Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.analchem.5b04333

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aqueous solution and living cells. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.analchem.5b04333

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Article
A broadly applicable strategy for the fluorescence based
detection and differentiation of glutathione and cysteine/
homocysteine: Demonstration in-vitro and in-vivo
Wenqiang Chen, Hongchen Luo, Xingjiang Liu, James Walter Foley, and Xiangzhi Song
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04333 • Publication Date (Web): 25 Feb 2016
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Analytical Chemistry
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A broadly applicable strategy for the fluorescence based detection
and differentiation of glutathione and cysteine/homocysteine:
Demonstration in-vitro and in-vivo
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Wenqiang Chen,‡a,b Hongchen Luo,‡a Xingjiang Liu,a James W. Foleyc and Xiangzhi Song*a
aCollege of Chemistry & Chemical Engineering, Central South University, 410083 Changsha, Hunan Province, P.R. China.
bCollege of Chemistry and Materials Science, Guangxi Teachers Education University, 530001 Nanning, Guangxi, P. R.
China.
cRowland Institute at Harvard , Harvard University, 02142, 100 Edwin H. Land Blvd. Cambridge, MA, USA.
*Corresponding author, Fax: +86ꢀ731ꢀ88836954; Tel: +86ꢀ731ꢀ88836954; Eꢀmail: song@rowland.harvard.edu
ABSTRACT: Glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) are small biomolecular thiols that are present in all cells
and extracellular fluids of healthy mammals. It is well known that each plays a separate, critically important role in human physiolꢀ
ogy and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescenceꢀbased
methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between
GSH and Cys/Hcy. In this report we demonstrate a broadly applicable approach for the design of fluorescent probes that can
achieve this goal. The strategy we employ is to conjugate a fluorescenceꢀquenching 7ꢀnitroꢀ2,1,3ꢀbenzoxadiazole (NBD) moiety to
a selected fluorophore (Dye) through a sulfhydrylꢀlabile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of
GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a
fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage
product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy
compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearꢀ
rangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from
Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone,
Bodipy and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detecꢀ
tion of GSH over Cys/Hcy in both aqueous solution and living cells.
transsulfuration) and, additionally, in mammals is a precursor
of Cys.6ꢀ7 Because of their ubiquity in mammals, especially
INTRODUCTION SECTION
Sulfhydryl groups have many characteristics that have provꢀ
en to be highly beneficial to mammals, plants and a variety of
microscopic forms of life. The most prominent of these propꢀ
erties are the ability to (i) sequester inimical metal ions, (ii)
inactivate reactive free radicals, (iii) quench reactive oxygen
species and (iv) undergo reversible redoxꢀmediated dimerizaꢀ
tion reactions.1 Although evolutionary processes have proꢀ
duced a wide variety of thiol containing oligomers that are
often unique to individual species, three specific small bioꢀ
molecular thiols – glutathione (GSH), cysteine (Cys) and hoꢀ
mocysteine (Hcy) – are present in all mammalian species as
well as in some plants, bacteria and fungi.2 Examples of the
multiple roles each plays in mammalian physiology include:
GSH, a mercaptotripeptide, functions as a detoxifying antioxiꢀ
dant that protects cells against damage caused by inimical
heavy metals, peroxides and energetic free radicals as well as
being an endogenous regulator of apoptosis.3ꢀ4 Cys is a proꢀ
teinogenic amino acid that confers structural integrity to proꢀ
teins through the formation of crosslinking disulfide bonds and
is also a precursor to GSH.5 Hcy is a nonꢀproteinogenic amino
acid whose significance derives from its central role in three
metabolic pathways (remethylation, transmethylation and
humans, numerous studies have focused on establishing conꢀ
centration levels of each in cells and in blood plasma that is
required for good health. The findings of these studies indicate
that abnormal levels of these biothiols are closely associated
with a variety of health disorders including Alzheimer’s and
Parkinson’s diseases, heart diseases, skin lesions, liver damꢀ
age, cancer and HIV infection.8ꢀ10 Recognition of the correlaꢀ
tion each of these biothiols has with a particular subset of disꢀ
ease states has provided the incentive for researchers to develꢀ
op methods to qualitatively and quantitatively measure both
their presence and concentration inꢀvitro and inꢀvivo. Because
of the sensitivity of fluorescenceꢀbased methodologies,11 conꢀ
siderable effort has been expended in the development of
probes that can detect and measure these physiologically imꢀ
portant mercaptans. Several recent articles12ꢀ20 devoted to this
subject have noted that while a relative large number of innoꢀ
vative fluorescent probes have been discovered that can disꢀ
tinguish and measure sulfuryl containing biomolecules from
other natural amino acids and their oligomers, few can distinꢀ
guish GSH from Cys or Hcy.18ꢀ27 While being of considerable
value, most of the methods that can differentiate these three
species are subject to a variety limitations including spectral
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Analytical Chemistry
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overlap between multiple emitting channels, slow response, or mg) and trimethylamine (0.15 mmol, 20 µL) were dissolved in
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relatively poor selectivity.
anhydrous acetonitrile (1 mL) and the resulting mixture was
stirred at room temperature for 2 h. After removal of the solꢀ
vent, the crude product was purified using column chromatogꢀ
raphy (silica gel; DCM: PE=1:1, v/v) to afford pure NBD-S-
In this report, we present a generic strategy for the design
of fluorescent probes that can simultaneously detect and
measure GSH in the absence and presence of Cys/Hcy and
vice versa in vitro and inꢀvivo.
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nPr as a yellow solid (13 mg, 56%). H NMR (500 MHz,
CDCl3) δ 8.42 (d, J = 7.9 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H),
3.28 (t, J = 7.3 Hz, 2H), 1.92 (h, J = 7.3 Hz, 2H), 1.18 (t, J =
7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 149.2, 142.5,
142.1, 132.5, 130.7, 120.2, 33.7, 21.5, 13.5.
EXPERIMENTAL SECTION
Materials and Instruments: Unless otherwise noted, all
reagents were obtained from commercial suppliers and used
without further purification. Solvents were purified by standꢀ
ard methods prior to use. Twiceꢀdistilled water was used
throughout all experiments. NMR spectra were recorded on a
BRUKER 400/500 spectrometer with TMS as an internal
standard. All accurate mass spectrometric experiments were
performed on a micrOTOFꢀQ II mass spectrometer (Bruker
Daltonik, Germany). UVꢀVis absorption spectra were recorded
on a Shimadzu UVꢀ2450 spectrophotometer. Fluorescence
spectra were recorded at room temperature using a HITACHI
Fꢀ7000 fluorescence spectrophotometer with both the excitaꢀ
tion and emission slit widths set at 5.0 nm. HPLC chromatoꢀ
grams were obtained using a Shimadzu LCꢀ16 series instruꢀ
ment. TLC analysis was performed on silica gel plates and
column chromatography was conducted using silica gel (mesh
200ꢀ300), both of which were obtained from Qingdao Ocean
Chemicals, China.
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Synthesis of compound NBD-OF: In a 5 mL roundꢀbottom
flask, compound FOH28 (0.15 mmol, 45 mg), NBD-Cl (0.15
mmol, 30 mg) and trimethylamine (0.16 mmol, 22 µL) were
dissolved in anhydrous acetonitrile (2 mL) and the resulting
mixture was stirred overnight. The resulted precipitate was
filtered and washed with cold ethanol (2×5 mL) and airꢀdried
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to afford pure NBD-OF as a brown solid (37 mg, 55%). H
NMR (500 MHz, DMSOꢀd6) δ 8.61 (d, J = 8.4 Hz, 1H), 8.06
(dd, J = 7.9, 1.4 Hz, 1H), 7.93 – 7.81 (m, 3H), 7.60 (d, J = 8.9
Hz, 2H), 7.54 (t, J = 7.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H),
7.00 (d, J = 15.8 Hz, 1H), 6.71 (d, J = 9.0 Hz, 2H), 2.99 (s,
6H). 13C NMR (125 MHz, DMSOꢀd6) δ 170.1, 157.5, 155.2,
152.7, 152.4, 145.4, 144.83, 140.9, 138.2, 135.8, 134.9, 132.7,
131.0, 130.9, 125.8, 125.6, 124.1, 122.5, 118.8, 112.3, 111.4,
109.9, 107.8, 9.3. ESIꢀMS: [M+H]+ calcd for 471.1299, found
471.1302.
Synthesis of compound NBD-OBodipy: In a 5 mL roundꢀ
bottom flask, compound Bodipy-OH (0.04 mmol, 20 mg),
NBD-Cl (0.04 mmol, 8 mg) and potassium carbonate (0.15
mmol, 21 mg) were dissolved in anhydrous acetone (2 mL)
and the resulting mixture was stirred at room temperature for 4
h. After removal of the solvent, the residue was further puriꢀ
fied by silica gel chromatography (DCM: PE=1:1, v/v) to afꢀ
Cell Culture and Fluorescence Imaging: HeLa cells were
seeded in a 6ꢀwell plate in Dulbecco’s modified Eagle’s mediꢀ
um (DMEM) supplemented with 10% fetal bovine serum and
1% penicillin. The cells were incubated under an atmosphere
of 5% CO2 and 95% air at 37°C for 24 h. Cell imaging was
performed with a FV500 laser scanning confocal microscope.
Before each experiment, cells were washed with PBS buffered
solution 3 times. The cells were then incubated with NBDꢀOF
(2.0 µM), or pretreated with GSH (1.0 mM, 30 min), Cys (1.0
mM, 30 min) or NEM (2.0 mM, 60 min) and further incubated
with NBD-OF (2.0 µM) at 37 °C (for 60 min). Before the
fluorescence imaging experiments were performed, cells were
washed with PBS buffer 3 times. The samples were excited at
488 nm. Emission was collected at 510ꢀ535 nm (green chanꢀ
nel) and 610ꢀ660 nm (red channel). With regard to NBD-
OBodipy, similar experimental procedures were used except
different excitation wavelengths were used (476 nm for green
channel and 633 nm for red channel).
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ford pure NBD-OBodipy as a black solid (24 mg, 94%). H
NMR (500 MHz, CDCl3) δ 8.53 (d, J = 8.3 Hz, 1H), 7.69 –
7.63 (m, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz,
2H), 7.28 – 7.19 (m, 2H), 6.96 (t, J = 7.4 Hz, 1H), 6.89 (d, J =
14.6 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 6.63 (d, J = 8.3 Hz,
1H), 6.57 (s, 1H), 5.98 (s, 1H), 5.72 (d, J = 12.2 Hz, 1H), 3.24
(s, 3H), 2.59 (s, 3H), 1.65 (s, 5H), 1.58 (s, 3H), 1.50 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 162.0, 156.6, 153.7, 153.2,
150.3, 145.2, 144.4, 144.1, 142.3, 139.2, 137.2, 136.8, 135.0,
133.7, 133.5, 133.2, 131.6, 131.3, 131.1, 130.4, 128.4, 128.0,
121.6, 121.3, 120.9, 119.8, 118.4, 113.6, 108.0, 107.0, 98.5,
46.6, 46.4, 29.6, 29.3, 28.7, 15.0, 14.2. ESIꢀMS: [M+H]+ calcd
for 687.2697, found 687.2706.
Synthesis of compound NBD-OCoumarin:29 In a 10 mL
roundꢀbottom flask, compound CoumarinꢀOH (0.15 mmol, 27
mg), NBD-Cl (0.15 mmol, 30 mg) and trimethylamine (0.32
mmol, 44 µL) were dissolved in anhydrous ethanol (3 mL) and
the resulting mixture was stirred at room temperature for 8 h.
The resulted precipitate was filtered, washed with cold methaꢀ
nol (2×4 mL) and airꢀdried to afford pure NBD-OCoumarin
as brown solid (32 mg, 60%). 1H NMR (400 MHz, DMSOꢀd6)
δ 8.67 (d, J = 8.3 Hz, 1H), 7.99 (d, J = 8.7 Hz, 1H), 7.57 (d, J
= 2.4 Hz, 1H), 7.45 (dd, J = 8.7, 2.4 Hz, 1H), 7.00 (d, J = 8.3
Hz, 1H), 6.47 (s, 1H), 2.49 (s, 3H). 13C NMR (100 MHz,
DMSOꢀd6) δ 159.9, 156.1, 154.7, 153.4, 152.3, 145.9, 144.9,
135.6, 131.6, 128.3, 118.6, 117.2, 114.5, 112.3, 109.2, 18.7.
Synthesis of NBD-N-nBu: In a 5 mL roundꢀbottom flask,
4ꢀchloroꢀ7ꢀnitroꢀ2,1,3ꢀbenzoxadiazole (NBD-Cl) (0.1 mmol,
20 mg) and nꢀbutylamine (0.5 mmol, 50 µL) were dissolved in
anhydrous acetonitrile (1 mL). The resulting mixture was
stirred at room temperature for 2 h. The reaction mixture was
poured into water (20 mL) and extracted with dichloromethane
(2×20 mL). The organic layer was washed successively with
water and brine, and dried over anhydrous Na2SO4 overnight.
Following removal of the solvent, the crude product was puriꢀ
fied by silica gel chromatography (DCM: PE = 1:1, v/v) to
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afford pure NBD-N-nBu as a brown solid (20 mg, 90%). H
NMR (500 MHz, CDCl3) δ 8.50 (d, J = 8.6 Hz, 1H), 6.35 (s,
1H), 6.20 (d, J = 8.6 Hz, 1H), 3.53 (d, J = 5.8 Hz, 2H), 1.92 –
1.76 (m, 2H), 1.62 – 1.48 (m, 2H), 1.04 (t, J = 7.3 Hz, 3H). 13
C
NMR (125 MHz, CDCl3) δ 144.3, 144.0, 143.9, 136.6, 123.8,
98.6, 43.8, 30.5, 20.2, 13.7.
RESULTS AND DISCUSSION
Synthesis of NBD-S-nPr: In a 5 mL roundꢀbottom flask,
NBD-Cl (0.1 mmol, 20 mg), 1ꢀpropanethiol (0.15 mmol, 11
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Analytical Chemistry
The strategy we used to design fluorescent probes for the
ment product, NBD-S-Cys/Hcy(2a/2b), can undergo a further,
rapid Smiles rearrangement to give NBD-N-Cys/Hcy(3a/3b)
via a 5ꢀ or 6ꢀmember cyclic intermediate, respectively. Such a
transformation would be accompanied by the formation of a
second, intense emission band (B2) corresponding to that of
NBD-NHR. We note that we would expect aminoꢀbiothiols
such as GSH to participate in the initial SNAr displacement
reaction with NBD-O-Dye but not in the subsequent Smiles
rearrangement because to do so would require the intervention
of a 10ꢀmember cyclic transition state. Consequently, the sigꢀ
nature of such a reaction would be the formation of only a B1
band. In summary, with the expectation that the only signifiꢀ
cant aminoꢀbiothiols that would normally be encountered in
mammalian cells and blood are GSH, Cys and Hcy, treatment
of either cells or plasma with NBD-O-Dye would give (i) no
fluorescence signal in the absence of all three, (ii) a single B1
band if only GSH was present, and (iii) dual B1 + B2 signals
with prescribed relative intensities if only Cys and/or Hcy
were present; intermediate B1 + B2 relative band intensities
should, upon deconvolution, give the ratio of GSH to Cys/Hcy.
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simultaneous detection and quantitation of GSH and Cys/Hcy,
as depicted in Scheme 1, was based on wellꢀestablished conꢀ
cepts and experimental observations. Most important among
these were: (1) Sulfhydryl groups (more polarizable) are both
(i) more nucleophilic and (ii) better SNAr leaving groups than
are amino moieties (less polarizable);30ꢀ31 (2) In physiological
conditions (pH=7.4), the thiols are ionized, while the amino
groups are protonated. As a result, 4ꢀaryloxyꢀ7ꢀnitroꢀ2,1,3ꢀ
benzoxadiaꢀzoles (NBD-OAr) are susceptible to SNAr substiꢀ
tution by sulfhydryl alkanes but are resistant to attack by amiꢀ
no alkanes under this condition; (3) Intramolecular rearrangeꢀ
ments involving 5 and 6 membered rings are kinetically
strongly favored over those involving larger ring systems;32
(4) Fluorescence efficiency of 4ꢀsubstitutedꢀ7ꢀnitroꢀ2,1,3ꢀ
benzoxadiazoles (NBD-X) markedly depends on the electronꢀ
donating ability of X through effective ICT process (e.g. relaꢀ
tive fluorescence quantum yields in methanol: X = NHCH3:
62.7; SCH3: 0.6; OCH3, 0.0);33 (5) 7ꢀNitroꢀ2,1,3ꢀ
benzoxadiazole (NBD) can efficiently quench the fluorescence
of a variety of dyes via a strong photoinduced electron transfer
(PET) process.
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Scheme 2. Synthetic route to NBD-OF and chemical struc-
ture of control compounds NBD-N-NBu and NBD-S-nPr.
Scheme 1. Proposed mechanism for the discriminative de-
tection of GSH and Cys/Hcy for NBD-based fluorescent
probes.
In order to demonstrate a proofꢀofꢀconcept, we initially synꢀ
thesized NBD-OF wherein starting material FOH our previꢀ
ously reported long wavelength 3ꢀhydroxyflavone derivative
(Scheme 2).28 The selection of FOH as a NBDꢀquenchable
dye was based on the following considerations: (1) FOH disꢀ
plays many favorable optical properties such as large Stokes
shift, good photostability, and strong fluorescence in red specꢀ
tral region; (2) The absorption spectra of FOH and NBD-N-
nBu (455 nm for FOH, and 476 nm for NBD-N-nBu) (Figure
S2) were largely overlapped in PBS buffer (10.0 mM, pH 7.4,
containing 30% CH3CN, v/v), which enabled the simultaneous
excitation of both chromophores by a single excitation band,
which can simplify multiꢀchannel fluorescence microscopy
experiments.
With this knowledge in mind, we reasoned that conjugating
an NBD group to the OH moiety of a hydroxylꢀsubstituted
fluorescent dye (DyeOH) would result in the formation of a
nonfluorescent NBD-O-Dye. As noted above, in aqueous enꢀ
vironments the ether linkage of this type of conjugate should
be impervious to attack by amino nucleophiles, such as those
found in typical amino acids. Contrariwise, treatment of NBD-
O-Dye with a biothiol, such as a sulfhydryl containing amino
acid, was expected to rapidly cleave the ether bond via a bimoꢀ
lecular SNAr mechanism to afford NBD-SR and DyeOH; such
a reaction would be accompanied by the formation of a single,
highly fluorescence emission band (B1) corresponding to that
of the untethered dye. In the special case where the biothiol
also contains a suitably situated amino moiety, as is the case
for amino acids Cys and Hcy, the initially formed displaceꢀ
The timeꢀdependent fluorescence measurements of NBD-
OF toward Cys/Hcy/GSH were performed in PBS buffer (10.0
mM, pH 7.4, containing 30% CH3CN, v/v). As shown in Figꢀ
ure 1, NBD-OF was observed to be essentially nonꢀ
fluorescent due to the efficient PET process induced by the
NBD moiety. Upon the addition of either Cys or Hcy, the forꢀ
mation of two emission bands centered at 545 nm and 621 nm
were immediately observed, having nearly identical waveꢀ
lengths and band shapes expected for FOH and control comꢀ
pound NBD-N-nBu, respectively. Both emission bands inꢀ
creased as a function of time, levelling off after approximately
60 min (Figure 1), and the pseudoꢀfirstꢀorder rate constants for
Cys and Hcy were calculate to be 5.56×10ꢀ2 minꢀ1, 2.82×10ꢀ2
minꢀ1, respectively (Figure S4ꢀS5). This behavior was conꢀ
sistent with what was expected if Cys/Hcy had cleaved the
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Analytical Chemistry
ether bond in NBD-OF, releasing the fluorescent FOH
Page 4 of 8
1
(λmaxem = 621 nm) and the corresponding intermediates NBD-
S-Cys/Hcy, 2a/2b, which subsequently was transformed into
fluorescent NBD-N-Cys/Hcy, 3a/3b (λmaxem = 545 nm) (Figꢀ
ure S3). On the other hand, treatment of NBD-OF with GSH,
produced only a single intense fluorescence band with λmaxem
= 621 nm and a negligible fluorescence signal at λmaxem = 545
nm, suggesting that GSH induced a SNAr thiolysis of NBD-
OF to afford fluorescent FOH and the nonꢀfluorescent interꢀ
mediate 2c (Figure 1). Separately, we found that NBD-OF
was capable of discriminating Cys/Hcy from GSH simply by
using the naked eye to observe under the illumination of a UV
365 nm lamp (Figure 2). It is readily apparent that introduction
of Cys/Hcy to a solution of NBD-OF elicited a strong orange
emission. In contrast, the addition of GSH to a separate soluꢀ
tion of NBD-OF resulted in the emission of bright red phoꢀ
tons. We note that in order to ensure the expected generic
NBD-SR cleavage product produced an insignificant fluoresꢀ
cence signal in our solvent system (30% acetonitrile/70% waꢀ
ter), we prepared control compound, NBD-S-nPr and found
that, in deed, its behavior mirrored that reported for NBD-
SCH3 in methanol (vide supra). These experiments provide
strong support for the generic idea that NBD-O-Dye has the
potential to act as a fluorescent probe for the simultaneous
detection of GSH and Cys/Hcy.
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Figure 2. Fluorescence images of NBD-OF alone and in the
presence of GSH, Cys and Hcy under illumination by a UV
365 lamp.
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The fluorescence of NBD-OF in response to different conꢀ
centrations of Cys, Hcy and GSH were investigated in PBS
buffer (10.0 mM, pH 7.4, containing 30% CH3CN, v/v). For
Cys/Hcy, the fluorescence intensity was linearly related to the
concentrations Cys/Hcy in the range of 0.0ꢀ500.0 µM (Figure
S7ꢀS10). The detection limits for Cys and Hcy were calculated
to be 2.1×10ꢀ6 M and 2.7×10ꢀ6 M, respectively base on S/N =
3. In the case of GSH, the fluorescence intensity at 621 nm
was linear with the concentration of GSH in the range of 0.0ꢀ
600.0 µM (Figure S11ꢀS12); the detection limit was deterꢀ
mined to be 6.4×10ꢀ6 M based on S/N = 3. Moreover, we inꢀ
vestigated the fluorescence behavior of NBD-OF (5.0µM)
treated with 50 equiv. of GSH in the presence of different conꢀ
centrations (0.0 µM, 25.0 µM, 50.0 µM and 75.0 µM) of Cys
in PBS buffer (10.0 mM, pH 7.4, containing 30% CH3CN,
v/v). After incubation the mixture for 2 hours, the addition of
50 equiv. of GSH only induced a significant fluorescence sigꢀ
nal with a peak at 621 nm. However, the addition of Cys and
GSH induced the fluorescence enhancement at both 621 nm
and 545 nm. It is noteworthy that the fluorescence intensity at
545 nm was linearly proportional to the concentration of Cys
(Figure S13ꢀS14). The results indicated that NBD-OF could
detect Cys in the presence of GSH.
Figure 3. Fluorescence spectra of NBD-OF upon the addition
of 100.0 equiv. of various biological species: Cys, Hcy, GSH,
Ala, Arg, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Try, Tyr, Val, GSH and ascorbic acid in PBS buffer (10.0
mM, pH 7.4, containing 30% CH3CN, v/v). Each spectrum
was recorded after the addition of the corresponding species
for 120 min.
Figure 1. Timeꢀdependent fluorescence of NBD-OF (5.0 µM)
with 100 equiv. of Cys (A1, A2), Hcy (B1, B2) and GSH (C1,
C2) in PBS buffer (10.0 mM, pH 7.4, containing 30% CH3CN,
v/v). Excited at 458 nm.
To evaluate the selectivity of NBD-OF toward GSH and
Cys/Hcy over various relevant biomolecules, we examined the
fluorescence of NBD-OF in response to amino acids Cys,
Hcy, His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg, Pro,
Thr, Gln, Try, Ile, Lys as well as with GSH and ascorbic acid
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(Figure 3). The findings of this evaluation were that of this
Scheme 3. Structures of NBD-OBodipy and NBD-
OCoumarin.
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entire array of biomolecules, only GSH and Cys/Hcy induced
a significant fluorescence response. Thus, NBD-OF appears to
be a highly selective probe for GSH and Cys/Hcy.
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To determine whether NBD-OF can function under physioꢀ
logical conditions, we studied its response toward GSH and
Cys/Hcy as a function of pH. As is evident from inspection of
the data presented in Figure 4, in the absence of biothiols,
NBD-OF is very stable over the physiological relevant pH
range of 2ꢀ10. Importantly, NBD-OF displays a strong fluoꢀ
rescence enhancement in the presence of the biothiols of the
present study in the pH range of 7ꢀ8 which suggests that it can
function under physiological conditions.
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Figure 4. pH effect on the fluorescence intensity of NBD-OF
(5.0 µM) in absence and presence of 100.0 equiv. of Cys (λem
= 545 nm) (a) and GSH (λem = 621 nm) (b). Sample intensity at
each pH was measured after 120 min.
To demonstrate the broad applicability of our strategy for
designing fluorescent probes for simultaneous detection GSH
and Cys/Hcy, we developed another probe: NBD-OBodipy
(Scheme 3). The Bodipy derivative has an absorption maxiꢀ
mum at 650 nm and fluorescence in near infrared region with
λmax = 705 nm. As shown in Figures 5a and S15, the addition
of 10.0 equiv. of Cys/Hcy/GSH to NBD-OBodipy induced a
strong fluorescence B1 band centered at 705 nm (at 120
minutes a 726ꢀfold increase in intensity for Cys, 709ꢀfold for
Hcy and 720ꢀfold for GSH). The significant fluorescence enꢀ
hancement in the NIR region renders NBD-OBodipy especialꢀ
ly suitable for in vivo applications because NIR light can minꢀ
imized photodamage to biological samples, has better tissue
penetration than visible photons and has less interference from
background autofluorescence commonly encountered in living
systems.34 Moreover, the addition of Cys/Hcy triggered an
additional fluorescence B2 band at 545 nm (70ꢀfold intensity
enhancement for Cys and 80ꢀfold for Hcy) when excited at
476 nm (Figures 5b and S16). By sharp contrast, emission at
545 nm was negligible when NBD-OBodipy was treated with
GSH. As expected, NBD-OBodipy was able to discriminate of
Cys/Hcy from GSH by the presence or absence of a B2 band
at 545 nm.
Figure 5. Fluorescence spectra of NBD-OBodipy (5.0 µM)
upon the addition of various amino acids in PBS buffer (10.0
mM, pH 7.4, containing 1.0 mM CTAB) at room temperature.
(a) Excited at 650 nm; (b) Excited at 476 nm. Each spectrum
was recorded 120 min after incubation with corresponding
species. Amino acid: Cys, Hcy, GSH, Ala, Gln, Glu, His, Ile,
Leu, Lys, Phe, Pro, Ser, Thr, Try, Tyr, Val.
Pluth and coꢀworkers have reported a coumarinꢀbased fluoꢀ
rescent probe, NBD-OCoumarin, to discriminate H2S from
Cys/Hcy employing NBD moiety as the sensing group.29 Inꢀ
deed, NBD-OCoumarin in Pluth’s work also has the ability to
discrimination GSH from Cys/Hcy. We synthesized NBD-
OCoumarin and studied its fluorescence behavior in response
to biothiols. The addition of Cys, Hcy or GSH to NBD-
OCoumarin in PBS buffer (10 mM, pH 7.4, containing 30%
CH3CN, v/v) caused a significant fluorescence band to appear
at 445 nm when excited at 380 nm, reaching a maximum withꢀ
in 10 min (Figure S17). Moreover, Cys/Hcy induced a fluoresꢀ
cence band at 545 nm when excited at 476 nm. Due to the
large difference in their absorption spectra between Couma-
rin-OH and 3a/3b, NBD-OCoumarin was unable to work
with single excitation wavelength for the discrimination of
GSH from Cys/Hcy with naked eye. On the contrary, the abꢀ
sorption spectral overlap between the resulting fluorescent
fragments FOH and 3a/3b from NBD-OF is large and the
simultaneous excitation of both chromophores by a single
excitation band can be achieved, which can simplify multiꢀ
channel fluorescence microscopy experiments. Moreover, the
In order to evaluate its selectivity, the fluorescence behavꢀ
iours of NBD-OBodipy toward other biologically relevant
amino acids were investigated. As illustrated in Figure 5, no
significant fluorescence response was generate by a series of
biologically relevant amino acids whereas Cys, Hcy and GSH
induced a significant fluorescence band at 705 nm when excitꢀ
ed at 650 nm. In addition, with the exception of Cys and Hcy,
none of the tested chemicals, including GSH, produced signifꢀ
icant emission at 545 nm when excited at 476 nm. These reꢀ
sults demonstrate that NBD-OBodipy is highly selective for
the detection and discrimination of GSH from Cys/Hcy and all
three of these biothiols from a wide array of common amino
acids.
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emission of FOH is in red spectral region, which is desirable incremental amounts of Cys, the peak of NBD-OCoumarin
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for biological fluorescence detection due to the deeper peneꢀ
tration in biological tissues. Also, the intensities of two fluoꢀ
rescence signal from FOH and 3a/3b generated from NBD-
OF in PBS buffer are quite comparable when excited at 458
nm, which is desirable for the discriminatory detection.
was gradually disappeared; two new peaks were emerged with
the retention times at 3.90 min, which is assigned to comꢀ
pound 3a, and 6.06 min, which is assigned to Coumarin-OH
(Figure S18). Similar results were obtained for the reaction
mixture of NBD-OCoumarin with Hcy and GSH and the
retention times of compounds 3b and 2c were found to be 3.84
min and 3.67 min, respectively (Figure S19ꢀS20). Additionally,
HRMS analysis of the reaction mixture of NBD-OCoumarin
with Cys, Hcy and GSH confirmed the formation of Couma-
rin-OH (m/z = 199.0387, [M+Na]+) and other fragments (m/z
= 307.0153 for 3a, [M+Na]+; 299.0449 for 3b, [M+1]+; and
493.0768 for 2c, [M+Na]+) (Figure S21ꢀS23). These results
provided strong support for our proposal that GSH and
Cys/Hcy both induce thiolysis to release Coumarin-OH but
that only the NBD-SR compound derived from Cys/Hcy has
the ability to undergo a subsequent Smiles rearrangement to
form NBD-NHR.
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Figure 6. 1H NMR spectral comparison of NBD-OCoumarin,
Coumarin-OH,
OCoumarin with Cys and GSH. All H NMR spectra were
obtained in DMSOꢀd6ꢀD2O (4:1, v/v).
NBD-N-nBu,
NBD-S-nPr,
NBD-
1
To provide data supporting for our proposed mechanism of
1
action, we performed H NMR titration experiments designed
Figure 7. Confocal microscopic images of Cys and GSH in
HeLa cells. (A1ꢀA4) HeLa cells incubated with 2.0 µM NBD-
OF. (B1ꢀB4) HeLa cells preꢀtreated with 1.0 mM GSH and
then incubated with 2.0 µM NBD-OF. (C1ꢀC4) HeLa cells
preꢀtreated with 1.0 mM Cys and then incubated with 2.0 µM
NBD-OF. (D1ꢀD4) HeLa cells preꢀtreated with 1.0 mM NEM
and then incubated with 2.0 µM NBD-OF. Excitation waveꢀ
length: 488 nm. Emission was collected at 510ꢀ535 nm for
green channel and 610ꢀ660 nm for red channel. Scale bar: 40
µm.
to follow product formation resulting from the cleavage and
possible subsequent Smiles rearrangement of our probes.
Compound NBD-OCoumarin was utilized to perform this
experiment due to its good solubility in solution. Upon the
addition of excessive Cys or GSH to NBD-OCoumarin in
DMSOꢀd6ꢀD2O (4:1, v/v), signals corresponding to each of the
protons attributable to Coumarin-OH began to appear, indiꢀ
cating the release of Coumarin-OH whereas the proton sigꢀ
nals associated with NBD depended on whether the biothiol
reactant was Cys or GSH. When Cys was the reactant, the Ha
and Hb protons of the NBD moiety showed upfield shifts,
especially for Hb, which were expected if NBD-NHR was
being formed. However, when GSH was the reactant, the
growing signals for the Ha and Hb protons of NBD had chemꢀ
ical shifts that corresponded with those expected for NBD-SR.
HPLC is an effective method widely used in the separation
and analysis of mixtures of organic compounds. Thus, NBD-
OCoumarin, the reference dye Coumarin-OH and the reacꢀ
tion mixture of NBD-OCoumarin with Cys, Hcy and GSH
were subjected to HPLC analysis (Figure S18ꢀS20). NBD-
OCoumarin itself displayed a single peak with a retention
time at 11.07 min. Coumarin-OH displayed a single peak
with a retention time at 6.06 min. With the addition of the
Encouraged by the aforementioned results, we evaluated the
capability of NBD-OF to selectively image intracellular Cys
and GSH with the aid of confocal microscopy. The microꢀ
scope was fitted with both green and red light detection optics
which correspond to the emitted photons from NBD-NHR and
FOH, respectively; a single excitation band centered at 488
nm was used to excite both chromophores. When HeLa cells
were incubated with NBD-OF (2.0 µM), the fluorescence
signals from green and red channels were observed (Figure 7
A1ꢀA4). For the control experiment, HeLa cells were incubatꢀ
ed with either Cys (1.0 mM) or GSH (1.0 mM) and, after
changing the growth medium, were incubated with NBD-OF
(2.0 µM) whereupon the fluorescence signals from the green
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and red channels were recorded and viewed either separately
or merged together (Figure 7 B1ꢀB4 and C1ꢀC4). As a control,
batch of cells initially treated with 2.0 mM Nꢀ
ACKNOWLEDGMENT
1
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This research was supported by the State Key Laboratory of Fine
Chemicals (KF1202) and the State Key Laboratory for Powder
Metallurgy.
a
ethylmaleimide (NEM) to inactivate all intracellular thiol speꢀ
cies and then incubated with 2.0 µM NBD-OF showed faint
fluorescence signals in green and red channels (Figure 7 D1ꢀ
D4). Inspection of Figure 7 shows that cells treated with GSH
glowed bright red and but were nearly devoid of signal in the
green channel. In sharp contrast, cells treated with Cys exhibꢀ
ited strong fluorescence images in both the red and green
channels which, when merged, produced bright yellow imagꢀ
es. The NEM treated control cells did not generate significant
images in either the red or green channels. Taken together,
these experiments show that NBD-OF can interact with bioꢀ
thiols under intracellular physiological conditions in the same
manner as it does inꢀvitro. Therefore, NBD-OF has the potenꢀ
tial to be an inꢀvivo probe for the detection and differentiation
of Cys and GSH with a single excitation wavelength.
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Moreover, we utilized the NIR probe, NBD-OBodipy, to
image intracellular Cys and GSH in living HeLa cells. As
shown in Figure S24, similar results were obtained that bright
yellow signal, merged from the red and green channels, was
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only exhibited strong red fluorescence. These results suggest
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and GSH from different emission channel.
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CONCLUSIONS
In conclusion, we have developed a broadly approach to deꢀ
signing fluorescent probes for simultaneous detection of GSH
and Cys/Hcy both inꢀvivo and inꢀvitro. The strategy is based
on selective thiolysis and intramolecular rearrangement reacꢀ
tions when NBD is conjugated to lightꢀinduced electron donatꢀ
ing dyes through a labile ether linkage. As a proofꢀofꢀconcept,
we have explored three novel fluorescent probes, NBD-OF,
NBD-OBodipy and NBD-OCoumarin, which can discrimiꢀ
nate GSH from Cys/Hcy and all three from a wide array of
amino acids with good selectivity and sensitivity. Moreover,
we also demonstrated that probes, NBD-OF, NBD-OBodipy,
were capable of simultaneously imaging intracellular GSH and
Cys in living cells. This novel strategy reported herein has the
potential to inspire the development of new fluorescent probes
with desired properties for the discriminatory detection of
GSH and Cys/Hcy.
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ASSOCIATED CONTENT
Supporting Information
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Chemical structures of compounds, additional spectra data, confoꢀ
cal microscopy images, 1H, 13C and HRMS spectra of compounds.
The Supporting Information is available free of charge via the
Intenet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: song@rowland.harvard.edu
Author Contributions
‡These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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32 Niu, L. Y.; Guan, Y. S.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.;
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