Synthesis of Resorufin Based Chromogenic and Fluorescent Probe for Detection of Mercury

Milind Shamrao Thakare1* and Pravin Madhavrao Yeole 2

1 &* Synthetic Organic Research lab, Department of chemistry, Pratap college Amalner, Maharashtra, INDIA

2 P. G. Research Lab, R. L. College Parola, Maharashtra, INDIA

* Correspondence: E-mail:

(Received 18 Aug, 2018; Accepted 09 Sept, 2018; Published 19 Sept, 2018 )

ABSTRACT: Mercury in its lipophilic and inorganic form is a major threat to the environment and to human health. It is therefore highly necessary to develop a chemosensor for mercury detection. A novel ?uorescent probe for mercury, 7-(vinyloxy)-3H-phenoxazin-3-one has been designed and synthesized by anchoring resoru?n (helping as a ?uorophore ) with vinylic ether moiety. In present work we have designed a ?uorescence strategy based on the p-electrophilicity. The Probe is essentially nonfluorescent which upon reaction with Hg(II) undergoes oxymercuration of vinylic doublebond followed by cleavage, complemented by the generation of strong ?uorescence to detect traces of mercury in biological samples. The resorufin based probe responds selectively to mercury species, while reluctant or little responsive toward other analyte metal ions. Significant colorimetric and fluorescence change from colourless to pink through the anchoring and unanchoring process is being observed.

Keywords: Mercury; Resorufin; Chemosensor; Chromogenic and Fluorescence.

INTRODUCTION: Mercury contamination is still a universal problem due to geological events and the extensive use of mercury species in modern human activities.1-4 Mercury exists in numerous forms metallic, organic, and inorganic to which people may be exposed through their occupation.5-10 Elemental and lipophilic organo-mercury are toxic to the central and peripheral nervous systems. The inhalation of mercury vapours can produce damaging effects on the nervous, digestive and immune systems, lungs and kidneys,11-14 and may be fatal. The inorganic salts of mercury are corrosive to the skin, eyes and gastrointestinal tract, and may induce kidney toxicity if consumed .Ingestion of methylmercury-contaminated fish and grain triggered the epidemics in Japan and in Iraq specifically known as Minamata disease. 15-16 The toxicity of Hg2+ in human caused by the coordination with biological ligands such as proteins, DNA and enzymes 17-18 due to its affinity towards thiol group. Its significant hazards to public health because of its presence in drinking water.

Considering the facts associated with mercury poisoning, efficient detection and quantification tools for mercury species which have been developed for evaluating their contamination in the environment and living species. For instance, mercury is generally quanti?ed by Cold vapor atomic absorption spectroscopy19-20 or Inductively coupled plasma mass spectroscopy21-22 or Gas chromatography 23. Although these methods are quantitative and powerful, the analyses require large and expensive instruments, highly trained personnel, and tedious maintenance. Other methods which have been reported were based on various strategies like reaction based strategy and complexation based strategies, with different fluorophores like Fluorescein, Coumarin , Rhodamine, Pyrene.24-33 The development of rapid, cost-effective colorimetric sensors for the easy and fast detection of toxic metal ions by the naked eye without resorting to any expensive instruments is still an active ongoing research area.

A chemodosimetric approach based on mercury-triggered reaction has emerged as a powerful tool in this area. These reaction-based probes can provide superior selectivity towards Hg2+ with large spectroscopic changes. In the present work, we devised a Hg-responsive fluorogenic probe whose molecular composition is elaborately designed to make the inner chemical environment optimal for the sensing mercury.

In continuation of our work on metal sensing using fluorescent probes. 34 We herewith described a colorimetric and fluorometric mercury assay system which is visible to the naked-eye, and this method can be used as Hg-monitoring system. Probe was designed based on resorufin as the chromophore, vinylic ether group as the cleavable group through mercury initiated hydrolysis reaction. Resorufin is selected as the chromophore based on its excellent chromogenic and fluorogenic signal behaviors. Resorufin is a strong pink fluorescent dye with maximal emission, is visible to the naked-eye as a pink color, has good water-solubility, and can be applied in biological analysis with easy signal readout and nontoxic properties, and is used to analyze intracellular processes without cell damage. The alkylation of the hydroxy of resorufin effectively quenches its fluorescence emission.

MATERIAL AND METHODS: All reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified and dried by standard methods. Redistilled water was used throughout all experiments. All reactions were carried out in oven or flame dried vials with magnetic stirring under nitrogen atmosphere. Dried solvents and liquid reagents were transferred by oven-dried syringes or hypodermic syringe cooled to ambient temperature in a desiccators. All experiments were monitored by analytical thin layer chromatography (TLC). TLC was performed on pre-coated silica gel plates. After elution, plate was visualized under UV chamber. Further visualization was achieved by staining KMnO 4 and charring on a hot plate.

1 H NMR spectra and 13C NMR spectra were recorded with Bruker AV, 200/400/500 MHz spectrometers in appropriate solvents using TMS as internal standard or the solvent signals as secondary standards and the chemical shifts are shown in d scales. Multiplicities of 1H NMR signals are designated as s (singlet), br. s (broad singlet), d (doublet), dd (doublet of doublet), t (triplet) and m (multiplet) All measurements were carried out at room temperature. Fluorescence spectra measurements were performed with a Perkin–Elmer UV/Vis Spectrophotometer and a Photon Technology International, Quanta Master 400 Spectrofluorometer, respectively, in degassed spectral grade solvents.

Design and Synthesis of Probe: Alkylation of resorufin (Scheme: 1) followed by base catalyzed dehydrohalogenation reaction (Scheme: 2) offers desired probe which on mercury promoted hydrolysis (Scheme 3) selectively converts nonfluorescent molecule to fluorescent molecule.

General procedure for synthesis of Probe:

Step 1: Synthesis of 7-(2-bromoethoxy)-3H-phenoxazin-3-one (Compound-A) - To a solution of resorufin (1 mmol) and K2CO3 (2 mmol) in DMF was added 1,2-dibromopropane (1 mmol) in one portion under an atmosphere of N2. The reaction mixture was stirred at 60°C overnight. After evaporation of the solvent, the resulting light brown material was dissolved in DCM (100 mL). After removal of insoluble materials by Celite filtration, the filtrate was concentrated to give the product from which desired product was isolated as a solid by silica-gel column chromatography (eluent: hexane:EA = 1:1) in 60 % yield.


NMR (CDCl3, 500 MHz): d (ppm) = 3.63 (2 H, t, J = 6.3 Hz,), 3.72 (2 H, t, J = 5.7 Hz), 6.33 (1 H, d, J = 1.8 Hz), 6.82-6.86 (2 H, m), 6.95 (1 H, dd, J = 9.1, 1.8 Hz), 7.43 (1 H, d, J = 9.7 Hz), 7.72 (1 H, d, J = 9.1 Hz) ,13C NMR (CDCl3, 125 MHz): d (ppm) = 29.6 , 66.5, 100.9, 107.0, 114.1, 128.7, 131.8, 134.5, 134.9, 145.8, 145.9, 150.0, 162.8, 186.5; FT-IR 1606.7 cm–1 (C=O). M.S.:[M + Na]+: 353.0. Found: 353.1.

Step 2: Synthesis of 7-(vinyloxy)-3H-phenoxazin-3-one (probe) - To a stirred solution of compound A (1mmol) in CH3CN (10 ml) was added DBU(1,8 diazabicyclo[5.4.0]undec-7-ene) (2 mmol). The mixture was stirred at 90°C for 12 h under N2 where the reaction was completed by TLC monitored. Then the solvent was evaporated by vacuum. Water (20 ml) was added and the solution was extracted with DCM (20 ml*3). The combined organic layers was washed with brine, dried over Na 2SO4 and evaporated under vacuum. The residue was purified by silica gel column chromatography to give dsired probe as paly solid.

1H NMR (400 MHz, CDCl3) d 7.74 (d, J = 8.7 Hz, 1H), 7.36 (d, J = 9.8 Hz, 1H), 7.18 (s, 1H), 7.15 (dd, J = 8.7, 2.5 Hz, 1H), 6.80 (dd, J = 9.8, 2.0 Hz, 1H), 6.27 (d, J = 2.0 Hz, 1H), 6.02-5.88 (m, 1H), 5.44-5.36 (m, 1H), 5.31 (dd, J = 10.4, 1.1 Hz, 1H), 13C NMR (101 MHz, CDCl3) d 186.24, 153.57, 152.40, 149.26, 144.37, 135.24, 134.80, 131.31, 131.23, 130.65, 120.14, 118.54, 109.09, 107.33 HRMS (ESI): calcd for (M+Na)+ 262.23.


Spectral Characterization of Compound-A and Probe:


Figure 1: 1H NMR of compound A.


Figure 2: 13C NMR of compound A.


Figure 3: 1H NMR of Probe.


Figure 4: 13 C NMR of probe.


Response of probe 1 to mercury sensing experiment: Probe 1 was prepared by the reaction of the 7-hydroxy group of resorufin 1,2 dibromoethane. This on later treatment with DBU undergoes 1-2 elimination to produce desired probe in good to moderate yield. The absorption and fluorescence spectra of probe before and after reaction with Mercury are shown in Figure 5 .Probe 1 exhibited a very weak absorption in the long wavelength region. However, upon reaction with Hg++ (Scheme 3), its solution not only displayed a distinctive color change from nearly colorless to pink (Figure 5), but also produced strong signal at 575 nm. Moreover, probe 1 itself was weakly fluorescent, this low background signal was due to the strong quenching effect of the vinylic ether unit. However, its reaction with mercury in (Figure 6) fluorescence enhancement. The fluorescence enhancement of probe was attributed to the mercury-triggered cleavage reaction causing the release of free resorufin ? emission 595nm.



Figure 5: Absorption spectra at 250 C in PBS buffer (10 mmol /L, pH 7.4, 30%CH3CN, v/v).clearly shows that the probe alone with PBS buffer merely shows negligible absorbance but on addition of Hg++ there is tremendous increase in absorption at 575 nm.


Figure 6: Fluorescence changes of probe 1 in presence of Hg (0-100 µM) in buffer ?emission 595nm,linearity plot of fluorescence emission after gradual addition of Hg++.

Mechanistic study:


CONCLUSION: In conclusion, we have proposed a new approach consisting of protection and deprotection of fluorophore for the recognition of Hg(II), which now a days a widely spread contamitant in ecosystem. The present probe is highly sensitive and selective enough in determination of mercury .We strongly believe that this approach of detection of notorious Hg (II) will definitely catch the attention scientific community and henceforth many probes based on this strategy may appear in near future The system described herein is highly efficient and highly sensitive in recognition of Hg(II) which represents a simple vinylic ether anchored Resorufin based fluorescent probe for Hg(II) ions, it combines the selectivity of Hg(II) ef?cient cleavage of vinylic ether bonds with the high sensitivity of ?uorescence detection. In summary, the novel chromogenic and fluorescent probe based on the release of resorufin upon addition of Hg(II),the probe displayed drastic changes in UV-vis absorption and fluorescence emission intensities selectively for Hg(II).We expect that this probe shall be further useful in identification of Hg(II) at cellular level.

ACKNOWLEDGEMENT: The author sincerely thanks Principal Pratap College, Amalner and Principal, R. L. College, Parola for providing necessary facilities.


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