Fluorescent Recognition of Cu2+ and Cys in Aqueous Medium

Turn “OffOn” Fluorescent Recognition of Cu2+ and Cys in Aqueous Medium: Implementation of Molecular Logic Gate and Cell Imaging Studies Virendra Kumar,1 Ajit Kumar,1 Uzra Diwan,1 Manish Kumar Singh,2 and K. K. Upadhyay*1 1

Department of Chemistry (Centre of Advanced Study), Faculty of Science, Banaras Hindu University, Varanasi 221005, India 2

Department of Zoology (Centre of Advanced Study), Faculty of Science, Banaras Hindu University, Varanasi 221005, India E-mail: [email protected] Received: December 14, 2015; Accepted: January 11, 2016; Web Released: January 20, 2016

K. K. Upadhyay K. K. Upadhyay received his Ph.D. in Inorganic Chemistry from Banaras Hindu University in1994. He is now working as Professor in Department of Chemistry, Banaras Hindu University. His current interest ranges from optical sensors for ionic analytes and self-assembly through supramolecular architectures.

Abstract The present communication incorporates utilization of a simple Schiff base (RO) fruitfully for the turn “off-on” successive fluorescent recognition of Cu2+ and Cys in aqueous media. The single crystal of RO-Cu2+ ensemble revealed a binding pattern which ultimately leads to quenching of the green fluorescence of RO. Interestingly the quenched fluorescence of the RO-Cu2+ ensemble was revived selectively in the presence of Cys via Cu2+ displacement approach. The selective “off-on” behavior of RO towards Cu2+ and Cys was consequently used as inputs to build up an implication (IMP) logic gate. Moreover, the RO exhibited successful bio-imaging of Cu2+ and Cys also in E. coli cells.

Introduction Developing an efficient and smart chemosensor for the effortless and selective marking of ionic/neutral analytes of biological importance is a burgeoning area of research.1 The Cu2+ ion which is the third most abundant element found in the human body plays a pivotal role in numerous biological, environmental and chemical systems.2 On the other hand, excess copper ion can generate reactive oxygen species (ROS) by catalyzing the oxygen molecule in the living cell.3 This transformation of oxygen molecule into ROS may harm nucleic acids and proteins upon their prolonged exposure and may further lead to several dreadful diseases like cardiovascular disorders and neurodegenerative diseases, including Menkes and Wilson disease.4 Thus, the Janus-faced properties of Cu2+ make recognition of the same very essential. 754 | Bull. Chem. Soc. Jpn. 2016, 89, 754–761 | doi:10.1246/bcsj.20150427

Albeit various sensing techniques including atomic absorption/emission spectroscopy, inductively coupled plasma mass spectrometry, electrochemical methods, etc. have been reported in the literature,5 optical detection methods through chemosensors are one of the easiest as well as cheapest methods coming up over the last couple of decades. Among the optical sensors, the fluorescent ones are extensively sought and employed due to their high sensitivity, rapidity, and simplicity over the last few years.6 Numerous fluorescent probes for Cu2+ have been reported in the literature with a turn “off ” response exploiting the paramagnetic nature of the same.7 Though these types of sensors are very frequent and have several desirable features also yet some common inherent lacuna such as multiion detection, slow response, moderate selectivity, low water solubility, etc. make them a poor choice of users.2c,8 In perpetuation to our sincere efforts in developing new sensors,9 we recently explored a coumarinrhodanine conjugate RC which underwent a chemical transformation into a highly fluorescent compound RO in the selective presence of methanol.10 In the present communication we successfully extended the use of RO as a highly sensitive and biocompatible fluorescent probe for turn “offon” detection of Cu2+ and Cys in aqueous media. In RO, the pendent arm of N,N-diethyl upon coumarin was employed in a bid to have better water compatibility of the resulting chemosensor. Nevertheless, the RO with N, O, and S as a donor set served the purpose of binding unit. As per our speculation the strong binding between Cu2+ and RO resulted in rapid quenching of the green fluorescence of RO in aqueous medium. Furthermore, while exploring the literature, we came across some metal complexes/ensembles (R-Mn+) exploited as sen-

© 2016 The Chemical Society of Japan

sors for the detection of specific analytes.11 The same are considered to be an ideal tactic for neutral/anionic analyte recognition in aqueous solutions and are still attracting much attention. As mentioned above, in the present case for RO-Cu2+ ensemble we observed a non-fluorescent state. The effective stripping of Cu2+ ions from the ensemble by a copper-binding analyte like cysteine triggered a fluorescence state of the sensor.12 Taking the above technical approach we further exploited the RO-Cu2+ ensemble as a turn “on” fluorescent probe for Cys, as the latter one is a potent nucleophile and plays an important role in several biological systems by exhibiting specific metal ion binding. For instance, Cys acts as a precursor of GSH, acetyl Co-A and taurine, a source of sulfide in ironsulfur clusters, a potential neurotoxin,13 and a biomarker for various medical conditions. The deficiency of the same can lead to several dreadful diseases, such as a hematopoiesis decrease, leucocyte loss, lethargy, liver damage, etc.14 The detection of cysteine via RO-Cu2+ was also very selective, even the foremost interfering analytes in the case of Cys detection such as GSH/homocysteine were unable to interfere. The detection of Cys through RO-Cu2+ involved the complexation of Cys with the Cu2+, as a result the RO got set free which finally resulted in a fluorescence state. This two-stage signal transduction mechanism in RO is shown in Figure 1. The switching “off on” response of RO during sequential addition of Cu2+ and Cys was beautifully translated in the form of an IMPLICATION (IMP) logic gate at the molecular level. Furthermore, RO was also utilized for live cell imaging of these analytes in E. coli bacteria cells. Thus, the RO being presented through this communication has its own worth. On one hand, Cu2+ rapidly and selectively quenched the fluorescence intensity of RO with nanomolar detection ability (8.44 © 10¹9 M) in aqueous medium. On the other hand, the revival of fluorescence of RO-Cu2+ ensemble selectively in the presence of Cys even in the presence of other similar and foremost interfering analytes including Hcy, GSH further demonstrates its efficiency. Finally, the single crystal of ROCu2+ complex, implementation of molecular logic gate (IMP) as well as the bio-imaging of Cu2+ and Cys, further

Figure 1. Chemical structure of RC and RO and two-stage signal transduction mechanism in RO with Cu2+ and Cys. Bull. Chem. Soc. Jpn. 2016, 89, 754–761 | doi:10.1246/bcsj.20150427

makes the present work lucrative in view of the present context in the same field. Experimental Materials and Measurements. All the reagents for synthesis were purchased from Sigma-Aldrich and were used without further purification. All the organic solvents were of analytical grade. All titration experiments were carried out at room temperature. All aqueous solutions were prepared using freshly deionized water. All the metal ions and anions were used as their chloride and tetrabutylammonium (TBA) salts in aqueous solution. All the titration experiments were carried out in HEPES buffered (10 mM, pH 7.4) water at 25 °C. Instruments. IR spectra were recorded with a Perkin-Elmer spectrometer using KBr pellets. The corresponding 1H NMR spectra were recorded in CDCl3 with a JEOL AL 300 FT NMR Spectrometer instrument using tetramethylsilane (Si(CH3)4) as an internal standard. 1H chemical shifts are reported in parts per million (ppm) relative to the residual proton signal of the deuterated solvents. Mass spectrometric analysis was carried out on a MDS Sciex API 2000 LCMS spectrometer while HRMS of RO and RO-Cu2+ was recorded with a Water-Q-Tof Premier-HAB213. The electronic spectra and UVvisible titrations were carried out room temperature (298 K) on a UV1700/1800 Pharmaspec spectrophotometer with quartz cuvette (path length = 1 cm). The emission spectra were recorded on a JY HORIBA Fluorescence spectrophotometer. Synthesis of RO and RO-Cu2+ Complex. Synthesis of RO (Scheme 1): RO was synthesized by adding 2.0 mM methenolic solution of 7-(diethylamino)-2-oxo-2H-chromene3-carbaldehyde to an equimolar methenolic solution of 3aminorhodanine and refluxing with constant stirring for three hours. A brick red solid was precipitated which was filtered and washed with diethylether and finally dried under vacuum over anhydrous CaCl2. RO was characterized through various spectroscopic techniques like IR, 1H NMR spectral studies along with mass determination through HRMS. Spectroscopic Characterization Data: IR (cm¹1): 3182, 2973, 2927, 2869, 2852, 1747, 1701, 1614, 1596, 1571, 1516, 1421, 1375, 1356, 1293, 1255, 1191, 1163, 1130, 1097, 1030, 878, 823, 769, 674; 1H NMR in CDCl3: ¤ 1.25 (t, 6H, CH3, J = 6.9 Hz), 3.46 (q, 4H, CH2, J = 6.7 Hz), 3.792 (s, 3H, OCH3), 4.177 (s, 2H, CH2), 6.602 (s, 1H, Ar-H), 6.76 (d, 1H, Ar-H, J = 8.4 Hz), 7.42 (d, 1H, Ar-H, J = 8.7 Hz), 8.074, (s, 1H, Ar-H), 8.325 (s, 1H, CH=N), 10.157 (s, 1H, NH); HRMS: m/z calculated for [C18H21N3O4S2Na]+ = 430.0871; found = 430.0888. Synthesis of [RO-Cu2+] Complex: A 10 mL of methanol solution of CuCl2¢2H2O (1.0 mmol) was added slowly to a magnetically stirred solution of RO (1 mmol) in methanol (10 mL). The mixture was further stirred for ca. 4 h at room temperature. A brown colored solid was precipitated out which was filtered, washed several times with water and dried under vacuum over anhydrous CaCl2. The single crystals of RO-Cu2+ complex were developed in methanol within a few days. Spectroscopic Characterization Data: IR (cm¹1): 2971, 2929, 1741, 1709, 1620, 1598, 1563, 1513, 1483, 1428, 1379, 1350, 1311, 1295, 1259, 1233, 1188, 1133, 1029, 955, 904, 875, 835, 796, 655, 637, 577, 471; HRMS: m/z calculated for [C18H20CuN3O4S2]+ = 469.0180; found = 469.0194. © 2016 The Chemical Society of Japan | 755

Scheme 1. Synthesis of RO.

Theoretical Calculations. Geometric and energy optimizations were performed with the Gaussian 03 program based on the density functional theory (DFT) method.15 Becke’s three parameter hybrid functional with the LeeYangParr correlation functional (B3LYP) was employed for all the calculations. The LANL2DZ basis set was used to treat the copper atom, whereas the 3-21G** basis set was used to treat all other atoms. Calculation of Detection Limit. The detection limit was calculated from the fluorescence titration data using IUPAC method. The standard deviation for blank measurement was determined by the fluorescence emission spectra of RO and RO-Cu2+ for 10 times. In order to gain the slope the ratio of fluorescence intensity at ca. 520 nm was plotted against varying concentration of Cu2+ and cysteine respectively. The detection limit was calculated using the following equation: Detection limit ¼ 3Sd1=S

ð1Þ

where Sdl is the standard deviation of blank measurement and S is the slope of the calibration curve. Calculation of Quantum Yield. Here, the quantum yield Φ was measured using the following equation: ¼ ref ðFsamp =Fref Þ ðAref =Asamp Þ ð©samp =©ref Þ2 ð2Þ where samp and ref indicate the unknown and standard solution respectively, Φ: quantum yield, F: Integrated area under the emission curve, A: absorbance at the excitation wavelength, and ©: refractive index of the solvent. Here Φref measurements were performed using Fluorescein in water as a standard (Φ = 0.95). X-ray Diffraction Studies. Single-crystal X-ray diffraction measurements were carried out on a Bruker SMART APEX CCD diffractometer using graphite-monochromated Mo Kα radiation (k = 0.71073 ¡). All the determinations of unit cell and intensity data were performed with graphite-monochromated Mo Kα radiation ( = 0.71073 ¡). Data for the metal complex was collected at liquid nitrogen temperature. The structures were solved by direct methods, using Fourier techniques, and refined by full-matrix least-squares on F 2 using the SHELXTL-97 program package.16 Crystal data and details of the structure determination for RO-Cu2+ complex are summarized in Table S1. Cell Imaging Studies. E. coli strains (DH5-α) were grown in LB media at 37 °C overnight in shaker incubator. The cells were collected in sterile water and vortexed to make the suspension homogeneous. These cell cultures were incubated with RO and RO-Cu2+ (10 ¯M) from 1.0 mM stock in 50 mM phosphate buffer (pH 7.54) for 1 hour. The treated cells were examined by the excitation range from 450490 nm and emission range from 500560 nm on a fluorescence microscope (Nikon-E800, Japan). 756 | Bull. Chem. Soc. Jpn. 2016, 89, 754–761 | doi:10.1246/bcsj.20150427

Figure 2. UVvisible spectrum of RO and RO-Cu2+ in HEPES buffer (10 mM; pH 7.4; H2O:MeOH = 99:1) at 10 ¯M.

Results and Discussion Fluorescence Quenching of RO with Cu2+. The synthesized and well-characterized RO (ESI; Figures S1S3) was subjected to photophysical studies. As RO possesses a good number of donor atoms (O, S, N), we checked its binding capability and possible applicability as an optical probe for a number of metal ions viz. K+, Na+, Ca2+, Mn2+, Ba2+, Mg2+, Cr3+, Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+, Pb2+, and Al3+ (ESI; Figure S4). Although, the UVvisible spectrum of RO (10 ¯M) in HEPES buffer (10 mM pH 7.4, H2O:MeOH = 99:1) showed change in the max from 455 to 493 nm for Cu2+ (Figure 2) and slight hypochromic shift for Hg2+ ion. However, the same were not discriminated via a naked-eye observation (ESI; Figure S5). Here it is worth mentioning that the max of RO at 455 nm is slightly red-shifted (ca. 10 nm) in comparison to that of our earlier observation due to solvent variation from methanol to buffer solution in the present study.10 The non-conclusive results in the UVvisible spectrum as well as through naked-eye observation (ESI; Figures S4 and S5) persuaded us to check the fluorescence response of RO with the above chosen metal ions. The emission spectrum of RO (0.5 ¯M) in HEPES buffer (10 mM; pH 7.4; H2O:MeOH = 99:1) exhibited a green fluorescence at 520 nm (ex = 455 nm) with high fluorescence quantum yield (Φ = 0.67).17 As reported earlier the strong fluorescent nature of RO was due to the suppression of PET imposed by the planarity in the structure as revealed by DFT and single crystal XRD studies.10 The green fluorescence of RO however, got quenched selectively in the presence of Cu2+ (Φ = 0.016) while the same remained almost silent towards other chosen cations (ESI; Figure S6).


The fluorescence titration profile (Figure 3) showed that sequential addition of 05 equiv of Cu2+ efficiently quenches the fluorescent peak of RO at 520 nm (quenching efficiency at 520 nm, (Io I)/Io © 100% = 92). The fluorescence quenching upon chelation of the Cu2+ is a consequence of CHEQ process as the result of electron transfer from the ligand to the metal d-orbital and/or ligand to metal charge transfer (LMCT).18 To further enumerate the quenching efficiencies of RO with increasing concentration of Cu2+ the fluorescent intensity at 520 nm was analyzed using the SternVolmer equation: Io/I = 1 + Ksv [Cu2+], where Io and I are the initial and final emission intensity of RO and Ksv is the SternVolmer quenching constant. The SternVolmer plot between RO and Cu2+ ion was found to be linear with Ksv value 3.13 © 107 M¹1 for RO (ESI; Figure S7). The SternVolmer constant (Ksv) value suggested that the Cu2+ ion possesses a strong quenching ability towards the RO which is comparable to the other probes reported for Cu2+ ion sensing.19 The complexation of Cu2+ with RO was established through IR, HRMS (ESI; Figures S8 and S9), and single crystal XRD studies. The HRMS spectrum of RO in the presence of Cu2+ exhibited a peak at m/z = 469.0888 which was assignable to

Figure 3. Fluorescence titration spectrum of RO (0.5 ¯M) with increasing conc. of Cu2+ (0 to 5.0 equiv) in HEPES buffer (10 mM; pH 7.4; H2O:MeOH = 99:1), (ex = 455 nm).

(a)

[RO-CuCl¢H2O] (m/z calc. = 469.0887) supports 1:1 binding stoichiometry. The same was finally confirmed through single crystal XRD studies (Figure 4). The RO-Cu2+ assembly crystalizes in the monoclinic system, with C2/c space group and Z = 8. The complex exhibited distorted square planar geometry with four different set of donor atoms (N, O, S, Cl) in a nearly coplanar fashion. The bond lengths for Cu(1)N(2), Cu(1) O(2), Cu(1)S(1), and Cu(1)Cl(1) are 1.962(3), 1.990(3), 2.231(1), and 2.214(1) ¡, respectively. The square planar angles between the copper(II) and the equatorial coordinating atoms O(2)Cu(1)N(2), S(1)Cu(1)N(2), Cl(1)Cu(1)O(2), and S(1)Cu(1)Cl(1) are found to be 90.7(1), 86.1(1), 89.89(9), and 93.64(4)°, respectively (ESI; Table S1). In order to have further insight into the quenching effect of Cu2+ towards RO, we observed the electrochemical redox behavior of the RO as well as of RO-Cu2+ complex (Figure 5a) through cyclic voltammetry (CV) at the Pt electrode in acetonitrile solution (1 mM) containing 0.1 M tetrabutylammonium perchlorate (TBAP) as the supporting electrolyte. The voltammograms obtained in a potential range of ¹1.0 to 1.0 V with varying scan rates have been shown in Figure 1. In the anodic domain, we have two bands at 0.65 and 0.89 V with reference to Ag/AgCl and have assigned them as ligand-based oxidation band and Cu2+ to Cu3+ (MLCT) oxidation band respectively. On the other hand in the cathodic domain we have a band at ¹0.78 V with reference to Ag/AgCl and this was assigned as Cu2+ to Cu+ (LMCT). We also recorded the CV pattern of ROCu2+ by varying the scan rates where we observed a continuous change in the cathodic and anodic peak separation value (Figure 5b).

Figure 4. ORTEP plot (thermal ellipsoids are shown at 50% level) of the ROCu2+ with the atom numbering scheme.

(b)

Figure 5. Cyclic voltammetry curve; (a) for RO and RO-Cu2+ and (b) RO-Cu2+ at different scan rate. Bull. Chem. Soc. Jpn. 2016, 89, 754–761 | doi:10.1246/bcsj.20150427

© 2016 The Chemical Society of Japan | 757

Figure 6. The relevant frontier molecular orbitals (FMOs) of the RO-Cu2+ complex.

The same was further substantiated by the TD-DFT study. The calculations showed that the contribution of β-HOMO to β-LUMO+1 is ca. 79% (ESI; Table S3). The same is well demonstrated through charge distribution in β-HOMO and β-LUMO+1 of RO-Cu2+ ensemble in Figure 6. Hence the CHEQ process was thus considered as the consequence of the LMCT from the excited state of the fluorophore to the metal ion via a nonradiative pathway eventually causing fluorescent quenching. Furthermore, for practical applicability of RO towards detection of Cu2+ we also studied the various analytical aspects of the same. The strong binding of Cu2+ with RO was reflected in terms of binding constant which was found to be 2.4 « 0.09 © 107 M¹1 (R2 = 0.998) (ESI; Figure S10) using nonlinear curve fitting method.20 The detection limit obtained from the fluorescence titration study and was found to be 8.44 © 10¹9 M (ESI; Figure S11) hence sufficiently sensitive for its practical usefulness in determination of Cu2+ in environmental and bio samples. The selectivity is one of the most essential key features of any excellent chemosensor. Hence for having an insight into the selectivity of RO towards Cu2+, its fluorescence was examined with various metal ions upon exciting it at 455 nm. The RO exhibited a strong emission band at 520 nm and separate additions of 10 equiv of different metal ions such as Na+, K+, Mg2+, Ca2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Fe3+, Cr3+, Al3+, Hg2+, and Pb2+ had no obvious influence on the fluorescence emission spectrum while with the addition of 1.0 equiv of Cu2+ to the solution of RO (0.5 ¯M), almost complete fluorescent quenching was observed (ESI; Figure S12). We also deliberated the reaction time profile of RO towards Cu2+ at 520 nm intensity. The graph presented in ESI; Figure S13 clearly showed that 5 equiv of Cu2+ was sufficient to cause fluorescence quenching in RO within ca. 30 s. Since a wide pH range for any chemosensor is desirable not only for the biological studies but also for many industrial and environmental applications. Hence we evaluated the pH range of RO towards Cu2+ by monitoring its pH from 2 to 12 at 520 nm fluorescence intensity. As shown in ESI; Figure S14, RO efficiently detected Cu2+ in aqueous solution with the pH range from 6 to 10, where the fluorescence of RO was completely quenched. At the same time the stability of RO was not affected in anyway in spite of the presence of an imine group. Thus, the above observations show sensitivity, selectivity, as well as quick response of the RO with a wide range of pH towards Cu2+ detection in aqueous media. Revival of Fluorescence of RO-Cu2+ with Cys. There have also been reports on the use of metal ion (Cu2+ or Hg2+) 758 | Bull. Chem. Soc. Jpn. 2016, 89, 754–761 | doi:10.1246/bcsj.20150427

Figure 7. Fluorescence titration spectrum of ROCu2+ ensemble with Cys in HEPES buffer (10 mM; pH 7.4; H2O:MeOH = 99:1) at 0.5 ¯M.

complexes as sensors for thiol-containing amino acid exploiting the high affinity of biothiols for metal ions.11,12 Thus we examined the changes in the absorbance/fluorescence spectra of RO-Cu2+ solutions after the separate additions of various thiol-containing amino acids i.e. glutathione (GSH), homocysteine (Hcy), cysteine (Cys) as well as a few non-thiolcontaining amino acids such as phenylalanine (Phe), glutamic acid (Glu), glutamine (Gln), tryptophan (Trp), alanine (Ala), arginine (Arg), glycine (Gly), lysine (Lys), tyrosine (Tyr), and histidine (His). In the UVvisible spectrum, the absorption maximum of RO-Cu2+ centred at ca. 493 nm remains unaltered in the case of the above-mentioned thiols, except for Cys that caused a blue shift of 37 nm in the absorption maximum (ESI; Figure S15). This newly formed band at ca. 455 nm matched well with the absorbance characteristics of RO. The same indicated the removal of Cu2+ from the RO-Cu2+ ensemble as the result of unique binding ability of Cys with Cu2+. The emission spectrum of the RO also revived (turn “on”) upon treatment of RO-Cu2+ complex with Cys. The fluorescence titration profiles of RO-Cu2+ (0.5 ¯M) with concomitant addition of Cys (0100 equiv) showed a large fluorescence enhancement at ca. 528 nm (Figure 7). The other amino acids however remain inactive even at higher concentrations (Figure 8). In fact other similar and foremost interfering analytes including Hcy, GSH were not able to modulate either the UVvisible or the fluorescence spectrum of RO-Cu2+.


To analyze the binding behavior of RO-Cu2+ with Cys we carried out ESI-mass spectrometric studies (ESI; Figure S16). The same showed a peak at m/z 302.2 and 408.1 assignable for (2Cys-Cu2+) complex and sensor (RO) respectively. These results demonstrated that the selective and strong binding of Cys with that of Cu2+ set the sensor RO free from the RO-Cu2+ ensemble, whereupon the corresponding UVvisible and fluorescence spectral pattern of RO is almost recovered. The detection limit of RO-Cu2+ towards Cys was determined using the fluorescence titration data and found to be 4.23 © 10¹7 M with a linearity range of 3.0 © 10¹5 to 9.0 © 10¹5 M for Cys (ESI; Figure S17). Reversibility of RO towards Cu2+/Cys. The reversible “offon” switching of RO in the presence of Cu2+ and Cys was checked over successive cycles by the consecutive additions of Cu2+ followed by Cys. The quenching of the fluorescence emission of RO at ca. 520 nm upon addition of Cu2+ and regeneration of the same by adding Cys was successfully achieved up to several cycles (Figure 9). Thus the “onoffon” behavior of RO in the successive absence and presence of Cu2+ and of Cys proves its worth as a fluorescent sensor. Implementation of IMP Molecular Logic Gate. Taking advantage of “offon” behavior of RO in the presence of two input signals viz. Cu2+ (input A) and Cys (input B), we tried to translate the same in the form of molecular logic gate by monitoring the emission of RO at ca. 520 nm. The presence of the guest species was marked as “1” while their absence were taken as “0” input. As discussed above the addition of Cu2+ causes quenching in the fluorescence emission of RO while addition of Cys regenerates it. However, when Cys was added alone to the RO, no effect was observed on its fluorescence emission. Thus the above possible combination of inputs and outputs as shown in a truth table gives rise to an IMP logic gate (Figure 10). Cell Imaging Studies. We further explored the ability of the RO to monitor Cu2+ and Cys in living cells. For this purpose, fluorescence microscopic analysis was carried upon Bull. Chem. Soc. Jpn. 2016, 89, 754–761 | doi:10.1246/bcsj.20150427

Intensity, a.u. at 520 nm

Figure 8. Bar graph showing selectivity of ROCu2+ ensemble towards Cys and other amino acids.

Cu2+

Cys

Cu2+

Cys

Cu2+

Cys

Cu2+

Cys

Cu2+

Cys

Figure 9. Reversible and recyclable behavior of ROCu2+ upon the sequential addition of Cu2+ followed by Cys.

E. coli cells which were incubated with 10 ¯M of RO for 20 min. The green fluorescence was observed at ca. 520 nm. After washing the incubated cells with phosphate buffer for the removal of extra cellular probe, these cells were again incubated with LB media containing CuCl2¢2H2O (1.0 equiv) for 20 min at 37 °C and washed extensively with buffer before imaging (Figure 11). The presence of Cu2+ ion in this solution showed a complete quenching of the green fluorescence of RO. However, the addition of Cys after addition of Cu2+ to RO resulted in regeneration of the green fluorescence. Thus, ROCu2+ ensemble can also be used for the intracellular imaging of cysteine. Conclusion In summary, we have successfully extended the use of RO as a highly sensitive and biocompatible fluorescent probe for turn “offon” detection of Cu2+ and Cys in aqueous medium. We successfully demonstrated the structure of RO-Cu2+ complete-


Figure 10. IMP logic gate representation and its transformation with corresponding truth table.

Figure 11. Fluorescent imaging of Cu2+ and Cys. in E. coli cells with RO (upper row bright field image of the cells) and lower row fluorescence image of the cells.

ly through single crystal XRD studies and further fortified this through theoretical calculations at the DFT level. In addition, RO-Cu2+ ensemble was utilised as a sensitive turn “on” fluorescent probe for Cys over various similar type of analytes including homocysteine (Hcy) and glutathione (GSH) in aqueous solution under mild conditions. Finally these spectral observations of sensing events were translated into a molecular logic gate (IMP). Moreover, the bio-imaging of Cu2+ and Cys makes the present work further lucrative. KKU and VK are thankful to CSIR, New Delhi for financial assistance [01(2709)/13/EMR-II]. UD acknowledges UGCBSR for granting meritorious fellowship. Authors are thankful to Prof. S.C. Lakhotia, Department of Zoology, Faculty of Science, Banaras Hindu University for providing access to the fluorescence microscope. Supporting Information The various spectroscopic characterizations viz. NMR, IR, and ESI mass spectrum of RO has been shown in Figures S1, S2, and S3 respectively. Respective IR and ESI mass of ROCu2+ are given in Figures S8 and S9, whereas ESI-MS of ROCu2+ ensemble with Cys has been shown in Figure S16. Other optical observations and various calculations based on observed optical data are shown in Figures S3S7, S10S13, S15, and S17. Effect of pH on fluorescence intensity is given in 760 | Bull. Chem. Soc. Jpn. 2016, 89, 754–761 | doi:10.1246/bcsj.20150427

Figure S14. Details of selected bond angles, distances and other X-ray crystallography data and has been provided in Tables S1 and S2. Theoretical calculation for absorption maxima RO-Cu2+ in gaseous state is provided in Table S3. This material is available on http://dx.doi.org/10.1246/bcsj. 20150427. References 1 a) C. Núñez, S. M. Santos, E. Oliveira, H. M. Santos, J. L. Capelo, C. Lodeiro, ChemistryOpen 2014, 3, 190. b) T. Chen, W. Zhu, Y. Xu, S. Zhang, X. Zhang, X. Qian, Dalton Trans. 2010, 39, 1316. c) U. Pischel, Angew. Chem., Int. Ed. 2007, 46, 4026. 2 a) J. A. Cowan, Inorganic Biochemistry: An Introduction, Wiley-VCH, New York, NY, 1997, 133. b) J. J. R. F. d. Silva, R. J. P. Williams, The Biological Chemistry of Elements: The Inorganic Chemistry of Life, Clarendon, Oxford, 1993, 388. c) S. A. Lee, J. J. Lee, J. W. Shin, K. S. Min, C. Kim, Dyes Pigm. 2015, 116, 131. 3 a) P. Venkatesan, S.-P. Wu, RSC Adv. 2015, 5, 42591. b) S.-P. Wu, T.-H. Wang, S.-R. Liu, Tetrahedron 2010, 66, 9655. 4 a) D. J. Waggoner, T. B. Bartnikas, J. D. Gitlin, Neurobiol. Dis. 1999, 6, 221. b) C. Vulpe, B. Levinson, S. Whitney, S. Packman, J. Gitschier, Nat. Genet. 1993, 3, 7. c) U. Diwan, A. Kumar, V. Kumar, K. K. Upadhyay, P. K. Roychowdhury, Sens. Actuators, B 2014, 196, 345. 5 a) N. R. Chereddy, P. S. Korrapati, S. Thennarasu, A. B.


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