Simultaneous Nucleophilic-Substituted and Electrostatic Interactions

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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-dinitro1′,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.

S

dimensional surface of gold nanoparticles attributed to the ability of zwitterionic merocyanines to bind with charged molecules.18 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 electrostatic interactions, were utilized by our group18 and Yao et al.19 Although these strategies presented a potential approach for determination of dipolar molecules, drawbacks toward a practical sensor for selective detection of a particular analyte exist.23,24 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 complex biological environment,25 so how to set up a practical

piropyrans, an important class of photoswitchable molecules, are an attractive starting point in construction of optical probes,1−4 because they show reversibly structural conversion between the closed spiropyran (spiro-) form and the opened merocyanine (mero-) form upon external optical, thermal, or chemical stimulations.5−8 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 metal ions9−13 and anions14,15 to amino acids16−19 and even nucleobases20 and nucleic acid molecules.21 Complementary electrostatic interaction between the zwitterionic 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 bilayers and membranes.22 Later, Thomas and co-workers presented the photoswitchable self-assembly of various amino acid derivatives by anchoring spiropyrans onto the three© 2012 American Chemical Society

Received: January 5, 2012 Accepted: April 30, 2012 Published: April 30, 2012 4732

dx.doi.org/10.1021/ac203494e | Anal. Chem. 2012, 84, 4732−4738

Analytical Chemistry

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gold nanorods,37 and quantum dots.38 Although these approaches have led to significant contributions to the thiolcontaining 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 acids reported in the literature,32−38 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 thiolcontaining amino acids is improved; and finally, the system is operated in aqueous solution, which has a good prospect of application.

sensor for specific detection of the corresponding analyte has been a great challenge. 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 acceptors.26 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, and disulfide27 and analyzing F− and CN− by unique desilylation reaction28 and nucleophilic addition reaction,29 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-substituted 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 is catalyzed by a primary thiol in solution,30 which would greatly enhance the conversion of spiropyran form to merocyanine form (Scheme 1). To test the feasibility of this

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EXPERIMENTAL SECTION Spiropyran probes of 1, 2, and 3 were prepared as reported by Buback and Brixner.39 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 absorption 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). For colorimetric measurement of GSH, 1.0 mL Tris−HCl buffer solution (pH 8.0, 20 mM) containing 5.0 × 10−7 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 spectrophotometer. 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 25 °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

Scheme 1. Proposed Scheme for Merocyanine Formation of 1 Induced by GSH Based on Simultaneous NucleophilicSubstituted Reaction and Complementary Electrostatic Interaction

ln[(A max − A t )/A max ] = −Kobst

(1)

where At and Amax are the absorbance at 480 nm at time t and the maximum value obtained after the reaction was complete. Kobs is the pseudo-first-order rate constant.

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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 human diseases.31 Due to their important roles in physiological process, various molecular probes and sensors have been developed for the detection of thiol-containing amino acids, including organic small molecules,32−34 metal complexes,35,36

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 nucleophilic aromatic substitution process.41 Moreover, it has been found that the cleavage of C−O bond of dinitrophenyl ethers is promoted by a primary thiol in solution,30 whereas agents with a secondary thiol is deficient in ability of nucleophilic attack. These observations imply that the 4733



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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, 6,8dinitro-1′, 3′, 3′-trimethylspiro [2H-1-benzopyran-2, 2′-indoline] (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 × 10−6 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. Simultaneously, metal ions and anions (50 equiv) with physiological functions, such as K+, Ca2+, Cu2+, Zn2+, Hg2+, Cl−, Br−, AcO−, CO32−, and PO43−, were added to the solution of 1 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 GSHinduced 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 Electrostatic 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 C2H5SH, C2H5OH, C2H5NH2, and CH3COOH, for an interaction time of 5 min, separately. As expected, no significant variations in the absorbance at 480 nm were found in the presence of substrates other than C2H5SH,

two strong electron withdrawing nitro groups in the 6- and 8positions, 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 interaction 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 mainly as the closed spiro form. As the polarity of H2O is much 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 presented in the solution of 1 (5.0 × 10−6 M), an orangeyellow 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 4734



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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 (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 480 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. 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 ESIMS. After addition of 10 μM GSH to the solution containing 1 and incubation for 5 min at room temperature, a new peak at 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. Enzyme-Catalyzed Path of GSH-Induced Ring-Opening. 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 adducts.42−44 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

while 1 evidently displays an absorbance enhancement in the presence of C2H5SH. The dependence of photochromic 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 ringopening 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

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.

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. 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 interactions, 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 bisubstituted 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. 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 1 at 480 nm as a function of pH in the absence and presence of GSH. The absorption spectra were measured directly in 4735



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change. Thus, formation of 1a can be described by km, and 1b− GSH can be corresponded to kcat.45 Namely, km and kcat represent k+1 and k+2, respectively. Hence, the relation of kobs, km, and kcat in the presence of GSH can be expressed as eq 2. Kobs

Km 1 1 1 = + − K nor Kcat − K nor [1] 0 Kcat − K nor

(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 × 10−6 M. The process could be analyzed according to the Michaelis−Menten kinetic model as eq 3 and Figure 3. 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 − A0)/A0 of 1 as a function of the concentrations of GSH. Where A0 and A are the absorbance of 1 at 480 nm in the 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 (BSA), was also examined (Figure S8, Supporting Information), 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 interfering agents.46,47

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.

K 1 1 1 = m + V0 Vmax [1] Vmax

(3)

The derived km and Vmax values at 25 °C correspond to 1.46 × 10−4 M−1 and 2.17 × 10−7 M s−1, respectively. The value of kcat was calculated to be 0.45 s−1 from eq 3, and the rate ratio of kcat/knor was 2.25 × 104. The corresponding dynamic parameters of catalyzing ring-opening by different analytes were listed in Table S2, Supporting Information. 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 0.8 μM. The regression equation was ΔA/A0 = 1.283 + 8.526 C (μM) with a linear coefficient efficient of 0.997, where ΔA = A − A0 and A0 and A are the absorbance of 1 at 480 nm in the 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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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 4736



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Table 1. Comparison of Spiropyran Probes for Assay of Amino Acids

a

method

analyte

signal output

solvent

response time

detection limit

ref 16 ref 17 ref 19 ref 18a ref 18b this work

basic amino acids L-Try/L-Tyr His Cys GSH GSH

absorbance absorbance absorbance absorbance fluorescence absorbance

acetonitrile/water methanol water ethanol/water ethanol/water Tris−HCl

>0.5 h 1.5 h 1−6 h --a 15 min 3 min

--a --a --a 4.0 × 10−8 M --a 1.0 × 10−8 M

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, 5919−5924. (5) Bertelson, R. C. In Photochromism; Brown, G. H.; Ed.; WileyInterscience: New York, 1971; pp 45−431, and references therein. (6) Guglielmetti, R. In Photochromoism: Molecules and Systems, Studies in Organic Chemistry; Dürr, 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, 1741−1753. (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. 1998, 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, 7294−7303. (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.; 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. (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− 2454. (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, 11, 3482−3485. (b) Shiraishi, Y.; Sumiya, S.; Hirai, T. Chem. Commun. 2011, 47, 4953−4955. (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, 125, 7174−7175. (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. (19) Liu, Y.; Fan, M.; Zhang, S.; Sheng, X.; Yao, J. New J. Chem. 2007, 31, 1878−1881. (20) (a) Inouye, M.; Kim, K.; Kitao, T. J. Am. Chem. Soc. 1992, 114, 778−780. (b) Takase, M.; Inouye, M. Chem. Commun. 2001, 2432− 2433. (21) Andersson, J.; Li, S.; Lincoln, P.; Andréasson, J. J. Am. Chem. Soc. 2008, 130, 11836−11837. (22) Sunamoto, J.; Iwamoto, K.; Mohri, Y.; Kominato, T. J. Am. Chem. Soc. 1982, 104, 5502−5504. (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. (25) Müller-Dethlefs, K.; Hobza, P. Chem. Rev. 2000, 100, 143−168.

Figure 5. The selectivity of 1 toward various amino acids. Gray bars: the ratio of (A − A0)/A0 of 1 in the presence of various amino acids; Black bars: the ratio of (A − A0)/A0 of 1 in the presence of the mixture 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; 15, His; 16, Ser; 17, Ala; 18, Thr; 19, Try; 20, Cys; 21, Hcy.

more sensitive because of dual interaction modes. The ringopening 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.

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ASSOCIATED CONTENT

* Supporting Information S

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

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-731-8882 2523. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC (21075032, 21005026, and 21135001), DFMEC (20100151120008, 20110161110006), and ‘973’ National Key Basic Research Program (2011CB911000)

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Simultaneous Nucleophilic-Substituted and Electrostatic Interactions

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