Bioorganic & Medicinal Chemistry 21 (2013) 7964–7970
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A new peptidyl fluorescent chemosensors for the selective detection of mercury ions based on tetrapeptide Ponnaboina Thirupathi, Keun-Hyeung Lee ⇑ Bioorganic Chemistry Laboratory, Department of Chemistry and Chemical Engineering, Inha University, 100 Inha-ro, Nam-Gu, Incheon 402-751, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 September 2013 Revised 23 September 2013 Accepted 24 September 2013 Available online 10 October 2013 Keywords: Peptidyl sensor Fluorescence Selective Hg(II) Chemosensor Peptide sensor
a b s t r a c t A novel peptidyl chemosensor (PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1) was synthesized by incorporation of two pyrene (Py) fluorophores into the tetrapeptide using sulfonamide group. Compound 1 exhibited selective fluorescence response towards Hg(II) over the other metal ions in aqueous buffered solutions. Furthermore, 1 with the potent binding affinity (Kd = 120 nM) for Hg(II) detected Hg(II) without interference of other metal ions such as Ag(I), Cu(II), Cd(II), and Pb(II). The binding mode of 1 with Hg(II) was investigated by UV absorbance spectroscopy, 1H NMR titration experiment, and pH titration experiment. The addition of Hg(II) induced a significant decrease in both excimer and monomer emissions of the pyrene fluorescence. Hg(II) interacted with the sulfonamide groups and the imidazole group of His in the peptidyl chemosensor and then two pyrene fluorophores were close to each other in the peptide. The decrease of both excimer and monomer emission was mainly due to the excimer/monomer emission change by dimerization of two pyrene fluorophores and a quenching effect of Hg(II). Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The design and synthesis of fluorescent chemosensors for the detection and quantification of low level contamination of heavy and transition metal (HTM) ions have received considerable attention because these types of metal ions are toxic for humans and other living organisms.1,2 Among the various HTM ions, mercury is most toxic and hazardous. The contamination of mercury occurs through many ways and it became a worldwide environmental problem.3 In general, the oxidized form of mercury, Hg(II) and methyl mercury enters the food chain through the contaminated water and accumulates in higher organisms.3c,4,5 Even accumulation of low concentration of mercury in human body causes a variety of diseases such as prenatal brain damage, serious cognitive, and motion disorders.6,7 Hence, it is highly recommended to develop selective and sensitive ways for Hg(II) in aqueous solutions. Among the various analytical detection techniques for Hg(II), fluorescence has received attention because of its inexpensive instrument, high sensitivity, convenient, rapid and accurate detection of small amounts of analytes in the sample.3c,8 Thus, in recent years, various types of fluorescent chemosensors for Hg(II) have been reported. In general, the fluorescent chemosensor consists of a ligand-binding site (receptor), responsible for recognizing analytes and a signal transduction site (fluorophore), converting the recognition events into fluorescent signals.9 Various type of
⇑ Corresponding author. E-mail address: [email protected] (K.-H. Lee). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.09.058
scaffolds such as calixarene,10a,b hydroxyquinolines,10c,d azines,10e azadiene,10f dioxaoctane-diamide,10g cyclams,10h and crown ethers,10i–l have been reported as a receptor part for the detection of Hg(II) in fluorescent chemosensors. However, most of them suffer from the cross-sensitivity or interference from other metal ions, for example, Cu(II), Ag(I), Cd(II) and Pb(II) due to the similar size or softness of these metal ions to Hg(II).10 Therefore, it is highly desirable to develop fluorescent chemosensors based on new scaffolds as a receptor for the selective and sensitive detection of heavy and transitional metal (HTM) ions in aqueous solutions. In recent years, the peptide scaffolds have been exploited as the basic molecular framework for fluorescent chemosensors in the construction of the binding site due to its structural diversity, good solubility in water, and biological and environmental compatibility.11,12 We and other research groups have successfully demonstrated that the fluorescent chemosensors based on the peptide scaffolds showed sensitive responses to heavy metal ions in aqueous solutions.11,12 However, almost all peptidyl chemosensors except the peptide sensors based on copper-binding motif originating from the amino terminal Cu and Ni binding (ATCUN) site showed cross-sensitivity to other heavy metal ions or suffered from the interference of other heavy metal ions.12 Alternatively, the fluorescent peptidyl chemosensors based on copper-binding motif and similar sequences of ATCUN site such as GGH, GHK, GGHG, and HGGG showed highly selective response only to Cu(II).11a,c–e,h Thus, it is highly challenging and important to synthesize a highly selective peptidyl chemosensor for a specific metal ion except Cu(II) and to investigate the binding mode of the peptidyl chemosensor for the specific metal ion.
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Considering the binding mode of the reported chemosensors based on amino acids and peptides,12,13 we designed and synthesized a new fluorescent chemosensor based on the tetrapeptide scaffold and investigated the fluorescent response to metal ions. Two pyrene fluorophores were incorporated into the tetrapeptide (PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1) using sulfonamide group because the pyrene fluorophore has an interesting photophysical properties such as high fluorescence quantum yield, chemical stability, and dual fluorescence emissions (monomer and excimer)14 and the sulfonamide group in several chemosensors based on amino acids acted as a ligand for specific metal ions.13a–g A His residue was included in the peptidyl chemosensor because His as a metal chelating amino acid played a critical role in the metal recognition in several metalloenzymes.12a,d,e,15 As shown in Scheme 2, we assumed that when the peptidyl chemosensor might interact with metal ions, it might fold and His and two pyrene fluorophores with sulfonamide group might be closer or far to each other, resulting in the change of the pyrene monomer as well as the excimer emissions. Gly residue was introduced in the sequence of the peptidyl chemosensor 1 for increasing flexibility of the peptide between two pyrene flourophores.12c Peptidyl chemosensor 1 exhibited a selective and sensitive response to Hg(II) over the other metal ions in aqueous solution. Furthermore, we investigated the binding mode of 1 for Hg(II) by using 1H NMR titration, fluorescent titration, and pH titration experiment to understand the potent binding affinity for Hg(II) and to explain the decrease of monomer emission intensity as well as the decrease of excimer emission intensity. 2. Results and discussion Two pyrene labeled peptidyl chemosensor 1 was easily synthesized in solid phase synthesis with a high yield (89.4%) using Fmoc chemistry (Scheme 1).16 Details on synthesis and characterization of 1 are described in the Section 4 (Figs. S1–S7). We measured the fluorescent spectrum of 1 in aqueous solution (10 mM HEPES at pH 7.4) containing DMSO (Fig. 1). Interestingly, the fluorescence behavior of 1 was dependent on the amount of DMSO in aqueous solutions. The spectrum of 1 measured in aque-
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ous solution containing 0.1% (v/v) DMSO displays a strong excimer emission at 494 nm, originated from the p–p-stacking between two pyrene moieties, and weak monomer emissions at 381 and 400 nm. Upon increasing the amount of DMSO in aqueous solution (10 mM HEPES at pH 7.4), the decrease of excimer emission and the considerable increases of monomer emissions were observed, respectively. The higher amount of DMSO in aqueous solution (10 mM HEPES at pH 7.4) induced a strong monomer emission intensity and the weak excimer emission intensity. The p–p interactions between the two pyrene moieties of the peptide sensor may depend on the hydrophobicity of solvent system. Small amounts of DMSO in aqueous solutions provided more hydrophilic environments for the peptide and the p–p interactions between the two pyrene moieties increased. Thus, two pyrene moieties of the peptide sensor 1 might come closer to each other due to strong p–p stacking between the two pyrene moieties, which leaded to the strong excimer emission at 494 nm. Large amounts of DMSO in aqueous solutions provided more hydrophobic environments and the p–p interactions between the two pyrene moieties relatively decreased. As the decrease of the p–p stacking interactions between the two pyrene moiety, the decrease of excimer emission and the increase of monomer emission intensity were observed. Among the various solvent conditions, aqueous solution containing 30% DMSO (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4) was chosen for further studies because the peptide sensor 1 showed a significant changes in fluorescence emission intensities in the presence of Hg(II) in this solvent condition (Fig. 1). As shown in Figure 2a, peptidyl chemosensor 1 displayed both typical monomer emission and excimer emission. Upon addition of Hg(II), the intensities of monomer and excimer emissions significantly decreased whereas both monomer and excimer emissions of 1 were not changed upon addition of other tested metal ions including Na(I), K(I), Mg(II), and Al(III) as chloride anion and Ca(II), Co(II), Cr(III), Fe(III), Mn(II), Ni(II), Pb(II) and Zn(II) as perchlorate anion. Figure 2b shows the gradual emission intensity change of peptidyl chemosensor 1 upon addition of Hg(II). The intensities of monomer emission at 381 and 400 nm and excimer emission at 494 nm decreased significantly. The monomer emission
Scheme 1. Solid phase synthesis of 1.
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Scheme 2. A proposed binding mode of peptidyl chemosensor 1 with Hg(II).
Figure 1. Fluorescence emission spectra of 1 (2 lM) (—) with 2 equiv Hg(II) (———) in aqueous solution, (a) 99.9:0.1, (b) 9:1, (c) 8:2, (d) 7:3 (e) 6:4 and (f) 5:5 (H2O/DMSO, v/v, 10 mM HEPES at pH 7.4) kex = 352 nm and slit = 15/3.
Figure 2. (a) Fluorescence response of 1 (2 lM) in the presence of various metal ions (2 equiv, except Na(I), K(I), Mg(II), and Mg(II) which were used 100 equiv) and (b) fluorescence changes of 1 (2 lM) in the presence of various concentrations of Hg(II) (0, 0.2, 0.4, 0.6, 0.8, . . . and 2.6 equiv) in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4; kex = 352 nm, slit 15/3).
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intensity of 1 (2 lM) as a function of Hg(II) concentration was plotted in Figure 2b. A complete change in the emission intensity required about 2.0 equiv of Hg(II). We investigated the binding stoichiometry of the complex between the chemosensor 1 and Hg(II) using Job’s plot analysis. Job’s plots that displayed a maximum at 0.5 mol fraction for Hg(II) indicates that peptidyl chemosensor 1 may form a 1:1 complex with Hg(II) (Fig. 3a). Assuming the formation of a 1:1 complex, the dissociation constant of 1 for Hg(II) was determined as 120 nM (R2 = 0.98) (Fig. 3b).17 This value indicates that 1 has a potent binding affinity for Hg(II) in an aqueous buffered solution. The sensitivity of 1 for Hg(II) was determined based on the linear relationships between the monomer emission intensity at 381 nm and the concentration of Hg(II). The detection limit of 1 was calculated as 31.2 nM (R2 = 0.99) by using 3r/m, where r is the standard deviation of the blank measurements, and m is the slope (sensitivity) of the intensity versus sample concentration in the plot (Fig. S8). This confirms that 1 can be used to detect qualitatively small amounts of Hg(II) in aqueous solution. To investigate the interference effect of other metal ions on the detection ability of 1, we measured the fluorescence response of 1 to Hg(II) in the presence of other metal ions (Fig. 4). The fluorescence response of 1 to Hg(II) was not changed in the presence of other metal ions (1 equiv) such as Zn(II), Fe(III), Cd(II), Pb(II), Ag(I), Al(III), Cr(III), Cu(I), Cu(II), Mg(II), and Na(I). In general, almost all chemosensors for Hg(II) suffered from the crosssensitivity of other heavy and transition metal ions such as Cu(II), Ag(I), Cd(II), and Pb(II).10,12f As shown in Figure 5, the visible emission change of 1 (2 lM) in the presence of various metal ions (1 equiv) was monitored under UV light (kem = 365 nm) using UV lamp. Peptidyl sensor 1 displayed a pale green color in the presence or absence of the tested metal ions except Hg(II), whereas 1 displayed a pale blue color in the presence of Hg(II). Peptidyl sensor 1 may provide a simple and easy detection way for Hg(II) using UV lamp. The pH influence on the fluorescence response of peptidyl sensor 1 to Hg(II) was examined at different pH to investigate the role of the functional groups of 1 for the binding of Hg(II) (Fig. S9). As pH increased above 9.5, the intensity of the monomer emission of free 1 slightly decreased and the excimer emission intensity of free 1 considerably decreased. This may be due to the deprotonation of the sulfonamide group (pKa 10)12d,13a,c,f,g because the deprotonated sulfonamide groups in this pH range may act as a quencher for the pyrene fluorophores. The considerable decrease of the excimer emission intensity can be explained by quenching
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effect of both deprotonated sulfonamide groups on the pyrene fluorophores. Interestingly, the emission intensity of 1 in the presence of Hg(II) showed a dependence on pH. In acidic pH (3.5–5.5), 1 showed no fluorescence response to Hg(II) due to the protonation of the imidazole group (pKa 6) of the His residue. In the pH range of 6.5–7.5, the peptidyl sensor began to show a fluorescence response to Hg(II) and the change of emission intensity of 1 to Hg(II) increased as pH increased, which strongly supports the critical role of the His residue for the recognition of Hg(II). The pH titration experiment reveals that 1 is suitable for monitoring Hg(II) in the wide range of pH values (6.5–11.5) and the His residue plays a vital role for the recognition of Hg(II) in the neutral and basic pH. 1 H NMR titration experiment provided additional information for the detailed binding mode of peptidyl sensor 1 with Hg(II). 1 H NMR experiments were carried out in D2O/DMSO-d6 (7:3, v/v) and the pH value of the sample solution was adjusted to seven with 1% NaOD because peptidyl chemosensor 1 showed fluorescence response to Hg(II) in neutral and basic pH. When 1 equiv of Hg(ClO4)2 was added, the downfield shifts of H-26 (D0.06 ppm) and H-24 (D0.04 ppm) corresponding to imidazole protons were observed, respectively (Fig. 6). The shifts was attributed to the co-ordination between Hg(II) and the imidazole moiety. Similarly, the aromatic protons of the pyrene fluorophores such as H-8 (D0.08 ppm), H-28 (D0.09 ppm), H-9 (D0.06 ppm), and H-29 (D0.09 ppm) were also downfield shifted in the presence of Hg(II). The shifts of the pyrene group can be explained by the chelation of Hg(II) with the sulfonamide group of the pyrene moiety because the binding modes of the previously reported chemosensors for Hg(II) based on dansyl labeled amino acids revealed that the NH of sulfonamide group of the dansyl moiety interacted with Hg(II) and the downfield shifts of napthyl protons of the dansyl fluorophore were explained by the chelation of Hg(II) with the sulfonamide moiety.13c,e–g Even though the amide proton peaks of 1 was not observed due to D2O in solvent system, the binding modes of the previously reported chemosensors based on amino acids and peptides suggest the possible interactions between the amide groups of 1 with Hg(II).12d,13a–c,e The binding mode of 1 with Hg(II) and the reason for the decrease of pyrene excimer and monomer emissions in the presence of Hg(II) were proposed using UV and fluorescent spectroscopy experiment, pH titration experiment, and 1H NMR titration experiment. The Job’s plot analysis suggested a 1:1 complex between 1 and Hg(II). As shown in Figure S10, the UV–visible absorption spectrum of peptidyl sensor 1 with gradual additions of Hg(II) exhibited a hypochromic shift with a significant decrease of the absorbance
Figure 3. (a) A Job’s plot analysis for 1 with Hg(II). (b) Nonlinear fitting of the fluorescence intensity change at 381 nm versus concentration of Hg(II) (0.0–4.0 lM in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4; kex = 352 nm, slit 15/3).
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Figure 4. Emission intensity of 1 (2 lM) in the presence of Hg(II) ions (1 equiv) and other metal ions (1 equiv) in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4, kex = 352 nm).
Figure 5. Visible emission color changes of 1 (2 lM) upon addition of 1 equiv of metal ions under UV light (k = 365 nm) in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4).
Figure 6. Partial 1H NMR spectra (400 MHz) of 1 (6.4 mM) in the (a) absence and (b) presence of 1 equiv of Hg(ClO4)2 in D2O/DMSO-d6 (7:3, v/v, pH 7).
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band at 352 nm. This suggests the formation of pyrene dimer in the ground state in the presence of Hg(II).12a,d,13a Considering the overall results, we could propose the binding mode of 1 with Hg(II), as shown in Scheme 2. In the absence of Hg(II), two major conformations of 1 were possible; two pyrene fluorophores were far away in the one major conformer, which exhibited a strong monomer emission intensity, whereas two pyrene fluorophores were close to each other in the other major conformer, which exhibited a considerable excimer emission intensity and a weak monomer emission intensity. In the presence of Hg(II), two sulfonamide groups of 1 interacted with Hg(II) and then the distance between the two pyrenes became shorter because of –SO2NH–Hg(II)–NHSO2– binding. The significant decrease of the absorbance band at 352 nm in UV– visible absorption spectrum also indicated the formation of pyrene dimer in the presence of Hg(II). Thus, the intensity of monomer emission should decrease and the intensity of excimer emission should increase by intra-dimerization of the two pyrene fluorophores. However, as the distance between Hg(II) and the pyrene was short enough to quench the pyrene fluorophores, Hg(II) bound in the peptide quenched the excimer emission. Thus, the decrease of both excimer and monomer emissions of 1 in the presence of Hg(II) could be explained by dimerization of two pyrene fluorophores and subsequently by quenching of Hg(II). In recent years, several research groups have reported that the decrease of monomer or/and excimer emission of pyrene fluorophore in the presence of Hg(II) was explained by quenching effect of Hg(II).18 The similar decrease of monomer emission as well as excimer emission of Calix[4]crown fluoroionophore containing two pyrene fluorophores in the presence of Cu(II) was reported and the decreasing of the excimer emission was explained by quenching of Cu(II).19 The pH titration experiment and 1H NMR titration experiment revealed that the His residue of the peptide sensor was essential for the recognition of Hg(II) because the peptide sensor did not show fluorescence response to Hg(II) in acidic pH 3.5–5.5. In general, as we mentioned in introduction section, the fluorescent peptidyl chemosensors based on copper-binding sequences such as GGH, GHK, GGHG, and HGGG showed highly selective responses only to Cu(II). However, peptidyl chemosensor 1 based on HGGK employed in this study showed highly selective response to Hg(II) without interference of Cu(II). It is very interesting property because the other peptide sensor containing His residue showed cross-selectivity for Cu(II) or interference of Cu(II). 3. Conclusions In summary, we synthesized a novel peptidyl chemosensor (PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1) for Hg(II) with a high yield using solid phase synthesis. The peptidyl chemosensor showed highly selective and sensitive fluorescence response to Hg(II) ion among tested metal ions in aqueous solutions. Peptidyl chemosensor 1 with the potent binding affinity (Kd = 120 nM) for Hg(II) detected Hg(II) without interference of other metal ions. The imidazole group and two sulfonamide group of 1 play a vital role in the co-ordination of Hg(II). The decrease of both excimer and monomer emissions of the pyrene fluorescence of 1 in the presence of Hg(II) was due to the excimer/monomer emission change by dimerization of two pyrene fluorophores and the quenching effect of Hg(II). 4. Experimental 4.1. Reagents Fmoc-Lys(Alloc)-OH, Fmoc-Gly-OH, Fmoc-His(Trit)-OH, N,Ndiisopropylcarboiimide (DIC), 1-hydroxybenzotriazole (HOBt),
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and rink amide MBHA resin were purchased from Advanced ChemTech. Other reagents for solid phase synthesis including trifluroacetic acid (TFA), triethylamine, N,N-dimethylformamide (DMF), triisopropylsilane (TIS), piperidine, phenylsilane and Pd(PPh3)4 were purchased from Aldrich. 1-Pyrenesulfonyl chloride was synthesized from 1-pyrenesulfonic acid (purchased from Aldrich). 4.1.1. Solid phase synthesis: general experimental procedure PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1 was efficiently synthesized in solid-phase synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (Scheme 1).16 Diisopropylcarbodiimide (DIC) and 1-hydroxylbenzotriazole (HOBt) in situ activation method was used for the coupling reactions. The amino acid, Fmoc-Lys(Alloc)-OH with Fmoc as protecting group (0.3 mmol, 0.3 equiv) was loaded to rink amide MBHA resin were (0.1 mmol, 0.1 equiv) according to the reported procedure. After deprotection of Fmoc group (with 20% piperidine in N,N-dimethylformamide (DMF)) of resin bound Lys, Fmoc-L-Gly-OH (0.3 mmol, 0.3 equiv), Fmoc-LGly-OH (0.3 mmol, 0.3 equiv), Fmoc-L-His(Trt)-OH (0.3 mmol, 0.3 equiv) were coupled sequentially (Scheme 1). After washing, drying, and deprotecting the Fmoc group with 20% piperidine in N,N-dimethylformamide (DMF) of tetrapeptide (H2N-His-Gly-GlyLys(alloc)-resin). The 1-pyrenesulfonyl chloride (0.3 mmol, 0.3 equiv) was coupled in the presence of triethylamine (0.6 mmol, 0.6 equiv). Then, the deprotection of alloc group on lysine side chain and conjugation of another 1-pyrenesulfonyl chloride were achieved with help of phenylsilane (0.3 mmol, 0.3 equiv) and Pd(PPh3)4 (0.02 mmol, 0.02 equiv) in dichloromethane and 1-pyrenesulfonyl chloride (0.3 mmol, 0.3 equiv) and triethylamine (0.6 mmol, 0.6 equiv), respectively. Finally, the cleavage of 1 from the resin was accomplished with CF3COOH/TIS/H2O (TFA/TIS/ water, 95:2.5:2.5, v/v) at room temperature for 3 h. Following vacuum filtration and removal of TFA with N2 blow-off, crude product was precipitated from cold ether. The solid precipitate was centrifuged, washed with ether, and lyophilized under vacuum. The crude product was further purified with semi preparative HPLC using water (0.1% TFA)/acetonitrile (0.1% TFA) gradient. The retention time of 1 is 62 min, respectively. The 89.4% of 1 was isolated and characterized by IR, 1H and 13C NMR and ESI-mass data. The characterization data of 1 are given below. Compound 1: Pale yellow solid, mp 162–163 °C; IR (KBr): 3430, 2926, 1670, 1314, 1198, 1126 cm1; 1H NMR (400 MHz, DMSO6) d 8.96 (d, J = 8.6 Hz, 1H), 8.85 (d, J = 8.6 Hz, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.55 (d, J = 8.4 Hz, 1H), 8.53 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 8.47–8.43 (m, 4H), 8.39 (d, J = 8.4 Hz, 1H), 8.40–8.36 (m, 2H), 8.35–8.33 (m, 2H), 8.27–8.23 (m, 2H), 8.19 (t, J = 8.4 Hz, 2H), 8.16 (m, 2H), 7.99 (t, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.24 (br s, 1H), 7.14 (s, 1H), 6.96 (br s, 1H), 4.24–4.20 (m, 1H), 4.12–4.06 (m, 1H), 3.64–618 (m, 4H), 3.41 (dd, J = 7.6, 2.0 Hz, 1H), 3.26 (dd, J = 7.6, 2.0 Hz, 1H), 2.85 (dd, J = 7.4, 2.0 Hz, 1H), 2.84–2.78 (m, 1H), 2.77–2.70 (m, 2H), 1.52–1.43 (m, 1H), 1.26–1.22 (m, 2H), 1.20–1.16 (m, 1H); 13C NMR (100 MHz, DMSO6) d 173.4, 169.4, 168.6, 168.2, 134.0, 133.8, 132.5, 132.1, 131.6, 130.5, 130.0, 129.9, 129.7, 129.6, 129.4, 129.3, 127.1, 127.0, 126.9, 126.8, 126.6, 124.3, 124.2, 123.9, 123.4, 123.2, 123.0, 117.1, 107.8, 55.7, 52.2, 42.1, 42.0, 41.9, 31.8, 28.9, 26.0, 22.6; ESI-mass (m/z): [M+H]+ calculated for C48H45N8O8S2: 925.27, observed: 925.42. 4.1.2. General fluorescence measurements A stock solution of 1 with the concentration of 1.653 103 M was prepared in DMSO and stored in a cold and dark place. This stock solution was used for all spectrofluoremetric experiments after appropriate dilution. The fluorescence experiments were carried out using the above referred solution after maintaining the pH of the solution to 7.4 using 10 mM HEPES buffer solution. Fluorescence emission spectrum of a sample in a 10 mm path length
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quartz cuvette was measured in 10 mM HEPES buffer solution at pH 7.4 using a Perkin Elmer luminescence spectrophotometer (model LS 55). Emission spectra (370–600 nm) of 1 in the presence of several metal ions (Na(I), K(I), Mg(II), and Al(III) as chloride anion and Ca(II), Co(II), Cr(III), Cu(II), Fe(III), Mn(II), Ni(II), Pb(II), and Zn(II) as perchlorate anion) were measured by excitation with 352 nm The slit size for excitation and emission were 15 and 3.0 nm, respectively. The concentration of 1 was confirmed by UV absorbance at 342 nm for pyrene group. The molar extension coefficient (e) of 1 is 16,000 cm1 M1. 4.1.3. Determination of dissociation constant The dissociation constant for 1:1 complex was calculated based on the titration curve of the 1 with with Hg(II). Dissociation constants was determined by a nonlinear least squares fitting of the data with the following equation as referenced elsewhere.17 The fluorescence signal, F, is related to the equilibrium concentration of the complex (HL) between Host (H) and metal ion (L) by the following expression:
F ¼ F 0 þ DF ½HL 1=2
½HL ¼ 0:5 ½K D þ LT þ HT fðK D LT HT Þ2 4LT HT g
where F0 is the fluorescence of the probe only and DF is the change in fluorescence due to the formation of HL. Acknowledgments This work was supported by a Grant (2012R1AB3000574) from the Basic Research Program of the National Research Foundation and a grant (201200054 0013) from the Korea Environmental Industry and Technology Institute and Dr. P.T. also acknowledges to the Department of Chemistry and Chemical Engineering, Inha University, Incheon, Republic of Korea. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2013.09.058. References and notes 1. (a) Amendola, V.; Fabbrizzi, L.; Forti, F.; Licchelli, M.; Mangano, C.; Pallavicini, P.; Poggi, A.; Sacchi, D.; Taglieti, A. Coord. Chem. Rev. 2006, 250, 273; (b) Valeur, B.; Leray, I. Coord. Chem. Rev. 2000, 205, 3; (c) DeSilva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515. 2. DeSilva, A. P.; Fox, D. B.; Huxley, A. J. M.; Moody, T. S. Coord. Chem. Rev. 2000, 205, 41. 3. (a) Benoit, J. M.; Fitzgerald, W. F.; Damman, A. W. Environ. Res. 1998, 78, 118; (b) Renzoni, A.; Zino, F.; Franchi, E. Environ. Res. 1998, 77, 68; (c) Malm, O. Environ. Res. 1998, 77, 73; (d) Mercury Update: Impact on Fish Advisories; EPA Fact Sheet EPA-823-F-01-001; Environmental Protection Agency, Office of Water: Washington, DC, 2001, p 1. 4. (a) Boening, D. W. Chemosphere 2000, 40, 1335; (b) Harris, H. H.; Pickering, I. J.; George, G. N. Science 2003, 301, 1203.
5. (a) Scoullos, G. H.; Vonkeman, M. J.; Thorton, L.; Makuch, Z. In Mercury, Cadmium, and Lead: Handbook for Sustainable Heavy Metals Policy and Regulation (Environment & Policy); Kluwer Academic: Norwell, MA, 2001; Vol. 31,; (b) Richardson, S D.; Temes, T. A. Anal. Chem. 2005, 77, 3807. 6. Grandjean, P.; Weihe, P.; White, R. F.; Debes, F. Environ. Res. 1998, 77, 165. 7. Harada, M. Crit. Rev. Toxicol. 1995, 25, 1. 8. (a) Krata, A.; Jedral, W.; Bulska, E. Spectrochim. Acta, Part B 2007, 62, 269; (b) Ashrafi, A. M.; Vytras, K. Talanta 2011, 85, 2700; (c) Zhu, X.; Alexandratos, S. D. Microchem. J. 2007, 86, 37; (d) Clevenger, W. L.; Simth, B. W.; Winefordner, J. D. Crit. Rev. Anal. Chem. 1997, 27, 1; (e) McShane, W. J.; Pappas, R. S.; McElprang, V. W.; Paschal, D. Spectrochim. Acta, Part B 2008, 63, 638. 9. (a) Hong, S. H.; Maret, W. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 2255; (b) Shults, M. D.; Pearce, D. A.; Imperiali, B. J. Am. Chem. Soc. 2003, 125, 10591; (c) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1996, 118, 3053; (d) Godwin, H. A.; Berg, J. M. J. Am. Chem. Soc. 1996, 118, 6514. 10. (a) Metivier, R.; Leray, I.; Valeur, B. Chem. Eur. J. 2004, 10, 4480; (b) Lee, Y. H.; Lee, M. H.; Zhang, J. F.; Kim, J. S. J. Org. Chem. 2010, 75, 7159; (c) Moon, S. Y.; Cha, N. R.; Kim, Y. H.; Chang, S. K. J. Org. Chem. 2004, 69, 181; (d) Zhang, H.; Han, L. F.; Zachariasse, K. A.; Jiang, Y. B. Org. Lett. 2005, 7, 4217; (e) Martinez, R.; Espinosa, A.; Tarraga, A.; Molina, P. Org. Lett. 2005, 7, 5869; (f) Zhou, Y.; Zhu, C.Y.; Gao, X.-S.; You, X.-Y.; Yao, C. Org. Lett. 2010, 12, 2566; (g) Kim, J. S.; Choi, M. G.; Song, K. C.; No, K. T.; Ahn, S.; Chang, S.-K. Org. Lett. 2007, 9, 1129; (h) Mahato, P.; Ghosh, A.; Saha, S.; Mishra, S.; Mishra, S. K.; Das, A. Inorg. Chem. 2010, 49, 11485; (i) Chen, T.; Zhu, W.; Xu, Y.; Zhang, S.; Zhang, X.; Qian, X. Dalton Trans. 2010, 39, 1316; (j) Tian, M.; Ihmels, H. Chem. Commun. 2009, 3175; (k) Lee, H. N.; Kim, H. N.; Swamy, K. M. K.; Park, M. S.; Kim, J.; Lee, H.; Lee, K. H.; Park, S.; Yoon, J. Tetrahedron Lett. 2008, 49, 1261; (l) Kim, H. N.; Ren, W. X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2012, 41, 3210. 11. (a) Torrado, A.; Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1998, 120, 609; (b) Deo, S.; Godwin, A. J. Am. Chem. Soc. 2000, 122, 174; (c) Zheng, Y.; Huo, Q.; Kele, P.; Andreopoulos, F. M.; Pham, S. M.; Leblanc, R. M. Org. Lett. 2001, 3, 3277; (d) Zheng, Y.; Gattas-Asfura, K. M.; Veeranjaneyulu, K.; Leblanc, R. M. Chem. Commun. 2002, 2350; (e) Zheng, Y.; Cao, X.; Orbulescu, J.; Veeranjaneyulu, K.; Andreopoulos, F. M.; Pham, S. M.; Leblanc, R. M. Anal. Chem. 2003, 75, 1706; (f) Li, Y.; Li, L.; Pu, X.; Ma, G.; Wang, E.; Kong, J.; Liu, Z.; Liu, Y. Bioorg. Med. Chem. Lett. 2012, 22, 4014; (g) White, B. R.; Holcombe, J. A. Talanta 2007, 71, 2015; (h) Wu, S.-P.; Liu, S.-R. Sens. Actuators, B 2009, 141, 187. 12. (a) Neupane, L. N.; Thirupathi, P.; Jang, S.; Jang, M. J.; Kim, J. H.; Lee, K. H. Talanta 2011, 85, 1566; (b) Joshi, B. P.; Park, J.; Lee, W. I.; Lee, K. H. Talanta 2009, 78, 214; (c) Wang, B.; Li, H.-W.; Gao, Y.; Zhang, H.; Wu, Y. J. Fluoresc. 2011, 21, 1921; (d) Jang, S.; Thirupathi, P.; Neupane, L. N.; Seong, J.; Lee, H.; Lee, W. I.; Lee, K. H. Org. Lett. 2012, 14, 4746; (e) Kim, J. M.; Lohani, C. R.; Neupane, L. N.; Choi, Y.; Lee, K. H. Chem. Commun. 2012, 3012; (f) Neupane, L. N.; Park, J. Y.; Park, J.; Lee, K. H. Org. Lett. 2013, 15, 254. 13. (a) Yang, M. H.; Thirupathi, P.; Lee, K. H. Org. Lett. 2011, 13, 5028; (b) Yang, M. H.; Lohani, C. R.; Cho, H. J.; Lee, K. H. Org. Biomol. Chem. 2011, 9, 2350; (c) Lohani, C. R.; Kim, J. M.; Lee, K. H. Tetrahedron 2011, 67, 4130; (d) Joshi, B. P.; Lohani, C. R.; Lee, K. H. Org. Biomol. Chem. 2010, 8, 3220; (e) Neupane, L. N.; Kim, J. M.; Lohani, C. R.; Lee, K. H. J. Mater. Chem. 2012, 22, 4003; (f) Li, H.; Li, Y.; Dang, Y.; Ma, L.; Wu, Y.; Hou, G.; Wu, L. Chem. Commun. 2009, 4453; (g) Chen, L.; Yang, L.; Li, H.; Gao, Y.; Deng, D.; Wu, Y.; Ma, L. J. Inorg. Chem. 2011, 50, 10028; (h) Kwon, S. K.; Kim, H. N.; Rho, J. H.; Swamy, K. M. K.; Shanthakumar, S. M.; Yoon, J. Bull. Korean Chem. Soc. 2009, 30, 719. 14. (a) Winnik, F. M. Chem. Rev. 1993, 93, 587; (b) Yao, C.; Kraatz, H.-B.; Steer, R. P. Photochem. Photobiol. Sci. 2005, 4, 191; (c) Birks, J. B. Rep. Prog. Phys. 1975, 38, 903. 15. Raina, J. Y. S.; Cram, E. D.; Czolij, R.; Matthews, J. M.; Crossley, M.; Mackay, J. P. J. Biol. Chem. 2003, 278, 28011. 16. (a) Wang, S. S. J. Org. Chem. 1975, 40, 1235; (b) Wang, S. S. J. Am. Chem. Soc. 1973, 95, 1328; (c) Fields, G. B. Methods in Enzymology; Academic Press: New York, 1997. p 289; (d) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161. 17. Reddi, A. R.; Guzman, T. R.; Breece, R. M.; Tierney, D. L.; Gibney, B. R. J. Am. Chem. Soc. 2007, 129, 12815. 18. (a) Xiaopeng, H.; Juan, X.; Guorong, C.; Kaixian, C. Chin. J. Chem. 2012, 30, 2874; (b) Zheng, J.; Nie, Y.; Hu, Y.; Li, J.; Li, Y.; Jiang, Y. R. Yang Chem. Commun. 2013, 6915; (c) Banerjee, A.; Karak, D.; Sahana, A.; Guha, S.; Lohar, S.; Das, D. J. Hazard. Mater. 2011, 186, 738. 19. Choi, J. K.; Juyoung Yoon, J.; Lee, K. H.; Bartsch, R. A.; Kim, J. S. J. Org. Chem. 2006, 71, 8011.
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
A new peptidyl fluorescent chemosensors for the selective detection of mercury ions based on tetrapeptide Ponnaboina Thirupathi, Keun-Hyeung Lee ⇑ Bioorganic Chemistry Laboratory, Department of Chemistry and Chemical Engineering, Inha University, 100 Inha-ro, Nam-Gu, Incheon 402-751, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 September 2013 Revised 23 September 2013 Accepted 24 September 2013 Available online 10 October 2013 Keywords: Peptidyl sensor Fluorescence Selective Hg(II) Chemosensor Peptide sensor
a b s t r a c t A novel peptidyl chemosensor (PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1) was synthesized by incorporation of two pyrene (Py) fluorophores into the tetrapeptide using sulfonamide group. Compound 1 exhibited selective fluorescence response towards Hg(II) over the other metal ions in aqueous buffered solutions. Furthermore, 1 with the potent binding affinity (Kd = 120 nM) for Hg(II) detected Hg(II) without interference of other metal ions such as Ag(I), Cu(II), Cd(II), and Pb(II). The binding mode of 1 with Hg(II) was investigated by UV absorbance spectroscopy, 1H NMR titration experiment, and pH titration experiment. The addition of Hg(II) induced a significant decrease in both excimer and monomer emissions of the pyrene fluorescence. Hg(II) interacted with the sulfonamide groups and the imidazole group of His in the peptidyl chemosensor and then two pyrene fluorophores were close to each other in the peptide. The decrease of both excimer and monomer emission was mainly due to the excimer/monomer emission change by dimerization of two pyrene fluorophores and a quenching effect of Hg(II). Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The design and synthesis of fluorescent chemosensors for the detection and quantification of low level contamination of heavy and transition metal (HTM) ions have received considerable attention because these types of metal ions are toxic for humans and other living organisms.1,2 Among the various HTM ions, mercury is most toxic and hazardous. The contamination of mercury occurs through many ways and it became a worldwide environmental problem.3 In general, the oxidized form of mercury, Hg(II) and methyl mercury enters the food chain through the contaminated water and accumulates in higher organisms.3c,4,5 Even accumulation of low concentration of mercury in human body causes a variety of diseases such as prenatal brain damage, serious cognitive, and motion disorders.6,7 Hence, it is highly recommended to develop selective and sensitive ways for Hg(II) in aqueous solutions. Among the various analytical detection techniques for Hg(II), fluorescence has received attention because of its inexpensive instrument, high sensitivity, convenient, rapid and accurate detection of small amounts of analytes in the sample.3c,8 Thus, in recent years, various types of fluorescent chemosensors for Hg(II) have been reported. In general, the fluorescent chemosensor consists of a ligand-binding site (receptor), responsible for recognizing analytes and a signal transduction site (fluorophore), converting the recognition events into fluorescent signals.9 Various type of
⇑ Corresponding author. E-mail address: [email protected] (K.-H. Lee). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.09.058
scaffolds such as calixarene,10a,b hydroxyquinolines,10c,d azines,10e azadiene,10f dioxaoctane-diamide,10g cyclams,10h and crown ethers,10i–l have been reported as a receptor part for the detection of Hg(II) in fluorescent chemosensors. However, most of them suffer from the cross-sensitivity or interference from other metal ions, for example, Cu(II), Ag(I), Cd(II) and Pb(II) due to the similar size or softness of these metal ions to Hg(II).10 Therefore, it is highly desirable to develop fluorescent chemosensors based on new scaffolds as a receptor for the selective and sensitive detection of heavy and transitional metal (HTM) ions in aqueous solutions. In recent years, the peptide scaffolds have been exploited as the basic molecular framework for fluorescent chemosensors in the construction of the binding site due to its structural diversity, good solubility in water, and biological and environmental compatibility.11,12 We and other research groups have successfully demonstrated that the fluorescent chemosensors based on the peptide scaffolds showed sensitive responses to heavy metal ions in aqueous solutions.11,12 However, almost all peptidyl chemosensors except the peptide sensors based on copper-binding motif originating from the amino terminal Cu and Ni binding (ATCUN) site showed cross-sensitivity to other heavy metal ions or suffered from the interference of other heavy metal ions.12 Alternatively, the fluorescent peptidyl chemosensors based on copper-binding motif and similar sequences of ATCUN site such as GGH, GHK, GGHG, and HGGG showed highly selective response only to Cu(II).11a,c–e,h Thus, it is highly challenging and important to synthesize a highly selective peptidyl chemosensor for a specific metal ion except Cu(II) and to investigate the binding mode of the peptidyl chemosensor for the specific metal ion.
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Considering the binding mode of the reported chemosensors based on amino acids and peptides,12,13 we designed and synthesized a new fluorescent chemosensor based on the tetrapeptide scaffold and investigated the fluorescent response to metal ions. Two pyrene fluorophores were incorporated into the tetrapeptide (PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1) using sulfonamide group because the pyrene fluorophore has an interesting photophysical properties such as high fluorescence quantum yield, chemical stability, and dual fluorescence emissions (monomer and excimer)14 and the sulfonamide group in several chemosensors based on amino acids acted as a ligand for specific metal ions.13a–g A His residue was included in the peptidyl chemosensor because His as a metal chelating amino acid played a critical role in the metal recognition in several metalloenzymes.12a,d,e,15 As shown in Scheme 2, we assumed that when the peptidyl chemosensor might interact with metal ions, it might fold and His and two pyrene fluorophores with sulfonamide group might be closer or far to each other, resulting in the change of the pyrene monomer as well as the excimer emissions. Gly residue was introduced in the sequence of the peptidyl chemosensor 1 for increasing flexibility of the peptide between two pyrene flourophores.12c Peptidyl chemosensor 1 exhibited a selective and sensitive response to Hg(II) over the other metal ions in aqueous solution. Furthermore, we investigated the binding mode of 1 for Hg(II) by using 1H NMR titration, fluorescent titration, and pH titration experiment to understand the potent binding affinity for Hg(II) and to explain the decrease of monomer emission intensity as well as the decrease of excimer emission intensity. 2. Results and discussion Two pyrene labeled peptidyl chemosensor 1 was easily synthesized in solid phase synthesis with a high yield (89.4%) using Fmoc chemistry (Scheme 1).16 Details on synthesis and characterization of 1 are described in the Section 4 (Figs. S1–S7). We measured the fluorescent spectrum of 1 in aqueous solution (10 mM HEPES at pH 7.4) containing DMSO (Fig. 1). Interestingly, the fluorescence behavior of 1 was dependent on the amount of DMSO in aqueous solutions. The spectrum of 1 measured in aque-
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ous solution containing 0.1% (v/v) DMSO displays a strong excimer emission at 494 nm, originated from the p–p-stacking between two pyrene moieties, and weak monomer emissions at 381 and 400 nm. Upon increasing the amount of DMSO in aqueous solution (10 mM HEPES at pH 7.4), the decrease of excimer emission and the considerable increases of monomer emissions were observed, respectively. The higher amount of DMSO in aqueous solution (10 mM HEPES at pH 7.4) induced a strong monomer emission intensity and the weak excimer emission intensity. The p–p interactions between the two pyrene moieties of the peptide sensor may depend on the hydrophobicity of solvent system. Small amounts of DMSO in aqueous solutions provided more hydrophilic environments for the peptide and the p–p interactions between the two pyrene moieties increased. Thus, two pyrene moieties of the peptide sensor 1 might come closer to each other due to strong p–p stacking between the two pyrene moieties, which leaded to the strong excimer emission at 494 nm. Large amounts of DMSO in aqueous solutions provided more hydrophobic environments and the p–p interactions between the two pyrene moieties relatively decreased. As the decrease of the p–p stacking interactions between the two pyrene moiety, the decrease of excimer emission and the increase of monomer emission intensity were observed. Among the various solvent conditions, aqueous solution containing 30% DMSO (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4) was chosen for further studies because the peptide sensor 1 showed a significant changes in fluorescence emission intensities in the presence of Hg(II) in this solvent condition (Fig. 1). As shown in Figure 2a, peptidyl chemosensor 1 displayed both typical monomer emission and excimer emission. Upon addition of Hg(II), the intensities of monomer and excimer emissions significantly decreased whereas both monomer and excimer emissions of 1 were not changed upon addition of other tested metal ions including Na(I), K(I), Mg(II), and Al(III) as chloride anion and Ca(II), Co(II), Cr(III), Fe(III), Mn(II), Ni(II), Pb(II) and Zn(II) as perchlorate anion. Figure 2b shows the gradual emission intensity change of peptidyl chemosensor 1 upon addition of Hg(II). The intensities of monomer emission at 381 and 400 nm and excimer emission at 494 nm decreased significantly. The monomer emission
Scheme 1. Solid phase synthesis of 1.
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Scheme 2. A proposed binding mode of peptidyl chemosensor 1 with Hg(II).
Figure 1. Fluorescence emission spectra of 1 (2 lM) (—) with 2 equiv Hg(II) (———) in aqueous solution, (a) 99.9:0.1, (b) 9:1, (c) 8:2, (d) 7:3 (e) 6:4 and (f) 5:5 (H2O/DMSO, v/v, 10 mM HEPES at pH 7.4) kex = 352 nm and slit = 15/3.
Figure 2. (a) Fluorescence response of 1 (2 lM) in the presence of various metal ions (2 equiv, except Na(I), K(I), Mg(II), and Mg(II) which were used 100 equiv) and (b) fluorescence changes of 1 (2 lM) in the presence of various concentrations of Hg(II) (0, 0.2, 0.4, 0.6, 0.8, . . . and 2.6 equiv) in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4; kex = 352 nm, slit 15/3).
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intensity of 1 (2 lM) as a function of Hg(II) concentration was plotted in Figure 2b. A complete change in the emission intensity required about 2.0 equiv of Hg(II). We investigated the binding stoichiometry of the complex between the chemosensor 1 and Hg(II) using Job’s plot analysis. Job’s plots that displayed a maximum at 0.5 mol fraction for Hg(II) indicates that peptidyl chemosensor 1 may form a 1:1 complex with Hg(II) (Fig. 3a). Assuming the formation of a 1:1 complex, the dissociation constant of 1 for Hg(II) was determined as 120 nM (R2 = 0.98) (Fig. 3b).17 This value indicates that 1 has a potent binding affinity for Hg(II) in an aqueous buffered solution. The sensitivity of 1 for Hg(II) was determined based on the linear relationships between the monomer emission intensity at 381 nm and the concentration of Hg(II). The detection limit of 1 was calculated as 31.2 nM (R2 = 0.99) by using 3r/m, where r is the standard deviation of the blank measurements, and m is the slope (sensitivity) of the intensity versus sample concentration in the plot (Fig. S8). This confirms that 1 can be used to detect qualitatively small amounts of Hg(II) in aqueous solution. To investigate the interference effect of other metal ions on the detection ability of 1, we measured the fluorescence response of 1 to Hg(II) in the presence of other metal ions (Fig. 4). The fluorescence response of 1 to Hg(II) was not changed in the presence of other metal ions (1 equiv) such as Zn(II), Fe(III), Cd(II), Pb(II), Ag(I), Al(III), Cr(III), Cu(I), Cu(II), Mg(II), and Na(I). In general, almost all chemosensors for Hg(II) suffered from the crosssensitivity of other heavy and transition metal ions such as Cu(II), Ag(I), Cd(II), and Pb(II).10,12f As shown in Figure 5, the visible emission change of 1 (2 lM) in the presence of various metal ions (1 equiv) was monitored under UV light (kem = 365 nm) using UV lamp. Peptidyl sensor 1 displayed a pale green color in the presence or absence of the tested metal ions except Hg(II), whereas 1 displayed a pale blue color in the presence of Hg(II). Peptidyl sensor 1 may provide a simple and easy detection way for Hg(II) using UV lamp. The pH influence on the fluorescence response of peptidyl sensor 1 to Hg(II) was examined at different pH to investigate the role of the functional groups of 1 for the binding of Hg(II) (Fig. S9). As pH increased above 9.5, the intensity of the monomer emission of free 1 slightly decreased and the excimer emission intensity of free 1 considerably decreased. This may be due to the deprotonation of the sulfonamide group (pKa 10)12d,13a,c,f,g because the deprotonated sulfonamide groups in this pH range may act as a quencher for the pyrene fluorophores. The considerable decrease of the excimer emission intensity can be explained by quenching
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effect of both deprotonated sulfonamide groups on the pyrene fluorophores. Interestingly, the emission intensity of 1 in the presence of Hg(II) showed a dependence on pH. In acidic pH (3.5–5.5), 1 showed no fluorescence response to Hg(II) due to the protonation of the imidazole group (pKa 6) of the His residue. In the pH range of 6.5–7.5, the peptidyl sensor began to show a fluorescence response to Hg(II) and the change of emission intensity of 1 to Hg(II) increased as pH increased, which strongly supports the critical role of the His residue for the recognition of Hg(II). The pH titration experiment reveals that 1 is suitable for monitoring Hg(II) in the wide range of pH values (6.5–11.5) and the His residue plays a vital role for the recognition of Hg(II) in the neutral and basic pH. 1 H NMR titration experiment provided additional information for the detailed binding mode of peptidyl sensor 1 with Hg(II). 1 H NMR experiments were carried out in D2O/DMSO-d6 (7:3, v/v) and the pH value of the sample solution was adjusted to seven with 1% NaOD because peptidyl chemosensor 1 showed fluorescence response to Hg(II) in neutral and basic pH. When 1 equiv of Hg(ClO4)2 was added, the downfield shifts of H-26 (D0.06 ppm) and H-24 (D0.04 ppm) corresponding to imidazole protons were observed, respectively (Fig. 6). The shifts was attributed to the co-ordination between Hg(II) and the imidazole moiety. Similarly, the aromatic protons of the pyrene fluorophores such as H-8 (D0.08 ppm), H-28 (D0.09 ppm), H-9 (D0.06 ppm), and H-29 (D0.09 ppm) were also downfield shifted in the presence of Hg(II). The shifts of the pyrene group can be explained by the chelation of Hg(II) with the sulfonamide group of the pyrene moiety because the binding modes of the previously reported chemosensors for Hg(II) based on dansyl labeled amino acids revealed that the NH of sulfonamide group of the dansyl moiety interacted with Hg(II) and the downfield shifts of napthyl protons of the dansyl fluorophore were explained by the chelation of Hg(II) with the sulfonamide moiety.13c,e–g Even though the amide proton peaks of 1 was not observed due to D2O in solvent system, the binding modes of the previously reported chemosensors based on amino acids and peptides suggest the possible interactions between the amide groups of 1 with Hg(II).12d,13a–c,e The binding mode of 1 with Hg(II) and the reason for the decrease of pyrene excimer and monomer emissions in the presence of Hg(II) were proposed using UV and fluorescent spectroscopy experiment, pH titration experiment, and 1H NMR titration experiment. The Job’s plot analysis suggested a 1:1 complex between 1 and Hg(II). As shown in Figure S10, the UV–visible absorption spectrum of peptidyl sensor 1 with gradual additions of Hg(II) exhibited a hypochromic shift with a significant decrease of the absorbance
Figure 3. (a) A Job’s plot analysis for 1 with Hg(II). (b) Nonlinear fitting of the fluorescence intensity change at 381 nm versus concentration of Hg(II) (0.0–4.0 lM in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4; kex = 352 nm, slit 15/3).
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Figure 4. Emission intensity of 1 (2 lM) in the presence of Hg(II) ions (1 equiv) and other metal ions (1 equiv) in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4, kex = 352 nm).
Figure 5. Visible emission color changes of 1 (2 lM) upon addition of 1 equiv of metal ions under UV light (k = 365 nm) in aqueous solution (H2O/DMSO = 7:3, v/v, 10 mM HEPES at pH 7.4).
Figure 6. Partial 1H NMR spectra (400 MHz) of 1 (6.4 mM) in the (a) absence and (b) presence of 1 equiv of Hg(ClO4)2 in D2O/DMSO-d6 (7:3, v/v, pH 7).
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band at 352 nm. This suggests the formation of pyrene dimer in the ground state in the presence of Hg(II).12a,d,13a Considering the overall results, we could propose the binding mode of 1 with Hg(II), as shown in Scheme 2. In the absence of Hg(II), two major conformations of 1 were possible; two pyrene fluorophores were far away in the one major conformer, which exhibited a strong monomer emission intensity, whereas two pyrene fluorophores were close to each other in the other major conformer, which exhibited a considerable excimer emission intensity and a weak monomer emission intensity. In the presence of Hg(II), two sulfonamide groups of 1 interacted with Hg(II) and then the distance between the two pyrenes became shorter because of –SO2NH–Hg(II)–NHSO2– binding. The significant decrease of the absorbance band at 352 nm in UV– visible absorption spectrum also indicated the formation of pyrene dimer in the presence of Hg(II). Thus, the intensity of monomer emission should decrease and the intensity of excimer emission should increase by intra-dimerization of the two pyrene fluorophores. However, as the distance between Hg(II) and the pyrene was short enough to quench the pyrene fluorophores, Hg(II) bound in the peptide quenched the excimer emission. Thus, the decrease of both excimer and monomer emissions of 1 in the presence of Hg(II) could be explained by dimerization of two pyrene fluorophores and subsequently by quenching of Hg(II). In recent years, several research groups have reported that the decrease of monomer or/and excimer emission of pyrene fluorophore in the presence of Hg(II) was explained by quenching effect of Hg(II).18 The similar decrease of monomer emission as well as excimer emission of Calix[4]crown fluoroionophore containing two pyrene fluorophores in the presence of Cu(II) was reported and the decreasing of the excimer emission was explained by quenching of Cu(II).19 The pH titration experiment and 1H NMR titration experiment revealed that the His residue of the peptide sensor was essential for the recognition of Hg(II) because the peptide sensor did not show fluorescence response to Hg(II) in acidic pH 3.5–5.5. In general, as we mentioned in introduction section, the fluorescent peptidyl chemosensors based on copper-binding sequences such as GGH, GHK, GGHG, and HGGG showed highly selective responses only to Cu(II). However, peptidyl chemosensor 1 based on HGGK employed in this study showed highly selective response to Hg(II) without interference of Cu(II). It is very interesting property because the other peptide sensor containing His residue showed cross-selectivity for Cu(II) or interference of Cu(II). 3. Conclusions In summary, we synthesized a novel peptidyl chemosensor (PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1) for Hg(II) with a high yield using solid phase synthesis. The peptidyl chemosensor showed highly selective and sensitive fluorescence response to Hg(II) ion among tested metal ions in aqueous solutions. Peptidyl chemosensor 1 with the potent binding affinity (Kd = 120 nM) for Hg(II) detected Hg(II) without interference of other metal ions. The imidazole group and two sulfonamide group of 1 play a vital role in the co-ordination of Hg(II). The decrease of both excimer and monomer emissions of the pyrene fluorescence of 1 in the presence of Hg(II) was due to the excimer/monomer emission change by dimerization of two pyrene fluorophores and the quenching effect of Hg(II). 4. Experimental 4.1. Reagents Fmoc-Lys(Alloc)-OH, Fmoc-Gly-OH, Fmoc-His(Trit)-OH, N,Ndiisopropylcarboiimide (DIC), 1-hydroxybenzotriazole (HOBt),
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and rink amide MBHA resin were purchased from Advanced ChemTech. Other reagents for solid phase synthesis including trifluroacetic acid (TFA), triethylamine, N,N-dimethylformamide (DMF), triisopropylsilane (TIS), piperidine, phenylsilane and Pd(PPh3)4 were purchased from Aldrich. 1-Pyrenesulfonyl chloride was synthesized from 1-pyrenesulfonic acid (purchased from Aldrich). 4.1.1. Solid phase synthesis: general experimental procedure PySO2-His-Gly-Gly-Lys(PySO2)-NH2, 1 was efficiently synthesized in solid-phase synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (Scheme 1).16 Diisopropylcarbodiimide (DIC) and 1-hydroxylbenzotriazole (HOBt) in situ activation method was used for the coupling reactions. The amino acid, Fmoc-Lys(Alloc)-OH with Fmoc as protecting group (0.3 mmol, 0.3 equiv) was loaded to rink amide MBHA resin were (0.1 mmol, 0.1 equiv) according to the reported procedure. After deprotection of Fmoc group (with 20% piperidine in N,N-dimethylformamide (DMF)) of resin bound Lys, Fmoc-L-Gly-OH (0.3 mmol, 0.3 equiv), Fmoc-LGly-OH (0.3 mmol, 0.3 equiv), Fmoc-L-His(Trt)-OH (0.3 mmol, 0.3 equiv) were coupled sequentially (Scheme 1). After washing, drying, and deprotecting the Fmoc group with 20% piperidine in N,N-dimethylformamide (DMF) of tetrapeptide (H2N-His-Gly-GlyLys(alloc)-resin). The 1-pyrenesulfonyl chloride (0.3 mmol, 0.3 equiv) was coupled in the presence of triethylamine (0.6 mmol, 0.6 equiv). Then, the deprotection of alloc group on lysine side chain and conjugation of another 1-pyrenesulfonyl chloride were achieved with help of phenylsilane (0.3 mmol, 0.3 equiv) and Pd(PPh3)4 (0.02 mmol, 0.02 equiv) in dichloromethane and 1-pyrenesulfonyl chloride (0.3 mmol, 0.3 equiv) and triethylamine (0.6 mmol, 0.6 equiv), respectively. Finally, the cleavage of 1 from the resin was accomplished with CF3COOH/TIS/H2O (TFA/TIS/ water, 95:2.5:2.5, v/v) at room temperature for 3 h. Following vacuum filtration and removal of TFA with N2 blow-off, crude product was precipitated from cold ether. The solid precipitate was centrifuged, washed with ether, and lyophilized under vacuum. The crude product was further purified with semi preparative HPLC using water (0.1% TFA)/acetonitrile (0.1% TFA) gradient. The retention time of 1 is 62 min, respectively. The 89.4% of 1 was isolated and characterized by IR, 1H and 13C NMR and ESI-mass data. The characterization data of 1 are given below. Compound 1: Pale yellow solid, mp 162–163 °C; IR (KBr): 3430, 2926, 1670, 1314, 1198, 1126 cm1; 1H NMR (400 MHz, DMSO6) d 8.96 (d, J = 8.6 Hz, 1H), 8.85 (d, J = 8.6 Hz, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.55 (d, J = 8.4 Hz, 1H), 8.53 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 8.47–8.43 (m, 4H), 8.39 (d, J = 8.4 Hz, 1H), 8.40–8.36 (m, 2H), 8.35–8.33 (m, 2H), 8.27–8.23 (m, 2H), 8.19 (t, J = 8.4 Hz, 2H), 8.16 (m, 2H), 7.99 (t, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.24 (br s, 1H), 7.14 (s, 1H), 6.96 (br s, 1H), 4.24–4.20 (m, 1H), 4.12–4.06 (m, 1H), 3.64–618 (m, 4H), 3.41 (dd, J = 7.6, 2.0 Hz, 1H), 3.26 (dd, J = 7.6, 2.0 Hz, 1H), 2.85 (dd, J = 7.4, 2.0 Hz, 1H), 2.84–2.78 (m, 1H), 2.77–2.70 (m, 2H), 1.52–1.43 (m, 1H), 1.26–1.22 (m, 2H), 1.20–1.16 (m, 1H); 13C NMR (100 MHz, DMSO6) d 173.4, 169.4, 168.6, 168.2, 134.0, 133.8, 132.5, 132.1, 131.6, 130.5, 130.0, 129.9, 129.7, 129.6, 129.4, 129.3, 127.1, 127.0, 126.9, 126.8, 126.6, 124.3, 124.2, 123.9, 123.4, 123.2, 123.0, 117.1, 107.8, 55.7, 52.2, 42.1, 42.0, 41.9, 31.8, 28.9, 26.0, 22.6; ESI-mass (m/z): [M+H]+ calculated for C48H45N8O8S2: 925.27, observed: 925.42. 4.1.2. General fluorescence measurements A stock solution of 1 with the concentration of 1.653 103 M was prepared in DMSO and stored in a cold and dark place. This stock solution was used for all spectrofluoremetric experiments after appropriate dilution. The fluorescence experiments were carried out using the above referred solution after maintaining the pH of the solution to 7.4 using 10 mM HEPES buffer solution. Fluorescence emission spectrum of a sample in a 10 mm path length
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quartz cuvette was measured in 10 mM HEPES buffer solution at pH 7.4 using a Perkin Elmer luminescence spectrophotometer (model LS 55). Emission spectra (370–600 nm) of 1 in the presence of several metal ions (Na(I), K(I), Mg(II), and Al(III) as chloride anion and Ca(II), Co(II), Cr(III), Cu(II), Fe(III), Mn(II), Ni(II), Pb(II), and Zn(II) as perchlorate anion) were measured by excitation with 352 nm The slit size for excitation and emission were 15 and 3.0 nm, respectively. The concentration of 1 was confirmed by UV absorbance at 342 nm for pyrene group. The molar extension coefficient (e) of 1 is 16,000 cm1 M1. 4.1.3. Determination of dissociation constant The dissociation constant for 1:1 complex was calculated based on the titration curve of the 1 with with Hg(II). Dissociation constants was determined by a nonlinear least squares fitting of the data with the following equation as referenced elsewhere.17 The fluorescence signal, F, is related to the equilibrium concentration of the complex (HL) between Host (H) and metal ion (L) by the following expression:
F ¼ F 0 þ DF ½HL 1=2
½HL ¼ 0:5 ½K D þ LT þ HT fðK D LT HT Þ2 4LT HT g
where F0 is the fluorescence of the probe only and DF is the change in fluorescence due to the formation of HL. Acknowledgments This work was supported by a Grant (2012R1AB3000574) from the Basic Research Program of the National Research Foundation and a grant (201200054 0013) from the Korea Environmental Industry and Technology Institute and Dr. P.T. also acknowledges to the Department of Chemistry and Chemical Engineering, Inha University, Incheon, Republic of Korea. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2013.09.058. References and notes 1. (a) Amendola, V.; Fabbrizzi, L.; Forti, F.; Licchelli, M.; Mangano, C.; Pallavicini, P.; Poggi, A.; Sacchi, D.; Taglieti, A. Coord. Chem. Rev. 2006, 250, 273; (b) Valeur, B.; Leray, I. Coord. Chem. Rev. 2000, 205, 3; (c) DeSilva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515. 2. DeSilva, A. P.; Fox, D. B.; Huxley, A. J. M.; Moody, T. S. Coord. Chem. Rev. 2000, 205, 41. 3. (a) Benoit, J. M.; Fitzgerald, W. F.; Damman, A. W. Environ. Res. 1998, 78, 118; (b) Renzoni, A.; Zino, F.; Franchi, E. Environ. Res. 1998, 77, 68; (c) Malm, O. Environ. Res. 1998, 77, 73; (d) Mercury Update: Impact on Fish Advisories; EPA Fact Sheet EPA-823-F-01-001; Environmental Protection Agency, Office of Water: Washington, DC, 2001, p 1. 4. (a) Boening, D. W. Chemosphere 2000, 40, 1335; (b) Harris, H. H.; Pickering, I. J.; George, G. N. Science 2003, 301, 1203.
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