Simultaneous nucleophilic-substituted and electrostatic interactions for thermal switching of spiropyran: A new approach for rapid and selective colorimetric detection of thiol-containing amino acids

Article abstract of DOI:10.1021/ac203494e

Complementary electrostatic interaction between the zwitterionic merocyanine and dipolar molecules has emerged as a common strategy for reversibly structural conversion of spiropyrans. Herein, we report a concept-new approach for thermal switching of a spiropyran that is based on simultaneous nucleophilic-substitution reaction and electrostatic interaction. The nucleophilic-substitution at spiro-carbon atom of a spiropyran is promoted due to electron-deficient interaction induced by 6- and 8-nitro groups, which is responsible for the isomerization of the spiropyran by interacting with thiol-containing amino acids. Further, the electrostatic interaction between the zwitterionic merocyanine and the amino acids would accelerate the structural conversion. As proof-of-principle, we outline the route to glutathione (GSH)-induced ring-opening of 6,8-dinitro-1′,3′,3′- trimethylspiro [2H-1-benzopyran-2,2′-indoline] (1) and its application for rapid and sensitive colorimetric detection of GSH. In ethanol-water (1:99, v/v) solution at pH 8.0, the free 1 exhibited slight-yellow color, but the color changed clearly from slight-yellow to orange-yellow when GSH was introduced into the solution. Ring-opening rate of 1 upon accession of GSH in the dark is 0.45 s^-1, which is 4 orders of magnitude faster in comparison with the rate of the spontaneous thermal isomerization. The absorbance enhancement of 1 at 480 nm was in proportion to the GSH concentration of 2.5 × 10 ^-8-5.0 × 10^-6 M with a detection limit of 1.0 × 10^-8 M. Furthermore, due to the specific chemical reaction between the probe and target, color change of 1 is highly selective for thiol-containing amino acids; interferences from other biologically active amino acids or anions are minimal.

Full text of DOI:10.1021/ac203494e

Article
pubs.acs.org/ac
Simultaneous Nucleophilic-Substituted and Electrostatic Interactions
for Thermal Switching of Spiropyran: A New Approach for Rapid and
Selective Colorimetric Detection of Thiol-Containing Amino Acids
Yinhui Li, Yu Duan, Jishan Li, Jing Zheng, Huan Yu, and Ronghua Yang*
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University,
Changsha, 410082, China
*
S Supporting Information
ABSTRACT: Complementary electrostatic interaction between the zwitterionic merocyanine and dipolar molecules has
emerged as a common strategy for reversibly structural conversion of spiropyrans. Herein, we report a concept-new approach for
thermal switching of a spiropyran that is based on simultaneous nucleophilic-substitution reaction and electrostatic interaction.
The nucleophilic-substitution at spiro-carbon atom of a spiropyran is promoted due to electron-deficient interaction induced by
6
- and 8-nitro groups, which is responsible for the isomerization of the spiropyran by interacting with thiol-containing amino
acids. Further, the electrostatic interaction between the zwitterionic merocyanine and the amino acids would accelerate the
structural conversion. As proof-of-principle, we outline the route to glutathione (GSH)-induced ring-opening of 6,8-dinitro-
1
′,3′,3′-trimethylspiro [2H-1-benzopyran-2,2′-indoline] (1) and its application for rapid and sensitive colorimetric detection of
GSH. In ethanol−water (1:99, v/v) solution at pH 8.0, the free 1 exhibited slight-yellow color, but the color changed clearly from
slight-yellow to orange-yellow when GSH was introduced into the solution. Ring-opening rate of 1 upon accession of GSH in the
dark is 0.45 s , which is 4 orders of magnitude faster in comparison with the rate of the spontaneous thermal isomerization. The
−1
−8
−6
absorbance enhancement of 1 at 480 nm was in proportion to the GSH concentration of 2.5 × 10 −5.0 × 10 M with a
−8
detection limit of 1.0 × 10 M. Furthermore, due to the specific chemical reaction between the probe and target, color change of
is highly selective for thiol-containing amino acids; interferences from other biologically active amino acids or anions are
1
minimal.
piropyrans, an important class of photoswitchable mole-
dimensional surface of gold nanoparticles attributed to the
ability of zwitterionic merocyanines to bind with charged
S
cules, are an attractive starting point in construction of
1
−4
18
optical probes,
because they show reversibly structural
molecules. For a spiropyran acceptor, only one additional
interaction is available for binding when the spiropyran unit is
opened. For advance, multipoint electrostatic interactions
between mono-spiropyran or double-spiropyran receptor and
amino acids, as well as simultaneous metal-ligation and
conversion between the closed spiropyran (spiro-) form and
the opened merocyanine (mero-) form upon external optical,
5
−8
thermal, or chemical stimulations.
During the past few
decades, a number of spiropyran-based probes have been
designed and utilized for colorimetric and fluorescent
detections of chemical or biological species from inorganic
18
electrostatic interactions, were utilized by our group and
19
9
−13
14,15
16−19
Yao et al. Although these strategies presented a potential
approach for determination of dipolar molecules, drawbacks
toward a practical sensor for selective detection of a particular
metal ions
and anions
to amino acids
and even
2
0
21
nucleobases and nucleic acid molecules.
Complementary electrostatic interaction between the zwit-
terionic merocyanine and dipolar molecules has emerged as a
common approach for thermal switching of spiropyran. As early
as 1982, Sunamoto et al. reported the zwitterionic merocyanine
structure of a spiropyran can bind to a polar amino acid
molecule via electrostatic interaction, and the approach was
utilized for the photocontrolled transfer of amino acids across
23,24
analyte exist.
Specifically, this type of sensor has generally
displayed moderate sensitivity and specificity over other
competitive species, even serious interference, which could
not adequately satisfy direct quantification of target in a
25
complex biological environment, so how to set up a practical
22
bilayers and membranes. Later, Thomas and co-workers
presented the photoswitchable self-assembly of various amino
acid derivatives by anchoring spiropyrans onto the three-
Received: January 5, 2012
Accepted: April 30, 2012
Published: April 30, 2012
©
2012 American Chemical Society
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Article
3
7
38
sensor for specific detection of the corresponding analyte has
been a great challenge.
gold nanorods, and quantum dots. Although these
approaches have led to significant contributions to the thiol-
containing amino acids assay, there is still plenty of room for
improvement in term of selectivity, sensitivity, rapid assay time,
and performance with a new interaction mechanism. Compared
to the known molecular sensors for thiol-containing amino
Molecular recognitions based on chemical reaction have
attracted attention by researchers due to the target specific
reactive functional groups on both the analytes and the
2
6
acceptors. Furthermore, specific chemical reactions induced
by target show high capacity of resisting interference in a
complex system. In recent years, molecular recognition and
detection based on irreversibly chemical reaction coupled to
absorption or fluorescence emission variations have been
developed, including qualitatively and quantitatively analyzing
thiols by Michael addition, cyclization with aldehyde, and
cleavage of sulfonamide/sulfonate ester, selenium−nitrogen,
32−38
acids reported in the literature,
the proposed approach
possesses three remarkable advantages: First, this nucleophilic
substitution mechanism is the wheel of the reaction and the
electrostatic interaction as accelerator that enhances the
transition of intermediate molecule to merocyanine formation;
second, by incorporating chemical reaction and supramolecular
interaction, the selectivity and the sensitivity for sensing thiol-
containing amino acids is improved; and finally, the system is
operated in aqueous solution, which has a good prospect of
application.
2
7
−
−
and disulfide and analyzing F and CN by unique
29
28
desilylation reaction and nucleophilic addition reaction,
respectively.
Inspired by the success of the aforementioned research and
to address the limitations of spiropyran probes based on
noncovalent interactions, herein, we would present a previously
undescribed mechanism via simultaneous nucleophilic-substi-
tuted reaction and complementary electrostatic interaction for
sensing of dipolar molecules. Our design strategy is based on
the finding that cleavage of C−O bond of dinitrophenyl ethers
EXPERIMENTAL SECTION
■
Spiropyran probes of 1, 2, and 3 were prepared as reported by
39
Buback and Brixner. All the solvents and other chemicals
were of analytical reagent grade and were supplied by Alfa
Aesar or Sigma Aldrich. Mass spectra were obtained on an
LCQ/Advantage HPLC-Mass spectrotometer. UV−vis absorp-
tion spectra were recorded in 1.0 cm path length quartz
cuvettes on a Hitachi U-4100 UV/vis spectrophotometer
Kyoto, Japan). pH was measured by model 868 pH meter
Orion).
30
is catalyzed by a primary thiol in solution, which would
greatly enhance the conversion of spiropyran form to
merocyanine form (Scheme 1). To test the feasibility of this
(
(
Scheme 1. Proposed Scheme for Merocyanine Formation of
For colorimetric measurement of GSH, 1.0 mL Tris−HCl
1
Induced by GSH Based on Simultaneous Nucleophilic-
−7
buffer solution (pH 8.0, 20 mM) containing 5.0 × 10 M 1
was first introduced to a quartz cuvette and placed in dark for 5
min. Varying concentrations of GSH were added to the
solution of 1. The addition was limited to 20 μL so that the
volume change was insignificant. After incubating at 25 °C for 3
min, the absorbance was recorded on the UV/vis spectropho-
tometer.
Substituted Reaction and Complementary Electrostatic
Interaction
To study the kinetics and time dependence of the
interactions between 1 and GSH, the 480 nm band of the
mero form was recorded in Tris−HCl buffer solution. The
absorption spectra of 500 μL of 1 (5 μM) was monitored for 5
min. Then, GSH was added to the probe buffer with different
concentrations, UV−vis absorption spectra were measured at
2
5 °C.
The pseudo-first-order rate constant for the reaction of 1
(
5.0 μM) with analytes in the Tris−HCl buffer solution was
determined by fitting the absorbance of the samples to the
pseudo-first-order equation:
40
ln[(Amax − A )/Amax] = −Kobst
(1)
t
where A and A are the absorbance at 480 nm at time t and
t
max
the maximum value obtained after the reaction was complete.
Kobs is the pseudo-first-order rate constant.
design mechanism, we demonstrate here how decisive this
thermal switching of a spiropyran molecule is to achieve the
expected sensitivity and selectivity for glutathione (GSH).
Of the 20 amino acids used as building blocks for proteins,
the thiol-containing amino acids play crucial roles in biological
systems. For example, cysteine (Cys), homocysteine (Hcy),
and GSH have been proven to be associated with various
RESULTS AND DISCUSSION
■
Design Concept. In previous literature, an intramolecular
charge transfer-based fluorescent probe for thiols was reported,
on the basis of cleavage of sulfonamide and sulfonate ester of
2,4-dinitrobenzenesulfonyl derivate by thiols through a
41
nucleophilic aromatic substitution process. Moreover, it has
3
1
human diseases. Due to their important roles in physiological
process, various molecular probes and sensors have been
developed for the detection of thiol-containing amino acids,
35,36
been found that the cleavage of C−O bond of dinitrophenyl
30
ethers is promoted by a primary thiol in solution, whereas
agents with a secondary thiol is deficient in ability of
nucleophilic attack. These observations imply that the
32−34
including organic small molecules,
metal complexes,
4
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negatively charged thiol plays a key role in the thermal
switching process of a spiropyran by cleaving the dinitrophenyl
ether moiety of the spiropyran molecule via nucleophilic
aromatic substitution. By taking advantage of the unique
reactivity profile and strong electron-withdrawing capacity of
the 2,4-dinitrobenzene moiety, a spiropyran molecule, 6,8-
dinitro-1′, 3′, 3′-trimethylspiro [2H-1-benzopyran-2, 2′-indo-
line] (1), has been designed (Chart 1). 1 was substituted by
Chart 1. Molecular Structures of the Spiropyran Probes of 1,
2
, and 3
Figure 1. Absorption spectra changes of 1 (5.0 μM) in Tris−HCl
solution (pH 8.0, 20 mM) under different conditions: free 1 (black);
irradiation with UV light for 5 min (blue); and upon addition of GSH
(red), Cys (cyan), Hcy (pink), and GSSG (green). The concentrations
of all substrates were 5 μM, respectively. Inset: Color change of 1 in
Tris−HCl solution (left) upon addition of 5 μM GSH (right).
original value by GSH (5.0 × 10− M). Under the same
conditions, a similar response was also observed in the presence
of Cys or Hcy. As for GSSG, no absorption peak at 480 nm
could be observed, indicating that the secondary thiol did not
support the nucleophilic substitution reaction. We noticed that
the final absorption spectra of the reaction system, composed of
GSH and 1 and that of mero form of spiropyran 1 upon UV
light irradiation in aqueous solution, were essentially identical,
indicating the formation of merocyanine in the reaction.
Namely, the increasing absorption bands were attributed to the
increasing proportion of the merocyanine form to that of the
spiropyran form by interaction with GSH.
6
two strong electron withdrawing nitro groups in the 6- and 8-
positions, respectively. The choice of the recognition element
was guided by the considerations of the high affinity and
specificity to the target. We thus anticipate that introduction of
two nitro groups into the molecule backbone of 1 could
significantly weaken the C−O bond of 1, and it is possible that
the negatively charged thiol could play a key role in thermal
switching process of 1 by cleaving the dinitrophenyl ether
moiety of the spiropyran molecule via nucleophilic substitution
reaction, affording the substitution adduct of mero form and
thiol-containing amino acids. In addition, electrostatic inter-
action between the zwitterionic merocyanine and the dipolar
molecule could further promote the conversion from the spiro
form to the mero form. To validate the necessity of two nitro
groups, mononitro spiropyrans 2 and 3, which were substituted
by nitro group individually in 6- or 8-position were synthesized
as control compounds. The ability of electron withdrawing of
mononitro group is weaker than that of two nitro groups, so the
spiro C−O bonds of 2 and 3 are more stable than that of 1,
which is not conducive to nucleophilic substitution reaction.
Photophysical Properties of Probe 1. To ensure the ring
closure state, 1 was first irradiated in ethanol solution for 30
min by visible light with λ > 500 nm. In organic solvents, the
free 1 displayed the maximal absorption wavelength around 330
nm (Figure S1A, Supporting Information), reflecting the nearly
complete presence of the spiro-component. Upon irradiating by
ultraviolet light, the two nitro groups of the ring-open form are
positioned so that one large planar π-system emerges, leading
to the strong absorption band appearing in the visible range
from 540 to 600 nm (Figure S1B, Supporting Information). In
Tris−HCl buffer solution and in the dark, 1 showed a
maximum centered around 352 nm concomitant with a slight
yellow color of the solution (Figure 1), indicating that 1 exists
Simultaneously, metal ions and anions (50 equiv) with
+
2+
2+
2+
2+
−
physiological functions, such as K , Ca , Cu , Zn , Hg , Cl ,
−
−
2−
3−
Br , AcO , CO , and PO4 , were added to the solution of 1
3
and the changes in absorption spectra were monitored. As
shown in Figure S2 (Supporting Information), all of them did
not induced any absorption changes of 1, which indicated that
1 could keep the ring closure state stable in the presence of
physiological relevant ions.
To gain insight into the role of the nitro group on GSH-
induced ring-opening, analogue compounds of 2 and 3 which
were substituted by monomer nitro group in 6- or 8-position
were investigated. Both spiropyrans were screened for
absorption intensity change at the open state (mero form)
toward GSH in the Tris−HCl buffer solution. After interaction
of 2 and 3 with GSH, respectively, for 5 min, no change at their
corresponding absorption band of mero form was observed
(Figure S3, Supporting Information). The results demonstrated
that both nitro groups in 6- and 8-position were crucial for
thiols as essential nucleophiles in the substitution reaction
because of the strong electron-withdrawing ability of
bisubstituted nitro groups.
Simultaneous Nucleophilic-Substituted and Electro-
static Interactions for GSH-Induced Ring-Opening. To
investigate the interaction mechanism between thiol-containing
amino acids and 1, we tested the effects of substrates containing
different functional groups on the absorption spectra of 1.
Figure S4 (Supporting Information) shows the absorbance
enhancements of 1 at 480 nm upon additions of selected
substrates, such as C H SH, C H OH, C H NH , and
mainly as the closed spiro form. As the polarity of H O is much
2
higher in comparison to the organic solvent, a new absorption
band was major blue-shift appearing at 480 nm after being
irradiated by ultraviolet light.
Then, to demonstrate the feasibility of this design, the
absorption responses of 1 toward GSH, Cys, Hcy, and GSSG
were examined (Figure 1). As expected, when GSH is
2
5
2
5
2
5
2
−6
presented in the solution of 1 (5.0 × 10 M), an orange-
yellow appeared with a strong absorption peak at 480 nm. The
absorbance of 1 at 480 nm was increased to 9.6-fold that of the
CH COOH, for an interaction time of 5 min, separately. As
3
expected, no significant variations in the absorbance at 480 nm
were found in the presence of substrates other than C H SH,
2
5
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while 1 evidently displays an absorbance enhancement in the
different pH of the tested buffer solution. In the absence of
GSH, the absorbance at 480 nm changed slightly with pH range
from 3.0 to 6.0, as 1 converted to its protonated mero-form
presence of C H SH. The dependence of photochromic
2
5
behaviors of 1 on substrate structure preliminary suggested
that, in a given medium, sulfhedryl group was the key factor for
triggering the structure conversion from spiro form to mero
form. The obvious absorbance enhancements after addition of
mercaptoethylamine (MEA) and thioglycolic acid (TA) further
confirmed the speculation.
To further evaluate the role of sulfhedryl group on ring-
opening of 1, time-dependent absorption changes of 1 at 480
nm were monitored in the absence and presence of GSH and
GSSG (Figure 2). Without GSH, no absorption at 480 nm is
+
(HME ), leading to blue shift of the absorption band compared
with the mero form 1c. While, at pH > 7.0, the spiro form of 1
could exist stably at first, the absorbance at 480 nm enhanced
slowly with the increase of pH, due to the high concentration of
−
OH in solution that nucleophilically attacked the weak C−O
bond. Upon addition of GSH, GSH had minor effect on the
absorption spectra under acidic medium, because the formation
+
of the open mero-form of 1 has been induced by H by forming
+
protonated HME , but in an alkaline environment, as pH
−
affects the concentration of deprotonated thiol anion (GS ) in
−
solution, the concentration of GS increased with the increase
of pH, which resulted in an obvious increase of absorbance at
−
4
80 nm. When GS was saturated at high concentrations, the
absorbance at 480 nm increased no further with higher pH. The
results clearly indicted that the concentration of GS− in
solution affected the rate of nucleophilic substitution reaction.
According to the pH effect on 1 in the absence and presence of
GSH, knor of the spontaneous thermal reaction of 1 from the
spiro form to the mero form with different pH values and kobs
of 1 as a function of pH in the presence of GSH were obtained
in Table S1, Supporting Information. From Figure S5 and
Table S1 (Supporting Information), one can see that the pH
range of 7.0−9.0 is practical for GSH sensing.
Figure 2. The time courses of ring-opening reaction of 1 in the
absence (a) and the presence of GSSG (b) and GSH (c) in Tris−HCl
solution at room temperature. The absorbance was recorded at 480
nm at 25 °C.
The reaction mechanism was further confirmed using
electrospray ionization mass spectrometry (ESI-MS) (Figure
+
S6, Supporting Information). The m/z formula (M + H) of
the free 1 was found to be 307.9 (calculated: 307.5) in its ESI-
MS. After addition of 10 μM GSH to the solution containing 1
and incubation for 5 min at room temperature, a new peak at
observed within 5 min, which indicates the nearly complete
absence of the merocyanine component. On the contrary, in
the presence of GSH, the thermal ring-opening reaction was
accelerated markedly. The formation of merocyanine proceeds
rapidly at first, followed by more gradual increase in the
absorption. After 5 min, the absorption of merocyanine
achieved a constant, while there was only a negligible
enhancement in the absorbance at 480 nm after incubation
with GSSG for 5 min.
+
m/z 674.9, which is assigned to [1 + GSH] , is clearly observed,
indicating the formation of a 1:1 complex between 1 and GSH.
It is worth noting that, from Figure S6 (Supporting
Information), the peak at 674.9 increased first and then
decreased gradually in intensity with an increase of the time.
+
The relative abundance changes of [1 + GSH] indicated that
GSH was released gradually, revealing the eventual product
should be 1c.
The corroborated results suggest that the sulfhydryl group
would be responsible for ring-opening of 1, and the
electrostatic interaction between zwitterionic amino acids and
zwitterionic mero form converted by sulfhedryl group would
accelerate the structure conversion. We thus can draw a
conclusion that the interaction between 1 and GSH would be
simultaneous nucleophilic-substituted and electrostatic inter-
actions, as shown in Scheme 1. The nucleophilic reaction at
spiro carbon atom is facilitated because of electron deficiency
resulting from strong electron-withdrawing ability of bisub-
stituted nitro-groups at 6- and 8-positions. Closed 1 is activated
by the nucleophilic reaction that cleaves the C−O bond by
sulfhydryl group to give a free oxygen anion, generating the
nucleophilic substitution products of 1a and 1b. Then, the
electrostatic interaction between the zwitterionic mero form
and zwitterionic GSH promotes the electron rearrangement of
the isolated electron pair at nitrogen atom to get quaternary
ammonium cation, resulting in the C−S to be severed and
release of the thiol to afford 1c.
Enzyme-Catalyzed Path of GSH-Induced Ring-Open-
ing. To further characterize the efficiency of GSH-induced
ring-opening, we used the absorption changes of 1 at 480 nm to
measure the isomerization kinetics as a function of GSH
concentration. At a constant pH of 8.0, the rate of
isomerization to mero-form initially increases linearly with the
GSH concentration (Figure S7, Supporting Information),
indicating a transition from first-order to zeroth-order kinetics.
With a further increase of the GSH concentration, the rate of
reaction gradually leveled off.
With a combination of the proposed mechanism and rate
constant kobs, initially, GSH participates in the chemical
reaction and then is released from the adduct attributed to
electron rearrangement, so it can be considered that GSH plays
the role as an enzyme to catalyze the isomerization process
from spiro form to mero form. We therefore could put forward
a kinetic scheme (Scheme S1, Supporting Information) in
which 1 reversibly forms an encounter complex with GSH
before a light-driven chemical reaction generates 1a and 1b
The pH dependence of the ring-opening reaction of 1
supports the nucleophilic aromatic substitution reaction. Figure
S5 (Supporting Information) depicts the absorbance changes of
42−44
adducts.
Due to the fact that enzyme reactions for a single
substrate show an identical type of equation no matter how
many intermediates are involved, only the physical significance
of the Michaelis constant km and catalytic constant kcat will
1
at 480 nm as a function of pH in the absence and presence of
GSH. The absorption spectra were measured directly in
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change. Thus, formation of 1a can be described by k , and 1b−
m
45
GSH can be corresponded to kcat. Namely, km and kcat
represent k+1 and k , respectively. Hence, the relation of k ,
+2
obs
km, and kcat in the presence of GSH can be expressed as eq 2.
1
Km
1
1
=
+
Kobs − Knor
Kcat − Knor [1]0
Kcat − Knor
(2)
The rate of the reaction from 1 to 1c levels off as the
concentration of 1 increases at a constant GSH concentration
of 5 × 10− M. The process could be analyzed according to the
Michaelis−Menten kinetic model as eq 3 and Figure 3.
6
Figure 4. Absorption spectra of 1 (5 μM) in the presence of increasing
amounts of GSH in the Tris−HCl solution. The arrow indicates the
signal changes as increases in GSH concentrations (0, 0.025, 0.05, 0.1,
0
.2, 0.3, 0.5, 0.8, 1.0, 1.2, 1.4, 1.8, 2.0, 2.5, 3.0, 3.5, and 4.0 μM). Inset:
The ratio of (A − A )/A of 1 as a function of the concentrations of
0
0
GSH. Where A and A are the absorbance of 1 at 480 nm in the
0
absence and in the presence of GSH, respectively. The magnitude of
the error bars was calculated from the uncertainty given by three
independent measurements.
of various amino acids were measured (Figure 5). 1 exhibited a
strong absorption response at 480 nm toward Hcy or Cys, with
an evident color change from slight-yellow to orange-yellow.
On the other hand, treatment of up to 50 equivalents of the
other amino acids (Tyr, Gly, Phe, Met, Leu, Arg, Pro, Lys, Glu,
Gln, Asp, Iso, Ile, Val, His, Ser, Ala, Thr, Try, Asp) with 1 did
not induce any obvious change in the absorption spectra.
Competitive experiments for the reaction of 1 with various
amino acids in the presence of GSH were studied. When GSH
was added to the mixture solution of each amino acid and 1,
significant variations in the absorbance of 1 were found as
shown in (Figure 5), suggesting that the approach was not only
insensitive to other biologically related amino acids but also
selective toward thiol-containing amino acids in the presence of
other amino acids. A biological protein, bovine serum albumine
Figure 3. Kinetic analysis of the GSH catalyzed isomerization of l to lc
according to the Michaelis−Menten model in the Tris−HCl solution
for 5 min at 25 °C. Absorbance was recorded at 480 nm.
1
V0
K
1
[1]
+ V1
m
= V
(3)
max
max
The derived k and V values at 25 °C correspond to 1.46 ×
m
max
−7
−
4
−1
−1
1
0
M
and 2.17 × 10 M s , respectively. The value of k
was calculated to be 0.45 s from eq 3, and the rate ratio of
cat
−
1
4
k /k
cat nor
was 2.25 × 10 . The corresponding dynamic
parameters of catalyzing ring-opening by different analytes
were listed in Table S2, Supporting Information.
(
BSA), was also examined (Figure S8, Supporting Informa-
tion), but the proportion of the mero form by BSA was
substantially lower in comparison with that by GSH, which
could be due to the steric hindrance that prevented the
sulfhydryl from attacking the C−O bond so as to affect the rate
of ring-opening. These results established that 1 can serve as a
selective and sensitive probe for quantification of intracellular
thiols with sample pretreatment to remove biologically relevant
Colorimetric Sensing of GSH. To demonstrate the
applicability of the proposed approach for quantitative
detection of GSH, we measured the absorption spectra of 1
in the Tris−HCl buffer solution containing GSH with varying
concentrations (Figure 4). Upon the addition of GSH to the
solution of 1 for 5 min, a significant absorption band at 480 nm
was realized compared to that in the absence of GSH, and the
absorbance increased considerably with the increase of GSH
concentration, approximately up to 1.0 equiv relative to the
host concentration, which displayed a concentration-dependent
manner. There was a good linearity between the absorbance
enhancement and GSH concentrations in the range of 0.05 to
46,47
interfering agents.
CONCLUSION
■
In summary, we have presented a new strategy for the
colorimetric assay of thiol-containing amino acids. The
absorption spectra, kinetics, and mass spectrometry analyses
suggest that the ring-opening state of 1 is formed by both
thiolysis of dinitrophenyl ether via nucleophilic attack and
intramolecular electron-rearrangement of the isolated electron
pair at nitrogen atom caused by electrostatic interaction. To the
best of our knowledge, this is the first find that small molecule
thiols could catalyze the spiro form to be converted to the mero
form. Also, it is the first time that the use of simultaneous
nucleophilic-substituted and electrostatic interactions designs a
molecular sensor. Compared with the spiropyrans receptor for
amino acids based on electrostatic interaction, this approach is
0
.8 μM. The regression equation was ΔA/A = 1.283 + 8.526 C
0
(μM) with a linear coefficient efficient of 0.997, where ΔA = A
−
A and A and A are the absorbance of 1 at 480 nm in the
0
0
absence and the presence of GSH, respectively. The detection
limit that was taken to be 3 times the standard derivation of a
blank solution was estimated to be 10 nM, which is lower than
that of other spiropyran probes for amino acids based on
supramolecular interactions (Table 1).
To examine the selectivity of 1 toward thiol-containing
amino acids, changes in the absorption spectra of 1 by addition
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Table 1. Comparison of Spiropyran Probes for Assay of Amino Acids
method
analyte
signal output
solvent
response time
detection limit
a
a
a
ref 16
basic amino acids
absorbance
absorbance
absorbance
absorbance
fluorescence
absorbance
acetonitrile/water
methanol
>0.5 h
1.5 h
--
--
--
ref 17
L-Try/L-Tyr
His
ref 19
water
1−6 h
a
−8
ref 18a
ref 18b
this work
Cys
ethanol/water
ethanol/water
Tris−HCl
--
4.0 × 10
M
M
a
GSH
15 min
3 min
--
−
8
GSH
1.0 × 10
a
Not mentioned.
(
4) Byrne, R.; Kevin, J.; Fraser, K. J.; Izgorodin, E.; MacFarlane, D.
R.; Forsyth, M.; Diamond, D. Phys. Chem. Chem. Phys. 2008, 10,
919−5924.
5) Bertelson, R. C. In Photochromism; Brown, G. H.; Ed.; Wiley-
Interscience: New York, 1971; pp 45−431, and references therein.
6) Guglielmetti, R. In Photochromoism: Molecules and Systems, Studies
in Organic Chemistry; Durr, H., Bouas-Laurent, H., Eds.; Elsevier:
Amsterdam, 1990; Chapter 8 and 23, and references therein.
7) Berkovic, G.; Krongauz, V.; Weiss, V. Chem. Rev. 2000, 100,
5
(
(
̈
(
1
(
(
(
(
741−1753.
8) Minkin, V. I. Chem. Rev. 2004, 104, 2751−2776.
9) Inouye, M. Coord. Chem. Rev. 1996, 148, 265−283.
10) Byrne, R.; Diamond, D. Nat. Mater. 2006, 5, 421−426.
11) (a) Winkler, J. D.; Bowen, C. M.; Michelet, V. J. Am. Chem. Soc.
Figure 5. The selectivity of 1 toward various amino acids. Gray bars:
the ratio of (A − A )/A of 1 in the presence of various amino acids;
0
0
1
998, 120, 3237−3242. (b) Shao, N.; Zhang, Y.; Cheung, S.; Yang, R.
H.; Chan, W. H.; Mo, T.; Li, K. A.; Liu, F. Anal. Chem. 2005, 77,
294−7303. (c) Shao, N.; Jin, J. Y.; Wang, H.; Zhang, Y.; Yang, R. H.;
Chan, W. H. Anal. Chem. 2008, 80, 3466−3475.
12) (a) Evans, L.; Collins, G. E.; Shaffer, R. E.; Michelet, V.;
Black bars: the ratio of (A − A )/A of 1 in the presence of the mixture
0
0
of each amino acid and GSH: 1, Tyr; 2, Gly; 3, Phe; 4, Met; 5, Leu; 6,
Arg; 7, Pro; 8, Lys; 9, Glu; 10, Gln; 11, Asp; 12, Iso; 13, Ile; 14, Val;
7
15, His; 16, Ser; 17, Ala; 18, Thr; 19, Try; 20, Cys; 21, Hcy.
(
Winkler, J. D. Anal. Chem. 1999, 71, 5322−5327. (b) Leaustic, A.;
Dupont, A.; Yu, P.; Clement, R. New J. Chem. 2001, 25, 1297−1301.
(c) Kopelman, R. A.; Snyder, S. M.; Frank, N. L. J. Am. Chem. Soc.
2003, 125, 13684−13685.
more sensitive because of dual interaction modes. The ring-
opening reaction of 1 is enhanced markedly by 4 orders of
magnitude by GSH which shows enzyme-like activity.
Furthermore, the present combination of GSH and 1 suggests
that each thiol-containing amino acid or peptide can in
principle act as an enzyme for catalyzing thermal switching of
spiropyrans, which may open up a new field of application of
photochromic molecules.
(13) (a) Kimura, K.; Kaneshige, M.; Yamashita, T.; Yokoyama, M. J.
Org. Chem. 1994, 59, 1251−1256. (b) Kimura, K.; Utsumi, T.;
Teranishi, T.; Yokoyama, M.; Sakamoto, H.; Okamoto, M.; Arakawa,
R.; Moriguchi, H.; Miyaji, Y. Angew. Chem., Int. Ed. 1997, 36, 2452−
2
454. (c) Salhin, A. M. A.; Tanaka, M.; Kamada, K.; Ando, H.; Ikeda,
T.; Shibutani, Y.; Yajima, S.; Nakamura, M.; Kimura, K. Eur. J. Org.
Chem. 2002, 655−662.
(14) (a) Shiraishi, Y.; Adachi, K.; Itoh, M.; Hirai, T. Org. Lett. 2009,
ASSOCIATED CONTENT
Supporting Information
■
11, 3482−3485. (b) Shiraishi, Y.; Sumiya, S.; Hirai, T. Chem. Commun.
*
S
2
011, 47, 4953−4955.
Synthesis of spiropyran 1−3, calculation of kinetic parameters,
ESI-MS measurements of 1 in the presence of GSH, and other
additional spectroscopic data. This material is available free of
charge via the Internet at http://pubs.acs.org
(15) Shao, N.; Wang, H.; Gao, X. D.; Yang, R. H.; Chan, W. H. Anal.
Chem. 2010, 82, 4628−4636.
(16) Tsubaki, K.; Mukoyoshi, K.; Morikawa, H.; Kinoshita, T.; Fuji,
K. Chiral 2002, 14, 713−715.
(17) Ipe, B. I.; Mahima, S.; Thomas, K. G. J. Am. Chem. Soc. 2003,
1
(
25, 7174−7175.
AUTHOR INFORMATION
Corresponding Author
E-mail: Yangrh@pku.edu.cn. Fax: +86-731-8882 2523.
■
18) (a) Shao, N.; Jin, J. Y.; Cheung, S. M.; Yang, R. H.; Chan, W. H.;
Mo, T. Angew. Chem., Int. Ed. 2006, 45, 4944−4948. (b) Shao, N.; Jin,
J.; Wang, H.; Zheng, J.; Yang, R.; Chan, W.; Abliz, Z. J. Am. Chem. Soc.
2010, 132, 725−736.
*
Notes
The authors declare no competing financial interest.
(19) Liu, Y.; Fan, M.; Zhang, S.; Sheng, X.; Yao, J. New J. Chem.
2
007, 31, 1878−1881.
ACKNOWLEDGMENTS
This work was supported by NSFC (21075032, 21005026, and
1135001), DFMEC (20100151120008, 20110161110006),
and ‘973’ National Key Basic Research Program
(20) (a) Inouye, M.; Kim, K.; Kitao, T. J. Am. Chem. Soc. 1992, 114,
78−780. (b) Takase, M.; Inouye, M. Chem. Commun. 2001, 2432−
433.
■
2
7
2
(
21) Andersson, J.; Li, S.; Lincoln, P.; Andrea
Soc. 2008, 130, 11836−11837.
22) Sunamoto, J.; Iwamoto, K.; Mohri, Y.; Kominato, T. J. Am.
́
sson, J. J. Am. Chem.
(
2011CB911000)
(
Chem. Soc. 1982, 104, 5502−5504.
REFERENCES
■
(23) Frieden, E. J. Chem. Educ. 1975, 52, 754−761.
(24) Karshikoff, A. Non-covalent Interactions in Proteins; World
Scientific Publishing Company: Singapore; Imperial College Press:
London, 2006; Chapter 1, and references therein.
(
(
(
1) Fischer, E.; Hirshberg, Y. J. Chem. Soc. 1952, 4522−4524.
2) Day, J. H. Chem. Rev. 1963, 63, 65−80.
3) Taylor, L. D.; Nicholson, J.; Davis, R. B. Tetrahedron Lett. 1967,
8, 1585−1588.
(25) Mu
̈
ller-Dethlefs, K.; Hobza, P. Chem. Rev. 2000, 100, 143−168.
4
737
dx.doi.org/10.1021/ac203494e | Anal. Chem. 2012, 84, 4732−4738

Analytical Chemistry
Article
(
26) Bao, Y.; Liu, B.; Wang, H.; Tian, J.; Bai, R. Chem. Commun.
011, 47, 3957−3959.
27) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Chem. Soc. Rev. 2010, 39,
120−2135.
28) (a) Kim, S. Y.; Hong, J. Org. Lett. 2007, 9, 3109−3112.
b) Huang, Y.; Xiong, S.; Liu, G.; Zhao, R. Chem. Commun. 2011, 47,
2
(
2
(
(
8
(
1
(
1
(
319−8321.
29) Xu, Z.; Chen, X.; Kim, H. N.; Yoon, J. Chem. Soc. Rev 2010, 39,
27−137.
30) Lin, W. Y.; Long, L.; Tan, W. Chem. Commun. 2010, 46, 1503−
505.
31) (a) Staal, F. J. T.; Ela, S. W.; Roederer, M.; Ansderson, M. T.;
Herzenberg, L. A. Lancet 1992, 339, 909−913. (b) McCully, K. S. Nat.
Med. 1996, 2, 386−389.
(
32) (a) Rusin, O.; St. Luce, N. N.; Agbaria, R. A.; Escobedo, J. O.;
Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. J. Am.
Chem. Soc. 2004, 126, 438−439. (b) Wang, W.; Rusin, O.; Xu, X.;
Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.; Lowry,
M.; Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I.
M.; Strongin, R. M. J. Am. Chem. Soc. 2005, 127, 15949−15958.
(
33) (a) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki,
K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922−2925. (b) Ros-Lis,
J. V.; Garcaa, B.; Jimenez, D.; Martanez-Manez, R.; Sancenon, F.; Soto,
J.; Gonzalvo, F.; Valldecabres, M. C. J. Am. Chem. Soc. 2004, 126,
̊
́
̊
́
̃
́
4
2
(
064−4065. (c) Ahn, Y. H.; Lee, J. S.; Chang, Y. T. J. Am. Chem. Soc.
007, 129, 4510−4511.
34) (a) Julia Guy, J.; Caron, K.; Dufresne, S.; Michnick, S. W.;
Skene, W. G.; Jeffrey, J. W. J. Am. Chem. Soc. 2007, 129, 11969−11977.
(
(
b) Novak, M.; Lin, J. J. Am. Chem. Soc. 1996, 118, 1302−1308.
c) Jing, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. Angew. Chem., Int. Ed.
2
(
007, 46, 8445−8448.
35) Chow, C. F.; Chiu, B. K. W.; Lam, M. H. W.; Wong, W. Y. J.
Am. Chem. Soc. 2003, 125, 7802−7803.
(
(
36) Han, S. M.; Kim, D. H. Tetrahedron 2004, 60, 11251−11257.
37) Sudeep, P. K.; Joseph, S. T.; Thomas, K. G. J. Am. Chem. Soc.
2
(
5
(
005, 127, 6516−6517.
38) Han, B. Y.; Yuan, J. P.; Wang, E. K. Anal. Chem. 2009, 81,
569−5573.
39) Buback, J.; Kullmann, M.; Langhojer, F.; Nuernberger, P.;
Schmidt, R.; Wurthner, F.; Brixner, T. J. Am. Chem. Soc. 2010, 132,
6510−16519.
40) (a) Dale, T. J.; Rebek, J. J. J. Am. Chem. Soc. 2006, 128, 4500−
501. (b) Jo, J.; Lee, D. J. Am. Chem. Soc. 2009, 131, 16283−16291.
41) (a) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki,
1
(
4
(
K.; Itoh, N. Angew. Chem. 2005, 117, 2982−2985. (b) Jiang, W.; Fu,
Q.; Fan, H.; Ho, J.; Wang, W. Angew. Chem., Int. Ed. 2007, 46, 8445−
8448. (c) Lu, J.; Sun, C.; Chen, W.; Ma, H.; Shi, W.; Li, X. Talanta
2
(
011, 83, 1050−1056.
42) Pfeifer-Fukumura, U. J. Photochem. Photobiol., A 1997, 111,
1
45−156.
43) Pfeifer, U.; Fukumura, H.; Misawa, H.; Kitamura, N.; Masuhara,
H. J. J. Am. Chem. Soc. 1992, 114, 4417−4418.
44) Willner, I.; Blonder, R.; Dagan, A. J. Am. Chem. Soc. 1994, 116,
121−3122.
45) (a) Segel, I. H. Enzyme Kinetics; Wiley: New York, 1993.
b) Dixon, M.; Webb, E. C. Enzymes, 3rd ed.; Academic Press: New
York, 1979.
46) Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.;
Cho, B. R. J. Am. Chem. Soc. 2011, 133, 11132−11135.
47) Fujikawa, Y.; Urano, Y.; Komatsu, T.; Hanaoka, K.; Kojima, H.;
Terai, T.; Inoue, H.; Nagano, T. J. Am. Chem. Soc. 2008, 130, 14533−
4543.
(
(
3
(
(
(
(
1
4
738
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