channel (Fig. 5a, see
zoomed images). The staining in the red channel implicated that NAP-3
has access to Fe3+ present in LIP of wild type C. elegans. In order to
confirm that the fluorescence in the red channel is due to the formation
of Fe3+ complex and not by NAP-3 alone, we measured the
fluorescence of NAP-3 at 561 nm excitation and no fluorescence in red
channel was observed (Fig. S25, ESI†). These results implicated the
Fig. 4 (a) HepG2 cells incubated with NAP-3 (3 μM, 24 h). (b) HepG2 cells
incubated with NAP-3 after ferric citrate (5 × 10-4 M, 24 h) supplementation. (c)
Ratiometric quantification of relative fluorescence intensity of confocal
microscopy images by image J software, ***p < 0.001. Green channel: λex = 405
nm, λem = 505–550 nm. Red channel: λex = 561 nm, λem = 575 nm long pass. Error
bars are + s.e.m. (n = 3). Scale bar: 10 μm.
The selectivity studies of NAP-3 in the presence of high
concentration (20 equiv.) of perchlorate salts of Li+, Na+, K+, Cs+, Mg2+,
Ca2+, Ba2+, Cr3+, Co2+, Mn2+, Fe2+, Fe3+, Cu2+, Al3+, Zn2+, Pb2+, Cd2+,
Hg2+, Ni2+ revealed that only Fe3+ triggered a dramatic increase in the
emission ratio I605/I544, while other metal ions were either silent or
induced minor ratiometric response (Fig. 3b, Fig. S17, ESI†). In order
to confirm the selectivity of NAP-3 for Fe3+ over Fe2+, we conducted
experiments under reducing (Fe3+ and hydroxylamine) and oxidizing
(Fe2+ and hydrogen peroxide) environments (Figs. S18 and S19, ESI†).
To our delight, NAP-3 successfully discriminated Fe3+ over Fe2+. We
further conducted competitive experiments of NAP-3 with a known
strong Fe3+ chelator deferoxamine (DFO). We observed that in the
presence of DFO, NAP-3 could not bind to Fe3+; while in the presence
detection of Fe3+ present in the LIP by the probe NAP-3
.
On exogenous Fe3+ supplementation, there was no increase in the
fluorescence intensity in the red channel as compared with only NAP-3
treated groups (data not shown). These results indicated that to maintain
iron homeostasis, worms might have triggered iron regulatory
mechanisms associated with ferritins (FTNꢀ1 and FTNꢀ2), which are
transcriptionally regulated by iron.24 Thereby we repeated the
experiments with ftn-1 silenced C. elegans (RNAi induced silencing of
gene). Interestingly at this time we observed significantly enhanced
signals in the red channel (Fig. 5b) as compared to wild type strain (Fig.
5a). These results supported the previous findings25 suggesting that
reduced level of ferritins causes elevation of free iron content in the
cytosol. Furthermore, the ratio of the intensities (IRed/IGreen) was
increased by 3 fold (p < 0.001) (Fig. 5c). The confocal imaging results
showed a marked staining pattern in the worm body, posterior to
pharyngeal region particularly concentrated around the gut region. It
has been reported that ftn-1 is upꢀregulated in the presence of
exogenous iron supplementation, and downꢀregulated on treatment with
an iron chelator.26 Therefore to crossꢀexamine whether NAP-3 detected
Fe3+ present in endogenous iron pool, we carried out quantitative real
time PCR (qPCR) assays to quantify the levels of ftn-1 mRNA in
worms. We observed 2.8 fold (p < 0.01) downꢀregulation and 3.7 fold
(p < 0.01) upꢀregulation of ftn-1 mRNA levels in worms from NAP-3
treated and Fe3+ꢀtreated groups respectively (Fig. 5d). Upon treatment
with both NAP-3 and Fe3+, a 1.5 fold reduction (p < 0.05) in levels of
ftn-1 mRNA was observed as compared to ironꢀtreated group (Fig. 5d).
These results unambiguously confirmed that ratiometric probe NAP-3
is capable of detecting labile iron pool in C. elegans. This is the first
report to visualize LIP in an organism with remarkably high selectivity
and in analytically advantageous ratiometric manner.
of NAP-3, DFO failed to remove Fe3+ from the complex (NAPꢀ3 Fe3+)
.
possibly due to steric hindrance (Fig. S20, ESI†).
Promising selectivity of NAP-3 for Fe3+ in both reducing and
oxidizing environments and aqueous compatibility encouraged us to
detect and visualize labile Fe3+ in living systems. Since small molecules
like cysteine and glutathione present in cellular environment may
interact with the probe, we examined the fluorescence response of
NAP-3 in the presence of these molecules (Fig. S21, ESI†).
Gratifyingly, no change in the fluorescence spectra of NAP-3 probe
was observed. Thereafter confocal fluorescence imaging experiments
were carried out with HepG2 cells considering liver as a major iron
storage organ of the body.8 We observed that treatment of cells with
different concentrations (nanomolar to micromolar) of NAP-3 exhibited
a concentrationꢀdependent staining pattern with 3 ꢁM showing the best
results (Fig. 4a, S22, ESI†). The cells incubated with NAP-3 (3 ꢁM) for
24 h exhibited intense intracellular fluorescence in the green channel
(505–550 nm) but no fluorescence in the red channel (Long pass 575
nm) was observed (Fig. 4a). However, HepG2 cells pretreated with
ferric citrate (5 × 10ꢀ4 M, 24 h) followed by incubation with NAP-3 (3
ꢁM, 24 h) showed significant increase in intracellular fluorescence in
the red channel due to the formation of the complex of NAP-3 in situ
with exogenously supplemented Fe3+ (Fig. 4b). Furthermore, NAP-3
exhibited significant intracellular ratiometric fluorescence response
(Fig. 4c). NAP-3 showed no cytotoxicity under the conditions used for
In summary, we have designed and synthesized a novel Fe3+
selective dual colorimetric and ratiometric fluorescent probe NAP-3 for
detection and visualization of exogenous Fe3+ in in vitro (HepG2 cell
line) and for the first time in in vivo (Wild type and ftn-1 silenced C.
elegans) models. The potential of NAP-3 for binding to labile iron pool
in wild type C. elegans was further established by monitoring the
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 3

ChemComm
Page 4 of 4
VJieowuArrtnicalelONnlainme e
COMMUNICATION
DOI: 10.1039/C4CC09798J
Fig. 5 (a) Wild type (N2) C. elegans treated with 3 μM NAP-3 (3 μM, 24 h). (b) ftn-1 silenced C. elegans treated with 3 μM NAP-3. (c) Ratiometric quantification of
relative fluorescence intensity of confocal microscopy images by image J software, ***p < 0.001. (d) Quantitative Real Time PCR analysis of ftn-1 gene (fold change) in
various groups using wild type strain of C. elegans. *p < 0.05, **p < 0.01 (with respect to OP50 treated control). Green channel: λex = 405 nm, λem = 505–550 nm. Red
channel: λex = 561 nm, λem = 575 nm long pass. Each experiment was repeated three independent times. Error bars are + s.e.m. (n = 3). Scale bar: 10 μm.
10. P. Li, L. Fang, H. Zhou, W. Zhang, X. Wang, Na Li, H. Zhong, and
mRNA levels of iron responsive gene ftn-1 via qPCR analysis. The high
selectivity of NAP-3 towards Fe3+ and its biocompatibility open new
avenues to unravel the mysteries of the cellular dynamics of iron
homeostasis and the role of labile iron pools associated with oxidative
stress produced during Fenton chemistry. Further studies in these
directions are underway.
B. Tang, Chem. Eur. J., 2011, 17, 10520.
11. Y. Wei, Z. Aydin, Y. Zhang, Z. Liu and M. Guo, ChemBioChem
,
2012, 13, 1569.
12. (a) E. A. Lemke and C. Schultz, Nat. Chem. Biol., 2011, 7, 480; (b)
S. Maruyama, K. Kikuchi, T. Hirano, Y. Urano and T. Nagano, J.
Am. Chem. Soc., 2002, 124, 10650.
13. G. Accorsi, A. Listorti, K. Yoosaf and N. Armaroli, Chem. Soc.
Rev., 2009, 38, 1690.
14. A. Goel, S. Umar, P. Nag, A. Nazir, L. Kumar, Shamsuzzama, J. R.
The work is supported by the Council of Scientific and Industrial
Research, New Delhi (BSC0102 and BSC0114). Authors thank K.
Mitra and K. Singh for confocal fluorescence imaging of the samples.
The CDRI communication number is 8873.
Gayen, and Z. Hossain, Substituted Naphtho[2,1ꢀb][1,10]ꢀ
phenanthrolineꢀbased fluorescent dyes and application thereof.
CSIR Patent application No. 1942/DEL/2014 (India, 2014).
15. (a) A. Goel, A. Sharma, M. Rawat, R. S. Anand, and R. Kant J.
Org. Chem. 2014, 79, 10873; (b) A. Goel, V. Kumar, S. P. Singh,
Notes and References
aMedicinal and Process Chemistry Division, bDepartment of Toxicology
cDepartment of Pharmacokinetics and Metabolism, CSIR-Central Drug
Research Institute, Lucknow 226031, India; fax: +91-522-2771941; Tel.:
+91-522-2772450; e-mail: atul_goel@cdri.res.in
A. Sharma, S. Prakash, C. Singh and R. S. Anand, J. Mater. Chem.
,
2012, 22, 14880; (c) A. Goel, S. Chaurasia, M. Dixit, V. Kumar, S.
Prakash, B. Jena, J. K. Verma, M. Jain, R. S. Anand and S. S.
Manoharan, Org. Lett., 2009, 11, 1289.
†Electronic Supplementary Information (ESI) available: [Spectroscopic
Characterization data, 1H, 13C NMR spectra and absorption, and
fluorescence spectra of all the compounds 9a-d and 10a-d. Cytotoxicity data,
materials and methods]. See DOI: 10.1039/b000000x/
16. A. Goel, A. Sharma, M. Kathuria, A. Bhattacharjee, A. Verma, P.
R. Mishra, A. Nazir and K. Mitra, Org. Lett., 2014, 16, 756.
17. B. Zhu, C. Gao, Y. Zhao, C. Liu, Y. Li, Q. Wei, Z. Ma, B. Du and
X. Zhang, Chem. Commun., 2011, 47, 8656.
18. H. Zheng, X.ꢀJ. Zhang, X. Cai, Q.ꢀN. Bian, M. Yan, G.ꢀH. Wu, X.ꢀ
W. Lai and Y.ꢀB. Jiang, Org. Lett., 2012, 14, 1986.
1. M. W. Hentze, M. U. Muckenthaler and N. C. Andrews, Cell, 2004,
117, 285.
19. L. N. Neupane, J.ꢀY. Park, J. H. Park and K.ꢀH. Lee, Org. Lett.
2013, 15, 254.
,
2. P. A. Frey and G. H. Reed, ACS Chem. Biol., 2012,
3. Breuer, M. Shvartsman, and Z. I. Cabantchik, Int J Biochem
Cell Biol., 2008, 40, 350.
4. H. J. H. Fenton, J. Chem. Soc. Trans, 1894, 65, 899.
5. (a) S. J. Dixon and B. R. Stockwell, Nat. Chem. Biol., 2014, 10, 9;
(b) D. B. Kell, BMC Medical Genomics, 2009, , 1.
6. D. W. Domaille, E. L. Que and C. J. Chang, Nat. Chem. Biol.
2008, , 168.
7, 1477.
W
.
20. (a) G. J. Zhao and K. L. Han, Acc. Chem. Res., 2012, 45, 404; (b)
G.Y. Li, G. J. Zhao, Y.H. Liu, K. L. Han and G. Z. He, J. Comput.
Chem., 2010, 31, 1759; (c) G. J. Zhao, J. Y. Liu, L. C. Zhou and K.
L. Han, J. Phys. Chem. B, 2007, 111
21. (a) M. Arifuzzaman, T. A. Siddiquee, M. R. Karim, A. H. Mirza
and M. A. Ali, Cryst Struct Theory Applicat., 2013, , 159; (b) J.
Burgess, S. Radulović, F. Sánchez, Transition Met Chem., 1987,12
529; (c) R. Mayilmurugan, M. Sankaralingam, E. Suresh and M.
Palaniandavar, Dalton Trans. 2010, 39 9611; (d) M.
, 8940.
2
,
2
4
,
7. (a) S. Epsztejn, O. Kakhlon, H. Glickstein, W. Breuer and Z. I.
Cabantchik, Anal. Biochem., 1997, 248, 31; (b) F. Petrat, U. Rauen
and H. D. Groot, Hepatology, 1999, 29, 1171.
,
,
Balamurugan, P. Vadivelub and M. Palaniandavar, Dalton Trans.
,
8. (a) T. Hirayama, K. Okuda and H. Nagasawa, Chem. Sci., 2013, 4,
2014, 43, 14653.
125; (b) H. Y. AuꢀYeung, J. Chan, T. Chantarojsiri and C. J.
Chang, J. Am. Chem. Soc., 2013, 135, 15165.
22. C. P. Anderson and E. A. Leibold, Front. Pharmacol., 2014, 5, 1.
23. K. T. Pate, N. A. Rangel, B. Fraser, M. H. S. Clement and C.
Srinivasan, Anal. Biochem., 2006, 358, 199.
24. S. J. Romney, C. Thacker and E. A. Leibold, J. Biol. Chem., 2008,
283, 716.
25. Y.ꢀI. Kim, J. H. Cho, O. J. Yoo and J. Ahnn, J. Mol. Biol., 2004,
342, 421.
26. S. Valentini, F. Cabreiro, D. Ackerman, M. M. Alam, M. B. A.
Kunze, C. W. M. Kay and D. Gems, Mech. Ageing Dev., 2012,
133, 282.
9. (a) S. K. Sahoo, D. Sharma, R. K. Bera, G. Crisponi and J. F.
Callan, Chem. Soc. Rev., 2012, 41, 7195; (b) S. Sen, S. Sarkar, B.
Chattopadhyay, A. Moirangthem, A. Basu, K. Dharad and P.
Chattopadhyay, Analyst, 2012, 137, 3335; (c) R. Wang, F. Yu, P.
Liu and L. Chen, Chem. Commun., 2012, 48, 5310; (d) S.
Goswami, S. Das, K. Aich, D. Sarkar, T. K. Mondal, C. K. Quah
and H.K. Fun, Dalton Trans., 2013, 42, 15113; (e) S. Goswami, K.
Aich, S. Das, A. K. Das, D. Sarkar, S. Panja, T. K. Mondal and S.
Mukhopadhyay, Chem. Commun., 2013, 49, 10739.
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 4