Selective detection of the reduced form of glutathione (GSH) over the oxidized (GSSG) form using a combination of glutathione reductase and a Tb(III)-cyclen maleimide based lanthanide luminescent 'Switch On' assay

Article abstract of DOI:10.1021/ja300887k

The synthesis of a novel Tb(III) luminescent probe for the detection of thiols is presented. The probe 1.Tb, possessing a maleimide moiety, as its sulfhydryl acceptor, was poorly emitting in aqueous pH 7 solution in the absence of a thiol. However, upon addition of thiols such as glutathione (GSH), large enhancements were observed, particularly within the physiological pH range. In contrast no enhancements were observed in the presence of the oxidized form of glutathione (GSSG), except in the presence of the enzyme glutathione reductase and NADPH which enabled 1.Tb to be used to observe the enzymatic reduction of GSSG to GSH in real time.

Full text of DOI:10.1021/ja300887k

Communication
pubs.acs.org/JACS
Selective Detection of the Reduced Form of Glutathione (GSH) over
the Oxidized (GSSG) Form Using a Combination of Glutathione
Reductase and a Tb(III)-Cyclen Maleimide Based Lanthanide
Luminescent ‘Switch On’ Assay
Brian K. McMahon and Thorfinnur Gunnlaugsson*
School of Chemistry, Center for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland
S
* Supporting Information
Glutathione (γ-L-glutamyl-L-cysteineglycine, or GSH) is the
ABSTRACT: The synthesis of a novel Tb(III) lumines-
cent probe for the detection of thiols is presented. The
probe 1.Tb, possessing a maleimide moiety, as its
sulfhydryl acceptor, was poorly emitting in aqueous pH
7 solution in the absence of a thiol. However, upon
addition of thiols such as glutathione (GSH), large
enhancements were observed, particularly within the
physiological pH range. In contrast no enhancements
were observed in the presence of the oxidized form of
glutathione (GSSG), except in the presence of the enzyme
glutathione reductase and NADPH which enabled 1.Tb to
be used to observe the enzymatic reduction of GSSG to
GSH in real time.
most predominant tripeptide thiol found within the human
cellular system. Of all the thiol containing components of
blood, the GSH content makes up 90% of its entire
composition with previous literature stating that, due to this
extremely high concentration of GSH, all other potential thiol
molecules can be disregarded.8 Existing essentially in its
reduced form, GSH can, however, be rapidly oxidized to its
dimeric GSSG form in response to oxidative stress within cells.
Therefore changes in the intracellular GSH concentration or in
the GSH/GSSG ratio have become a key indicator in
monitoring the cells' overall health and their ability to resist
oxidative damage.9 Various analytical methods have been
developed to date for GSH detection, including the use of
HPLC,10 UV−vis/calorimetric assays11 and capillary electro-
phoresis.12 In the past, we have developed many examples of
lanthanide luminescent sensors, probes, and imaging agents for
biological applications.13,14 However, to the best of our
knowledge, the development of such systems for the detection
of thiol specific, or biothiols, and in particular GSH has not
been achieved to date. Consequently, we set out to develop
such a probe, which could display both high sensitivity and
selectivity for thiols and, in particular, enable the selective
detection of GSH over GSSG. Herein, we present 1.Tb, an
ulfhydryl-containing amino acids and peptides play a
Spivotal role in many biological processes such as in
reversible redox reactions and in important cellular functions
like detoxification and metabolism.1,2 Low cysteine (Cys) levels
have been linked to many health issues such as hematopoiesis
reduction, retarded growth, hair depigmentation, liver damage,
skin lesion development, and cancer.3 In contrast, homocys-
teine (HCys) is a common risk factor for disorders such as
Alzheimer’s disease, osteoporosis, thrombosis, and cardiovas-
cular disease.4,5 Hence, there currently exists a significant
interest within the field of supramolecular chemistry to develop
selective sensors and probes for the detection of such species.
The development of fluorescent probes for such detection has
recently been highlighted by Yoon et al.1a However, while being
highly sensitive, these sensors/probes usually consist of short
wavelength emitting and short lifetime-based fluorophores,
which is a real drawback for use in competitive biological
media. The development and use of specific analyte targeting
lanthanide based luminescent sensors/probes has recently
emerged for various biological applications.6,7 The lanthanides
possess many desirable photophysical properties, such as long
wavelength emissions (within the visible and the near-infrared
(NIR) regions), sharp line-like emission bands and long-lived
excited states (μs−ms time frame), allowing them to be easily
distinguished from shorter-lived (ns-based) autofluorescence
from biological material. This greatly improves the signal-to-
noise ratio of such time delayed sensors/probes, making them
desirable alternatives to fluorescence based systems.
Scheme 1. Synthesis of 1 (Free Ligand) and the
Corresponding Tb(III) Complex 1.Tb
octadentate macrocyclic Tb(III) cyclen conjugate possessing a
maleimide functionality, which, upon 1,4-Michael addition of a
thiol group to the alkene bond, displays large changes in the
Tb(III) centered emission. Moreover, we demonstrate the high
selectivity of 1.Tb for GSH and that, in combination with the
enzyme glutathione reductase, the formation of GSH from
GSSG can be monitored in real-time with high sensitivity.
Received: January 27, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja300887k | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
The synthesis of 1.Tb (Scheme 1) was achieved in three
steps from 2 (previously developed in our laboratory15), which
involved reaction with maleic anhydride, 3, in refluxing CH3CN
solution for 12 h. The resulting crude acid amide derivative was
treated with NaOAc in acetic anhydride at 100 °C for 2 h, and
the desired product 1, isolated in 27% yield after aqueous−
organic workup (see Supporting Information (SI)). The
corresponding Tb(III) complex, 1.Tb, was formed in 93%
yield by refluxing 1 with 1 equiv of Tb(CF3SO3)3 in freshly
1
distilled CH3CN for 15 h (see H SI Figure S1a-S1c)
Figure 1. Changes in the Tb(III) luminescence of 1.Tb (10 μM) upon
excitation of the phenyl antenna (λmax = 256 nm) as a function of GSH
in H2O (20 mM HEPES, 135 mM KCl, pH = 7.4). Inset: Plot of
intensity at 545 nm with equivalents of GSH added.
The design of 1.Tb allows for sensitized excitation of the
5
Tb(III) D4 excited state via the covalently attached phenyl
antenna, which upon relaxation to the 7FJ (J = 6, 5, 4, 3) ground
states gives rise to the characteristic line-like Tb(III) emission
bands occurring at long wavelengths. Indeed, upon excitation of
1.Tb at 256 nm (λmax of phenyl antenna) in an aqueous
buffered pH 7.4 (20 mM HEPES, 135 mM KCl) solution,
metal centered emission was observed with bands appearing at
490, 545, 586, and 622 nm (Figure S2). The excited state decay
was measured in both H2O and D2O (λex = 256 nm), and best
fit to a monoexponential decay profile, from which the excited
state lifetimes τH2O = 1.31 ms and τD2O = 1.99 ms, were
determined. From these lifetimes, the hydration state for 1.Tb
(the q value)16 was determined as 1 (Figure S3).
the observed emission changes in Figure 1 were indeed due to
the formation of the reduced form of 1.Tb (assigned here as
1.Tb.GSH) in solution, as it could be possible that GSH was in
fact interacting with 1.Tb in a noncovalent manner. To
investigate this, a small scale reaction, where 1.Tb was reacted
with 1 equiv of GSH in H2O, at rt for 2 h, was carried out. The
reaction mixture was then analyzed using ESMS without
carrying out any workup on the crude reaction mixture. The
results indeed confirmed that 1.Tb was converted successfully
under these experimental conditions to the desired 1.Tb.GSH
product, where the ESMS (+ mode) showed the presence of a
peak at m/z = 1119.38, corresponding to a species with the
chemical formula corresponding to the [1.Tb.GSH-3CF3SO3-
2H]+ complex, and an isotopic distribution pattern that
matched the calculated species (see SI, Figures S5 and S6).
To further analyze the changes in the Tb(III) emission
spectra in the presence of 1 equiv of GSH, the Tb(III) excited
state decay was determined and found to be best fit to a
monoexponential profile. From these lifetimes, the q value was
recalculated and shown to be unchanged (q = 0.9) (see SI,
Table S1), confirming that the observed increase in Tb(III)
emission of 1.Tb was not due to displacement of the bound
H2O molecule by GSH.
The principal reaction mechanism behind the luminescence
response of 1.Tb to GSH is shown in Scheme 2. As stated
Scheme 2. Reaction of 1.Tb with GSH Yielding the Adduct
1.Tb.GSH
above this reaction involves a 1,4-Michael addition of the thiol
functional group to the electron deficient alkene of the
maleimide moiety. We envisaged that this reaction and the
concomitant formation of the succinimide derivative would
effect the sensitization process of the Tb(III) excited state, thus
directly modulating the photophysical properties of the Tb(III)
ion. We consequently carried out several screening studies with
1.Tb, using various biologically relevant thiols. We initially used
GSH using the aforementioned experimental conditions. This
showed that changes in the absorption spectrum of 1.Tb (see
SI, Figure S4) upon addition of the thiol were minor and too
small to analyze accurately. Similarly the changes in the
fluorescence emission spectra of 1.Tb were minor. However,
and as anticipated, significant changes were seen in the metal
centered emission, where the Tb(III) emission was greatly
enhanced as a function of added GSH concentration. The
changes in the Tb(III) emission are shown in Figure 1, where
the Tb(III) emission was enhanced, or ‘switched on’ by ca. 500%
upon addition of GSH, signifying the formation of 1.Tb.GSH.
More importantly, the emission did not enhance after the
addition of 1 equiv of GSH as demonstrated as an inset in
Figure 1, where the intensity changes measured for the 550 nm
transition are plotted as a function of added equivalents of
GSH, signifying that the reaction was stoichiometric.
We next evaluated the photophysical behavior of 1.Tb (10
μM solutions) and its ability to react with GSH over a range of
pH values. As shown in Figure 2, the Tb(III) emission of 1.Tb
Figure 2. Effect of 1.Tb (10 μM) and its response to GSH as a
function of pH (individual 10 μM solutions of 1.Tb at each pH value
in 20 mM HEPES were used, followed by addition of 10 μM GSH).
was found to be relatively independent of pH, with only small
changes being observed within the pH range of 7−9 (in blue).
However, when the Tb-emission from 1.Tb was monitored in
the presence of 1 equiv of GSH it was shown to be highly pH
dependent (in red, Figure 2). The effect of pH on the Tb(III)
emission in the presence of GSH can be best described as
displaying a bell-shaped ‘Off−On−Off ’ emission behavior17 for
the 550 nm transition (similar changes were seen for all the 5D4
While this reaction has been widely used within the
literature, where it takes place, under physiological conditions,
in real time and with high yields, it was necessary to verify that
B
dx.doi.org/10.1021/ja300887k | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
7FJ deactivations), which mimics that often seen for
Communication
→
adenine dinucleotide phosphate (NADPH). This enzyme
catalyzes the reduction of GSSG to GSH under physiological
pH conditions, and it was anticipated that 1.Tb could be used
successfully to monitor the kinetics of this redox process. This
could be achieved by following the enhancement in the delayed
Tb(III) emission intensity upon GSH formation, as it would be
subsequently trapped by reacting with the maleimide unit as
shown above (Scheme 2). Indeed, this was found to be the case
where we observed the changes in the Tb(III) emission as a
function of GSH formation in the presence of the enzyme and
NADPH. Furthermore, optimization studies on this reaction
(see SI Figure S10) showed that 1.Tb could monitor this
conversion of GSSG to GSH at low NADPH concentrations,
with the optimum luminescence response being observed at 3.3
μM of NADPH. We next studied the kinetic effect on the
reduction of GSSG as a function of enzyme concentration, the
results of which are shown in Figure 4. These results
biological processes such as enzymatic reactions. As shown in
Figure 2, the Tb(III) emission was not affected by the presence
of GSH in either acidic (<pH 4) or basic (>pH 10) media.
However, within these two pH envelopes, the Tb(III) emission
was highly sensitive to the presence of GSH. With a maximum
Tb(III) emission enhancement being observed at pH ∼7, it was
demonstrated that this system could be employed as a probe
for the detection of GSH within the physiological pH range.
Having demonstrated that the Tb(III) emission was highly
sensitive to the presence of GSH within the physiological pH
range, we next investigated the response of 1.Tb to several
other thiol based biological molecules as well as amino acids, as
the latter could also potentially interact with the maleimide unit
in 1.Tb using the same 1,4-Michael addition. As expected, the
Tb(III) emission of 1.Tb (10 μM) was found to be modulated
in the presence of the thiol based amino acids cysteine (Cys)
and homocysteine (HCys) (see SI, Figures S7−S8). However,
no significant changes were observed in the Tb(III) emission
using other naturally occurring amino acids (40 μM) (Figure
3). Further investigations also showed very little variation in the
Figure 4. Luminescence response of 1.Tb (10 μM) as a result of the
enzymatic conversion of GSSG to its reduced GSH form using
NADPH (3.3 μM) and various concentrations of the enzyme
glutathione reductase. All enzymatic titrations were carried out in
aqueous pH 7.4 buffered solutions (0.1 M TRIS, 0.135 M KCl) at 37
°C.
Figure 3. Tb(III) luminescence response of 1.Tb (10 μM) in the
presence of various physiologically important amino acids (40 μM) in
H2O (20 mM HEPES, 135 mM KCl, pH 7.4). Red bars represent the
final emission intensity (IF) subtracting the initial intensity (I0) over I0
at λem = 545 nm. Blue bars represent the addition of the amino acids:
(1) Ala, (2) Asp, (3) His, (4) Arg, (5) Phe, (6) Ser, (7) Val, (8) IIe,
(9) GSSG, (10) GSH, (11) Pro, (12) Thr, (13) Tyr, (14) Gly, (15)
Leu, (16) Lys, (17) Met, (18) Glu, (19) Sar, (20) Asn. Red bars
represent the subsequent addition of GSH (40 μM) to these respective
solutions.
demonstrate that when the concentration of glutathione
reductase was doubled, the time associated with the redox
process was halved. Therefore, using an enzyme concentration
as low as 19.72 nM ensured that 1.Tb maximized its response
over a short period of 10 min, Figure 4. By monitoring the
changes in the UV−vis absorption spectra for these titrations
only minimal changes were observed for the π−π* absorbance
band of 1.Tb. However, a decrease in the absorption of the
NADPH band at 340 nm confirmed that the redox process was
occurring and that the modulation of the Tb(III) emission was
indeed a direct result of the subsequent formation of GSH and
the trapping by 1.Tb (Figures S11a−S11c, S12), which
concomitantly switched the Tb(III) emission on.
With the view of evaluating the competitiveness of this
reaction, the enzymatic reduction of GSSG, in the presence of
1.Tb, was also carried out in the presence of 40 equiv of each of
the amino acids tested above (excluding any thiol based amino
acids) at pH 7.4. The results (see SI Figure S13a−S13d)
showed on all occasions an intensity enhancement of ca. 400%
in the Tb(III) emission, clearly demonstrating the ability of
1.Tb to monitor the above process, even in competitive media.
Therefore, we decided to lower the concentration of the
enzyme in the presence of 1.Tb (10 μM) with the view of
further maximizing the potential of using this unique
combination of an enzyme and probe to observe a biological
process in real time. Various titrations indeed demonstrated the
feasibility of an enzyme concentration of 4.93 nM. The reaction
was observed to be completed within 12 min compared to the
1.Tb response to GSH in the presence of any of these amino
acids (see SI Figure S9a −S9b).18 However, the most significant
result emerging from this screening process was the ability of
1.Tb to selectively respond to GSH over the oxidized GSSG
form, which lacks the nucleophilic thiol moiety. As shown in
Figure 3, in the presence of a large excess of GSSG (40 equiv),
the Tb(III) emission was within experimental error, unchanged,
demonstrating that 1.Tb could be used for monitoring GSH in
the presence of GSSG.
The above results also suggested that 1.Tb could potentially
be employed to observe the conversion of GSSG to its reduced
GSH form in real time in competitive media; an event which is
highly desirable as being able to monitor such enzymatic
reactions in real time19 is of great current interest. The shift in
the concentration of the reduced form to the oxidized
glutathione form is often used as an indicator of cellular well-
being in vivo, as it can give a good indication of overall health
and any oxidative damage occurring.9 To demonstrate the
ability of 1.Tb to monitor the reduction of GSSG in solution, a
series of experiments were carried out, by using the enzyme
glutathione reductase and the reducing agent nicotinamide
C
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Journal of the American Chemical Society
Communication
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36 min observed previously, demonstrating the success of 1.Tb
in monitoring the enzymatic conversion in real time.
Lop
́
ez, J. A.; Andreu-de-Riquer, G.; Alvarado-Monzon
́
, J. C.; Ratnakar,
In summary, we have designed and developed the first
example of a lanthanide based GSH sensor, through
functionalization of the cyclen framework with a pendant arm
incorporating the maleimide moiety. We have demonstrated
the ability of 1.Tb to be used in monitoring thiol specific and in
particular the GSSG to GSH redox process using glutathione
reductase and the reducing agent NADPH. We are currently
developing similar systems using NIR emitting probes which
will allow for the sensitization of the lanthanide excited state
within the visible region.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and characterization of 1 and 1.Tb; Figures S1−S14.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
gunnlaut@tcd.ie
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the Science Foundation Ireland (SFI PI
Award 2010, SFI RFP Awards 2008 and 2009), the Irish
Research Council for Science, Engineering & Technology
(IRCSET, for an initial Postgraduate award to B.K.M.), and
TCD for financial support.
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