A turn-on NIR fluorescence and colourimetric cyanine probe for monitoring the thiol content in serum and the glutathione reductase assisted glutathione redox process

Article abstract of DOI:10.1039/c2ob27138a

We report a novel reaction-based thiol selective turn-on near-infrared (NIR) fluorescence and colourimetric dinitrobenzenesulfonyl-cyanine (DNBSCy) probe. In the presence of thiols such as glutathione (GSH), new absorption bands (476 and 581 nm) were observed, with the colour of the solution (10 mM PBS, pH = 7.4) changing from light green to blue. Interestingly, relatively non-fluorescent DNBSCy exhibited enhanced fluorescence emission around 700 nm in the NIR region. GSH reacted efficiently with the electron withdrawing sulfonyl ester moiety of DNBSCy, releasing the quinone embedded heptamethine cyanine (Cy-quinone) with extended π-electron conjugation responsible for the turn-on NIR fluorescence. Cy-quinone also displayed a conjugated π-electron push-pull character under physiological conditions. The DNBSCy probe was effectively employed to monitor the thiols in fetal bovine serum (FBS). The probe was capable of monitoring the oxidized glutathione (GSSG)/GSH redox process in the presence of glutathione reductase and NADPH with NIR fluorescence and colourimetric optical response. Thus, DNBSCy has the potential to measure the activity of glutathione reductase as a measure of oxidative stress. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2ob27138a

Organic&
BiomolecularChemistry
www.rsc.org/obc
Volume 11 | Number 13 | 7 April 2013 | Pages 2049–2214
ISSN 1477-0520
PAPER
Debabrata Maity and T. Govindaraju
A turn-on NIR fluorescence and colourimetric cyanine probe for monitoring the
thiol content in serum and the glutathione reductase assisted glutathione
redox process

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
A turn-on NIR fluorescence and colourimetric cyanine
probe for monitoring the thiol content in serum and
the glutathione reductase assisted glutathione redox
process†
Cite this: Org. Biomol. Chem., 2013, 11,
2098
Debabrata Maity and T. Govindaraju*
We report a novel reaction-based thiol selective turn-on near-infrared (NIR) fluorescence and colouri-
metric dinitrobenzenesulfonyl-cyanine (DNBSCy) probe. In the presence of thiols such as glutathione
(GSH), new absorption bands (476 and 581 nm) were observed, with the colour of the solution
(10 mM PBS, pH = 7.4) changing from light green to blue. Interestingly, relatively non-fluorescent
DNBSCy exhibited enhanced fluorescence emission around 700 nm in the NIR region. GSH reacted
efficiently with the electron withdrawing sulfonyl ester moiety of DNBSCy, releasing the quinone
embedded heptamethine cyanine (Cy-quinone) with extended π-electron conjugation responsible for
the turn-on NIR fluorescence. Cy-quinone also displayed a conjugated π-electron push–pull character
under physiological conditions. The DNBSCy probe was effectively employed to monitor the thiols in
fetal bovine serum (FBS). The probe was capable of monitoring the oxidized glutathione (GSSG)/GSH
redox process in the presence of glutathione reductase and NADPH with NIR fluorescence and colouri-
metric optical response. Thus, DNBSCy has the potential to measure the activity of glutathione reductase
as a measure of oxidative stress.
Received 2nd November 2012,
Accepted 11th December 2012
DOI: 10.1039/c2ob27138a
www.rsc.org/obc
one of the potential reasons for oxidative stress.6 In cells, the
enzyme glutathione reductase catalyses the reduction of GSSG
Introduction
The optical monitoring of thiols such as glutathione (GSH), to free GSH, thus preventing the occurrence of oxidative stress.
cysteine (Cys) and homocysteine (Hcy) is of significant interest Hence, it is very crucial to monitor the concentration of GSH
due to their crucial roles in maintaining the biological redox and the activity of glutathione reductase in physiological
homeostasis through the equilibrium of free thiols and oxi- media. Although several strategies have been developed to
dized disulfides in biological systems.1 These low molecular detect these mercapto-biomolecules, fluorescence detection
weight thiols (LMWTs) are essential for the growth of cells and has been recognized as the most convenient method due to its
tissues in living organisms.2 Dramatic changes in the intra- simplicity and low detection limit.7 In the past few years,
cellular thiol concentrations in biological fluids are implicated researchers have developed thiol-selective fluorescence probes
in a number of diseases including liver damage, heart disease, exploiting the strong nucleophilicity of the thiol group and
cancer, AIDS, inflammatory bowel disease, osteoporosis, Alz- their cation binding affinity.7a However, the existing fluore-
heimer’s and cardiovascular diseases.3 Among these LMWTs, scence probes for thiols are plagued by certain serious limit-
GSH is a major endogenous antioxidant produced in the cell ations like excitation and emission in the UV-visible region,
(0.5–10 mM).4 GSH protects the cells from oxidative stress by relatively time consuming procedures, and the use of organic–
trapping free radicals and reactive oxygen species (ROS); it also water mixed media.
maintains exogenous antioxidants in their reduced forms.5
NIR molecular probes have gained special interest in
The abnormal ratio of reduced GSH to oxidized glutathione is recent years due to several inherent advantages over the UV-
visible probes.8 Deeper penetration of the NIR radiation
(650–900 nm) enables the assessment of molecular and physio-
logical events several layers deep inside the analyte samples
and tissues. Moreover, optical response in the NIR region is
not affected by the autofluorescence generated from chromo-
Bioorganic Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for
Advanced Scientific Research, Jakkur P.O., Bangalore-560064, India.
E-mail: tgraju@jncasr.ac.in; Fax: +91 80 22082627; Tel: +91 80 22082969
†Electronic supplementary information (ESI) available: Experimental procedure,
phores and macromolecules present in the analyte or tissue
additional UV-Vis and fluorescence spectra. See DOI: 10.1039/c2ob27138a
2098 | Org. Biomol. Chem., 2013, 11, 2098–2104
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
samples.9 A few research groups have taken the initiative in protected phenolic moiety onto the conjugated cyanine back-
designing thiol-selective NIR sensors.10 However, many of bone.14 The DNBS-phenolic moiety incorporated hepta-
these thiol sensors work by means of reduction in NIR fluor- methine cyanine (DNBSCy) was designed, however, with a
escence. Therefore, a turn-on NIR fluorescence probe based on completely altered π-conjugation pattern and positive charges
the extended π-electron conjugated system that could instantly localised on both the nitrogen atoms (Scheme 1). The thiol
monitor thiols in complete physiological media is necessary.
mediated nucleophilic reaction on the DNBSCy probe releases
Our design strategy for the development of a turn-on NIR the thiol-dinitrobenzene (DNB) by-product, generating the Cy-
fluorescence probe relied on a reaction-based approach in phenolate which rearranges itself by neutralising the positive
which a thiol mediated bond-cleaving reaction converted a charge on a nitrogen atom to form stable Cy-quinone. This Cy-
weak or non-fluorescence probe into a robust NIR fluorescent quinone resumes the extended π-electron conjugation pattern
dye.11 In this full paper we report a highly sensitive turn-on of the cyanine dyes. Similar to parent heptamethine cyanine
NIR fluorescence and colourimetric probe for monitoring (Cy), the Cy-quinone has an odd number of carbon atoms
thiols, especially GSH, over GSSG in physiological media. The between two nitrogen atoms to facilitate conjugation along the
thiol assisted removal of 2,4-dinitrobenzenesulfonyl (DNBS) polymethine chain. It was thus expected to exhibit turn-on NIR
from the hydroxyl group moiety of a fluorophore has been fluorescence besides showing unique visible colour changes
found to have excellent selectivity towards thiol detection with due to the conjugated π-electron push–pull character.
off–on fluorescence signaling.12 Strong electron withdrawing
DNBS functionality would significantly disturb the internal
charge transfer (ICT) process, maximising the fluorescence
switching on–off process. Heptamethine cyanine (Cy), a NIR
Results and discussion
Synthesis of DNBSCy and its photophysical studies in the
fluorescent dye with high extinction coefficient and quantum
presence of thiol
yield, has been extensively used in NIR bioimaging.13 We
devised an interesting approach by incorporating the DNBS A two-step straightforward synthetic route was developed for
the preparation of the DNBSCy probe as outlined in Scheme 1.
The arenesulfonate ester moiety was synthesized by sulfonat-
ing the hydroxyl group of 4-hydroxy-1,3-benzenedicarboxalde-
hyde with 2,4-dinitrobenzenesulfonyl chloride. Good yields of
DNBSCy were obtained by condensing this dialdehyde-arene-
sulfonate ester intermediate with 2 equiv. of indolium-3-butyl-
sulfonate. All the compounds were characterized by NMR,
mass spectrometry, and elemental analysis. The arenesulfo-
nate ester moiety in the DNBSCy probe is highly reactive
towards thiolate, which releases the hydroxyl group of the
cyanine fluorophore backbone in the presence of free thiols.
The indolium-3-butyl-sulfonate chains in DNBSCy are chosen
to impart water solubility and to prevent aggregation of the
cyanine backbone in solution. DNBSCy is a yellow powder and
readily dissolves in water to form a light green solution.
DNBSCy exhibits an absorption band centred around 390 nm
with an extinction coefficient (ε) of 2.6 × 104 M−1 cm−1 in
10 mM PBS buffer (pH = 7.4) (Fig. S1, ESI†). The probe itself is
almost non-fluorescent, displaying a very weak emission band
around 700 nm (Fig. 1).
In the presence of GSH, the absorbance at 390 nm
decreased drastically while two new absorption bands
appeared at 476 nm and 581 nm (Fig. S1, ESI†). Concurrently,
the colour of the solution changed from light green to blue. As
anticipated upon 600 nm excitation (λex), the fluorescence
emission spectra displayed a switch-on peak around 700 nm in
the NIR region (Fig. 1). This large Stokes shift (∼119 nm)
between the absorbance and emission peaks is a highly desir-
able characteristic feature of a fluorescence probe that assists
in increasing the signal-to-noise ratio. Other free thiol contain-
ing amino acids like Cys and Hcy showed similar absorption
and emission spectral changes (Fig. S1, ESI†). However, the
Scheme 1 Synthesis of DNBSCy and molecular structure of Cy-quinone.
presence of amino acids with no thiol functionality or
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2098–2104 | 2099

View Article Online
Paper
Organic & Biomolecular Chemistry
maximum (>25 fold) over a period of 20 min. The GSH titration
study shows that DNBSCy could detect submicromolar concen-
trations of GSH efficiently (Fig. S2, ESI†). The GSH mediated
rapid nucleophilic reaction with the electron withdrawing sul-
fonyl ester moiety of the DNBSCy probe releases the Cy-phenol-
ate anion. The negative charge of Cy-phenolate rearranges
itself by neutralising the positive charge on one of the nitrogen
atoms to form stable Cy-quinone (Scheme 1). In the process,
Cy-quinone resumes a heptamethine cyanine-like extended
π-electron conjugation pattern which is responsible for the
observed characteristic turn-on NIR fluorescence. The conju-
gated π-electron push–pull character is also generated in the
Cy-quinone system that lends a unique blue colour to the solu-
tion. Further, DNBSCy was also found to be highly selective
for GSH over other competing non-thiol amino acids in the
buffer solution (Fig. S3, ESI†). Mass spectroscopic analysis con-
firmed that DNBSCy was converted to the Cy-quinone dye
under the experimental conditions used for GSH detection
(Fig. S4, ESI†). The ESI-mass spectra showed peaks at m/z =
705.2 (calculated for C38H43N2O7S2 + H+) corresponding to Cy-
quinone and at m/z = 474.0 (calculated for C16H19N5O10S) cor-
responding to the by-product γ-glutamyl-S-(2,4-dinitrophenyl)-
cysteinylglycine.
Fig. 1 Fluorescence response of the DNBSCy probe (10.0 μM) towards various
amino acids (1.0 mM) and metal ions (1.0 mM). Each spectrum was acquired
after 30 min incubation of the probe with an analyte in 10 mM PBS buffer (pH
7.4) with λex = 600 nm.
pH dependent study
The effect of pH on the GSH mediated arenesulfonate ester
bond cleavage was studied to understand the efficiency of the
process. DNBSCy alone was stable within the pH range of 5–8
while small changes were observed in the pH range of 8–10
(Fig. 3). However, the DNBSCy probe reacted selectively with
GSH in the biologically relevant pH range of 6–10 to form the
NIR fluorescence emitting Cy-quinone dye. The optimum NIR
fluorescence emission was observed in the commonly used
physiological pH range of 6.5–8.5, which makes the DNBSCy
probe convenient for the detection and monitoring of thiols
Fig. 2 Time-dependent NIR fluorescence spectra recorded when the DNBSCy
probe (10.0 μM) was treated with GSH (5.0 μM) in 10 mM PBS buffer (pH 7.4)
with λex = 600 nm. Inset: Fluorescence intensity changes at 695 nm recorded as
a function of time.
biologically relevant metal ions did not show any significant
spectral changes with DNBSCy (Fig. 1). Though thiols are
present in millimolar concentration inside cells, only 5.0 μM
GSH was found to be enough to display significant NIR fluore-
scence response within two minutes after reacting with the
arenesulfonate ester moiety of the DNBSCy probe (10 μM)
(Fig. 2). This clearly emphasised the sensitivity of the probe, as
DNBSCy responds quickly towards the detection of GSH as low
as three orders of magnitude less than the actual concen-
tration present in normal cells. On addition of GSH to the
DNBSCy solution, NIR fluorescence emission intensity at Emax
= 695 nm increased rapidly in the initial stage and reached the
Fig. 3 Effect of pH on the NIR emission of DNBSCy monitored at 695 nm.
Black trace: DNBSCy (10 μM) and red trace: DNBSCy (10.0 μM) with 5.0 μM of
GSH in 10 mM PBS buffer.
2100 | Org. Biomol. Chem., 2013, 11, 2098–2104
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Scheme 2 Efficient monitoring of free thiol generated from glutathione
reductase (in the presence of NADPH) catalysed GSSG/GSH conversion by coup-
ling the thiol responsive DNBSCy/Cy-quinone transformation with unique turn-
on NIR fluorescence and colourimetric optical response.
Fig. 4 NIR fluorescence spectra of DNBSCy (10.0 μM) upon addition of ali-
quots (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μL) of reduced fetal bovine
serum (λex = 600 nm; Emax = 695 nm).
without interference from the pH-dependent effects of the
physiological media.
to GSH in the presence of the reducing agent nicotinamide
adenine dinucleotide phosphate (NADPH) under physiological
conditions.16 Thus, we monitored the formation of GSH by
reducing the oxidized GSSG in the presence of glutathione
reductase by coupling our free thiol responsive DNBSCY/
Cy-quinone transformation with enzyme catalysed GSSG/GSH
conversion (Scheme 2). DNBSCy reacted instantaneously with
the free GSH generated from GSSG under the assay conditions
(10 mM PBS buffer, 1 unit per mL glutathione reductase,
0.4 mM NADPH) as indicated by the enhanced NIR emission
(>11 fold) within 2 min of adding the enzyme (Fig. S10, ESI†).
This preliminary experiment motivated us to study the kinetic
effect of the reduction of GSSG to GSH at different concen-
trations of the enzyme. This experiment revealed that higher
concentrations of glutathione reductase lowered the time
required for completion of the GSSG/GSH redox process and
its subsequent detection (Fig. 5). Maximum NIR emission
signal was achieved within 20 min when 10 mU mL−1 of gluta-
thione reductase was employed. However, more time (double)
was required to reach the maximum intensity when half the
concentration of glutathione reductase was used. In the
absorption spectra, the DNBSCy absorbance band at 385 nm
decreased while new bands appeared at 475 and 582 nm with
the addition of NADPH and glutathione reductase (Fig. 6).
A clear isosbestic point at 440 nm indicated the formation of
the Cy-quinone dye by the reaction of free GSH on DNBSCy.
The solution colour changed from light green to blue as
a result of formation of the cyanine dye with extended
conjugation.
Reduced thiol detection in fetal bovine serum (FBS)
To ascertain the practical applicability of our turn-on NIR fluo-
rescence probe, thiol content in the fetal bovine serum (FBS)
sample was studied. Thiol containing amino acids generally
occur in the disulfide form in blood serum. Estimation of the
thiol pool in blood serum is crucial for understanding the role
of thiols in the pathogenesis of cardiovascular diseases. We
first treated the plasma (FBS) with a reducing agent triphenyl-
phosphine to reduce all the oxidized disulfide to free thiols.15
Different aliquots of the reduced FBS were added to DNBSCy
in buffer solution (2 mL, 10 mM PBS, pH 7.4) at ambient temp-
erature. Fig. 4 shows the turn-on NIR fluorescence signal and
linear increase in the intensity of the signal with the addition
of reduced FBS. These data clearly suggest the presence of free
thiols that react rapidly with the DNBSCy probe to form the
NIR fluorescence emitting Cy-quinone dye. In addition, spon-
taneous colourimetric changes were observed in the absorp-
tion spectra (Fig. S9, ESI†). The visible light greenish colour of
the solution gradually changed to blue as the amount of
reduced FBS was increased in the analysis mixture under
study. This experiment demonstrates that DNBSCy is capable
of monitoring thiols in the serum sample by means of charac-
teristic turn-on NIR fluorescence as well as colourimetric
optical responses.
Selective detection of reduced GSH over oxidized GSSG using a
combination of glutathione reductase and DNBSCy
Normally, the glutathione reductase activity is monitored
Significant imbalance in the GSH/GSSG ratio is responsible for
oxidative stress and has direct implications for human health. with NADPH consumption by recording the decreased absor-
bance at 340 nm or the free GSH generated can be visualized
from the increased absorbance at 412 nm using Ellman’s
Therefore, the measure of glutathione reductase activity serves
as an indicator for the assessment of oxidative stress. The
enzyme glutathione reductase catalyses the reduction of GSSG reagent. Our DNBSCy probe, with dual optical response
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2098–2104 | 2101

View Article Online
Paper
Organic & Biomolecular Chemistry
serum and to measure the activity of glutathione reductase in
the GSSG/GSH redox process (Scheme 2).
Conclusions
In conclusion, we have successfully developed a thiol selective
water soluble turn-on NIR fluorescence probe DNBSCy. This
molecular probe readily reacts with thiols, in particular GSH,
to release the NIR fluorescence emitting cyanine dye over a
wide pH range. The probe was used effectively for the turn-on
NIR fluorescence and colourimetric monitoring of thiols in
fetal bovine serum (FBS). For the first time, we also demon-
strated the ability of the DNBSCy probe to be used in monitor-
ing the GSSG/GSH redox process in the presence of
glutathione reductase enzyme and the reducing agent NADPH
with the unique combination of NIR turn-on fluorescence and
colourimetric optical response. Therefore, this probe is
capable of monitoring the activity of glutathione reductase
Fig. 5 Glutathione reductase activity assay. NIR fluorescence response of
DNBSCy (10.0 μM) at 695 nm (λex = 600 nm) monitored as a function of time. In
the assay mixture glutathione reductase reduces GSSG to reduced free GSH
form using a reducing agent NADPH (100.0 μM). The activity of enzyme gluta- which in turn serves as a tool to assess the levels of oxidative
thione reductase was measured at three different concentrations (2.5, 5.0 and
10.0 mU mL−1) in 10 mM PBS buffer (pH 7.4).
stress. In general, DNBSCy can be used as a non-invasive tool
for the determination of thiol content in biological fluids,
measuring the activities of the enzymes involved in thiol
coupled redox processes and in vivo NIR fluorescence imaging
applications.
Experimental section
Materials and instruments
All the solvents and reagents were obtained from Sigma-
Aldrich and used as received unless otherwise mentioned.
1H and 13C NMR were recorded on a Bruker AV-400 spec-
trometer with chemical shifts reported as ppm (in DMSO-d6,
tetramethylsilane as an internal standard). Mass spectra were
obtained on a Shimadzu 2020 LC-MS. Elemental analysis was
carried out on a ThermoScientific FLASH 2000 Organic
Element Analyzer. UV/Vis spectra were recorded on a Perkin
Elmer Lambda 900 spectrophotometer and fluore-
scence spectra were recorded on a Perkin Elmer LS 55
spectrophotometer.
Fig. 6 Time-dependent UV/Vis absorption spectra of DNBSCy (10.0 μM). Assay
conditions: 10 mM PBS buffer (pH = 7.4), GSSG, NADPH (10.0 μm), glutathione
reductase (10 mU mL−1). Inset: Absorbance changes at 582 nm recorded as a
function of time.
Synthesis of 2,4-dinitrophenyl 2,4-diformylphenyl sulphate
A mixture of 4-hydroxy-1,3-benzenedicarboxaldehyde (150 mg,
1
mmol), 2,4-dinitrobenzenesulfonyl chloride (292 mg,
1.1 mmol) and triethylamine (0.15 mL, 1.1 mmol) in dichloro-
methane (10 mL) was stirred at 0 °C for 1 h under an inert
atmosphere. The reaction mixture was washed with water. The
organic phase was dried over Na2SO4 and the solvent was evap-
orated to dryness. The crude product was purified by column
chromatography on silica gel using chloroform as an eluent to
obtain a yellow solid (68%). 1H NMR (400 MHz, [d6]DMSO)
δppm 7.50 (1H, d, J = 8.4 Hz), 8.23 (1H, dd, J = 6.4 Hz, 2 Hz),
8.37 (1H, d, J = 8.8 Hz), 8.46 (1H, d, J = 2.0 Hz), 8.65 (1H, dd,
J = 6.4 Hz, 2.0 Hz), 9.14 (1H, d, J = 2.4 Hz), 10.11 (1H, s), 10.16
(1H, s). 13C NMR (100 MHz, [d6]DMSO) δppm 121.2, 124.2,
(turn-on NIR fluorescence and colourimetric response), pro-
vides a sophisticated, non-invasive and accurate way of moni-
toring the activity of glutathione reductase which in turn can
be a parameter for measuring oxidative stress. As an added
advantage, this probe allows the naked eye detection of GSSG
to GSH conversion in the presence of glutathione reductase
without the need for expensive instrumentation. To the best of
our knowledge, dual responsive DNBSCy is the first probe with
an excellent combination of turn-on NIR fluorescence and col-
ourimetric optical response to detect the free thiol content in
2102 | Org. Biomol. Chem., 2013, 11, 2098–2104
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
127.7, 129.3, 130.3, 131.4, 133.8, 135.6, 135.8, 148.0, 151.7,
152.0, 187.4, 191.5. LCMS: m/z
C14H8N2O9S. Elemental analysis: Found: C, 44.19; H, 2.12; N,
7.38, Calcd: C, 44.22; H, 2.12; N, 7.37 for C14H8N2O9S.
2007, 9, 3375; (d) S.-T. Huang, K.-N. Ting and K.-L. Wang,
Anal. Chim. Acta, 2008, 620, 120; (e) W. Jiang, Y. Cao, Y. Liu
and W. Wang, Chem. Commun., 2010, 46, 1944;
(f) H. S. Jung, J. H. Han, T. Pradhan, S. Kim, S. W. Lee,
J. L. Sessler, T. W. Kim, C. Kang and J. S. Kim, Biomaterials,
2012, 33, 945; (g) P. Wang, J. Liu, X. Lv, Y. Liu, Y. Zhao and
W. Guo, Org. Lett., 2012, 14, 520.
= 381.2 [M +
H]+ for
Synthesis of the dinitrobenzenesulfonyl-cyanine (DNBSCy)
probe
3 (a) L. A. Herzenberg, S. C. De Rosa, J. G. Dubs,
M. Roederer, M. T. Anderson, S. W. Ela, S. C. Deresinski
and L. A. Herzenberg, Proc. Natl. Acad. Sci. U. S. A., 1997,
94, 1967; (b) S. Shahrokhian, Anal. Chem., 2001, 73, 5972;
(c) D. M. Townsend, K. D. Tew and H. Tapiero, Biomed.
Pharmacother., 2003, 57, 145; (d) R. Njälsson and
S. Norgren, Acta Paediatr., 2005, 94, 132; (e) H. P. Wu,
C. C. Huang, T. L. Cheng and W. L. Tseng, Talanta, 2008,
76, 347; (f) H. S. Jung, J. H. Han, Y. Habata, C. Kang and
J. S. Kim, Chem. Commun., 2011, 47, 5142.
A
mixture of 2,4-dinitrophenyl-2,4-diformylphenyl sulfate
(76 mg, 0.2 mmol), indolium-3-butyl-sulfonate (118 mg,
0.4 mmol) and NaOAc (32 mg, 0.4 mmol) was dissolved in
3 mL Ac2O. The reaction mixture was stirred for 30 min at
80 °C under an argon atmosphere. After completion, the reac-
tion mixture was concentrated by evaporation under reduced
pressure. The crude product was diluted with 3.0 mL H2O,
3.0 mL ACN, 300 μL AcOH, and purified by preparative
RP-HPLC (grad. 10–90% ACN in water, 20 min) to obtain the
probe DNBSCy (130 mg, 70%) as a yellow powder. 1H NMR
(400 MHz, [d6]DMSO) δppm 1.79 (6H, s), 1.84 (6H, s), 1.86–1.91
(4H, m), 2.04–2.11 (4H, m), 2.62 (2H, t, J = 6.8 Hz), 2.69 (2H, t,
J = 6.7 Hz), 4.80–4.87 (4H, m), 7.41 (1H, d, J = 8.5 Hz),
7.65–7.69 (4H, m), 7.89–7.93 (2H, m), 8.03–8.12 (3H, m),
8.21–8.35 (3H, m), 8.48–8.53 (2H, m), 8.68 (1H, dd, J = 6.4 Hz,
2.2 Hz), 9.18 (1H, d, J = 2.2 Hz), 9.35 (1H, d, J = 1.5 Hz).
13C NMR (100 MHz, [d6]DMSO) δppm 22.0, 22.1, 25.3, 26.4,
26.6, 47.1, 47.4, 49.2, 49.4, 52.5, 53.0, 115.7, 115.9, 116.5,
118.1, 121.5, 123.1, 127.9, 129.0, 129.1, 129.3, 129.9, 130.1,
130.7, 131.1, 133.9, 135.1, 136.0, 140.6, 140.7, 142.4, 144.1,
144.2, 148.0, 149.4, 149.6, 151.7, 157.9, 158.3, 181.5, 181.9.
LCMS: m/z = 935.1 [M]+ for C44H46N4O13S3. Elemental analysis:
Found: C, 56.51; H, 4.95; N, 6.01, Calcd: C, 56.52; H, 4.96; N,
5.99 for C44H46N4O13S3.
4 A. Meister and M. E. Anderson, Annu. Rev. Biochem., 1983,
52, 711.
5 (a) R. H. Schirmer, J. G. Müller and R. L. Krauth-Siegel,
Angew. Chem., 1995, 107, 153; (b) T. P. Dalton,
H. G. Shertzer and A. Puga, Annu. Rev. Pharmacol. Toxicol.,
1999, 39, 67; (c) S. M. Kanzok, R. H. Schirmer,
I. Turbachova, R. Iozef and K. Becker, J. Biol. Chem., 2000,
275, 40180; (d) R. L. Krauth-Siegel, H. Bauer and
R. H. Schirmer, Angew. Chem., 2005, 117, 698; (e) X. Chen,
S.-K. Ko, M. J. Kim, I. Shin and J. Yoon, Chem. Commun.,
2010, 46, 2751.
6 (a) T. P. Akerboom, M. Bilzer and H. Sies, J. Biol. Chem.,
1982, 257, 4248; (b) O. W. Griffith, Free Radical Biol. Med.,
1999, 27, 922; (c) D. M. Townsend, K. D. Tew and
H. Tapiero, Biomed. Pharmacother., 2003, 57, 145;
(d) D. Han, N. Hanawa, B. Saberi and N. Kaplowitz,
Am. J. Physiol., 2006, 291, G1; (e) D. Ścibior, M. Skrzycki,
M. Podsiad and H. Czeczot, Clin. Biochem., 2008, 41, 852;
(f) H. S. Jung, K. C. Ko, G.-H. Kim, A.-R. Lee, Y.-C. Na,
C. Kang, J. Y. Lee and J. S. Kim, Org. Lett., 2011, 13, 1498;
(g) B. K. McMahon and T. Gunnlaugsson, J. Am. Chem. Soc.,
2012, 134, 10725.
Acknowledgements
We thank Prof. C. N. R. Rao, FRS for constant support and
encouragement, JNCASR and the Department of Science and
Technology (DST), India for financial support and CSIR,
New Delhi, India for awarding an SRF to D.M.
7 (a) X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev.,
2010, 39, 2120; (b) D. Maity and T. Govindaraju,
Eur. J. Inorg. Chem., 2011, 5479; (c) D. Maity and
T. Govindaraju, Chem. Commun., 2012, 48, 1039.
Notes and references
1 (a) Z. A. Wood, E. Schröder, J. Robin Harris and L. B. Poole,
Trends Biochem. Sci., 2003, 28, 32; (b) M. Zhang, M. Yu,
F. Li, M. Zhu, M. Li, Y. Gao, L. Li, Z. Liu, J. Zhang,
D. Zhang, T. Yi and C. Huang, J. Am. Chem. Soc., 2007, 129,
10322; (c) S.P. Wang, W.-J. Deng, D. Sun, M. Yan, H. Zheng
and J.-G. Xu, Org. Biomol. Chem., 2009, 7, 4017; (d) V. Hong,
A. A. Kislukhin and M. G. Finn, J. Am. Chem. Soc., 2009,
131, 9986; (e) X. Yang, Y. Guo and R. M. Strongin, Angew.
Chem., Int. Ed., 2011, 50, 10690.
2 (a) A. P. de Silva, H. Q. N. Gunaratne and T. Gunnlaugsson,
Tetrahedron Lett., 1998, 5077; (b) D. H. Kim and M. S. Han,
Bioorg. Med. Chem. Lett., 2003, 13, 2543; (c) T. Matsumoto,
Y. Urano, T. Shoda, H. Kojima and T. Nagano, Org. Lett.,
8 (a) G. Patonay and M. D. Antoine, Anal. Chem., 1991, 63,
321A; (b) R. B. Thompson, Top. Fluoresc. Spectrosc., 1994, 4,
151; (c) S. Stoyanov, Pract. Spectrosc., 2001, 25, 35;
(d) W. M. Leevy, S. T. Gammon, J. R. Johnson,
A. J. Lampkins, H. Jiang, M. Marquez, D. Piwinica-Worms
and B. D. Smith, Bioconjugate Chem., 2008, 19, 686;
(e) D. Maity and T. Govindaraju, Chem.–Eur. J., 2011, 17,
1410; (f) D. Maity, A. K. Manna, D. Karthigeyan,
T. K. Kundu, S. K. Pati and T. Govindaraju, Chem.–Eur. J.,
2011, 17, 11152.
9 (a) J. Fabian, H. Nakazumi and M. Matsuoka, Chem. Rev.,
1992, 92, 1197; (b) J. V. Frangioni, Curr. Opin. Chem. Biol.,
2003, 7, 626; (c) B. A. Smith, W. J. Akers, W. M. Leevy,
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2098–2104 | 2103

View Article Online
Paper
Organic & Biomolecular Chemistry
A. J. Lampkins, S. Z. Xiao, W. Wolter, M. A. Suckow, 12 (a) H. Maeda, H. Matsuno, M. Ushida, K. Katayama,
S. Achilefu and B. D. Smith, J. Am. Chem. Soc., 2010, 132,
67; (d) J. M. Baumes, J. J. Gassensmith, J. Giblin, J.-J. Lee,
A. G. White, W. J. Culligan, W. M. Leevy, M. Kuno and
B. D. Smith, Nature Chem., 2010, 2, 1025.
K. Saeki and N. Itoh, Angew. Chem., Int. Ed., 2005, 44, 2922;
(b) H. Maeda, K. Katayama, H. Matsuno and T. Uno, Angew.
Chem., Int. Ed., 2006, 45, 1810; (c) W. Jiang, Q. Fu,
H. Fan, J. Ho and W. Wang, Angew. Chem., Int. Ed.,
2007, 46, 8445; (d) J. Bouffard, Y. Kim, T. M. Swager,
R. Weissleder and S. A. Hilderbrand, Org. Lett., 2008, 10,
37; (e) A. Shibata, K. Furukawa, H. Abe, S. Tsuneda and
Y. Ito, Bioorg. Med. Chem. Lett., 2008, 18, 2246;
(f) S. Ji, J. Yang, Q. Yang, S. Liu, M. Chen and J. Zhao,
J. Org. Chem., 2009, 74, 4855; (g) H. Guo, Y. Jing, X. Yuan,
S. Ji, J. Zhao, X. Li and Y. Kan, Org. Biomol. Chem., 2011, 9,
3844; (h) J. Shao, H. Sun, H. Guo, S. Ji, J. Zhao, W. Wu,
X. Yuan, C. Zhang and T. D. James, Chem. Sci., 2012, 3,
1049.
10 (a) S. Sreejith, K. P. Divya and A. Ajayaghosh, Angew. Chem.,
Int. Ed., 2008, 47, 7883; (b) H. S. Hewage and E. V. Anslyn,
J. Am. Chem. Soc., 2009, 131, 13099; (c) Z. Guo, S. W. Nam,
S. Park and J. Yoon, Chem. Sci., 2012, 3, 2760; (d) R. Wang,
L. Chen, P. Liu, Q. Zhang and Y. Wang, Chem.–Eur. J., 2012,
18, 11343.
11 (a) B. Tang, Y. Xing, P. Li, N. Zhang, F. Yu and G. Yang,
J. Am. Chem. Soc., 2007, 129, 11666; (b) M. M. Pires and
J. Chmielewski, Org. Lett., 2008, 10, 837; (c) J. H. Lee,
C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am. Chem.
Soc., 2010, 132, 1216; (d) X. Li, S. Qian, Q. He, B. Yang, J. Li 13 (a) W. M. Leevy, S. T. Gammon, H. Jiang, J. R. Johnson,
and Y. Hu, Org. Biomol. Chem., 2010, 8, 3627; (e) B. Zhu,
X. Zhang, Y. Li, P. Wang, H. Zhang and X. Zhuang, Chem.
Commun., 2010, 46, 5710; (f) C. Zhao, Y. Zhou, Q. Lin,
L. Zhu, P. Feng, Y. Zhang and J. Cao, J. Phys. Chem. B, 2011,
115, 642; (g) X. Cao, W. Lin and Q. Yu, J. Org. Chem., 2011,
76, 7423; (h) K. Cui, Z. Chen, Z. Wang, G. Zhang and
D. Zhang, Analyst, 2011, 136, 191; (i) L. Long, W. Lin,
B. Chen, W. Gao and L. Yuan, Chem. Commun., 2011, 47,
893; ( j) C. S. Lim, G. Masanta, H. J. Kim, J. H. Han,
D. J. Maxwell, E. N. Jackson, M. Marquez, D. Piwnica-
Worms and B. D. Smith, J. Am. Chem. Soc., 2006, 128,
16476; (b) K. Kiyose, H. Kojima and T. Nagano,
Chem.–Asian. J., 2008, 3, 506; (c) S. Atilgan, T. Ozdemir and
E. U. Akkaya, Org. Lett., 2008, 10, 4065; (d) X. Chen,
S. W. Nam, G. H. Kim, N. Song, Y. Jeong, I. Shin, S. K. Kim,
J. Kim, S. Park and J. Yoon, Chem. Commun., 2010, 46,
8953; (e) Z. Guo, W. Zhu, M. Zhu, X. Wu and H. Tian,
Chem.–Eur. J., 2010, 16, 14424.
H. M. Kim and B. R. Cho, J. Am. Chem. Soc., 2011, 133, 14 N. Karton-Lifshin, E. Segal, L. Omer, M. Portnoy, R. Satchi-
11132; (k) M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya,
J. Y. Kim, J. L. Sessler, C. Kang and J. S. Kim, J. Am. Chem.
Fainaro and D. Shabat, J. Am. Chem. Soc., 2011, 133,
10960.
Soc., 2012, 134, 1316; (l) Q. Lin, C. Bao, S. Cheng, Y. Yang, 15 W. Lin, L. Yuan, Z. Cao, Y. Feng and L. Long, Chem.–Eur. J.,
W. Ji and L. Zhu, J. Am. Chem. Soc., 2012, 134, 5052; 2009, 15, 5096.
(m) D. Maity, V. Kumar and T. Govindaraju, Org. Lett, 2012, 16 L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew.
14, 6008.
Chem., Int. Ed., 2009, 121, 4094.
2104 | Org. Biomol. Chem., 2013, 11, 2098–2104
This journal is © The Royal Society of Chemistry 2013