Synthesis of a dihydroquinoline based fluorescent cyanine for selective, naked eye, and turn off detection of Fe³⁺ ions

Abstract

A cyanine dye was synthesized by condensing 6-formylated 1,2,2,4-tetramethyl-1,2-dihydroquinoline (TMDQ) and 1,7-dimethyl-2-phenylimidazo[1,2-a]pyridin-1-ium iodide. The probe was a selective sensor for ferric ions in aqueous ethanolic solution. The sensor was fluorescent and the fluorescence was quenched by Fe³⁺ ions. The decrease in the fluorescence was quick and linear with respect to Fe³⁺ ions. The quenching was selective and no other metal ions tested showed this effect. The colour change could be detected visually and hence it is a naked eye and turn-off fluorescence detector for Fe³⁺ ions.

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Introduction

In industrial, environmental, medical, and biological applications, quantitative measurement and real-time monitoring are necessary for analytes such as metal ions,¹ and toxic chemicals. For this sake, fluorescence detection is very useful as it has high sensitivity, selectivity, and a fast response time. Furthermore, fluorescence detection does not lead to any photodamage of cells and hence has recently attracted substantial bio-analytical interest.

In biology, metals play a vital role. A key element for electron transport in the respiratory system is iron.² Iron is a necessary component in hemes, many enzymatic reactions, and iron sulfur clusters. In ensuring the quality of water, measuring the level of ferric ions (Fe³⁺) is important. The major portion of bioiron is firmly connected with enzymes and specialized transport and storage proteins.³ A minor fraction of bioiron is bound relatively loosely to species like organic anions (phosphates and carboxylates), polyfunctional ligands (chelates, siderophores, and polypeptides), or extracellular matrix (glycans and sulfonates).⁴ The latter is labile iron and associated with important functions such as a source of iron for incorporation into proteins, target for chelators or metal scavengers. Labile bioiron is chelatable and therefore metal-sensing devices bearing chelating moiety can detect them.⁴

In human being, iron level should be balanced. If the iron content increases, it spoils the biological system by catalyzing the generation of highly reactive oxygen species. This is called as iron poisoning, with symptoms such as diarrhea, vomiting, stomach pain, and heart/liver damage.⁵ On the other hand, iron deficiency leads to anemia.⁶ Hence quantification of iron is very important in biology. Techniques like ICP-MS⁷ and FAAS⁸ and spectrophotometric detection using organic dyes⁹ or quantum dots¹⁰ are used for iron quantification. These techniques have disadvantages like sophisticated instrumentation, complicated synthetic procedures for the fluorescent detecting probe, tiresome buffer making procedures etc. Fluorescent probes are preferred over others since they are rapid and reliable and can offer on-site and real-time detection of the analyte which is under investigation. There are numerous reports on ferric ion sensing.^11–23 But only few are on fluorescent probes for ferric ion.

Cyanines have conjugated structure and were initially used in photography as developers. Later on they have been used for varied applications.²⁴ Many cyanines contain quinoline moiety.²⁵ Recently, we reported a dihydroquinoline based merocyanine which is a probe for highly toxic hydrazine.²⁶ Our continued work on cyanines led to a new dihydroquinoline based cyanine which is a rapid, selective, and sensitive probe for Fe³⁺.

Herein we would like to report the synthesis of (E)-1-methyl-2-phenyl-7-(2-(1,2,2,4-tetramethyl-1,2-dihydroquinolin-6-yl)vinyl)imidazo[1,2-a]pyridin-1-ium iodide (8) which is a new cyanine dye. It is a conjugate of 1,2,2,4-tetramethyldihydroquinoline and 1,7-dimethyl-2-phenylimidazo[1,2-a]pyridin-1-ium iodide. The cyanine effectively and proficiently senses ferric ions. It acts as a fluorescent sensor for ferric ion in an aqueous alcoholic medium. It is easy to synthesize, eco-friendly, sensitive, and selective. The sensing can be done simply in an ethanol–water system. It does not involve any complicated buffer-making.

Results and discussion

2-Amino-4-picoline (1) and phenacyl bromide (2) were condensed in ethanol in the presence of concentrated HCl to obtain 7-methyl-2-phenylimidazo[1,2-a]pyridine (3). 3 was quaternized using methyl iodide under microwave irradiation to obtain 1,7-dimethyl-2-phenylimidazo[1,2-a]pyridin-1-ium iodide (4). Aniline was condensed with acetone to obtain 1,2,2,4-tetramethyl-1,2-dihydroquinoline (5). It was methylated using methyl iodide in DMF to obtain 6 which was formylated using DMF/POCl₃ according to the reported procedure to 1,2,2,4-tetramethyl-1,2-dihydroquinoline-6-carbaldehyde (7).^27,28 4 and 7 were condensed in the presence of piperidine in n-butanol to obtain probe 8 in 65% yield (Scheme 1). The structure of probe 8 was confirmed by ¹H NMR and ¹³C NMR as well as by ESI-MS. It was an orange coloured powder and soluble in many organic solvents.

Scheme 1 Synthesis of (E)-1-methyl-2-phenyl-7-(2-(1,2,2,4-tetramethyl-1,2-dihydroquinolin-6-yl)vinyl)imidazo[1,2-a]pyridin-1-ium iodide (8).

Fluorescence study

The cyanine probe 8 offered longer electronic conjugation together with rigid structure. A solution of the probe in aqueous ethanolic medium was highly fluorescent. The advantage with the probe was that it could detect ferric ions in an aqueous solution; biological systems need aqueous medium. It was completely soluble in ethanol–water (9 : 1 v/v) medium and the sensing studies could be done in the same.

A 100 ppm solution of probe 8 was highly fluorescent in UV (365 nm). When FeCl₃ was added to probe 8 in aqueous ethanolic medium, fluorescence quenching was observed. With 10 equivalents of FeCl₃, complete quenching took place (Fig. 1). The quenching was selective to Fe³⁺ ions and did not occur with any other metal ions. This change in fluorescence was monitored by using fluorescence spectrometer.

Fig. 1 Visible fluorescence change of probe 8 inside UV chamber by the addition of Fe³⁺ ions.

When aq. Fe³⁺ solution was added to a solution of probe 8 in aq. ethanol, the fluorescence of probe 8 was lost. We studied the fluorescence quenching effect of Fe³⁺ using fluorescence spectroscopy. The excitation wavelength was 460 nm. To a fixed concentration (9.13 × 10⁻⁷ M) of probe 8 in aq. ethanol, aq. solutions with increasing concentration of Fe³⁺ ions were added (Fig. 2a). Fluorescence of probe 8 decreased with addition of Fe³⁺ ions and the decrease was linear with respect to concentration of Fe³⁺ ions; complete quenching took place with 7 equivalents of Fe³⁺ ions. Fluorescence quenching happened immediately after mixing Fe³⁺ ions with the probe. Initial fluorescence studies were done at two instants; one immediately after addition of Fe³⁺ and the other 2 h after addition of Fe³⁺ ions. There was negligible change in the emission pattern. Hence, all further fluorescence studies were done immediately after addition of ferric ions.

Fig. 2 (a) Fl. spectra of probe-8 (9.13 × 10⁻⁷ M or 50 ppm) with increasing conc. of Fe³⁺ (0.2 to 10 equiv.) and (b) corresponding (I⁰₅₉₉–I₅₉₉)/I⁰₅₉₉ vs. Fe³⁺ concentration with the linear equation and R² value calculated till 7.0 equivalents of Fe³⁺; error bars are shown using standard deviations from the mean.

The ratiometric change in the fluorescence intensity of probe 8 reached a point of negligible deviation with 7 equivalents (6.5 μM) of Fe³⁺ (Fig. 2b). A linear relationship was found between the emission intensity at 599 nm and the Fe³⁺ concentration over 1.8 × 10⁻⁷ M (0.2 equiv. of Fe³⁺) to 7.38 × 10⁻⁶ M (7 equiv. of Fe³⁺) range (Fig. 2) with a correlation coefficient of 0.9897.

The turning-off of the fluorescence of probe 8 by Fe³⁺ ions was due to complexation of Fe³⁺ ions with probe 8. Stern–Volmer plot was drawn for understanding the quenching process (Fig. 3). A non-linear Stern–Volmer plot was obtained consistent with the static fluorescence quenching rather than dynamic quenching. This is a kind of static fluorescence quenching in which the probe acted as an iron chelator resulting in loss of fluorescence properties. Such non-linear Stern–Volmer plots are reported in the literature.^29–31

Fig. 3 Stern–Volmer plot of the fluorescence response of probe-8 to increasing concentrations of ferric ions.

The maximum level of Fe³⁺ permitted in drinking water by the U.S. Environmental Protection Agency (EPA) is 0.3 mg l⁻¹.³² To establish the detection limit of our probe, a graph of minimum equivalents of Fe³⁺ versus fluorescence intensity was plotted (ESI†). The detection limit³³ of probe 8 for Fe³⁺ ions was estimated to be of 0.3 mg l⁻¹ or 18.3 × 10⁻⁹ M which is equal to the permitted level of Fe³⁺ ions by US EPA.

The selectivity towards Fe³⁺ ions was investigated by recording the fluorescence spectrum of the probe 8 by mixing the solutions of different metal ions. The metals tested were from the corresponding compounds of AgNO₃, AlCl₃, BaCl₂·2H₂O, CaCl₂, CdCl₂·5H₂O, CoCl₂·2H₂O, CrCl₃·6H₂O, CsCl, CuCl, CuCl₂, FeCl₂·4H₂O, HgCl₂, InCl₃, KCl, LiCl, MgCl₂·6H₂O, MnCl₂·2H₂O, NaCl, NiCl₂·6H₂O, PbCl₂, SbCl₃, SnCl₂·2H₂O, SrCl₂·6H₂O, and ZnCl₂. Even if the ferric ion sensing happened immediately after addition, for selectivity study, the fluorescence spectra were recorded after 5.0 min of incubation with other metal ions. The data presented in Fig. 4 were obtained with probe 8 concentration of 9.13 × 10⁻⁷ M and 10 equivalents of all metal ions. This study confirmed high selectivity of the probe towards Fe³⁺ among selected set of metals. Other metal ions had insignificant quenching effect on the fluorescence of the probe 8.

Fig. 4 Fluorescence response of probe-8 (9.13 × 10⁻⁷ M) to 10 equivalents of various metal ions and ferric. Excitation wavelength = 460 nm.

UV-vis study

There was instantaneous visual colour change from orange to brown upon addition of ferric ions to probe 8 (Fig. 5a). Thus, 8 is a ‘naked-eye’ colorimetric sensor for ferric ion. To understand the absorbance pattern, we did UV-vis study for probe 8. The probe showed two absorption bands at 287 nm and 477 nm (Fig. 5b). There was slight increase in absorbance with increasing quantity of ferric ions into a fixed concentration (9.13 × 10⁻⁷ M) of 8. A new peak at 357 nm appeared which might be due to complexation of the ferric ions with probe 8. This led to a change in the absorbance of the probe. This happened only with the ferric ions and hence 8 is selective to ferric ion (ESI†).

Fig. 5 (a) Visual colour change upon adding 10 equivalents of Fe³⁺ into probe 8 (9.13 × 10⁻⁷ M) (b) UV absorption spectrum of probe 8 with increasing concentrations of Fe³⁺.

DFT studies

For better understanding of the chromo/fluorophore, DFT study was done for probe 8. All theoretical calculations were carried out using the Gaussian 09 program.³⁴ The B3LYP exchange correlation functional under 6-31G +, p, d basis set was performed to calculate HOMO and LUMO levels. The geometries were optimized, and electron distribution in the FMOs was calculated (Fig. 6). The MO contours were plotted using Gauss view 5.0.8. The HOMO–LUMO orbital distribution showed that the electron cloud was spread from the quinoline moiety to the imidazopyridine moiety. The major contribution for HOMO was from the quinoline moiety whereas in case of LUMO the contribution was from quinoline as well as imidazopyridine moieties.

Fig. 6 HOMO, LUMO orbital energies and the optimized structures for probe 8.

Conclusions

We describe a cyanine probe that gives very good sensitivity for detecting ferric ions without needing any advanced, complex readout equipment or complex buffer making procedure. The detection limit of the probe for ferric ion is 0.3 ppm and shows a linear relationship in the Fe³⁺ concentration range of 1.8 × 10⁻⁷ M (0.2 equiv. of Fe³⁺) to 7.38 × 10⁻⁶ M (7 equiv. of Fe³⁺). The probe is a selective ‘naked eye’ detector for the ferric ion. The synthesis of the probe is easy.

Experimental section

General information

All chemicals were commercially available. They were obtained from S.D. Fine Chem. All solvents were directly used from commercial sources without further purification. Nuclear magnetic resonance spectra were recorded on Bruker 400 MHz or Varian 300 MHz spectrometer. Fluorescence was measured using Shimadzu spectrofluorimeter RF-5301. UV-vis absorption spectra were recorded with a Shimadzu UV-Vis spectrophotometer – 1650.

Preparation of 1,2,2,4-tetramethyl-1,2-dihydroquinoline (6)

A mixture of TDQ (10.0 g), potassium carbonate (10.0 g) and iodomethane (5.5 ml) in 80 ml of DMF was maintained at 110 °C under inert atmosphere for 24 hours. The reaction mixture was cooled to room temperature and poured into water, and extracted with ethyl acetate. After removal of solvent, the product was purified silica column using hexane/ethyl acetate as mobile phase. Yield of 6 was 82%. ¹H-NMR (400 MHz, CDCl₃): δ_H 7.20 (t, J = 7.2 Hz, 1H), 7.1 (d, J = 7.6 Hz, 1H), 6.7 (t, J = 7.2 Hz, 1H), 6.5 (d, J = 8.0 Hz, 1H), 5.3 (s, 1H), 2.8 (s, 3H), 1.98 (s, 3H), 1.3 (s, 6H).

Preparation of 1,2,2,4-tetramethyl-1,2-dihydro-6-quinoline carbaldehyde (7)

POCl₃ (8.1 ml) was slowly added to dry DMF (100 ml) at 5–10 °C under inert atmosphere and heated at 50–60 °C for 30 min. 6 (10 g) in dry DMF (50 ml) was added slowly over 10 min. The temperature was raised to 90 °C and maintained overnight under nitrogen. The mass was cooled to 30 °C, poured into ice water (400 ml), and basified with aq. NaOH. It was extracted with ethyl acetate and the organic layer was washed with water and dried over anhydrous Na₂SO₄. After removal of solvent, the product was purified on silica column using hexane/ethyl acetate as mobile phase. Yield of 7 is 60%. ¹H-NMR (300 MHz, CDCl₃): δ 9.80 (s, 1H), 7.6 (d, J = 8.7 Hz, 1H), 7.6 (s, 1H), 6.5 (d, J = 8.4 Hz, 1H), 5.3 (s, 1H), 2.9 (s, 3H), 2.0 (s, 1H), 1.37 (s, 6H).

Preparation of 7-methyl-2-phenylimidazo[1,2-a]pyridine (3)

4-Methylpyridin-2-amine (1) (0.02 m) and bromoaceto phenone (2) (0.02 m) were dissolved in ethanol (30 ml) and refluxed for 5 h. 2 ml of conc. HCl was added and refluxed further for 3 h. The solution was concentrated and aq. NaHCO₃ was added to basify the mass (pH = 10). White solid was filtered, and washed aq. ethanol, and dried under vacuum to obtain 3 (80%). ¹H-NMR (400 MHz, CDCl3): δ_H 7.90 (m, 3H), 7.77 (s, 1H), 7.28–7.46 (m, 4H), 6.59 (d, J = 6.8 Hz, 2H), 2.40 (s, 3H). ¹³C-NMR (400 MHz, CDCl₃): 146.1, 145.5, 135.6, 133.9, 128.7, 127.8, 126.0, 124.8, 115.9, 115.07, 107.5, 21.4.

Preparation of 1,7-dimethyl-2-phenylimidazo[1,2-a]pyridin-1-ium iodide (4)

3 (1.0 g) and iodomethane (0.85 g) were dissolved in dioxan (10 ml) in a microwave vial. It was heated under microwave conditions at 120 °C for 30 min, cooled to 30 °C, filtered and the solid was washed with dioxan followed by diethyl ether. The yield of 4 was 70%. ¹H-NMR (400 MHz, CDCl₃): δ_H 9.25 (d, J = 8 Hz, 1H), 8.66 (s, 1H), 7.92 (s, 1H), 7.56–7.62 (m, 5H), 7.22 (d, J = 8.0 Hz, 1H), 4.03 (s, 3H), 2.65 (s, 3H). ¹³C-NMR (400 MHz, CDCl₃): 146.9, 140.2, 137.7, 131.0, 130.0, 129.5, 128.9, 124.7, 120.0, 112.6, 110.2, 33.1, 22.3.

Protocol for the synthesis of (E)-1-methyl-2-phenyl-7-(2-(1,2,2,4-tetramethyl-1,2-dihydroquinolin-6-yl)vinyl)imidazo[1,2-a]pyridin-1-ium iodide (8)

A solution of 4 (500 mg), piperidine (20 mg), and 7 (338 mg) were mixed in n-butanol (10 ml) was refluxed for 16 h and cooled to 30 °C. The solution was allowed to stand at 10 °C for 20 h. The orange red solid was filtered, washed with isopropyl alcohol, and dried in vacuum at 60 °C for 3 h to obtain 8 (yield = 65%). ¹HNMR (400 MHz, CDCl₃): δ_H 9.12 (d, J = 4.0 Hz, 1H), 8.45 (s, 1H), 7.96 (s, 1H), 7.37–7.54 (m, 8H), 7.30 (s, 1H), 7.00 (d, J = 16 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 5.35 (s, 1H), 4.01 (s, 3H), 2.87 (s, 3H), 2.08 (s, 3H), 1.37 (s, 6H). ¹³C-NMR (400 MHz, CDCl₃): 147, 144.8, 140.9, 137.8, 137.5, 130.9, 130.1, 129.7, 129.5, 128.9, 127.5, 124.8, 122.8, 122.7, 118.0, 115.2, 112.4, 110.5, 104.9, 57.1, 33.2, 31.0, 28.2, 18.9. MS (ESI MS): (m/z, %): 547 [M⁺, 100%].

Acknowledgements

The authors are thankful to CIL-GJUS&T, Hisar, India for providing NMR spectra. KV is thankful for CSIR, New Delhi, India for a fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06642a

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