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A new fluorescent pyrene–pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2+ in pure aqueous medium and in live cells
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A new fluorescent pyrene-pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2 þ in pure aqueous medium and in live cells Vikram Singh, Priyanka Srivastava, Shiv PrakashVerma, Arvind Misra, Parimal Das, Nanhai Singh www.elsevier.com/locate/jlumin
PII: DOI: Reference:
S0022-2313(14)00319-6 http://dx.doi.org/10.1016/j.jlumin.2014.05.027 LUMIN12712
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Journal of Luminescence
Received date: 29 January 2014 Accepted date: 26 May 2014 Cite this article as: Vikram Singh, Priyanka Srivastava, Shiv PrakashVerma, Arvind Misra, Parimal Das, Nanhai Singh, A new fluorescent pyrene-pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2 þ in pure aqueous medium and in live cells, Journal of Luminescence, http://dx.doi.org/10.1016/j. jlumin.2014.05.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A new fluorescent pyrene-pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2+ in pure aqueous medium and in live cells Vikram Singh,a Priyanka Srivastava,a Shiv PrakashVerma,b Arvind Misraa,Parimal Das,b and Nanhai Singh*a a
Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi221005, India.
b
Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi221005, India Email: [email protected]
Abstract A new pyrene-pyridine dithiocarbamate based fluorescent chemodosimeter, potassium
(pyren-1-ylmethyl)(pyridin-2-ylmethyl)dithiocarbamate
(L1)
has
been
designed and synthesized. The chemodosimeter shows high selectivity and sensitivity (5.2 ppb) for Hg2+ in pure aqueous medium in which emission intensity was quenched by ≈ 80% due to the formation of new cyclized species, 1. The probe behaves as a chemodosimeter for Hg2+ ions and forms Hg2+ triggered cyclised imidazoline species with approximate detection time of 50 seconds and exhibits both colorimetric and fluorometric changes on detection of Hg2+ ion. Color of the probe (L1) changed from green to colorless visible to the naked eye and from green to dark blue upon the addition of Hg2+ ions under UV light. The Hg2+ triggered cyclization reaction was confirmed by spectral data analysis and a single crystal structure determination of the cyclised entity 2 obtained
from
the
model
compound
potassium
benzyl(pyridin-2-ylmethyl)
dithiocarbamate (L2). L1 finds its application for detection of Hg2+ ions on paper strips, and in BSA (Bovine serum albumin) medium. L1 is also applicable for the monitoring of Hg2+ ion in NIH3T3 live cells. Keywords: Chemodosimeter, Imidazole, Fluorescence imaging
2
1. Introduction Hg2+ has long been considered to be the most toxic heavy metal ion and pollutant that presents severe risks for human health and environment [1,2]. Mercury contamination is widespread in the environment due to anthropogenic actions and industrial releases [3]. The bioaccumulation of such toxic material in the living tissues of human and animal bodies via the food chain causes mercury poisoning and lethal diseases [1,2]. Once the inorganic mercury is converted into toxic lipophilic methylmercury it can easily accumulate within living tissues via the food chain to cause mercury poisoning, [4] neurological damage [5] and diseases like minamata [6]. The Environmental Protection Agency (EPA) has set the maximum allowable level of inorganic mercury in drinking water at 2 ppb [4]. Thus, the detection and measurement of Hg2+ is of great concern among chemists, biologists and environmentalists. Among the various analytical techniques to detect toxic analytes fluorescence based methods are well established as having many merits including economy, sensitivity and selectivity as well as being easy to carry out rapidly in real time [7]. The design and synthesis of good chemosensors for Hg2+ present challenges because HTMs (heavy transition metals) generally induce nonspecific fluorescence quenching due to spin–orbit coupling [8a] and electron transfer mechanism [8b,c]. Additionally, aqueous medium compatibility [9] and properties such as, pH, polarity, temperature, and concentration need to be optimized for a sensing system to become good analytical tool. Therefore, there is a great need for efficient systems that are compatible to aqueous or partial aqueous media that can detect Hg2+ ions via optical signals [10]. The colorimetric and fluorescence methods are important and effective ways to detect metal ions [11,12] because once a metal ion is coordinated to a specifically designed organic molecule significant modulation may occur in the absorption and emission spectra. Moreover, methods based on irreversible selective chemical reactions are particularly appealing because they can result dependent on the analyte concentration in the formation of new entities that can be readily detected [10]. This can be easily achieved by the use of a suitable chemodosimeter to detect specific metal ions through a chemical reaction [1314]. Chemodosimeters generally work with high detection limit to sense specific metal 3
ions such as Hg2+ and in turn, avoid the possibility of interference of closely related ions such as, Ag(I) and Cu(II) [15,16]. Recently, some chemodosimeters have been successfully utilized with high sensitivity to detect Hg2+ as well as other metal ions in solution [17-19] and biological media [20]. Chemodosimeters involving mercurytriggered desulfurization reactions are also established in the literature [12,17-19]. Most of the chemodosimeters developed so far exhibit changes in emission properties upon interaction with metal ions however, those inducing color changes accompanied with shifts in absorption and emission patterns in aqueous medium are very limited in number [11, 19]. With this perspective we herein present the first report concerning a pyrenepyridine dithiocarbamate based potential fluorescent chemodosimeter to detect Hg2+ ions in pure aqueous medium. The dithiocarbamate was purposely introduced into this sensor because of the high thiophilic nature of Hg2+ ions. Secondly, it has been assumed that sulfur containing L1 upon interaction with Hg2+ would undergo a desulfurization reaction to generate an electrophilic centre that would ultimately facilitate an attack by the lone pair electrons present at the neighboring pyridine nitrogen atom to form a new fivemembered imidazole derivative. Monoanionic dithiocarbamates (dtc-) are known to be potential bidentate ligands that promote complexation with heavy metal ions and the structural environment of dtc in a particular receptor system will encourage discriminative binding affinities toward a specific cation [21]. Dithiocarbamates have also been used as a strong chelator for heavy metals and have been extensively used as a colorimetric, extracting and/or masking agent in quantitative or qualitative analyses of cations such as Ni2+, Cu2+, Zn2+, and Hg2+ [22]. The sensing properties of the dithiocarbamate are virtually unexplored for the detection of heavy metal ions [23]. Influenced by these observations herein we have synthesized a novel pyrene-picolyl based probe, having heterodonor aoms (N, S, S), the (L1) for selective Hg2+ sensing in pure aqueous medium (Scheme 1). On interaction with Hg2+ ions, L1 formed a new cyclised entity, 2-(pyren-1-ylmethyl)imidazo[1,5-a]pyridine-3(2H)-thione (1), which was characterized by the 1H NMR titration experiment and ESI-MS. In order to support the formation
of
1,
a
model
compound
potassium
benzyl(pyridin-2-ylmethyl)
dithiocarbamate (L2) was synthesized following a procedure similar to that for L1 by 4
reacting the picolyl-2-amine and benzaldehyde. The reaction of Hg2+ with L2 yielded a cyclised product benzyl-2H-imidazo[1,5-a]pyridine-3-thione (2) which was characterized by single crystal X-ray [24a]. The results of these investigations are described in this contribution. 2. Experimental Section 2.1. Instrumentation and chemicals All chemicals were of analytical grade obtained from commercial sources and used without further purification. The experimental details pertaining to the elemental analysis, IR (KBr), UV-vis. 1H and 13C NMR spectra and single crystal X-ray diffraction of 2 are the same as described previously [24b]. The ESI-Mass spectra of complexes 1 was obtained on Agilent 6310 Ion Trap mass spectrometer. Fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Varian) having a slit width of 5 nm. Stock solution of probe (c = 1x10-2 M) was prepared in 10 mM HEPES buffer in water. For each experiment 12.5 μL of stock solution was taken and diluted to 2.5 mL to make the concentration of 50 µM for each experiment in 10 mM HEPES buffer solution. For interaction study, 0.1 M solutions of different metal ions were used. The 1H NMR titration experiment was performed by addition of a solution of Hg(NO3)2 into the 1x10-2 M L1 in DMSO-d6 solution. To explore the possibility of Hg2+ ion detection in living cells or biological fluids, the NIH3T3 live cells (mouse fibroblast cell line, NCCS, Pune, India) and confocal microscopy for imaging experiments were used. NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma), supplemented with 10% fetal bovine serum (FBS Sigma), 100 units/ml penicillin (Sigma), 100 µg/ml streptomycin (Sigma) and 5% CO2 with humidity. The limit of detection [11a-d, 25-26] was calculated by using equation (1) LOD = 3σ / m
(1)
Where σ stands for the standard deviation of blank solution of L1 and m stands for calibration sensitivity towards Hg2+ ions in aqueous-ethanol solution of L1. The association constant for a stoichiometry was estimated by Benesi-Hildebrand methods [27] using equation (2) for 1:1 stoichiometry. 1/(I - Io) = 1/(I - If) + 1/K (I - If) [M]
(2)
5
Where K is the association constant, I is intensity of free L1, Io is the observed intensity after BSA addition and If is the intensity at saturation point. The fluorescence quenching of the fluorescent probe was estimated by Stern-Volmer equation (3). Fo / F = 1 + KS [Q]
(3)
2.2. Synthesis of Fluorescent Probes 2.3 Potassium-N-methylpyrene-N-methylpyridine-2-dithiocarbamate (L1) The probe (L1) was synthesized according to literature method [24b]. 1pyrenecarboxaldehyde (0.230 g, 1 mmol) and picolyl-2-amine (0.108 g, 1 mmol) were dissolved in 5 mL ethanol and the reaction mixture was refluxed for 6 h. After the reaction was complete (monitored on TLC) the solvent was removed under reduced pressure. The desired product was taken in methanol and reduced with NaBH4 (0.190 g, 5 mmol) in an inert atmosphere of N2 ice cold conditions with continuous stirring for 2 h. The product was extracted into dichloromethane (50 mL) and washed thrice with water (2×25 mL) and organic layer was dried over anhydrous sodium sulphate to obtain an oily yellow color compound N-methylpyrene-N-methylpyridyl-2-amine. The oily yellow amine (0.198, g) thus obtained was dissolved in THF/ ACN (50:50 v/v, 10 mL) and KOH (0.056 g, 1 mmol) and CS2 (0.076 g, 1 mmol) was added under ice cold conditions. The reaction mixture was further stirred for 2-3 h. The solvent was removed under vacuum and the product was washed with diethylether (3 x 15 mL) to afford a green colored solid compound L1. Yield: 0.348 g, 80 %. 1HNMR (400.MHz, DMSO-d6, ppm): δ 8.11-8.10 (d, 1H), 7.39 (s, 2H) 7.36-7.34 (m, 1H), 7.17-7.14 (m, 1H), 7.01-6.99 (m, 1H), 6.87-6.85 (m, 2H), 6.82-6.69 (m, 1H), 6.66-6.64 (m, 4H), 5.36 (s, 4 H).
13
C NMR
(75.45 MHz, DMSO-d6, ppm): δ 214.9 (CS2), δ 155.4, 152.5, 139.6, 136.8, 130.0, 129.6, 126.8, 124.4, 122.3, 122.2, 119.4, 46.3. 2.4. Formation of cyclized entity 1, 2-(pyren-1-ylmethyl)imidazo[1,5-a]pyridine-3(2H)thione (C24H16N2S) (1) The cyclized product 1 which is formed during titration experiment was synthesized, by treating a methanolic solution of L1 (0.218 g, 0.5 mmol) with an aqueous solution of mercuric nitrate (0.159 g, 0.5 mmol). The black precipitate of HgS formed 6
during the reaction was filtered off and the filtrate was evaporated under reduced pressure and dried. Yield: 0.118 g, 65%, m.p. 176-178 °C. Elemental analysis calcd for C24H16N2S: C 79.09, H 4.42, N 7.69, S 8.80; found C 78.88, H 4.67, N 7.92, S 8.97; 1H NMR (300.40 MHz, DMSO-d6, ppm ): δ 8.43-8.41 (d, 1H), 8.23-8.21, (m, 2H), 8.08-8.06 (m, 2H), 7.83-7.81 (m, 1H), 7.28-7.26 (m, 4H), 7.10-7.09 (m, 1H), 6.84-6.73 (m, 2H), 6.18 (s, 1H), 5.69 (s, 2H); 13C NMR (75.45 MHz, DMSO-d6, ppm): δ 210.8 (CS2), 148.8, 147.6, 135, 134.2, 132.5, 129.1, 126.1, 123.1, 120.7, 108, 107.7, 100.6, 53.3. ESI-MS: m/z 365.4. 2.5. Potassium N-benzyl-N-methyl-2-pyridyl dithiocarbamate (L2) The green L2 was synthesized by a procedure similar to that for L1 by the reaction of the picolyl-2-amine (0.108 g, 1 mmol) and benzaldehyde (0.108 g, 1 mmol). Yield: 0.265 g, 85%. 1H NMR (300.40 MHz, DMSO-d6, ppm): δ 8.43-8.41 (m, 1H), 7.72-7.70 (m, 1H), 7.33-7.17 (m, 8H), 5.44 (s, 2H), 5.37 (s, 2H). 13C NMR (75.45 MHz, DMSO-d6): δ 216.6 (CS2), 158.6, 148.5, 138.7, 136.4, 136.1, 128.5, 128.4, 128.0, 127.8, 127.4, 126.4, 124.3, 122.1, 121.6, 121.4, 55.7, 54.2; νmax/ (KBr, cm-1) 1325 (C=N), 975, 987 (C-S). 2.6. Formation of 2-benzylimidazo[1,5-a] pyridine-3-thione (C14H13N2S) (2) The cyclised entity 2 was synthesized by adopting a procedure similar to that for 1 but using L2 (0.312g, 1mmol). The light blue coloured crystals were obtained within 10-15 days (0.199 g, 82 % Yield), mp 172-174 °C. Elemental analysis calcd. for C14H11N2S; C, 69.96; H, 5.03; N, 11.65; S, 13.34; %. Found : C, 69.78; H, 5.13; N, 11.17; S,13.05%. 1
H NMR (300.40 MHz, DMSO-d6, ppm): δ 8.43-8.38 (m, 2H,), δ 7.69-7.67 (d, 1H), 7.30-
7.26 (m, 1H), 6.81-6.71 (m, 61H), 5.95 (s, 3H). 13C NMR (75.45 MHz, CDCl3), δ 210.5 (CS2), δ 148.8, 147.6, 147.0, 145.8,135.0,134.2,132.5, 123.2, 120.7, 108.0, 107.7 , 58.8; νmax/ (KBr, cm-1) 1420 (C=N), 1028 and 1076 (C-S), 3. Results and Discussion 3.1. UV-vis. and fluorescence spectra of L1 The absorption spectrum of L1 showed a low energy n-π* electronic transition band at, λmax = 372 nm (ε = 1.62 x 103 M-1cm-1) and high energy bands in the range of 283-306 nm assignable to π-π* electronic transitions. On excitation at 372 nm, L1 displayed a typical pyrene excimer emission band at 492 nm.
7
The photophysical behavior of L1 (50 µM) in the absence and presence of different cations was examined through absorption and emission spectroscopy in aqueous HEPES buffer (0.01 M; pH=7). Upon interaction with the test cations (10 equiv) Na+, K+, Ca2+, Zn2+, Cd2+, Co2+, Ni2+, Pb2+, Cu2+, Hg2+, Mg2+, Fe2+, Ag2+, Ba2+ and Sr2+, significant change only with Hg2+ ions was observed where two new broad absorption bands at 354 and 277 nm were obtained (Fig. 1a). Similarly the emission spectra of L1 (50 µM) upon interaction with different cations showed fluorescence quenching only with Hg2+ in which the relative emission intensity centered at 492 nm is decreased by ~ 80% (Fig. 1b). Furthermore to understand the metal ion selectivity, interference studies have been performed by the addition of the test cations in excess (20 equiv) to a solution of L1Hg2+. The observed insignificant changes in the absorption and emission spectra confirm the selectivity of L1 for Hg2+ (Fig. 2). Color changes in the solution were observed although from green to colorless observed with the naked eye, green to dark blue was only apparent under UV light (Fig. 3). This mode of interaction between L1 and Hg2+ ions was then shown to be irreversible as the strong chelating agent EDTA was added to a solution of L1+Hg2+ but no change in the electronic spectra was observed (Fig. S12S13, ESI). The binding affinity of L1 with Hg2+ has been analyzed by absorption and emission titration experiments in HEPES buffer solution. Upon gradual addition of Hg2+ ions (0-1 equiv) to a solution of L1 (50 µM), the low energy absorption band centered at 372 nm increased gradually with a hypsochromic shift while new bands appeared at 354 nm and 277 nm and the high energy bands centered in the range of 283-306 nm were diminished (Fig. 4a). An isosbestic point observed at 315 nm suggested the presence of more than one species in the medium. Further addition of Hg2+ ions (2-10 equiv.) led to more hypsochromic shifts in the absorption spectra. The respective changes observed can be attributed to the formation of a new moiety in the medium. Similarly, upon increasing the concentration of Hg2+ ions (0-1 equiv) the emission titration spectra showed a gradual decrease in the relative emission intensity along with a blue shift of 2 nm (Fig. 4b). It is noteworthy that the maximum decrease in the relative emission intensity, ~ 80% of L1 occurred only upon addition of 1 equiv of Hg2+, while further addition of Hg2+ ions (2-10
8
equiv.) made no further change (Fig. S14, ESI). The resultant Jobs plot revealed consistently a 1:1 stoichiometry for the interaction between L1 and Hg2+ ions (Fig. 5a). The fluorescence quenching constant, KS-V has been estimated 71000 /M from a Stern-Volmer plot shown in Fig. 5b. The detection limit of L1 for Hg2+ ion was estimated by our previously reported method [4] as 5.2 ppb (Fig. 6), thus confirming that the probe has sufficient high sensitivity to detect Hg2+ ions in pure aqueous medium. Additionally, the emission titration spectra acquired at 315 nm excitation (isosbestic point) (Fig. S14, ESI), show fluorescence quenching and the emission band shifted from 496 nm to 408 nm (Δλ = 88 nm) with significant loss of intensity, thus indicating the formation of a new species, probably 1 in the medium. 3.2. Excitation Spectrum The excitation spectrum of L1 at 492 nm exhibits a broad band at 360-372 nm. The gradual addition of Hg2+ ions (0-1 equiv.) leads to quenching in the emission band and forms two new bands at 328 and 368 nm accompanied with a shoulder at 395 nm (Fig. S15, ESI). Similar to the emission spectra, the excitation spectra becomes saturated after addition of 1 equiv of Hg2+ ions. 3.3. 1H NMR titration of L1 To obtain further information concerning the formation of the proposed Hg2+ triggered cyclization reaction, 1H NMR titration spectra were carried out in DMSO-d6 (Fig. 7). The spectrum of L1 as shown in Fig. 7, showed resonances attributable to Hd and Hb H4 protons at δ 8.11-8.10 (d, J = 4 Hz) and δ 7.39 ppm respectively. The resonating signals that appeared between δ 7.36-7.34 (d, J = 8Hz), 7.17-7.14, 7.01-6.99 and 6.87-6.85 ppm may be attributed to H6, H5, H9, Ha Hc protons respectively. The resonances attributable to H10, H2, H3, H7, H8 protons merged to appear in the range of δ 6.82-6.64 ppm. A singlet appeared at δ 5.36 ppm has been assigned for H1’ H2’ (aliphatic -CH2) resonances. Upon the gradual addition of Hg2+ ions (0-1 equiv.) to a solution of L1 (Fig. 7) most of the resonating signals shifted toward the downfield region. However the Ha and Hc protons of the picolyl unit were separated at δ 6.84 and 6.68 ppm (∆δ = 0.2-0.03) respectively while, H5 and H6 resonances of the pyrene unit merged at δ 7.98 ppm (∆δ=0.62). Similarly, H2’ and H1’resonances separated from δ 5.36 ppm to appear at δ 6.15 (∆δ=0.79) and 5.74 (∆δ=0.38) ppm respectively. These results suggest 9
that a cyclization reaction has occurred in the presence of Hg2+ to generate a new derivative 1. Subsequently compound 1 was isolated and characterized by spectral data analysis (Fig. S5-S7, ESI). 13
C NMR spectra of L1 showed typical resonance of thiol (–C=S) carbon at δ
214.9 ppm. The pyridine ring carbons a e, c, d and b were resonated at, δ 155.4, 152.9, 122.2 and 119.4 ppm respectively. The resonances appeared in the region of 139.6 122.3 ppm attributed to a pyrene ring carbons and the carbons of –CH2 (1’ and 2’) linker arm appeared at δ 46.3 ppm respectively (Fig. S2, ESI). Further the 13C NMR spectrum of cyclised species 1 showed resonance at 210.8 ppm attributed to thiol carbon (–C=S), while the CH2 1’’ and 2’’carbons appeared at the same place 46.3 ppm in L1 gets separated to appear at 100.6 and 53.5 ppm due to 2’ and 1’ carbons respectively. The carbons of pyridine ring c d and a appears at 148.8, 147.6 ppm respectively while e and b carbons appeared at 108 and 107.7 ppm respectively. Pyrene ring carbons resonate in the region of 135-120.7 ppm (Fig. S6, ESI). A molecular ion peak (ESI-MS) observed at m/z 365.4 (Fig. S7, ESI) further supports the formation of cyclised entity 1. 3.4. UV-vis. and fluorescence spectra of Model compound L2 L2 exhibits n-π* transition band at 360 nm (ε =1.16 x 103 M-1cm-1) and π-π* transition band at λmax 297 nm (ε = 3.9 x 103 M-1cm-1) respectively. When excited at λex 360 nm show an emission band at 502 nm. The spectral study of L2 shows similar changes on interaction of Hg2+ ions. L2 shows selective changes with only Hg2+ metal ions (1 equiv) and forms a new band at 335 nm by merging of two absorption bands and in the emission spectra, exhibits 94% quenching in the emission intensity with a blue shift of 4 nm (Fig. 8, S16-17, ESI). The emission intensity of L2 shows linear decrease upto 1 equiv and with further addition of Hg2+ ions (2-10 equiv.) the emission intensity remains constant (Fig. S18a). The estimation of quenching efficiency was calculated by a Stern-Volmer plot and the value of quenching constant was found to be 2.5 x 105 M-1 (Fig. S18b, ESI). Jobs plot analysis also revealed a 1:1 stoichiometry for the interaction of L2 with Hg2+ ions like in L1 (Inset, Fig. 8). Reversibility experiment with EDTA shows the irreversible nature of binding of L2 and with Hg2+ ions (Fig. S19-20, ESI). The single crystal X-ray analysis revealed the formation of a new cyclised species (2) (Fig. S10, ESI) in case of L2. The formation of the cyclised entity were attributed due to the 10
probable attack of the nitrogen atom on the electron deficient carbon atom linked to sulphur atoms at position-2 of the pyridine moiety (L1 and L2) with the elimination of HgS as shown in Scheme 2. Initially the interaction of the 2- substituted probes L1 and L2 with Hg2+ ions, may involves two units, which favors the stereo chemically facile nucleophillic attack of pyridine lone pair with elimination of HgS to the electron deficient carbon. Further the charge of the ring was stabilized by the formation of double bond between the carbon of five and six membered rings and leads to perturbation in the electronic environment of picolyl unit which was evidenced by the crystal structure in case of 2 (Fig. S10, ESI) and by 1H NMR titration experiment and ESI-MS spectral analysis in case of 1. 3.5. Strips of Probe To confirm the practical application of L1 for the Hg2+ detection the cellulose paper strips (WhatmanTM) of L1 were prepared at three different concentrations (2.5 mM, 1 mM and 0.5 mM) were prepared (1.5 x 0.5 cm2) in water and dried in air. The dried test strips were dipped separately into different concentrations (3 x 10-6, 3 x 10-7 and 3 x 10-8 M) of Hg(NO3)2 solution in water for 10 min and dried in air. The fluorescent green strips changed to light blue under UV light at 365 nm (Fig. 10) thus indicating that the synthesized probe could also detect Hg2+ ions on cellulose paper strips. 3.6. Detection of Hg2+in BSA (Bovine serum albumin) To test the applicability of L1 for detection of Hg2+ ions in protein medium we have added BSA (Bovine serum albumin) to an aqueous solution of L1. The gradual addition of BSA (0-20 µM) leads to an enhancement ~ 2.5 fold in the emission intensity of L1 with a blue shift to appearing at 472 nm (Δλ = 18 nm) (Fig. 11a). This enhancement in emission intensity on addition of BSA was attributed to the movement of L1 to the hydrophobic cavity of BSA [28]. Job’s plot analysis by continuous variation method shows 1:1 stoichiometry (Fig. 11b) for L1 and BSA and the B-H plot (Fig. 11c) for this binding gives the value of Kass =1.73 x 104 M-1 (Binding Constant). Further the addition of Hg2+ ions to a solution of L1 + BSA (5 µM) decreases the emission intensity at 490 nm thereby showing two shoulders at 402 and 424 nm which corresponds to the monomer emission of the pyrene (Fig. 12). The addition of 5 µM of BSA to L1 leads to 1.6 fold enhancement. The addition of Hg2+ ions to this solution decreased the emission 11
intensity to nearly half in comparison to L1 thus indicating Hg2+ triggered cyclization of L1 in the protein medium. 3.7. Optical circuits to construct Logic Gate To perform the logic operations, the Hg2+ and BSA were considered as inputs for the present system that leads to the construction of TRANSFER logic gate at 490 nm wavelength. For the construction of this logic gate, presence and absence of inputs were indicated by the ‘1’ and ‘0’ digits entry in the truth table. We have chosen 0.5 ratio of maximum intensity as a threshold value on 490 nm, emission band of the probe. The values of relative emission intensities at or above 50 % were assigned as ‘1’ digital value (high) while below 50% were assigned as ‘0’ digital values (low). In the absence of both inputs (0 0) or in the presence of only BSA, the relative intensities remain high (1) above 50 % at 490 nm (Fig. 12, Table), while in the presence of only Hg2+ ions or both inputs BSA and Hg2+ ions, low output fluorescence was observed. The values of the corresponding output of relative intensity of the input combination fit to the truth table of TRANSFER logic gate. Thus the designed probe L1 exhibits TRANSFER logic gate with the applied inputs of BSA and Hg2+ ions in aqueous medium. As L1 is a chemodosimeter for Hg2+ ions, the constructed TRANSFER gate is applicable for one time in solution of L1 by the applied inputs of BSA and Hg2+ ions and is not reusable. 3.8. Fluorescence imaging for NIH3T3 cells The excellent selectivity and sensitivity of L1 towards Hg2+ in aqueous solution displays its potential to monitoring Hg2+ in living cells. Therefore for imaging experiments, cells were trypsinized and plated on glass coverslips. After two days cultured cells were exposed to 200 µM Hg(NO3)2·H2O and 50 µM Probe in DMEM for 15 minutes; after that cells were mounted in PBS. Fluorescent imaging experiments of live NIH3T3 cells mounted in PBS were performed with Zeiss LSM 510 Meta confocal microscope with excitation at 488 nm and emission at 505-550 nm (Fig. 13a). Live NIH3T3 cells treated with this compound in DMEM for 15 minutes at 37 °C, showed intracellular cytoplasmic fluorescence (Fig. 13b) which was slightly quenched when incubated along with 200 µM Hg(NO3)2·H2O (Fig. 13c). These results demonstrate that L1 can be successfully used as a biosensor to detect Hg2+ ions in biological samples.
12
Conclusion Conclusively through the present contribution we have demonstrated the potential application of probe L1 as an efficient chemodosimeter to detect Hg2+ in pure aqueous medium. The probe has shown high sensitivity for Hg2+ (5.2 ppb) with approximate detection time of 50 seconds through an irreversible reaction in which mercury triggered cyclization led to the formation of a new imidazole moiety. The proposed mechanism of cyclization was confirmed by NMR and ESI-MS data as well as crystal structure of cyclised entity 2 of model compound L2. L1 is highly permeable to detect Hg2+ in living cells also. L1 also exhibits TRANSFER logic gate by applying Hg2+ and BSA as chemical inputs. Moreover, the studies widen the scope of dithiocarbamate based probes to detect and scavenge Hg2+ ions in aqueous medium. Acknowledgements We gratefully acknowledge the financial support from the Council of Scientific and Industrial Research (CSIR), New Delhi in the form of CSIR Scheme No: 01(2679)/12/EMR-II (NS) and SRF (V.S and P.S).
Supplementary data Synthesis, 1HNMR, 13CNMR, ESI-MS, UV-Vis. and fluorescence spectra.
13
References [1] A. W. Czarnik, Am Chem. Soc. Washington, D.C., 1993. [2] A. P. deSilva, D. B. Fox, A. J. M. Huxley, T. S. Moody, Coord. Chem. Rev. 205 (2000) 41-57. [3] A. Renzoni, F. Zino, E. Franchi, Envir. Res. 77 (1998) 68-72. [4] E.M. Nolan, S. J. Lippard, Chem. Rev. 108 (2008) 3443-3480. [5] D.W. Boening, Chemosphere 40 (2000) 1335-1351. [6] P. Grandjean, P. Weihe, R. F. White, F. Debes, Envir. Res. 77 (1998) 165-172. [7] W. S. Han, H. Y. Lee, S. H. Jung, S. J. Lee, J. H. Jung, Chem. Soc. Rev. 38 (2009) 1904–1915. [8] (a) D. S. McClure, J. Chem. Phys. 20 (1952) 682-686; (b) M. Yu, M. Shi, Z. Chen, F. Li, X. Li, Y. Gao, J. Xu, H. Yang, Z. .Zhou, T. Yi, C. Huang, Chem. Eur. J. 14 (2008) 6892-6900; (c) G.-K. Li, Z.-X. Xu, C.-F. Chen, Z. T. Huang, Chem. Commun. (2008) 1774-1776. [9] (a) R. Metiver, I. Leray, B. Valeur, Chem. Commun (2003) 996-997; (b) J. Y. Kwon, Y. J. Jang, Y. J. Lee, K. M. Kim, M. S. Seo, W. Nam, J. Yoon, J. Am. Chem. Soc. 127 (2005) 10107-10111. [10] J. Ros-Lis, M. D. Marcos, R. Mártinez-Mánez, K. Rurack, J. Soto, Angew. Chem. Int. Ed. 44 (2005) 4405-4407. [11] (a) P. Srivastava, R. Ali, S. S. Razi, M. Shahid, A. Misra, Sens. Actuators B 181 (2013) 584-595; (b) P. Srivastava, R. Ali, S. S. Razi, M. Shahid, S. Patnaik, A. Misra, Tetrahedron. Lett. 54 (2013) 3688-3693; (c) A. Misra, P. Srivastava, M. Shahid, Analyst 137 (2012) 3470-3478; (d) P. Srivastava, M. Shahid, A. Misra, Org. Biomol. Chem. 9 (2011) 5051-5055; (e) A. Misra, M. Shahid, P. Srivastava, Sens. Actuators B 169 (2012) 327-340. [12] (a) B. Liu, H. Tian, Chem. Commun. (2005) 3156–3158; (b) J.-S. Wu, I.-C. Hwang, K. S. Kim, J. S. Kim, Org. Lett. 9 (2007) 907-910. [13] D.-G. Cho, J. L. Sessler, Chem. Soc. Rev. 38 (2009) 1647-1662. [14] M. J. Choi, Y. H. Kim, J. E. Namgoong, S.-K. Chang, Chem. Commun. (2009) 3560-3562. 14
[15] J. Wang, X. Qian, J. Cui, J. Org. Chem. 71 (2006) 4308-4311. [16] Y. Zhou, C.-Y. Zhu, X-S. Gao, X.-Y. You, C. Yao, Org. Lett. 12 (2010) 2566-2569. [17] J. C. Manimala, E.V. Anslyn, Eur. J. Org. Chem. (2002) 3909-3922. [18] K. C. Song, J. S. Kim, S. M. Park, K.-C. Chung, S. Ahn, S.-K. Chang, Org. Lett. 8 (2006) 3413-3416. [19] J. Du, M. Hu, J. Fan, X. Peng, Chem. Soc. Rev. 41 (2012) 4511-4535. [20] B. Valeur, I. Leray, Coord. Chem. Rev. 205 (2000) 3-40. [21] (a) S.-M. Cheung, W.-H. Chan, Tetrahedron 62 (2006) 8379-8383; (b) C. Chen, R. Wang, L. Guo, N. Fu, H. Dong, Y. Yuan, Org. Lett. 13 (2011) 11621165. [22] T. Nagano, T. Yoshimura, Chem. Rev. 102 (2002) 1235-1269. [23] (a) M. Lerchi, E. Reitter, W. Simon, E. Pretsch, D. A. Chowdhury, S. Kamata, Anal. Chem. 66 (1994) 1713-1717; (b) J. Hatai, S. Pal, G. P. Jose, S. Bandyopadhyay, Inorg. Chem. 51 (2012) 10129-10135. [24] (a) X-ray crystallographic data and ccdc number were submitted for publication elsewhere; (b) V. Singh, A. Kumar, R. Prasad, G. Rajput, M. G. B. Drew, N. Singh, CrystEngComm 13 (2011) 6817-6826. [25] J. D. Ingle Jr, J. Chem. Educ. 51 (1974) 100. [26] G. L. Long, J. D. Winefordner, Anal. Chem. 55 (1983) 712A. [27] H. A. Benesi, J. H. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703-2707. [28] (a) A. Mallick, B. Haldar, N. Chattopadhyay, J. Phys. Chem B. 109 (2005) 14683; (b) A. Chakrabarty, A. Mallick, B. Haldar, P. Das, N. Chattopadhyay, Biomacromolecules 8 (2007) 920; (c) K. A. Willets, O. Ostroverkhova, M. He, R. J. Twieg, J. Am. Chem. Soc. 125 (2003) 1174; (d) A. Wu, Y. Xu and X. Qian, Bioorg. Med. Chem. 17 (2009) 592.
15
Figure captionsScheme 1. (1) EtOH/ Reflux (ii) NaBH4/ MeOH (iii) KOH/ CS2/0˚C (iv) Hg(NO3)2/ MeOH-H2O. Fig.1. Interaction study of L1 upon addition of different metal ions by (a) absorption (b) emission spectral changes in aqueous 0.01 M HEPES buffer (pH 7).Inset: Bar diagram of interaction study with different metal ions. Fig. 2. Interference study of L1 by (a) absorption (b) emission spectral changes upon addition of different metal ions in aqueous 0.01 M HEPES buffer (pH 7). Inset: Bar diagram of interference study of L1 + Hg2+with different metal ions. Fig. 3. (a) Change in color of solutions of L1 with the test metal ions ( Na+, K+, Ca2+,Zn2+, Cd2+, Co2+, Ni2+, Pb2+, Cu2+, Hg2+, Mg2+, Fe2+, Ag2+ ,Ba2+, Sr2+); (b) under 354 nm UV light. Fig. 4. (a) Absorption (b) Emission titration spectra of L1 upon addition of Hg2+ ions (01 equiv) in aqueous 0.01 M HEPES buffer (pH 7). Fig. 5. (a) Jobs plot analysis of L1 for Hg2+ ions (b) Stern-Volmer plot for the estimation of quenching constant of L1 with Hg2+ ions. Fig. 6. (a) Calibration curve of Intensity with respect to concentration of L1 (b) Plot of change in intensity with respect to the Hg2+ ions concentration (where ΔI shows the changes in the emission intensity on each addition). Fig. 7. 1H NMR titration spectra of L1 upon addition of Hg2+ ions (0- 1.0 equiv) in DMSO-d6. Fig. 8. (a) Absorption (b) Emission titration spectra of L2 upon addition of Hg2+ ions (01 equiv) in aqueous 0.01 M HEPES buffer (pH 7). Inset: Jobs plot analysis of L2 for Hg2+ ions. Fig. 9. Mechanism of cyclization of L1 to cyclised entity 1. Fig. 10. Fluorescent paper strips of L1 (a) 2.5 mM (b) 1 mM (c) 0.5 mM (Green) and after addition of Hg2+ (a) 3 × 10-6 (b) 3 × 10-7 (c) 3 × 10-8 M (non fluorescent). Fig. 11. (a) Emission titration experiment of L1 upon addition of BSA (0- 40 µM) (b) Jobs plot analysis of L1 for Hg2+ ions (c) B-H plot of L1 with BSA in aqueous 0.01 M HEPES buffer (pH 7). Fig. 12. Relative emission intensity of the applied inputs. Inset: Bar diagram of relative intensity of respective inputs at 490 nm wavelength in aqueous 0.01 M HEPES buffer (pH 7) and logic symbol and truth table of TRANSFER logic gate. 16
Fig. 13. Confocal fluorescence microscopic images of (a) NIH3T3 cells (b) NIH3T3 cells with 50 μM L1 and (c) with 200 μM Hg2+ and 50 μM L1.
17
Highlights • • • •
Efficient chemodosimeter to detect Hg2+ ions in pure aqueous medium. Hg2+ triggered cyclisation and formation of imidazoline species. Probe exhibit both colorimetric and fluorometric changes Probe is applicable to detect Hg2+ in live cells and on cellulose paper strips.
18
Scheme 1. (1) EtOH/ Reflux (ii) NaBH4/ MeOH (iii) KOH/ CS2/0˚C (iv) Hg(NO3)2/ MeOHH2O.
(a)
L1 + M ions
160 2+
Intensity ( a.u. )
Absorbance
L1 + Hg ions 0.5
0.0
300
350
400
450
Wavelength ( nm )
(b)
L1 + M ions 120
120
500
80
I
1.0
80
40 2+
L1 + Hg ions
40
0
0
420
480
540
600
K Na Ag Pb Zn Cu Ca Co Ni Sr BaMg Fe Hg Cd
660
Wavelength ( nm )
Fig.1. Interaction study of L1 upon addition of different metal ions by (a) absorption (b) emission spectral changes in aqueous 0.01 M HEPES buffer (pH 7).Inset: Bar diagram of interaction study with different metal ions.
L1
160 2+
Absorbance
L1 + Hg + M ions 0.5
0.0
350
400
450
Wavelength ( nm )
120 80 40 0
300
L1
(b)
,
(a)
Intensity ( a.u. )
1.0
500
2+
L1 + Hg + M ions
420
480
540
600
+J . 1D $J 3E =Q &X &D &R 1L 6U %D 0J )H &G
660
Wavelength ( nm )
Fig. 2. Interference study of L1 by (a) absorption (b) emission spectral changes upon addition of different metal ions in aqueous 0.01 M HEPES buffer (pH 7). Inset: Bar diagram of interference study of L1 + Hg2+with different metal ions.
L1
Na+ K+ Ca2+ Zn2+ Hg2+ Cu2+ Co2+ Ni2+ Ag2+Fe2+ Cd2+ Mg2+ Ba2+ Sr2+ Pb2+
(a)
L1 Na
K
Ca2 Zn2
Hg2+ Cu2
2+ 2+ 2+ 2+ Co2 Ni2 Ag2 Fe2 Cd Mg Ba Sr
Pb2
(b)
Fig. 3. (a) Change in color of solutions of L1 with the test metal ions ( Na+, K+, Ca2+,Zn2+, Cd2+, Co2+, Ni2+, Pb2+, Cu2+, Hg2+, Mg2+, Fe2+, Ag2+ ,Ba2+, Sr2+); (b) under 354 nm UV light.
160
(a)
2+
Hg
0.5
0.0
300
350
400
Wavelength ( nm )
Intensity ( a.u. )
Absorbance
1.0
450
(b) Hg
120
2+
80 40 0
420
480
540
600
660
Wavelength ( nm )
Fig. 4. (a) Absorption (b) Emission titration spectra of L1 upon addition of Hg2+ ions (0- 1 equiv) in aqueous 0.01 M HEPES buffer (pH 7).
5.0
Io/I
I
140 (a)
70
0 0.0
0.2
0.4 2+
0.6
0.8 2+
[ Hg ] / [ L1 + Hg ]
(b)
KS-V= 71000 / M
2.5
1.0
0
10
20
30
40
50
2+
[Hg ] M
Fig. 5. (a) Jobs plot analysis of L1 for Hg2+ ions (b) Stern-Volmer plot for the estimation of quenching constant of L1 with Hg2+ ions.
Fig. 6. (a) Calibration curve of Intensity with respect to concentration of L1 (b) Plot of change in intensity with respect to the Hg2+ ions concentration (where ΔI shows the changes in the emission intensity on each addition).
Fig. 7. 1H NMR titration spectra of L1 upon addition of Hg2+ ions (0- 1.0 equiv) in DMSO-d6.
Fig. 8. (a) Absorption (b) Emission titration spectra of L2 upon addition of Hg2+ ions (0-1 equiv) in aqueous 0.01 M HEPES buffer (pH 7). Inset: Jobs plot analysis of L2 for Hg2+ ions.
N N
S
S
Hg2+ N N
S
S N
N
S
H
N N
Hg(NO3)2
S
S LH H
L1
-HgS
N
N
S S
Hg2+
Fig. 9. Mechanism of cyclization of L1 to cyclised entity 1.
1
Fig. 10. Fluorescent paper strips of L1 (a) 2.5 mM (b) 1 mM (c) 0.5 mM (Green) and after addition of Hg2+ (a) 3 × 10-6 (b) 3 × 10-7 (c) 3 × 10-8 M (non fluorescent).
160
300 (a)
I
200
80
100 0.0
0.2
0.4
0.6
0.8
[BSA] / [ L1 + BSA]
0
420
480
540
(c)
0.08
120 1/I
BSA Intensity ( a.u. )
0.12 (b)
1.0
0.04
0.00 0.0
0.1
0.2
0.3
0.4
1 / BSA (M)
600
Wavelength ( nm ) Fig. 11. (a) Emission titration experiment of L1 upon addition of BSA (0- 40 µM) (b) Jobs plot analysis of L1 for Hg2+ ions (c) B-H plot of L1 with BSA in aqueous 0.01 M HEPES buffer (pH 7).
Hg2+ (In1) 0 1 0 1
BSA (In2) 0 0 1 1
O (490 nm) 1 1 0 0
Fig. 12. Relative emission intensity of the applied inputs. Inset: Bar diagram of relative intensity of respective inputs at 490 nm wavelength in aqueous 0.01 M HEPES buffer (pH 7) and logic symbol and truth table of TRANSFER logic gate.
Fig. 13. Confocal fluorescence microscopic images of (a) NIH3T3 cells (b) NIH3T3 cells with 50 μM L1 and (c) with 200 μM Hg2+ and 50 μM L1.
A new fluorescent pyrene-pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2 þ in pure aqueous medium and in live cells Vikram Singh, Priyanka Srivastava, Shiv PrakashVerma, Arvind Misra, Parimal Das, Nanhai Singh www.elsevier.com/locate/jlumin
PII: DOI: Reference:
S0022-2313(14)00319-6 http://dx.doi.org/10.1016/j.jlumin.2014.05.027 LUMIN12712
To appear in:
Journal of Luminescence
Received date: 29 January 2014 Accepted date: 26 May 2014 Cite this article as: Vikram Singh, Priyanka Srivastava, Shiv PrakashVerma, Arvind Misra, Parimal Das, Nanhai Singh, A new fluorescent pyrene-pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2 þ in pure aqueous medium and in live cells, Journal of Luminescence, http://dx.doi.org/10.1016/j. jlumin.2014.05.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A new fluorescent pyrene-pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2+ in pure aqueous medium and in live cells Vikram Singh,a Priyanka Srivastava,a Shiv PrakashVerma,b Arvind Misraa,Parimal Das,b and Nanhai Singh*a a
Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi221005, India.
b
Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi221005, India Email: [email protected]
Abstract A new pyrene-pyridine dithiocarbamate based fluorescent chemodosimeter, potassium
(pyren-1-ylmethyl)(pyridin-2-ylmethyl)dithiocarbamate
(L1)
has
been
designed and synthesized. The chemodosimeter shows high selectivity and sensitivity (5.2 ppb) for Hg2+ in pure aqueous medium in which emission intensity was quenched by ≈ 80% due to the formation of new cyclized species, 1. The probe behaves as a chemodosimeter for Hg2+ ions and forms Hg2+ triggered cyclised imidazoline species with approximate detection time of 50 seconds and exhibits both colorimetric and fluorometric changes on detection of Hg2+ ion. Color of the probe (L1) changed from green to colorless visible to the naked eye and from green to dark blue upon the addition of Hg2+ ions under UV light. The Hg2+ triggered cyclization reaction was confirmed by spectral data analysis and a single crystal structure determination of the cyclised entity 2 obtained
from
the
model
compound
potassium
benzyl(pyridin-2-ylmethyl)
dithiocarbamate (L2). L1 finds its application for detection of Hg2+ ions on paper strips, and in BSA (Bovine serum albumin) medium. L1 is also applicable for the monitoring of Hg2+ ion in NIH3T3 live cells. Keywords: Chemodosimeter, Imidazole, Fluorescence imaging
2
1. Introduction Hg2+ has long been considered to be the most toxic heavy metal ion and pollutant that presents severe risks for human health and environment [1,2]. Mercury contamination is widespread in the environment due to anthropogenic actions and industrial releases [3]. The bioaccumulation of such toxic material in the living tissues of human and animal bodies via the food chain causes mercury poisoning and lethal diseases [1,2]. Once the inorganic mercury is converted into toxic lipophilic methylmercury it can easily accumulate within living tissues via the food chain to cause mercury poisoning, [4] neurological damage [5] and diseases like minamata [6]. The Environmental Protection Agency (EPA) has set the maximum allowable level of inorganic mercury in drinking water at 2 ppb [4]. Thus, the detection and measurement of Hg2+ is of great concern among chemists, biologists and environmentalists. Among the various analytical techniques to detect toxic analytes fluorescence based methods are well established as having many merits including economy, sensitivity and selectivity as well as being easy to carry out rapidly in real time [7]. The design and synthesis of good chemosensors for Hg2+ present challenges because HTMs (heavy transition metals) generally induce nonspecific fluorescence quenching due to spin–orbit coupling [8a] and electron transfer mechanism [8b,c]. Additionally, aqueous medium compatibility [9] and properties such as, pH, polarity, temperature, and concentration need to be optimized for a sensing system to become good analytical tool. Therefore, there is a great need for efficient systems that are compatible to aqueous or partial aqueous media that can detect Hg2+ ions via optical signals [10]. The colorimetric and fluorescence methods are important and effective ways to detect metal ions [11,12] because once a metal ion is coordinated to a specifically designed organic molecule significant modulation may occur in the absorption and emission spectra. Moreover, methods based on irreversible selective chemical reactions are particularly appealing because they can result dependent on the analyte concentration in the formation of new entities that can be readily detected [10]. This can be easily achieved by the use of a suitable chemodosimeter to detect specific metal ions through a chemical reaction [1314]. Chemodosimeters generally work with high detection limit to sense specific metal 3
ions such as Hg2+ and in turn, avoid the possibility of interference of closely related ions such as, Ag(I) and Cu(II) [15,16]. Recently, some chemodosimeters have been successfully utilized with high sensitivity to detect Hg2+ as well as other metal ions in solution [17-19] and biological media [20]. Chemodosimeters involving mercurytriggered desulfurization reactions are also established in the literature [12,17-19]. Most of the chemodosimeters developed so far exhibit changes in emission properties upon interaction with metal ions however, those inducing color changes accompanied with shifts in absorption and emission patterns in aqueous medium are very limited in number [11, 19]. With this perspective we herein present the first report concerning a pyrenepyridine dithiocarbamate based potential fluorescent chemodosimeter to detect Hg2+ ions in pure aqueous medium. The dithiocarbamate was purposely introduced into this sensor because of the high thiophilic nature of Hg2+ ions. Secondly, it has been assumed that sulfur containing L1 upon interaction with Hg2+ would undergo a desulfurization reaction to generate an electrophilic centre that would ultimately facilitate an attack by the lone pair electrons present at the neighboring pyridine nitrogen atom to form a new fivemembered imidazole derivative. Monoanionic dithiocarbamates (dtc-) are known to be potential bidentate ligands that promote complexation with heavy metal ions and the structural environment of dtc in a particular receptor system will encourage discriminative binding affinities toward a specific cation [21]. Dithiocarbamates have also been used as a strong chelator for heavy metals and have been extensively used as a colorimetric, extracting and/or masking agent in quantitative or qualitative analyses of cations such as Ni2+, Cu2+, Zn2+, and Hg2+ [22]. The sensing properties of the dithiocarbamate are virtually unexplored for the detection of heavy metal ions [23]. Influenced by these observations herein we have synthesized a novel pyrene-picolyl based probe, having heterodonor aoms (N, S, S), the (L1) for selective Hg2+ sensing in pure aqueous medium (Scheme 1). On interaction with Hg2+ ions, L1 formed a new cyclised entity, 2-(pyren-1-ylmethyl)imidazo[1,5-a]pyridine-3(2H)-thione (1), which was characterized by the 1H NMR titration experiment and ESI-MS. In order to support the formation
of
1,
a
model
compound
potassium
benzyl(pyridin-2-ylmethyl)
dithiocarbamate (L2) was synthesized following a procedure similar to that for L1 by 4
reacting the picolyl-2-amine and benzaldehyde. The reaction of Hg2+ with L2 yielded a cyclised product benzyl-2H-imidazo[1,5-a]pyridine-3-thione (2) which was characterized by single crystal X-ray [24a]. The results of these investigations are described in this contribution. 2. Experimental Section 2.1. Instrumentation and chemicals All chemicals were of analytical grade obtained from commercial sources and used without further purification. The experimental details pertaining to the elemental analysis, IR (KBr), UV-vis. 1H and 13C NMR spectra and single crystal X-ray diffraction of 2 are the same as described previously [24b]. The ESI-Mass spectra of complexes 1 was obtained on Agilent 6310 Ion Trap mass spectrometer. Fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Varian) having a slit width of 5 nm. Stock solution of probe (c = 1x10-2 M) was prepared in 10 mM HEPES buffer in water. For each experiment 12.5 μL of stock solution was taken and diluted to 2.5 mL to make the concentration of 50 µM for each experiment in 10 mM HEPES buffer solution. For interaction study, 0.1 M solutions of different metal ions were used. The 1H NMR titration experiment was performed by addition of a solution of Hg(NO3)2 into the 1x10-2 M L1 in DMSO-d6 solution. To explore the possibility of Hg2+ ion detection in living cells or biological fluids, the NIH3T3 live cells (mouse fibroblast cell line, NCCS, Pune, India) and confocal microscopy for imaging experiments were used. NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma), supplemented with 10% fetal bovine serum (FBS Sigma), 100 units/ml penicillin (Sigma), 100 µg/ml streptomycin (Sigma) and 5% CO2 with humidity. The limit of detection [11a-d, 25-26] was calculated by using equation (1) LOD = 3σ / m
(1)
Where σ stands for the standard deviation of blank solution of L1 and m stands for calibration sensitivity towards Hg2+ ions in aqueous-ethanol solution of L1. The association constant for a stoichiometry was estimated by Benesi-Hildebrand methods [27] using equation (2) for 1:1 stoichiometry. 1/(I - Io) = 1/(I - If) + 1/K (I - If) [M]
(2)
5
Where K is the association constant, I is intensity of free L1, Io is the observed intensity after BSA addition and If is the intensity at saturation point. The fluorescence quenching of the fluorescent probe was estimated by Stern-Volmer equation (3). Fo / F = 1 + KS [Q]
(3)
2.2. Synthesis of Fluorescent Probes 2.3 Potassium-N-methylpyrene-N-methylpyridine-2-dithiocarbamate (L1) The probe (L1) was synthesized according to literature method [24b]. 1pyrenecarboxaldehyde (0.230 g, 1 mmol) and picolyl-2-amine (0.108 g, 1 mmol) were dissolved in 5 mL ethanol and the reaction mixture was refluxed for 6 h. After the reaction was complete (monitored on TLC) the solvent was removed under reduced pressure. The desired product was taken in methanol and reduced with NaBH4 (0.190 g, 5 mmol) in an inert atmosphere of N2 ice cold conditions with continuous stirring for 2 h. The product was extracted into dichloromethane (50 mL) and washed thrice with water (2×25 mL) and organic layer was dried over anhydrous sodium sulphate to obtain an oily yellow color compound N-methylpyrene-N-methylpyridyl-2-amine. The oily yellow amine (0.198, g) thus obtained was dissolved in THF/ ACN (50:50 v/v, 10 mL) and KOH (0.056 g, 1 mmol) and CS2 (0.076 g, 1 mmol) was added under ice cold conditions. The reaction mixture was further stirred for 2-3 h. The solvent was removed under vacuum and the product was washed with diethylether (3 x 15 mL) to afford a green colored solid compound L1. Yield: 0.348 g, 80 %. 1HNMR (400.MHz, DMSO-d6, ppm): δ 8.11-8.10 (d, 1H), 7.39 (s, 2H) 7.36-7.34 (m, 1H), 7.17-7.14 (m, 1H), 7.01-6.99 (m, 1H), 6.87-6.85 (m, 2H), 6.82-6.69 (m, 1H), 6.66-6.64 (m, 4H), 5.36 (s, 4 H).
13
C NMR
(75.45 MHz, DMSO-d6, ppm): δ 214.9 (CS2), δ 155.4, 152.5, 139.6, 136.8, 130.0, 129.6, 126.8, 124.4, 122.3, 122.2, 119.4, 46.3. 2.4. Formation of cyclized entity 1, 2-(pyren-1-ylmethyl)imidazo[1,5-a]pyridine-3(2H)thione (C24H16N2S) (1) The cyclized product 1 which is formed during titration experiment was synthesized, by treating a methanolic solution of L1 (0.218 g, 0.5 mmol) with an aqueous solution of mercuric nitrate (0.159 g, 0.5 mmol). The black precipitate of HgS formed 6
during the reaction was filtered off and the filtrate was evaporated under reduced pressure and dried. Yield: 0.118 g, 65%, m.p. 176-178 °C. Elemental analysis calcd for C24H16N2S: C 79.09, H 4.42, N 7.69, S 8.80; found C 78.88, H 4.67, N 7.92, S 8.97; 1H NMR (300.40 MHz, DMSO-d6, ppm ): δ 8.43-8.41 (d, 1H), 8.23-8.21, (m, 2H), 8.08-8.06 (m, 2H), 7.83-7.81 (m, 1H), 7.28-7.26 (m, 4H), 7.10-7.09 (m, 1H), 6.84-6.73 (m, 2H), 6.18 (s, 1H), 5.69 (s, 2H); 13C NMR (75.45 MHz, DMSO-d6, ppm): δ 210.8 (CS2), 148.8, 147.6, 135, 134.2, 132.5, 129.1, 126.1, 123.1, 120.7, 108, 107.7, 100.6, 53.3. ESI-MS: m/z 365.4. 2.5. Potassium N-benzyl-N-methyl-2-pyridyl dithiocarbamate (L2) The green L2 was synthesized by a procedure similar to that for L1 by the reaction of the picolyl-2-amine (0.108 g, 1 mmol) and benzaldehyde (0.108 g, 1 mmol). Yield: 0.265 g, 85%. 1H NMR (300.40 MHz, DMSO-d6, ppm): δ 8.43-8.41 (m, 1H), 7.72-7.70 (m, 1H), 7.33-7.17 (m, 8H), 5.44 (s, 2H), 5.37 (s, 2H). 13C NMR (75.45 MHz, DMSO-d6): δ 216.6 (CS2), 158.6, 148.5, 138.7, 136.4, 136.1, 128.5, 128.4, 128.0, 127.8, 127.4, 126.4, 124.3, 122.1, 121.6, 121.4, 55.7, 54.2; νmax/ (KBr, cm-1) 1325 (C=N), 975, 987 (C-S). 2.6. Formation of 2-benzylimidazo[1,5-a] pyridine-3-thione (C14H13N2S) (2) The cyclised entity 2 was synthesized by adopting a procedure similar to that for 1 but using L2 (0.312g, 1mmol). The light blue coloured crystals were obtained within 10-15 days (0.199 g, 82 % Yield), mp 172-174 °C. Elemental analysis calcd. for C14H11N2S; C, 69.96; H, 5.03; N, 11.65; S, 13.34; %. Found : C, 69.78; H, 5.13; N, 11.17; S,13.05%. 1
H NMR (300.40 MHz, DMSO-d6, ppm): δ 8.43-8.38 (m, 2H,), δ 7.69-7.67 (d, 1H), 7.30-
7.26 (m, 1H), 6.81-6.71 (m, 61H), 5.95 (s, 3H). 13C NMR (75.45 MHz, CDCl3), δ 210.5 (CS2), δ 148.8, 147.6, 147.0, 145.8,135.0,134.2,132.5, 123.2, 120.7, 108.0, 107.7 , 58.8; νmax/ (KBr, cm-1) 1420 (C=N), 1028 and 1076 (C-S), 3. Results and Discussion 3.1. UV-vis. and fluorescence spectra of L1 The absorption spectrum of L1 showed a low energy n-π* electronic transition band at, λmax = 372 nm (ε = 1.62 x 103 M-1cm-1) and high energy bands in the range of 283-306 nm assignable to π-π* electronic transitions. On excitation at 372 nm, L1 displayed a typical pyrene excimer emission band at 492 nm.
7
The photophysical behavior of L1 (50 µM) in the absence and presence of different cations was examined through absorption and emission spectroscopy in aqueous HEPES buffer (0.01 M; pH=7). Upon interaction with the test cations (10 equiv) Na+, K+, Ca2+, Zn2+, Cd2+, Co2+, Ni2+, Pb2+, Cu2+, Hg2+, Mg2+, Fe2+, Ag2+, Ba2+ and Sr2+, significant change only with Hg2+ ions was observed where two new broad absorption bands at 354 and 277 nm were obtained (Fig. 1a). Similarly the emission spectra of L1 (50 µM) upon interaction with different cations showed fluorescence quenching only with Hg2+ in which the relative emission intensity centered at 492 nm is decreased by ~ 80% (Fig. 1b). Furthermore to understand the metal ion selectivity, interference studies have been performed by the addition of the test cations in excess (20 equiv) to a solution of L1Hg2+. The observed insignificant changes in the absorption and emission spectra confirm the selectivity of L1 for Hg2+ (Fig. 2). Color changes in the solution were observed although from green to colorless observed with the naked eye, green to dark blue was only apparent under UV light (Fig. 3). This mode of interaction between L1 and Hg2+ ions was then shown to be irreversible as the strong chelating agent EDTA was added to a solution of L1+Hg2+ but no change in the electronic spectra was observed (Fig. S12S13, ESI). The binding affinity of L1 with Hg2+ has been analyzed by absorption and emission titration experiments in HEPES buffer solution. Upon gradual addition of Hg2+ ions (0-1 equiv) to a solution of L1 (50 µM), the low energy absorption band centered at 372 nm increased gradually with a hypsochromic shift while new bands appeared at 354 nm and 277 nm and the high energy bands centered in the range of 283-306 nm were diminished (Fig. 4a). An isosbestic point observed at 315 nm suggested the presence of more than one species in the medium. Further addition of Hg2+ ions (2-10 equiv.) led to more hypsochromic shifts in the absorption spectra. The respective changes observed can be attributed to the formation of a new moiety in the medium. Similarly, upon increasing the concentration of Hg2+ ions (0-1 equiv) the emission titration spectra showed a gradual decrease in the relative emission intensity along with a blue shift of 2 nm (Fig. 4b). It is noteworthy that the maximum decrease in the relative emission intensity, ~ 80% of L1 occurred only upon addition of 1 equiv of Hg2+, while further addition of Hg2+ ions (2-10
8
equiv.) made no further change (Fig. S14, ESI). The resultant Jobs plot revealed consistently a 1:1 stoichiometry for the interaction between L1 and Hg2+ ions (Fig. 5a). The fluorescence quenching constant, KS-V has been estimated 71000 /M from a Stern-Volmer plot shown in Fig. 5b. The detection limit of L1 for Hg2+ ion was estimated by our previously reported method [4] as 5.2 ppb (Fig. 6), thus confirming that the probe has sufficient high sensitivity to detect Hg2+ ions in pure aqueous medium. Additionally, the emission titration spectra acquired at 315 nm excitation (isosbestic point) (Fig. S14, ESI), show fluorescence quenching and the emission band shifted from 496 nm to 408 nm (Δλ = 88 nm) with significant loss of intensity, thus indicating the formation of a new species, probably 1 in the medium. 3.2. Excitation Spectrum The excitation spectrum of L1 at 492 nm exhibits a broad band at 360-372 nm. The gradual addition of Hg2+ ions (0-1 equiv.) leads to quenching in the emission band and forms two new bands at 328 and 368 nm accompanied with a shoulder at 395 nm (Fig. S15, ESI). Similar to the emission spectra, the excitation spectra becomes saturated after addition of 1 equiv of Hg2+ ions. 3.3. 1H NMR titration of L1 To obtain further information concerning the formation of the proposed Hg2+ triggered cyclization reaction, 1H NMR titration spectra were carried out in DMSO-d6 (Fig. 7). The spectrum of L1 as shown in Fig. 7, showed resonances attributable to Hd and Hb H4 protons at δ 8.11-8.10 (d, J = 4 Hz) and δ 7.39 ppm respectively. The resonating signals that appeared between δ 7.36-7.34 (d, J = 8Hz), 7.17-7.14, 7.01-6.99 and 6.87-6.85 ppm may be attributed to H6, H5, H9, Ha Hc protons respectively. The resonances attributable to H10, H2, H3, H7, H8 protons merged to appear in the range of δ 6.82-6.64 ppm. A singlet appeared at δ 5.36 ppm has been assigned for H1’ H2’ (aliphatic -CH2) resonances. Upon the gradual addition of Hg2+ ions (0-1 equiv.) to a solution of L1 (Fig. 7) most of the resonating signals shifted toward the downfield region. However the Ha and Hc protons of the picolyl unit were separated at δ 6.84 and 6.68 ppm (∆δ = 0.2-0.03) respectively while, H5 and H6 resonances of the pyrene unit merged at δ 7.98 ppm (∆δ=0.62). Similarly, H2’ and H1’resonances separated from δ 5.36 ppm to appear at δ 6.15 (∆δ=0.79) and 5.74 (∆δ=0.38) ppm respectively. These results suggest 9
that a cyclization reaction has occurred in the presence of Hg2+ to generate a new derivative 1. Subsequently compound 1 was isolated and characterized by spectral data analysis (Fig. S5-S7, ESI). 13
C NMR spectra of L1 showed typical resonance of thiol (–C=S) carbon at δ
214.9 ppm. The pyridine ring carbons a e, c, d and b were resonated at, δ 155.4, 152.9, 122.2 and 119.4 ppm respectively. The resonances appeared in the region of 139.6 122.3 ppm attributed to a pyrene ring carbons and the carbons of –CH2 (1’ and 2’) linker arm appeared at δ 46.3 ppm respectively (Fig. S2, ESI). Further the 13C NMR spectrum of cyclised species 1 showed resonance at 210.8 ppm attributed to thiol carbon (–C=S), while the CH2 1’’ and 2’’carbons appeared at the same place 46.3 ppm in L1 gets separated to appear at 100.6 and 53.5 ppm due to 2’ and 1’ carbons respectively. The carbons of pyridine ring c d and a appears at 148.8, 147.6 ppm respectively while e and b carbons appeared at 108 and 107.7 ppm respectively. Pyrene ring carbons resonate in the region of 135-120.7 ppm (Fig. S6, ESI). A molecular ion peak (ESI-MS) observed at m/z 365.4 (Fig. S7, ESI) further supports the formation of cyclised entity 1. 3.4. UV-vis. and fluorescence spectra of Model compound L2 L2 exhibits n-π* transition band at 360 nm (ε =1.16 x 103 M-1cm-1) and π-π* transition band at λmax 297 nm (ε = 3.9 x 103 M-1cm-1) respectively. When excited at λex 360 nm show an emission band at 502 nm. The spectral study of L2 shows similar changes on interaction of Hg2+ ions. L2 shows selective changes with only Hg2+ metal ions (1 equiv) and forms a new band at 335 nm by merging of two absorption bands and in the emission spectra, exhibits 94% quenching in the emission intensity with a blue shift of 4 nm (Fig. 8, S16-17, ESI). The emission intensity of L2 shows linear decrease upto 1 equiv and with further addition of Hg2+ ions (2-10 equiv.) the emission intensity remains constant (Fig. S18a). The estimation of quenching efficiency was calculated by a Stern-Volmer plot and the value of quenching constant was found to be 2.5 x 105 M-1 (Fig. S18b, ESI). Jobs plot analysis also revealed a 1:1 stoichiometry for the interaction of L2 with Hg2+ ions like in L1 (Inset, Fig. 8). Reversibility experiment with EDTA shows the irreversible nature of binding of L2 and with Hg2+ ions (Fig. S19-20, ESI). The single crystal X-ray analysis revealed the formation of a new cyclised species (2) (Fig. S10, ESI) in case of L2. The formation of the cyclised entity were attributed due to the 10
probable attack of the nitrogen atom on the electron deficient carbon atom linked to sulphur atoms at position-2 of the pyridine moiety (L1 and L2) with the elimination of HgS as shown in Scheme 2. Initially the interaction of the 2- substituted probes L1 and L2 with Hg2+ ions, may involves two units, which favors the stereo chemically facile nucleophillic attack of pyridine lone pair with elimination of HgS to the electron deficient carbon. Further the charge of the ring was stabilized by the formation of double bond between the carbon of five and six membered rings and leads to perturbation in the electronic environment of picolyl unit which was evidenced by the crystal structure in case of 2 (Fig. S10, ESI) and by 1H NMR titration experiment and ESI-MS spectral analysis in case of 1. 3.5. Strips of Probe To confirm the practical application of L1 for the Hg2+ detection the cellulose paper strips (WhatmanTM) of L1 were prepared at three different concentrations (2.5 mM, 1 mM and 0.5 mM) were prepared (1.5 x 0.5 cm2) in water and dried in air. The dried test strips were dipped separately into different concentrations (3 x 10-6, 3 x 10-7 and 3 x 10-8 M) of Hg(NO3)2 solution in water for 10 min and dried in air. The fluorescent green strips changed to light blue under UV light at 365 nm (Fig. 10) thus indicating that the synthesized probe could also detect Hg2+ ions on cellulose paper strips. 3.6. Detection of Hg2+in BSA (Bovine serum albumin) To test the applicability of L1 for detection of Hg2+ ions in protein medium we have added BSA (Bovine serum albumin) to an aqueous solution of L1. The gradual addition of BSA (0-20 µM) leads to an enhancement ~ 2.5 fold in the emission intensity of L1 with a blue shift to appearing at 472 nm (Δλ = 18 nm) (Fig. 11a). This enhancement in emission intensity on addition of BSA was attributed to the movement of L1 to the hydrophobic cavity of BSA [28]. Job’s plot analysis by continuous variation method shows 1:1 stoichiometry (Fig. 11b) for L1 and BSA and the B-H plot (Fig. 11c) for this binding gives the value of Kass =1.73 x 104 M-1 (Binding Constant). Further the addition of Hg2+ ions to a solution of L1 + BSA (5 µM) decreases the emission intensity at 490 nm thereby showing two shoulders at 402 and 424 nm which corresponds to the monomer emission of the pyrene (Fig. 12). The addition of 5 µM of BSA to L1 leads to 1.6 fold enhancement. The addition of Hg2+ ions to this solution decreased the emission 11
intensity to nearly half in comparison to L1 thus indicating Hg2+ triggered cyclization of L1 in the protein medium. 3.7. Optical circuits to construct Logic Gate To perform the logic operations, the Hg2+ and BSA were considered as inputs for the present system that leads to the construction of TRANSFER logic gate at 490 nm wavelength. For the construction of this logic gate, presence and absence of inputs were indicated by the ‘1’ and ‘0’ digits entry in the truth table. We have chosen 0.5 ratio of maximum intensity as a threshold value on 490 nm, emission band of the probe. The values of relative emission intensities at or above 50 % were assigned as ‘1’ digital value (high) while below 50% were assigned as ‘0’ digital values (low). In the absence of both inputs (0 0) or in the presence of only BSA, the relative intensities remain high (1) above 50 % at 490 nm (Fig. 12, Table), while in the presence of only Hg2+ ions or both inputs BSA and Hg2+ ions, low output fluorescence was observed. The values of the corresponding output of relative intensity of the input combination fit to the truth table of TRANSFER logic gate. Thus the designed probe L1 exhibits TRANSFER logic gate with the applied inputs of BSA and Hg2+ ions in aqueous medium. As L1 is a chemodosimeter for Hg2+ ions, the constructed TRANSFER gate is applicable for one time in solution of L1 by the applied inputs of BSA and Hg2+ ions and is not reusable. 3.8. Fluorescence imaging for NIH3T3 cells The excellent selectivity and sensitivity of L1 towards Hg2+ in aqueous solution displays its potential to monitoring Hg2+ in living cells. Therefore for imaging experiments, cells were trypsinized and plated on glass coverslips. After two days cultured cells were exposed to 200 µM Hg(NO3)2·H2O and 50 µM Probe in DMEM for 15 minutes; after that cells were mounted in PBS. Fluorescent imaging experiments of live NIH3T3 cells mounted in PBS were performed with Zeiss LSM 510 Meta confocal microscope with excitation at 488 nm and emission at 505-550 nm (Fig. 13a). Live NIH3T3 cells treated with this compound in DMEM for 15 minutes at 37 °C, showed intracellular cytoplasmic fluorescence (Fig. 13b) which was slightly quenched when incubated along with 200 µM Hg(NO3)2·H2O (Fig. 13c). These results demonstrate that L1 can be successfully used as a biosensor to detect Hg2+ ions in biological samples.
12
Conclusion Conclusively through the present contribution we have demonstrated the potential application of probe L1 as an efficient chemodosimeter to detect Hg2+ in pure aqueous medium. The probe has shown high sensitivity for Hg2+ (5.2 ppb) with approximate detection time of 50 seconds through an irreversible reaction in which mercury triggered cyclization led to the formation of a new imidazole moiety. The proposed mechanism of cyclization was confirmed by NMR and ESI-MS data as well as crystal structure of cyclised entity 2 of model compound L2. L1 is highly permeable to detect Hg2+ in living cells also. L1 also exhibits TRANSFER logic gate by applying Hg2+ and BSA as chemical inputs. Moreover, the studies widen the scope of dithiocarbamate based probes to detect and scavenge Hg2+ ions in aqueous medium. Acknowledgements We gratefully acknowledge the financial support from the Council of Scientific and Industrial Research (CSIR), New Delhi in the form of CSIR Scheme No: 01(2679)/12/EMR-II (NS) and SRF (V.S and P.S).
Supplementary data Synthesis, 1HNMR, 13CNMR, ESI-MS, UV-Vis. and fluorescence spectra.
13
References [1] A. W. Czarnik, Am Chem. Soc. Washington, D.C., 1993. [2] A. P. deSilva, D. B. Fox, A. J. M. Huxley, T. S. Moody, Coord. Chem. Rev. 205 (2000) 41-57. [3] A. Renzoni, F. Zino, E. Franchi, Envir. Res. 77 (1998) 68-72. [4] E.M. Nolan, S. J. Lippard, Chem. Rev. 108 (2008) 3443-3480. [5] D.W. Boening, Chemosphere 40 (2000) 1335-1351. [6] P. Grandjean, P. Weihe, R. F. White, F. Debes, Envir. Res. 77 (1998) 165-172. [7] W. S. Han, H. Y. Lee, S. H. Jung, S. J. Lee, J. H. Jung, Chem. Soc. Rev. 38 (2009) 1904–1915. [8] (a) D. S. McClure, J. Chem. Phys. 20 (1952) 682-686; (b) M. Yu, M. Shi, Z. Chen, F. Li, X. Li, Y. Gao, J. Xu, H. Yang, Z. .Zhou, T. Yi, C. Huang, Chem. Eur. J. 14 (2008) 6892-6900; (c) G.-K. Li, Z.-X. Xu, C.-F. Chen, Z. T. Huang, Chem. Commun. (2008) 1774-1776. [9] (a) R. Metiver, I. Leray, B. Valeur, Chem. Commun (2003) 996-997; (b) J. Y. Kwon, Y. J. Jang, Y. J. Lee, K. M. Kim, M. S. Seo, W. Nam, J. Yoon, J. Am. Chem. Soc. 127 (2005) 10107-10111. [10] J. Ros-Lis, M. D. Marcos, R. Mártinez-Mánez, K. Rurack, J. Soto, Angew. Chem. Int. Ed. 44 (2005) 4405-4407. [11] (a) P. Srivastava, R. Ali, S. S. Razi, M. Shahid, A. Misra, Sens. Actuators B 181 (2013) 584-595; (b) P. Srivastava, R. Ali, S. S. Razi, M. Shahid, S. Patnaik, A. Misra, Tetrahedron. Lett. 54 (2013) 3688-3693; (c) A. Misra, P. Srivastava, M. Shahid, Analyst 137 (2012) 3470-3478; (d) P. Srivastava, M. Shahid, A. Misra, Org. Biomol. Chem. 9 (2011) 5051-5055; (e) A. Misra, M. Shahid, P. Srivastava, Sens. Actuators B 169 (2012) 327-340. [12] (a) B. Liu, H. Tian, Chem. Commun. (2005) 3156–3158; (b) J.-S. Wu, I.-C. Hwang, K. S. Kim, J. S. Kim, Org. Lett. 9 (2007) 907-910. [13] D.-G. Cho, J. L. Sessler, Chem. Soc. Rev. 38 (2009) 1647-1662. [14] M. J. Choi, Y. H. Kim, J. E. Namgoong, S.-K. Chang, Chem. Commun. (2009) 3560-3562. 14
[15] J. Wang, X. Qian, J. Cui, J. Org. Chem. 71 (2006) 4308-4311. [16] Y. Zhou, C.-Y. Zhu, X-S. Gao, X.-Y. You, C. Yao, Org. Lett. 12 (2010) 2566-2569. [17] J. C. Manimala, E.V. Anslyn, Eur. J. Org. Chem. (2002) 3909-3922. [18] K. C. Song, J. S. Kim, S. M. Park, K.-C. Chung, S. Ahn, S.-K. Chang, Org. Lett. 8 (2006) 3413-3416. [19] J. Du, M. Hu, J. Fan, X. Peng, Chem. Soc. Rev. 41 (2012) 4511-4535. [20] B. Valeur, I. Leray, Coord. Chem. Rev. 205 (2000) 3-40. [21] (a) S.-M. Cheung, W.-H. Chan, Tetrahedron 62 (2006) 8379-8383; (b) C. Chen, R. Wang, L. Guo, N. Fu, H. Dong, Y. Yuan, Org. Lett. 13 (2011) 11621165. [22] T. Nagano, T. Yoshimura, Chem. Rev. 102 (2002) 1235-1269. [23] (a) M. Lerchi, E. Reitter, W. Simon, E. Pretsch, D. A. Chowdhury, S. Kamata, Anal. Chem. 66 (1994) 1713-1717; (b) J. Hatai, S. Pal, G. P. Jose, S. Bandyopadhyay, Inorg. Chem. 51 (2012) 10129-10135. [24] (a) X-ray crystallographic data and ccdc number were submitted for publication elsewhere; (b) V. Singh, A. Kumar, R. Prasad, G. Rajput, M. G. B. Drew, N. Singh, CrystEngComm 13 (2011) 6817-6826. [25] J. D. Ingle Jr, J. Chem. Educ. 51 (1974) 100. [26] G. L. Long, J. D. Winefordner, Anal. Chem. 55 (1983) 712A. [27] H. A. Benesi, J. H. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703-2707. [28] (a) A. Mallick, B. Haldar, N. Chattopadhyay, J. Phys. Chem B. 109 (2005) 14683; (b) A. Chakrabarty, A. Mallick, B. Haldar, P. Das, N. Chattopadhyay, Biomacromolecules 8 (2007) 920; (c) K. A. Willets, O. Ostroverkhova, M. He, R. J. Twieg, J. Am. Chem. Soc. 125 (2003) 1174; (d) A. Wu, Y. Xu and X. Qian, Bioorg. Med. Chem. 17 (2009) 592.
15
Figure captionsScheme 1. (1) EtOH/ Reflux (ii) NaBH4/ MeOH (iii) KOH/ CS2/0˚C (iv) Hg(NO3)2/ MeOH-H2O. Fig.1. Interaction study of L1 upon addition of different metal ions by (a) absorption (b) emission spectral changes in aqueous 0.01 M HEPES buffer (pH 7).Inset: Bar diagram of interaction study with different metal ions. Fig. 2. Interference study of L1 by (a) absorption (b) emission spectral changes upon addition of different metal ions in aqueous 0.01 M HEPES buffer (pH 7). Inset: Bar diagram of interference study of L1 + Hg2+with different metal ions. Fig. 3. (a) Change in color of solutions of L1 with the test metal ions ( Na+, K+, Ca2+,Zn2+, Cd2+, Co2+, Ni2+, Pb2+, Cu2+, Hg2+, Mg2+, Fe2+, Ag2+ ,Ba2+, Sr2+); (b) under 354 nm UV light. Fig. 4. (a) Absorption (b) Emission titration spectra of L1 upon addition of Hg2+ ions (01 equiv) in aqueous 0.01 M HEPES buffer (pH 7). Fig. 5. (a) Jobs plot analysis of L1 for Hg2+ ions (b) Stern-Volmer plot for the estimation of quenching constant of L1 with Hg2+ ions. Fig. 6. (a) Calibration curve of Intensity with respect to concentration of L1 (b) Plot of change in intensity with respect to the Hg2+ ions concentration (where ΔI shows the changes in the emission intensity on each addition). Fig. 7. 1H NMR titration spectra of L1 upon addition of Hg2+ ions (0- 1.0 equiv) in DMSO-d6. Fig. 8. (a) Absorption (b) Emission titration spectra of L2 upon addition of Hg2+ ions (01 equiv) in aqueous 0.01 M HEPES buffer (pH 7). Inset: Jobs plot analysis of L2 for Hg2+ ions. Fig. 9. Mechanism of cyclization of L1 to cyclised entity 1. Fig. 10. Fluorescent paper strips of L1 (a) 2.5 mM (b) 1 mM (c) 0.5 mM (Green) and after addition of Hg2+ (a) 3 × 10-6 (b) 3 × 10-7 (c) 3 × 10-8 M (non fluorescent). Fig. 11. (a) Emission titration experiment of L1 upon addition of BSA (0- 40 µM) (b) Jobs plot analysis of L1 for Hg2+ ions (c) B-H plot of L1 with BSA in aqueous 0.01 M HEPES buffer (pH 7). Fig. 12. Relative emission intensity of the applied inputs. Inset: Bar diagram of relative intensity of respective inputs at 490 nm wavelength in aqueous 0.01 M HEPES buffer (pH 7) and logic symbol and truth table of TRANSFER logic gate. 16
Fig. 13. Confocal fluorescence microscopic images of (a) NIH3T3 cells (b) NIH3T3 cells with 50 μM L1 and (c) with 200 μM Hg2+ and 50 μM L1.
17
Highlights • • • •
Efficient chemodosimeter to detect Hg2+ ions in pure aqueous medium. Hg2+ triggered cyclisation and formation of imidazoline species. Probe exhibit both colorimetric and fluorometric changes Probe is applicable to detect Hg2+ in live cells and on cellulose paper strips.
18
Scheme 1. (1) EtOH/ Reflux (ii) NaBH4/ MeOH (iii) KOH/ CS2/0˚C (iv) Hg(NO3)2/ MeOHH2O.
(a)
L1 + M ions
160 2+
Intensity ( a.u. )
Absorbance
L1 + Hg ions 0.5
0.0
300
350
400
450
Wavelength ( nm )
(b)
L1 + M ions 120
120
500
80
I
1.0
80
40 2+
L1 + Hg ions
40
0
0
420
480
540
600
K Na Ag Pb Zn Cu Ca Co Ni Sr BaMg Fe Hg Cd
660
Wavelength ( nm )
Fig.1. Interaction study of L1 upon addition of different metal ions by (a) absorption (b) emission spectral changes in aqueous 0.01 M HEPES buffer (pH 7).Inset: Bar diagram of interaction study with different metal ions.
L1
160 2+
Absorbance
L1 + Hg + M ions 0.5
0.0
350
400
450
Wavelength ( nm )
120 80 40 0
300
L1
(b)
,
(a)
Intensity ( a.u. )
1.0
500
2+
L1 + Hg + M ions
420
480
540
600
+J . 1D $J 3E =Q &X &D &R 1L 6U %D 0J )H &G
660
Wavelength ( nm )
Fig. 2. Interference study of L1 by (a) absorption (b) emission spectral changes upon addition of different metal ions in aqueous 0.01 M HEPES buffer (pH 7). Inset: Bar diagram of interference study of L1 + Hg2+with different metal ions.
L1
Na+ K+ Ca2+ Zn2+ Hg2+ Cu2+ Co2+ Ni2+ Ag2+Fe2+ Cd2+ Mg2+ Ba2+ Sr2+ Pb2+
(a)
L1 Na
K
Ca2 Zn2
Hg2+ Cu2
2+ 2+ 2+ 2+ Co2 Ni2 Ag2 Fe2 Cd Mg Ba Sr
Pb2
(b)
Fig. 3. (a) Change in color of solutions of L1 with the test metal ions ( Na+, K+, Ca2+,Zn2+, Cd2+, Co2+, Ni2+, Pb2+, Cu2+, Hg2+, Mg2+, Fe2+, Ag2+ ,Ba2+, Sr2+); (b) under 354 nm UV light.
160
(a)
2+
Hg
0.5
0.0
300
350
400
Wavelength ( nm )
Intensity ( a.u. )
Absorbance
1.0
450
(b) Hg
120
2+
80 40 0
420
480
540
600
660
Wavelength ( nm )
Fig. 4. (a) Absorption (b) Emission titration spectra of L1 upon addition of Hg2+ ions (0- 1 equiv) in aqueous 0.01 M HEPES buffer (pH 7).
5.0
Io/I
I
140 (a)
70
0 0.0
0.2
0.4 2+
0.6
0.8 2+
[ Hg ] / [ L1 + Hg ]
(b)
KS-V= 71000 / M
2.5
1.0
0
10
20
30
40
50
2+
[Hg ] M
Fig. 5. (a) Jobs plot analysis of L1 for Hg2+ ions (b) Stern-Volmer plot for the estimation of quenching constant of L1 with Hg2+ ions.
Fig. 6. (a) Calibration curve of Intensity with respect to concentration of L1 (b) Plot of change in intensity with respect to the Hg2+ ions concentration (where ΔI shows the changes in the emission intensity on each addition).
Fig. 7. 1H NMR titration spectra of L1 upon addition of Hg2+ ions (0- 1.0 equiv) in DMSO-d6.
Fig. 8. (a) Absorption (b) Emission titration spectra of L2 upon addition of Hg2+ ions (0-1 equiv) in aqueous 0.01 M HEPES buffer (pH 7). Inset: Jobs plot analysis of L2 for Hg2+ ions.
N N
S
S
Hg2+ N N
S
S N
N
S
H
N N
Hg(NO3)2
S
S LH H
L1
-HgS
N
N
S S
Hg2+
Fig. 9. Mechanism of cyclization of L1 to cyclised entity 1.
1
Fig. 10. Fluorescent paper strips of L1 (a) 2.5 mM (b) 1 mM (c) 0.5 mM (Green) and after addition of Hg2+ (a) 3 × 10-6 (b) 3 × 10-7 (c) 3 × 10-8 M (non fluorescent).
160
300 (a)
I
200
80
100 0.0
0.2
0.4
0.6
0.8
[BSA] / [ L1 + BSA]
0
420
480
540
(c)
0.08
120 1/I
BSA Intensity ( a.u. )
0.12 (b)
1.0
0.04
0.00 0.0
0.1
0.2
0.3
0.4
1 / BSA (M)
600
Wavelength ( nm ) Fig. 11. (a) Emission titration experiment of L1 upon addition of BSA (0- 40 µM) (b) Jobs plot analysis of L1 for Hg2+ ions (c) B-H plot of L1 with BSA in aqueous 0.01 M HEPES buffer (pH 7).
Hg2+ (In1) 0 1 0 1
BSA (In2) 0 0 1 1
O (490 nm) 1 1 0 0
Fig. 12. Relative emission intensity of the applied inputs. Inset: Bar diagram of relative intensity of respective inputs at 490 nm wavelength in aqueous 0.01 M HEPES buffer (pH 7) and logic symbol and truth table of TRANSFER logic gate.
Fig. 13. Confocal fluorescence microscopic images of (a) NIH3T3 cells (b) NIH3T3 cells with 50 μM L1 and (c) with 200 μM Hg2+ and 50 μM L1.