Synthesis, Photophysical Properties, and Metal-Ion Recognition Studies of Fluoroionophores Based on 1-(2-Pyridyl)-4-Styrylpyrazoles.

A convenient access toward novel fluoroionophores based on 1-(2-pyridyl)-4-styrylpyrazoles (PSPs) substituted at position 3 with donor or acceptor aryl groups is reported. The synthesis proceeds in two steps: the first one via Wittig olefination of the appropriate 4-formylpyrazole and then Mizoroki-Heck coupling to yield the desired products in an overall yield of up to 69%. Photophysical properties of products (4-styryl) and their intermediates (4-vinyl) were explored, finding that they have strong blue-light emission with high quantum yields (up to 66%) due to ICT phenomena. The 3-phenyl PSP was studied as a turn-off fluorescent probe in metal ion sensing, finding a high selectivity to Hg2+ (LOD = 3.1 × 10-7 M) in a process that could be reversed with ethylenediamine. The sensing mechanism and binding mode of the ligand to Hg2+ were established by HRMS analysis and 1H NMR titration tests.

Introduction

Recently, the synthesis of functional aza-heterocycles have attracted the attention of researchers in the chemical, technological, environmental, and biological fields[1,2] since their structural and electronic features favor the scope and applicability of such compounds. For instance, pyrazoles receive great interest due to their proved value in the preparation of diverse bioactive compounds, coordination complexes, and organic materials.[3,4] In particular, various functional π-extended pyrazole derivatives have been photophysically studied displaying a strong blue-light emission with high quantum yields;[5,6] thus, these fluorophores are increasingly recurrent in materials science mainly because their high synthetic versatility allows them to modulate the electronic properties.[3−6] These features are strictly related to the nature of their heteroatoms obviously by the effect of the π-extended substituents since the pyrazole ring has a pyridine-type nitrogen atom (C=N, cation receptor) and another pyrrole-type (electron donor) that favor charge-transfer (CT) phenomena.[3−7] Therefore, this important class of pyrazoles is already being used in the design of fluorescent probes for ion sensing by altering their π conjugation, which appears when chelation of metals or receiving of anions occurs at the recognition sites (Figure ).[8,9]

Figure 1

Structures of some functional π-extended pyrazoles. (a) Fluorescent push–pull system,[5] (b) cyanide detection probe,[8] and (c) aluminum detection probe.[9]

The charge transfer (CT) processes are frequent in fluorescent aza-heterocycles bearing diverse electron-donor (D) and electron-acceptor (A) groups.[1,2,10−12] These processes include internal (or intramolecular) charge transfer (ICT),[10] twisted internal charge transfer (TICT),[11] and metal–ligand (or ligand–metal) charge transfer (MLCT or LMCT).[12] CT phenomena involving metal ions can induce changes in fluorescent emission by chelation-enhanced fluorescence (CHEF) or chelation-enhanced fluorescence quenching (CHEQ) upon the chelation of a metal ion to the sensor molecule.[1,9,12,13] Accordingly, the synthesis and design of fluorescent probes for metals based on CT phenomena is an essential area of research due to their proved utility in selective recognition of biologically or environmentally relevant metal cations (i.e., Al3+, Mg2+, Cu2+, Ni2+, Pb2+, Hg2+, etc.).[9,11−17] For example, mercury (Hg) is one of the more severe environmental pollutants and is extremely harmful to humans. The methyl mercury yielded from the microbial biomethylation of Hg2+ is known to cause brain damage and other chronic diseases;[17,18] thus, research on rapid and sensitive analysis of mercury ions is very needed.

Considering the important electronic properties of pyrazole derivatives[3−9] together with our interest in the synthesis and design of fluorescent probes,[11,14,19,20] we proposed the synthesis of 1-(2-pyridyl)-4-styrylpyrazoles (PSPs) substituted at position 3 with donor or acceptor aryl groups (3a–3c). These compounds are novel π-extended bidentate trans-stilbene analogous[21] ligands containing two pyridine-type nitrogen atoms suitably located to achieve the formation of chelates. Consequently, we have hypothesized that PSPs 3a–3c have the appropriate molecular architecture for their use in metal ion sensing by fluorescence quenching based on LMCT phenomena.[1,12,21] The synthesis of 3a–3c was optimized proceeding via two steps: first, a Wittig olefination of the appropriate 4-formylpyrazole (1a–1c) to afford intermediates 4a–4c and second, a later Mizoroki–Heck reaction (Scheme a). We have already reported about the preparation of the key precursors 4-formyl-1-(2-pyridyl)pyrazoles 1a–1c to reach the ligands 3a–3c, starting from p-substituted acetophenones (7) by a two-step sequence involving a condensation with 2-pyridylhydrazine (8b) and then Vilsmeier–Haack reactions (formylation and cyclization) on the respective hydrazone (9a–9c) (see Scheme S2).[19] In this case, a fluorescent chemosensor (6c) for selective cyanide sensing could be synthesized and designed from 1a–1c (Scheme b).[19]

Scheme 1

Synthetic Approach toward Fluorescent 4-Alkenylpyrazoles 3, 4, and 6

Results and Discussion

Synthesis

Given our previous results regarding the synthesis and photophysical application of the 4-dicyanovynylpyrazoles 6a–6c obtained from 4-formyl-1-(2-pyridyl)pyrazoles 1a–1c, we pictured to expand the scope of these results by the preparation of the novel π-extended pyrazoles 6a–6c (Scheme ). The synthesis of the aldehydes 1a–1c as well as of 4-formyl-1-phenylpyrazole 1d was carried out by our established method (see Scheme S2 in the Supporting Information).[19] Reaction products from 1d cannot be used for metal chelation but can work to set up initial conditions in the synthesis of 6a–6c (1d is easier to access versus 1a–1c due to the disposition of the respective precursor hydrazine) and as well as a reference for our later photophysical studies. With the 4-formylpyrazoles 1a–1d in our hands, we envisaged the Wittig reaction using benzyltriphenylphosphonium bromide (2a) as a straight way to synthesize 4-styrylpyrazoles 3a–3d. Initially, we evaluated the synthesis of the alkene 3d by the reaction of 2a with the freshly synthesized aldehyde 1d and using n-BuLi-THF in accordance with that reported by Li et al.;[22] however, the reaction afforded a mixture of isomers (E)-3d and (Z)-3d with a very similar Rf in a 75% yield (Scheme and Figures S1 and S2). It is noteworthy that very few examples of 4-alkenylpyrazoles with substitution analogous to that in pyrazoles 3 and 4 (Scheme a) have been reported in the literature.

Scheme 2

Synthesis of 4-Styrylpyrazoles (Z)-3d and (E)-3d

Reaction conditions: 1d (0.5 mmol), 2a (0.5 mmol), and n-BuLi in hexane (0.2 mL, 2.5 M, 0.5 mmol) in THF (3.0 mL). The E:Z (∼1:3) ratio was determined by 1H NMR (CDCl3).

We continued our study using methyltriphenylphosphonium bromide (2b) under similar conditions to those in which the mixture (E/Z)-3d was obtained in order to form the 4-vinylpyrazoles 4a–4d that would then allow access to the desired products 3a–3d through a Mizoroki–Heck reaction. The synthesis of 4a–4d was developed using an excess of 2b and n-BuLi with respect to 1a–1d, and except for 4a, 4-vinylpyrazoles 4b–4d were isolated in good yields, probably from the low solubility in THF of their respective precursor 1a (Scheme ).

Scheme 3

Synthesis of 4-Vinylpyrazoles 4a–4d

Reaction conditions: 1a–1d (1.2 mmol), 2b (3.6 mmol), and n-BuLi/hexane (∼1.5 mL, 2.5 M, 3.6 mmol) in THF (14.0 mL).

Once 4-vinylpyrazoles 4a–4d were obtained, we stepped to set up the Mizoroki–Heck coupling reaction to form products 3a–3d using the 4-vinylpyrazole 4d and testing the halobenzene (different ratios of PhBr (5a) or PhI (5b) with respect to 4d), palladium catalyst (Pd(AcO)2 and PdCl2), phosphine ligand (PPh3 and (oMePh)3P), and reaction solvent (dimethylformamide (DMF), acetonitrile, water, and ethanol as highly polar solvents[23]). Likewise, the reactions were carried out under microwave radiation (MW) in order to accelerate the study to settle the best coupling conditions.[3,10,11] Pleasantly and after several attempts, the desired (E)-1-phenyl-4-styrylpyrazol (3d) was afforded in an 85% yield by using 10 mol % of Pd(AcO)2, 20 mol % (o-MePh)3P, ∼2 equiv of Et3N, and 2.5 equiv of iodobenzene (5b) in DMF. However, when the reaction was carried out with the 1-(2-pyridyl)pyrazole 4b under the same conditions, a mixture of three products was obtained possibly via Pd-substrate complexes (Scheme and Figures S3–S5). NMR spectra data and MS analysis (Figures S3–S5) suggested that the mixture could result from other coupling reactions favored by the pyridyl group in the substrate.

Scheme 4

Synthesis of 4-Styrylpyrazoles 3b and 3d

Reaction conditions (A): 4b and 4d (0.4 mmol), 5b (1.0 mmol), Pd(AcO)2 (10 mol %), (o-MePh)3P (20 mol %), and Et3N (0.1 mL) in dry DMF (3.0 mL) under microwave irradiation.

Relative conversion (%) determined by 1H NMR (CDCl3).

Consequently, we decided to carry out the reaction conditions’ optimization to form the desired PSPs 3a–3c starting from the substrate 4b by making a slight variation in the previous method in order to decrease or eliminate the formation of collateral products (Table ). Under the previously established conditions, 4b reacted completely (using only 2 equiv of PhI) but along with byproduct formation (3b′ and 3b″, Scheme b), which decreased under conventional heating (Table , entry 1 vs 2). More tests were made in order to evaluate the effect of time and excess of iodobenzene (5b), concluding that an increase in the reaction time favored the formation of product 3b, while with an excess of 5b, a greater formation of byproducts was observed (Table , entries 3–5). Notably, the formation of the desired product 3b was favored when Ph3P was used as a ligand (Table , entries 1–5 vs 6 and 7). When using bromobenzene (5a) instead of 5b, a considerable decrease of byproducts is found because of its lower reactivity, and the use of MW heating led to a greater formation of 3b (Table , entries 8 and 9). Finally, the catalyst effectiveness was evaluated when it is in greater quantity and by the direct use of a Pd(0) catalyst; in both cases, the 3b formation was inferior to that of the other tests (Table , entries 10 and 11). Therefore, it was concluded that the best reaction condition for the synthesis of 3b is entry 9 (Table ).

Table 1

Optimization of the Reaction Conditions for the Synthesis of 3ba

entry	ratio 4b:5, X	ligand	T (°C)	time, t	% 4b	% 3bc	% byproducts
1	1:2, I	(o-MePh)3P	180	90 mind	0	70	30
2	1:2, I	(o-MePh)3P	100	24 hb	36	52	12
3	1:2, I	(o-MePh)3P	180	5 min	39	41	20
4	1:2, I	(o-MePh)3P	180	7 min	22	56	22
5	1:4, I	(o-MePh)3P	180	7 min	16	52	32
6	1:2, I	Ph3P	180	90 mind	24	62	14
7	1:2, I	Ph3P	100	24 hb	14	69	17
8	1:2.5, Br	Ph3P	150	24 hb	19	75	6
9	1:2.5, Br	Ph3P	150	1.5 hd	7	80	13
10e	1:2.5, Br	Ph3P	150	24 hb	51	40	9
11f	1:2.5, Br		150	24 hb	90	10	0

Reaction conditions: 4b (0.2 mmol), Pd(AcO)2 (10 mol %), ligand (20 mol %), and Et3N (50.0 μL) in dry DMF (1.5 mL) under MW.

Conventional heating.

Relative conversion (%) determined by 1H NMR (CDCl3).

After three heating cycles of 30 min.

Pd(AcO)2 with Ph3P (15 and 30 mol %, respectively).

Pd(PPh3)4 (5 mol %).

Once the optimal reaction conditions for the PSP 3b formation were established, the novel (E)-1-(2-pyridyl)-4-styrylpyrazoles 3a–3c containing both electron-donor and electron-acceptor groups were successfully synthesized in good yields (Scheme ).

Scheme 5

Synthesis of (E)-4-Styrylpyrazoles 3a–3d

Reaction conditions (A): 4a–4d (0.4 mmol), 5b (1.0 mmol), Pd(AcO)2 (10 mol %), Ph3P (20 mol %), and Et3N (0.1 mL) in dry DMF (3.0 mL) under microwave irradiation.

Yields for (E)-4-styrylpyrazoles 3a–3d are shown.

Photophysical Properties of 4a–4d and 3a–3d

The 4-alkenylpyrazoles 4a–4d and 3a–3d were photophysically explored in order to establish their scope as fluorescent probes for metal cation detection. The 1-(2-pyridyl)-4-vinylpyrazoles (PVPs) 4a–4c and PSPs 3a–3c are substituted at position 3 with different D or A groups (i.e., a: 4-NO2Ph, b: Ph, and c: 4-MeOPh), while the N-phenylpyrazoles 4d and 3d were analyzed as reference in this study (Figure ). The UV–vis and fluorescence emission spectra of 4 and 3 were recorded at room temperature (∼20 °C) in solvents of different polarities such as dimethylsulfoxide (DMSO), acetonitrile (ACN), ethanol (EtOH), dichloromethane (DCM), and toluene (see Figures S6–S8 and Table S1 in the Supporting Information). The maximum absorption bands for the 4-vinylpyrazoles 4a–4d are around 290–325 nm, which were attributed to π → π* transitions. By comparing the maximum absorption bands between PVPs 4a–4c, bathochromic shifts were found in the following order 4c > 4b > 4a, indicating a greater charge transfer (CT) in 4c. The maximum absorption bands of 4b were compared with 4d in order to understand the effect of the 2-pyridyl group on the electronic nature of PVPs 4a–4c where hypsochromic shifts were found at 4d versus 4b (Figure S6). This confirms that the acceptor character of the 2-pyridyl group favors the D−π–A system and so increases the dipole moment in 4a–4c.[19,24] In a similar fashion to that of compounds 4a–4c, the PSPs 3a–3c showed the same trend in the maximum absorption bands with bathochromic shifts in the order 3c > 3b > 3a. The maximum absorption bands of 3d have also hypsochromic shifts versus 3b due to the absence of the 2-pyridyl group. In addition, the maximum absorption bands in 3a–3d have bathochromic shifts with respect to 4a–4d since the styryl group in 3 favors a higher π-conjugation than that of the vinyl group in 4 (Figure S7).

Figure 2

Structure of photophysically studied fluorophores 4a–4d and 3a–3d. Photographs were taken using 20.0 × 10–6 M solutions of each compound under a UV lamp (λex = 365 nm).

From the fluorescence emission spectra results, it can be seen that pyrazoles 4a–4d and 3a–3d are fluorophores with strong blue-light emission (quantum yields of up to 66%) in the range of 350–400 nm (Table S1). The pyrazoles 3a–3d have a bathochromic shift versus the derivatives 4a–4d due to the π-conjugation effect mentioned above (Figure S8). The heterocycles with electron-withdrawing substituents such as the NO2 group in pyrazoles 4a and 3a are characterized by having lower fluorescence emission than that of the rest of the pyrazoles due to an efficient crossing process between systems carried out by the existence of a low-energy transition n → π*. This phenomenon is the result of a high internal conversion rate of S1 → S0 that may be related to the high CT in the excited state to the NO2 group since this substituent has a strong electron-withdrawing character. Likewise, the N-phenylpyrazoles 4d and 3d have lower quantum yields compared with the N-(2-pyridyl)pyrazoles 4b and 4c and 3b and 3c, indicating that the 2-pyridyl group of these ligands has a great influence on the fluorescence emission favoring ICT phenomena. In contrast to fluorescent compounds 4c and 3c, the 4-formylpyrazole 1c does not fluoresce due to a similar internal conversion phenomenon to that occurring in nitrocompounds (Figure S8d). In this way, it can be concluded that pyrazoles must be functionalized with vinyl or styryl groups to increase their π-conjugation in order to render novel fluorophores.

Furthermore, the quantum yields in compounds 4a–4d and 3a–3d are dependent on the solvent polarity. The values reveal high dependence on solvent properties such as hydrogen-bonding donor ability and polarizability (Table S1). The quantum yields for most compounds were higher in ethanol (polar protic solvent), which may be due to a negative solvatokinetic phenomenon.[25−27] A solvatokinetic effect occurs when the quantum yield of fluorescence of a compound shows a slight variation with the change in the polarity of the solvent. In this effect, the quantum yield increases with an adequate improvement of the ICT that involves the configuration of the electronic n → π transition, while the reduction in the quantum yield due to a strong ICT is called a positive solvatokinetic effect.[25−27]

Subsequently, we evaluated the effect of water on the fluorescence emission of 3b (Ph) using EtOH:H2O mixtures due to its high performance in ethanol, although 3c (4-MeOPh) has a slightly higher quantum yield in ethanol (Table S1); however, the phenyl group of 3b is not affected with this protic mixture by not partaking in hydrogen bonding.[11] In this experiment, it can be observed that a higher water percentage leads to a decrease of the fluorescence emission intensity (Figure ). Only a range of 0–20% of water constant tendency in the emission of fluorescence is maintained. In the next step, cation detection studies in ethanol were performed, and the percentage of water should not exceed 20% in order for it not to interfere in the analysis quantification. Thus, compound 3b was used in further studies toward the design of a fluorescent probe for metal sensing in an EtOH:H2O mixture (9:1 v/v, pH = 7.14 at 20 °C).

Figure 3

(a) Fluorescence spectra of 3b (2.0 × 10–6 M in mixtures EtOH:H2O) at 20 °C (λex = 320 nm). (b) Integral of the respective fluorescence emission curves vs water percentage.

Fluorescence and UV–vis Response of Ligand 3b to Metal Ions

To evaluate the use of the PSP 3b in the metal ion detection, UV–vis spectra of 3b (4.0 × 10–6 M) were measured in the presence of alkaline (Na+ and K+), alkaline earth (Mg2+, Ca2+, and Ba2+), and heavy metals (Cr3+, Fe3+, Zn2+, Co2+, Ni2+, Cu2+, Cd2+, Hg2+, Al3+, and Pb2+) in EtOH:H2O (9:1). After the addition of a high cation excess (4.0 × 10–4 M, 100 equiv), we observed slight spectral changes of a hypsochromic shift (Na+, K+, Mg2+, Ca2+, Ba2+, Cu2+, and Cd2+) and a hyperchromic type (Cr3+, Fe3+, Zn2+, Co2+, Ni2+, and Pb2+) suggesting that 3b may be interacting with these metals (Figure S9). Additionally, a strong hyperchromic effect in the band around 250 nm was observed in the presence of Hg2+ that can be associated with 3b:metal interaction but could also be due to the absorbance of Hg2+ itself around 270 nm.[28] To understand the response in fluorescence emission from 3b (1.0 × 10–6 M) toward the metal ions evaluated by absorption spectroscopy, emission spectra of 3b were measured using a high concentration (1.0 × 10–4 M, 100 equiv) of the different cations (λex = 320 nm), achieving EtOH:H2O (∼9:1) final solutions (Figure ). Under these conditions, only the presence of Hg2+ induced notable fluorescence quenching, while with other metal ions, there was some change in the emission intensity versus the free ligand 3b, or an increase in fluorescence emission was observed in a nonselective manner (Figure ). For example, Ba2+, Cr3+, Fe3+, Co2+, Ni2+, Cd2+, and Al3+ showed to some extent an increase in intensity compared to 3b. However, this change in the fluorescence emission signal does not allow us to selectively identify the chemosensor response in the presence of these cations.

Figure 4

(a) Fluorescence spectra of 3b (1.0 × 10–6 M in EtOH:H2O, 9:1 v/v at 20 °C) in the presence of various metal ions (1.0 × 10–4 M, 100 equiv, λex = 320 nm). (b) Fluorescence intensity response of 3b to Hg2+ and other metals (λem = 390 nm). The photograph was taken under a UV lamp (λex = 365 nm).

These results suggest that the ligand 3b could only be a good fluorescence chemosensor for Hg2+. Next, we analyzed the preferential selectivity of 3b as a fluorescent chemosensor for Hg2+ detection in the presence of several competing ions for which the ligand 3b (2.5 × 10–6 M in EtOH:H2O, 9:1 v/v) was treated with Hg2+ in the presence of other metal ions (2.5 × 10–4 M, 100 equiv). From these results, it can be observed that the interference for Hg2+ sensing when Na+, K+, Mg2+, Ca2+, Ba2+, Cr3+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Al3+, and Pb2+ are present is relatively low; thus, the probe 3b could be used for Hg2+ sensing in the presence of these competing ions, which could appear in real samples (Figure ).

Figure 5

(a) Fluorescence spectra of 3b (2.5 × 10–6 M in EtOH:H2O, 9:1 v/v) at 20 °C (λex = 320 nm) in the presence of Hg2+ (2.5 × 10–4) and of different metallic ions (2.5 × 10–4). (b) Maximum fluorescence intensity of 3b in these experiments (values were at λem = 390 nm).

Measurements of fluorescence emission of 3b after an increase in the concentration of Hg2+showed that the fluorescence is completely turned off after the addition of 200 equiv of Hg2+. Additionally, a linear range between 0.5 × 10–6 and 4.5 × 10–6 M with a calculated limit of detection (LOD) of 3.1 × 10–7 M (R2 = 0.9950) was observed (Figure a–c), meaning that this probe is able to detect approximately 6.2 × 10–5 g of Hg2+ in 1 L of dissolution (62 ppb). The value of the LOD is higher than the permitted level in drinking water according to the EPA standard in the United States (2 ppb, 2.0 × 10–6 g/L)[29] and to the amount allowed in accordance with Resolution 2115 of 2007 of the Ministry of Environment of the Government of Colombia (1.0 × 10–6 g/L).[30] However, the LOD calculated in this work is a value that is in the same order of magnitude compared with other chemosensors recently reported in the literature.[31−33] In order to determine the stoichiometry of the complex, the Job’s plot study of the fluorescence emission for 3b–Hg2+ interaction was made. This curve showed a maximum around 0.3 indicating the formation of complex 3b:Hg2+ with a molar ratio that can be between 2:1 and 3:1 (Figure ).

Figure 6

(a) Fluorescence spectra of 3b (2.5 × 10–6 M in EtOH:H2O, 9:1 v/v) with increasing Hg2+ addition (0–500 × 10–6 M, λex = 320 nm). (b) Maximum fluorescence intensity of 3b at 390 nm. (c) LOD calculated by the selection of the lineal interval of fluorescence intensity data of 3b. (d) Job’s plot of the 3b:Hg2+ complex in EtOH:H2O (9:1 v/v) showing a stoichiometry of ∼2:1 (λex = 320 nm).

Additionally, it was found that the interaction of probe 3b with Hg2+ is reversible after having added ethylenediamine. In this way, two tests were carried out in which the minimum necessary amount of diamine to recover the fluorescence emission of 3b was evaluated after turn-off with Hg2+ (Figure ). In the first testing, an excess of Hg2+ was added (Figure a and Table ) where twice of the ethylenediamine molar ratio was required to recover the initial fluorescence emission of 3b. In the second testing, a greater amount of Hg2+ was added, and three times of the diamine molar ratio was needed to get back the initial fluorescence emission of 3b (Figure b and Table ). This study confirms that fluorescence quenching is effectively due to the interaction of the PSP 3b with Hg2+ and not any external agent. In this case, the turn-on fluorescence is a result of the high affinity of ethylenediamine toward Hg2+ (Ka = 2.0 × 1014 L/mol at 25 °C),[34,35] which resulted in the 3b:Hg2+ complex dissociation and releasing of the fluorophore 3b. Consequently, ligands having the 1-(2-pyridyl)-4-styrylpyrazolic moiety could be good fluorescent probe models for Hg2+ sensing, so we expected to extend our research toward a possible use in real samples and including more analytical details.

Figure 7

Fluorescence spectra of 3b (2.0 × 10–6 M EtOH:H2O, 9:1 v/v) at 20 °C (λex = 320 nm) with different molar ratios of Hg2+ and ethylenediamine. (a) 3b:Hg2+ (1:100) and (b) 3b:Hg2+ (1:200).

Table 2

Quantum Yields of 3b in the Reversibility Studya

3b:Hg2+	Hg2+:diamine	ϕFb
1:0	0:0	0.455
1:100	1:0	0.109
	1:1	0.228
	1:2	0.446
	1:3	0.446
1:200	1:0	0.013
	1:1	0.059
	1:2	0.266
	1:3	0.417

Molar ratios (in EtOH:H2O 9:1 v/v at 20 °C) of Hg2+ with respect to 3b (2.0 × 10–6 M) and ethylenediamine.

Relative quantum yields using quinine sulfate as a reference (ϕF = 0.59 in 1.5 × 10–1 M HClO4); excited at the maximum absorption wavelength.

Proposed Sensing Mechanism of 3b toward Hg2+

A plausible mechanism for Hg2+ detection using the probe 3b based on the above experimental results is proposed. In addition, 1H NMR titration experiments and HRMS analysis were employed to corroborate this mechanism and understand the binding mode of ligand 3b to Hg2+ (Figure and Figure S10). The titration was made in DMSO-d6 using a constant amount of ligand and gradually increasing the concentration of Hg2+ (a CHCl3 trace was used as a reference). The increase of the Hg2+ ratio provokes the corresponding signals to the pyridinic (H1–H4, green) and pyrazolic (H5, red) protons to be shifted downfield. In contrast to those, the characteristic signals of the alkenyl (H6–H7, blue) and phenyl groups (gray) protons are remained almost unchanged. These results suggest that both pyridinic and pyrazolic nitrogen atoms participate in the coordination of 3b with Hg2+ (Figure ).

Figure 8

1H NMR (DMSO-d) titration of 3b with an increase in the molar ratio of Hg(NO3)2 vs 3b at 20 °C.

Finally, the high-resolution mass spectrum (Figure S10) using electrospray ionization (HRMS-ESI) was recorded for the complex formed in situ between 3b and Hg(NO3)2·H2O in MeOH:H2O (1:1 v/v), which led to a quick fluorescence quenching. An ion peak at m/z = 865.2595 is found, which corresponds to the mass of the complex [2 3b:HgOH]+ (calculated, 865.2573). This result agrees with the formation of a 3b:Hg2+ complex with a molar ratio of ∼2:1 previously found by the Job method (Figure d). To corroborate that, the peak at m/z = 865.2595 corresponds to the mass of the complex [2 3b:HgOH]+ detected by HRMS. The isotopic distribution of the experimental mass spectrum was compared with that calculated resulting in a perfect match (Figure S10).[36]

With all the above results about 3b in metal probe designing and based on reported metal cation chemosensors,[9,12−18] we finally describe the proposed sensing mechanism of 3b toward Hg2+. In this way, upon addition of Hg2+ to dissolution of the bidentate ligand 3b, fluorescence quenching is observed as a result of the coordination 2 3b:Hg2+, which promotes a ligand-to-metal charge transfer (LMCT) process in the excited state. This interaction provides a pathway for the nonradiative deactivation of the excited state that manifests itself in the quenching of the fluorescence emission.[37,38] The sensing mechanism and the reversibility process through the complex 2 3b:Hg2+ are shown in Scheme .

Scheme 6

Proposed turn-off fluorescence mechanism for Hg2+ sensing using the probe 3b

Conclusions

In summary, both the 4-vinyl- and 4-styryl-pyrazoles substituted at position 3 with donor or acceptor groups were sequentially produced in good yields by Wittig olefination (from 4-formylpyrazoles) and then using the Mizoroki–Heck coupling reaction. These π-extended pyrazoles were characterized by spectroscopic and HRMS analysis, which allowed us to find that they are key fluorophores with strong blue-light emission and high quantum yields due to ICT phenomena. The presence of the styrene moiety in product 3 increases the π-conjugation inducing bathochromic shifts in both the UV–vis and fluorescence spectra. The ligand 3b was evaluated as a “turn-off” fluorescent probe for Hg2+ detection since its structure has two donor N atoms at pyridine and pyrazole rings suitably located to achieve the formation of chelates. The results showed a selective detection of Hg2+ versus other cations (LOD = 3.1 × 10–7 M, R2 = 0.995). Photophysical and HRMS analysis and 1H NMR titration experiments confirmed the formation of the complex 2 3b:Hg2+ with stoichiometry of 2:1. Reversibility studies confirmed the reignition of the probe 3b after adding ethylenediamine, indicating that the interaction of 3b with Hg2+ is the direct cause of the fluorescence dulling and not external agents. Therefore, the 4-styrylpyrazoles 3a–3c or even their precursors 4a–4c appear to be good models for designing novel fluorescent probes in metal ion detection.

Experimental Section

General Information

All reagents were purchased from commercial sources and used without further purification unless otherwise noted. All starting materials were weighed and handled in air at room temperature. The reactions were monitored by TLC visualized by a UV lamp (254 or 365 nm), which were performed on Merck TLC-plates aluminum silica gel 60 F254. Column and flash chromatography were performed on silica gel (70–230-mesh and 230–400-mesh, respectively). All reactions under MW irradiation were performed using a sealed reaction vessel (10 mL, max pressure = 300 psi) containing a Teflon-coated stirring bar (obtained from CEM). MW-assisted reactions were performed in a CEM Discover focused microwave (ν = 2.45 GHz) reactor equipped with a built-in pressure measurement sensor and a vertically focused IR temperature sensor; controlled temperature, power, and time settings were used for all reactions. NMR spectra were recorded at 400 MHz (1H) and 100 MHz (13C) at 298 K. NMR spectroscopic data were recorded in CDCl3 using as internal standards the residual nondeuteriated signal for 1H NMR and the deuteriated solvent signal for 13C NMR spectroscopy. DEPT spectra were used for the assignment of the carbon-type signals. Chemical shifts (δ) are given in parts per million, and coupling constants (J) are given in Hertz. The following abbreviations are used for multiplicities: s = singlet, d = doublet, t = triplet, and m = multiplet. Melting points were collected using a capillary melting point apparatus and are uncorrected. HPLC–high-resolution mass spectra (HRMS) data were recorded using a Q-TOF spectrometer via electrospray ionization (ESI, 4000 V). The mass spectrum of the byproduct 3b″ (Figure S5) was recorded using a DSQ II spectrometer (equipped with a direct inlet probe) operating at 70 eV. Noncommercially available 4-formylpyrazoles 1a–1d were prepared using our established protocol (see Scheme S2 in the Supporting Information).[19] The electronic absorption and fluorescence emission spectra were recorded in quartz cuvettes having a path length of 1 cm. UV–vis and fluorescence measurements were performed at room temperature (∼20 °C). For fluorescence measurements, both the excitation and emission slit widths were 5 nm.

Synthesis and Characterization

General Procedure for the Synthesis of 1,3-Diaryl-4-vinyl-1H-pyrazoles 4a–4d

To a solution of methyltriphenylphosphonium bromide (2b, 1.29 g, 3.60 mmol) in dry THF (14 mL) at −40 °C was added dropwise n-BuLi in hexane (1.45 mL, 2.5 M, 3.60 mmol). The mixture was stirred for 20 min. Then, a solution the appropriate 4-formylpyrazole (1a–1d) (1.2 mmol) in dry THF (8 mL) was added dropwise to the solution of phosphorus ylide at −40 °C. The resultant mixture was gradually heated to room temperature and stirred for 5 h. The reaction was quenched by addition of a saturated NH4Cl solution (20 mL), and it was extracted with diethyl ether (3 × 20 mL). The organic phases were combined and dried over anhydrous Na2SO4, and the solvent was removed in vacuo to give a residue, which was purified by column chromatography on silica gel (eluent:DCM) to give the expected 4-vinylpyrazoles 4a–4d in good yields.

2-(3-(4-Nitrophenyl)-4-vinyl-1H-pyrazol-1-yl)pyridine (4a)

Following the general procedure in the reaction with 3-(4-nitrophenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde (1a, 353 mg, 1.20 mmol), the 4-vinylpyrazole 4a was obtained as a light orange solid (192 mg, 55%). Mp: 112–114 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.75 (s, 1H), 8.44 (d, J = 4.9, 1H), 8.31 (d, J = 8.9 Hz, 2H), 8.07 (d, J = 8.3, 1H), 7.94 (d, J = 9.1 Hz, 2H), 7.85 (m, 1H), 7.23 (m, 1H), 6.69 (dd, J = 17.5 and 11.0 Hz, 1H), 5.68 (d, J = 17.5, 1H), 5.32 (d, J = 11.0, 1H) ppm. 13C{1H} NMR (100 MHz, CDCl3): δ = 151.0 (C), 149.5 (C), 148.2 (CH), 139.7 (C), 138.9 (CH), 138.8 (C), 128.9 (CH), 126.3 (CH), 125.6 (CH), 123.9 (CH), 122.0 (CH), 121.4 (C), 116.4 (CH2), 112.6 (CH). HRMS (ESI+): calcd for C16H13N4O2+, 293.1033 [M + H]+; found, 293.1033.

2-(3-Phenyl-4-vinyl-1H-pyrazol-1-yl)pyridine (4b)

Following the general procedure in the reaction with 3-(phenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde (1b, 300 mg, 1.2 mmol), the 4-vinylpyrazole 4b was obtained as a white solid (237 mg, 80%). Mp: 59–61 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.74 (s, 1H), 8.43 (d, J = 4.9 Hz, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.82 (m, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.49–7.38 (m, 3H), 7.19 (m, 1H), 6.71 (dd, J = 17.6, and 11.0 Hz, 1H), 5.64 (d, J = 17.6 Hz, 1H), 5.23 (d, J = 11.0 1H) ppm. 13C{1H} NMR (100 MHz, CDCl3): δ = 152.3 (C), 151.3 (C), 148.0 (CH), 138.7 (CH), 133.3 (C), 128.5 (CH), 128.4 (CH), 128.3 (CH), 127.0 (CH), 124.8 (CH), 121.4 (CH), 120.8 (C), 114.7 (CH2), 112.5 (CH). HRMS (ESI+): calcd for C16H14N3+, 248.1182 [M + H]+; found, 248.1179.

2-(3-(4-Methoxyphenyl)-4-vinyl-1H-pyrazol-1-yl)pyridine (4c)

Following the general procedure in the reaction with 3-(4-methoxyphenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde (1c, 335 mg, 1.2 mmol), the 4-vinylpyrazole 4c was obtained as a white solid (303 mg, 91%). Mp: 72–74 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.71 (s, 1H), 8.42 (d, J = 4.9 Hz, 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.80 (m, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 6.99 (d, J = 8.9 Hz, 2H), 6.69 (dd, J = 17.6 and 11.4 Hz, 1H), 5.62 (d, J = 17.6 Hz, 1H), 5.21 (d, J = 11.5 Hz, 1H), 3.86 (s, 3H) ppm. 13C{1H} NMR (100 MHz, CDCl3): δ = 159.8 (C), 152.1 (C), 151.4 (C), 148.0 (CH), 138.6 (CH), 129.7 (CH), 127.1 (CH), 125.6 (C), 124.7 (CH), 121.3 (CH), 120.6 (C), 114.6 (CH2), 114.0 (CH), 112.5 (CH), 55.4 (CH3). HRMS (ESI+): calcd for C17H16N3O+, 278.1288 [M + H]+; found, 278.1285.

1,3-Diphenyl-4-vinyl-1H-pyrazole (4d)

Following the general procedure in the reaction with 1,3-diphenyl-1H-pyrazole-4-carbaldehyde (1d, 300 mg, 1.2 mmol), the 4-vinylpyrazole 4d was obtained as a yellow oil (222 mg, 75%). Mp: 65–67 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.10 (s, 1H), 7.82–7.73 (m, 4H), 7.50–7.43 (m, 4H), 7.42 (m, 1H), 7.30 (m, 1H), 6.74 (dd, J = 17.6 and 11.0 Hz, 1H), 5.58 (d, J = 17.6 Hz, 1H), 5.22 (d, J = 11.0 Hz, 1H) ppm. 13C{1H} NMR (CDCl3, 100 MHz) δ = 151.4 (C), 140.0 (C), 133.2 (C), 129.4 (CH), 128.5 (CH), 128.5 (CH), 128.1 (CH), 127.2 (CH), 126.5 (CH), 124.7 (CH), 120.6 (C), 119.1 (CH), 114.1 (CH2) ppm. HRMS (ESI+): calcd for C17H15N2+, 247.1230 [M + H]+; found, 247.1227.

General Procedure for the Synthesis of (E)-4-Styryl-1H-pyrazoles 3a–3d

A mixture of the respective 4-vinylpyrazole 4a–4d (0.4 mmol), halobenzene 2a or 2b (1.0 mmol), Et3N (0.1 mL), 10 mol % of Pd(AcO)2, 20 mol % of phosphine (PPh3 or (o-MePh)3P), and 3 mL of dry DMF was irradiated with MW at 150–180 °C (100 W, monitored by an IR temperature sensor) and maintained at this temperature for 30 min (3 cycles) in a sealed tube containing a Teflon-coated magnetic stirring bar. The resulting crude was partitioned by addition of DCM and water. The organic layer was washed with water and dried over anhydrous Na2SO4. Subsequently, the solvent was removed under reduced pressure, and the residue was purified by flash chromatography (eluent: n-C6H14:AcOEt 20:1) to afford the (E)-4-styryl-1H-pyrazoles 3a–3d.

(E)-2-(3-(4-Nitrophenyl)-4-styryl-1H-pyrazol-1-yl)pyridine (3a)

Following the general procedure in the reaction with the 4-vinylpyrazole 4a (117 mg, 0.4 mmol), bromobenzene (5a), and PPh3 at 150 °C, the 4-styrylpyrazole 3a was obtained as a yellow solid (94 mg, 64%). Mp: 199–200 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.86 (s, 1H), 8.48 (d, J = 4.8 Hz, 1H), 8.35 (d, J = 8.8 Hz, 2H), 8.09 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.8 Hz, 2H), 7.86 (m, 1H), 7.46 (d, J = 7.4 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.32–7.22 (m, 2H), 7.04 (s, 2H) ppm. 13C{1H} NMR (CDCl3, 100 MHz) δ = 151.0 (C), 150.0 (C), 148.2 (CH), 147.6 (C), 139.7 (C), 138.9 (CH), 137.1 (C), 131.0 (CH), 129.0 (CH), 128.8 (CH), 127.9 (CH), 126.4 (CH), 125.3 (CH), 124.0 (CH), 122.0 (CH), 121.1 (C), 117.5 (CH), 112.6 (CH) ppm. HRMS (ESI+): calcd for C22H17N4O2+, 369.1346 [M + H]+; found, 369.1340.

(E)-2-(3-Phenyl-4-styryl-1H-pyrazol-1-yl)pyridine (3b)

Following the general procedure with the 4-vinylpyrazole 4b (99 mg, 0.4 mmol), bromobenzene (5a), and PPh3 at 150 °C, the 4-styrylpyrazole 3b was obtained as a white solid (100 mg, 77%). Mp: 104–105 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.85 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 8.10 (d, J = 8.3 Hz, 1H), 7.84–7.76 (m, 3H), 7.51–7.41 (m, 5H), 7.34 (t, J = 7.6 Hz, 2H), 7.26–7.19 (m, 2H), 7.05 (q, J = 16.3 Hz, 2H) ppm. 13C{1H} NMR (CDCl3, 100 MHz) δ = 152.8 (C), 151.3 (C), 148.1 (CH), 138.7 (CH), 137.6 (C), 133.1 (C), 129.5 (CH), 128.7 (CH), 128.7 (CH), 128.6 (CH), 128.4 (CH), 127.5 (CH), 126.3 (CH), 124.6 (CH), 121.5 (CH), 120.6 (C), 118.6 (CH), 112.6 (CH) ppm. HRMS (ESI+): calcd for C22H18N3+, 324.1495 [M + H]+; found, 324.1488.

(E)-2-(3-(4-Methoxyphenyl)-4-styryl-1H-pyrazol-1-yl)pyridine (3c)

Following the general procedure with the 4-vinylpyrazole 4c (110 mg, 0.4 mmol), bromobenzene (5a), and PPh3 at 150 °C, the 4-styrylpyrazole 3c was obtained as a white solid (99 mg, 70%). Mp: 125–126 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.82 (s, 1H), 8.44 (d, J = 4.9 Hz, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.81 (m, 1H), 7.70 (d, J = 8.9 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 7.18 (m, 1H), 7.08–6.98 (m, 4H), 3.88 (s, 3H) ppm. 13C{1H} NMR (CDCl3, 100 MHz) δ = 159.9 (C), 152.6 (C), 151.4 (C), 148.1 (CH), 138.7 (CH), 137.6 (C), 129.9 (CH), 129.4 (CH), 128.7 (CH), 127.4 (CH), 126.3 (CH), 125.7 (C), 124.5 (CH), 121.3(CH), 120.5 (C), 118.8 (CH), 114.2 (CH), 112.5 (CH), 55.4 (CH3) ppm. HRMS (ESI+): calcd for C23H20N3O+, 354.1601 [M + H]+; found, 354.1595.

(E)-1,3-Diphenyl-4-styryl-1H-pyrazole (3d)

Following the general procedure in the reaction with the 4-vinylpyrazole 4d (98 mg, 0.4 mmol), halobenzene (2a or 2b), and (o-MePh)3P at 150–180 °C, the 4-styrylpyrazole 3d was obtained as a white solid (110 mg, 85%). Mp: 121–122 °C. 1H NMR (CDCl3, 400 MHz): δ = 8.20 (s, 1H), 7.82–7.76 (m, 4H), 7.51–7.40 (m, 7H), 7.37–7.00 (m, 3H), 7.25 (m, 1H), 7.02 (d, J = 16.3 Hz, 1H), 6.87 (d, J = 16.3 Hz, 1H) ppm. 13C{1H} NMR (CDCl3, 100 MHz) δ = 151.9 (C), 139.9 (C), 137.6 (C), 133.2 (C), 129.5 (CH), 129.0 (CH), 128.7 (CH), 128.6 (CH), 128.5 (CH), 128.2 (CH), 127.4 (CH), 126.6 (CH), 126.2 (CH), 124.5 (CH), 120.3 (C), 119.1 (CH), 118.8 (CH) ppm. HRMS (ESI+): calcd for C23H19N2+, 323.1543 [M + H]+; found, 323.1535.

Chemosensor Design

UV–Vis Absorption and Fluorescence Studies

The solvochromic studies of 3a–3d and 4a–4d were carried out from 2.0 × 10–4 M stock solutions in toluene, DCM, ACN, DMSO, and EtOH. UV–vis spectra were recorded at 1.0 × 10–5 M and fluorescence spectra were recorded using different concentrations (10.0, 5.0, 2.5, and 1.0 × 10–6 M) and the respective maximum absorption wavelength.

Determination of the Relative Quantum Yields

The relative quantum yields of 3a–3d and 4a–4d were determined by using quinine sulfate (φF = 0.59 in 1.5 × 10–1 M HClO4) as reference and calculated according to the following equation.[39−41]where x and st indicate the sample and standard solution, respectively, φ is the quantum yield, F is the integrated area of the emission, A is the absorbance at the excitation wavelength, and η is the index of refraction of the solvents.

Response of the PSP 3b to Metal Ions

Initially, this study was carried out from solutions of 3b in EtOH:H2O (9:1 v/v, pH = 7.14 at 20 °C) 1.0 × 10–6 M (fluorescence) and 4.0 × 10–6 M (absorbance). Salts used in stock solutions of metal ions were NaCl, KNO3, Mg(NO3)2·6H2O, CaCl2, Ba(NO3)2, Cr(NO3)3·9H2O, Fe(NO3)3·9H2O, Co(NO3)2·6H2O, Ni(NO3)2·6H2O, Cu(NO3)2·2.5H2O, Zn(NO3)2·6H2O, CdCl2, Hg(NO3)2·H2O, Al(NO3)3·9H2O, and Pb(NO3)2. These salts were dissolved in deionized water to afford the respective aqueous solutions (1.0 × 10–3 M). In the selectivity and competition experiments of 3b toward Hg2+ and other metal ions, the fluorescence emission spectra were recorded at λex = 320 nm from 1.0 × 10–6 M of 3b in the presence of a high concentration (100 equiv) of different metal ions, achieving EtOH:H2O final solutions (∼9:1 v/v at 20 °C). Solutions were stirred and waited for 20 min before taking the respective measurements, but the response was immediate when using Hg2+ (fluorescence quenching observed under a UV lamp). The sensing studies were performed at pH 7.14 because it is close to the physiological pH (approximately 7–7.4). The fluorescence intensities were measured at λem = 390 nm. For the Job’s plot experiment of 3b and Hg2+, the total concentrations of 3b and Hg2+ were kept as 1.0 × 10–6 M. The fluorescence response in pictures was excited at 365 nm using a handheld UV lamp.

Determination of the Detection Limit

The detection limit (LOD) of 3b for Hg2+ was obtained by 3Sb/k where Sb is the standard deviation of the blank measurements (by 10 times, Sb = 0.8527) and k is the slope from the plot fluorescence intensity I versus [Hg2+].[42,43]

Reversible Study with Ethylenediamine

Once there was quenching fluorescence of 3b (2.0 × 10–6 M in EtOH:H2O, 9:1 v/v) with the respective quantity of Hg2+, the fluorescence emission spectra (λexc = 320 nm at 20 °C) were recorded upon addition of different quantities of ethylenediamine dissolved in EtOH:H2O (9:1 v/v). For a turn-off 1:100 (3b:Hg2+) solution, molar ratios from 1 to 3 of diamine with respect to Hg2+ were used to achieve a fluorescence turn-on, while for a 1:200 (3b:Hg2+) solution, molar ratios from 2 to 6 of diamine versus Hg2+ were used.