(1-(4-(5-Phenyl-1,3,4-oxadiazol-2-yl)phenyl)-1H-1,2,3-triazol-4-yl)-methylenyls α,ω-Bisfunctionalized 3- and 4-PEG: Synthesis and Photophysical Studies

Two new azaheterocycle-based bolas, such as (1-(4-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)-1H-1,2,3-triazol-4-yl)-methylenyls α,ω-bisfunctionalized PEGs, were prepared via Cu-catalyzed click reaction between 2-(4-azidophenyl)-5-(aryl)-oxadiazole-1,3,4 and terminal ethynyls derived from PEG-3 and PEG-4. Due to the presence of two heteroaromatic cores and a PEG linker, these bola molecules are considered as promising fluorescent chemosensors for electron-deficient species. As a result of a well-pronounced “turn-off” fluorescence response towards common nitro-explosive components, such as 2,4-dinitrotoluene (DNT) and 2,4,6-trinitrotoluene (TNT), hard-to-detect pentaerythritol tetranitrate (PETN), as well as Hg2+ cation was observed.


Introduction
One of the most important tasks of modern synthetic organic chemistry is to obtain new compounds that will find wide application in various industries and medicine. These could be fluorophores that contain cyclic azole and azine fragments as pharmacophores. The targeted synthetic design of functional bola-type scaffolds and the study of their response to conformational changes or the presence of (bio)analytes are of key importance for their application in a wide variety of fields. Typical bola scaffolds are composed of two functional units acting as receptors and/or fluorophores and connected by alkyl or heteroatom-containing chains [1]. Bola-type scaffolds have attracted a great deal of attention for their application in biology and pharmacy, including in drug and gene transport systems [2][3][4][5], chemical sensing, biomedical applications, and molecular recognition [6][7][8].
Chemosensor-assisted fluorescence detection is one of the most convenient techniques used for the recognition of various (bio)analytes, including explosives. Noticeable advantages of fluorescence methods are their simplicity, fast detection time, and the possibility of

Synthesis
The synthetic strategy for the preparation of target compounds 3a-b is presented below (Scheme 1). This strategy involves a copper-catalyzed azide-alkyne cycloaddition. Thus, an azide component, 4-azidophenyloxadiazole 2, was prepared by means of modified Sandmeyer reaction [21,22]. Additionally, the ethynyl components, such as alkyne-terminated ethylenglycols 1a-b, were prepared as reported previously [23] (Scheme 1). As a final step, Cu(I)-promoted azido-alkyne coupling (CuAAC) was carried out by means of the heating of compounds 1 and 2 in THF-H 2 O solution in the presence of CuI to afford the target products 3a-b in 30-75% yields. The structures of all the obtained compounds were confirmed by means of 1 H and 13 C NMR spectroscopy, mass spectrometry, and elemental analysis (see ESI for details).
Molecules 2023, 28, x FOR PEER REVIEW 2 of 14 vantages of fluorescence methods are their simplicity, fast detection time, and the possibility of the fine structural tuning of chemosensor/sensory material to improve selectivity and/or desired photophysical properties. In comparison with other physical methods, such as chromatographic methods [9], HPLC-electron capture detection (HPLC-ECD) [10], mass spectrometry [11,12], photoacoustic spectroscopy [13], and electrochemical methods [14,15], fluorescence-chemosensor-based methods are inexpensive, mobile, and accurate, which are crucial factors for their application in the development of highly affordable and reliable sensing devices. Among various types of bola-type scaffolds, arylsubstituted triazole-and oxadiazole-based scaffolds were selected by us due to a bright emission in the visible region [16][17][18]. Compared to other commonly used nitroaromatic explosives, the detection of aliphatic explosives, such as pentaerythritol tetranitrate (PETN)-one of the major components in plastic explosives-is a challenging task due to the low vapor pressure of PETN (7,17 ppt) and its less pronounced electron-deficient character (µ = 0.0033D) [19]. Another problem for the fluorescent detection of PETN is its high-lying LUMO energy level, and to ensure efficient "chemosensor-analyte" PET transfer, high-LUMO energy level chemosensors are required [20].
We recently reported the synthesis of several PAH-containing bola-type chemosensors for the fluorescent "turn-off" detection of explosives in aqueous solutions. In this manuscript, we wish to report our results on the synthesis of bola-type 1,2,3-triazolebased fluorophores/chemosensors and on the study of their photophysical properties and fluorescence response towards some nitro-explosives and metal cations.

Photophysical Studies
Next, the photophysical studies of the 3 compounds were carried out. At concentrations below 2 × 10 −5 M, both samples, 3a-b, were well soluble both in medium and high polar aprotic solvents (THF, dichloromethane, DMSO, acetonitrile) and in high-polar protic solvents such as methanol. In absorption spectra, intensive absorption maxima were observed around 300 nm. Upon excitation at 300 nm, an intensive fluorescence in a range from 325 to 425 nm was observed in emission spectra. The absorption and emission spectra of the samples are presented in Figures 1 and 2, respectively. high molecular absorption extinction coefficients (εM = 30,800-91,500 М -1 sm -1 ), as well as the high values of the oscillator strength (f = 1.47-1.62, Table S6), correspond well with strong π-π*-transitions, with little change in geometry, between electronic ground and excited states.
As shown in Figures 1a and 2a, the polarity of the solvent did not significantly influence the position of absorption peaks, indicating the weakly polar nature of POPOP-like 3 samples in a ground state. On the other hand, fluorescence spectra were more sensitive to solvent polarity. Thus, the increase in the polarity of the solvent resulted in the slight bathochromic shift of the emission maxima along with a significant increase in emission intensity (Figures 1b and 2b). At the same time, structured emission spectra of the samples 3a-b, with high PLQY > 60% and Stokes shifts < 100 nm (Δν = 50 ÷ 58 nm), led to our conclusion about the predominance of the state of fluorophores in local excitation (LE), which correlates well with the data of DFT calculations (see Section 2.3) [24]. As shown in Table 1, samples 3a-b had high molar extinction coefficients in the long wavelength region (εM > 80,000 M −1 cm −1 ) and fluorescence quantum yields above 60%, suggesting that 3a,b are good candidates for practical application as fluorescent sensors/probes.      Both compounds 3a-b can be considered as structural analogs of well-reported POPOP dye. The absorption and emission properties of both fluorophores and the POPOP dye (as a reference sample) in a solution of THF are presented in Table 1. Thus, both fluorophores exhibited 2 similar absorption bands, the high-energy band at λ max 216 nm and the lower energy band at λ max 300 nm, which correspond to the S 0 →S 2 and S 0 →S 1 transitions. Both samples had a dominative transition band of S 0 → S 1 with εM > 80,000 M −1 sm −1 . These high molecular absorption extinction coefficients ( εM = 30,800-91,500 M -1 sm -1 ), as well as the high values of the oscillator strength (f = 1.47-1.62, Table S6), correspond well with strong π-π*-transitions, with little change in geometry, between electronic ground and excited states.
As shown in Figures 1a and 2a, the polarity of the solvent did not significantly influence the position of absorption peaks, indicating the weakly polar nature of POPOP-like 3 samples in a ground state. On the other hand, fluorescence spectra were more sensitive to solvent polarity. Thus, the increase in the polarity of the solvent resulted in the slight bathochromic shift of the emission maxima along with a significant increase in emission intensity (Figures 1b and 2b). At the same time, structured emission spectra of the samples 3a-b, with high PLQY > 60% and Stokes shifts < 100 nm (∆ν = 50 ÷ 58 nm), led to our conclusion about the predominance of the state of fluorophores in local excitation (LE), which correlates well with the data of DFT calculations (see Section 2.3) [24]. As shown in Table 1, samples 3a-b had high molar extinction coefficients in the long wavelength region (ε M > 80,000 M −1 cm −1 ) and fluorescence quantum yields above 60%, suggesting that 3a,b are good candidates for practical application as fluorescent sensors/probes. Next, time-resolved fluorescence studies were carried out. Thus, for both samples, all the time-resolved fluorescence decays could be fitted with double exponential components whose amplitudes αi and time constants τ i , are presented in Table S1. We assume that for the samples 3a-b, the fast component may be related to the processes of rapid fluorescence quenching that does not reach the stabilized LE geometry.
To study the fluorescence "turn-off" response of the 3 fluorophores to nitro-containing analytes in various media, the most effective photoinduced electronic transition, LE, was selected [26]. To study the fluorescence response, the most common nitroaromatic explosive components, DNT and TNT, were selected, as well as low volatile and hard-to-to detect nitro-explosives such as pentaerythritol tetranitrate (PETN). Fluorescence studies were carried out in MeCN solution at 296 K by using concentrations of the chemosensors 3 of 10 -6 M in order to avoid self-quenching. To calculate the quenching efficiency of the chemosensors 3a-b, the Stern-Volmer mathematical model was used: where I 0 and I are fluorescence intensity before and after the addition of a nitroaromatic compound (quencher); Q is the concentration of the nitroaromatic compound, mol/L; and K sv is the value of the constant, M −1 .
Based on the results of fluorescence quenching experiments, the Stern-Volmer quenching constants (K sv ) of the "3a/3b sample-nitro-analyte" were estimated by means of measuring the slope of the SV plot, and the constants' value was found to be as high as 1.0 × 10 −4 M −1 for the nitroaromatic aromatic quenchers and 0.5 × 10 −4 M −1 for the PETN ( Table 2). The minimum limit of detection (LOD) was calculated based on the fluorometric titration data for 3a-b [27]. The high order of binding constants (~0.7 × 10 4 M −1 ), with detection limits of 3 × 10 −7 M (~95 ppb), identified the sample 3b as the most promising for the qualitative and quantitative detection of nitro compounds of various natures. The obtained data are comparable with values of quenching constants and LOD for the previously reported chromophores/chemosensors [28]. To confirm the selectivity of 3a,b towards nitroaromatic compounds, their fluorescent response to a nonexplosive compound (such as highly electron-deficient benzoquinone (BQ)) was studied and, as a result, no fluorescence quenching was observed.
The linear quenching response on the SV plots for the 3 chemosensors ( Figures S3-S26) suggests that only one quenching mechanism prevails. The static quenching assumes the formation of stable nonfluorescent complexes' "chemosensor-quencher". However, we did not observe the formation of such a complex between the chemosensor 3b and TNT during the UV-Vis spectrophotometric titration experiments ( Figure S27). The dynamic quenching model was also ruled out as, during the time-resolved fluorescence quenching, no decrease of the lifetime of the chemosensor 3b upon increasing the concentration of TNT was observed (Table S3, Figures S28 and S29). Based on these observations, a false static quenching model via photoinduced electron transfer (PET) was suggested [29]. In this case, unlike for the static quenching model [30], no stable nonfluorescence adduct formed, but fluorescence quenching takes place due to the presence of a quencher on a periphery (action volume) of a chemosensor/fluorophore [31].

DFT Calculations
To support the experimental results, DFT calculations were carried out at the CAM-B3LYP/6-31G*//PM6-level of theory by using the Gaussian-09 [32] program package. No symmetry restrictions were applied during the geometry optimization procedure. The solvent effects were taken into account using the SMD (solvation model based on density) continuum solvation model suggested by Truhlar and coworkers [33]. The Hessian matrices were calculated for all optimized model structures to prove the location of correct minima on the potential energy surface (no imaginary frequencies were found in all cases). The Cartesian atomic coordinates for all optimized equilibrium model structures are presented in the attached xyz-files. Hole-electron analysis was carried out in Multiwfn program, Version 3.7 [34].
As a first step, in order to evaluate the internal characteristics of the processes of electronic excitation, an analysis of the difference in electron density was carried out (Figure 3). According to Table S6, all the main absorption peaks of 3a-b belong to the S 0 -S 1 transition; therefore, the analysis of the electron density difference is built between the S 0 and S 1 states and refers to the π-π* transition.
As shown in Figure 3, blue and green isosurfaces (isovalue = 0.001) represent hole and electron distributions, respectively. The highly overlapped isosurfaces and the fact that the hole and electron occupy a similar spatial region prove that the S 0 -S 1 transition is a local excitation process, which agrees well with the experimental data. For both samples (3a,b), the electron density isosurfaces are concentrated within one aromatic part of the molecule, which is associated with the violation of the initial conjugation due to the presence of the PEG bridge. Thus, special optical properties of sensors are associated with different distributions of electronic excitation.
(3a,b), the electron density isosurfaces are concentrated within one aromatic part of the molecule, which is associated with the violation of the initial conjugation due to the presence of the PEG bridge. Thus, special optical properties of sensors are associated with different distributions of electronic excitation. In order to study the sensing mechanism, optimized configurations of adducts of interaction of samples with analytes, which are difficult to obtain from experiments, were considered in detail ( Figure S33). When optimizing the adduct for the sample 3a, the nitroanalyte molecules turned out to be oriented with respect to the polyethylene group. For the sample 3b, NAC were oriented to the 1,2,3-triazole and phenylene fragments, and PETN was oriented to the polyethylene group and the phenylene fragment. Figure 4 shows that in the optimized state, the closest distance between the hydrogen of the phenyl fragment of the sample 3b and the oxygen atoms of the PETN nitro groups was only 2.1 Å, and the distance between the hydrogen atoms of the methylene group and the oxygen atoms of the PETN nitro groups was 2.3 Å, which indicates a strong interaction between the sensor and the analyte. For comparison, the closest distance between the hydrogen atoms of the methylene group 3a and the oxygen atoms of the nitro group of PETN was only 2.3 Å (Figure 4, Table 3). Table 3. Change of the distance between NO2-and methylene moiety for the sample 3a (using the oxygen atom and hydrogen atom as a reference, d1H-O), between NO2-and triazole (using the oxygen atom and hydrogen atom as a reference, d2H-O), and between NO2-and phenyl moiety for 3b (using the oxygen atom and hydrogen atom as a reference, d3H-O).

Probes
Distance, Å DNT In order to study the sensing mechanism, optimized configurations of adducts of interaction of samples with analytes, which are difficult to obtain from experiments, were considered in detail ( Figure S33). When optimizing the adduct for the sample 3a, the nitroanalyte molecules turned out to be oriented with respect to the polyethylene group. For the sample 3b, NAC were oriented to the 1,2,3-triazole and phenylene fragments, and PETN was oriented to the polyethylene group and the phenylene fragment. Figure 4 shows that in the optimized state, the closest distance between the hydrogen of the phenyl fragment of the sample 3b and the oxygen atoms of the PETN nitro groups was only 2.1 Å, and the distance between the hydrogen atoms of the methylene group and the oxygen atoms of the PETN nitro groups was 2.3 Å, which indicates a strong interaction between the sensor and the analyte. For comparison, the closest distance between the hydrogen atoms of the methylene group 3a and the oxygen atoms of the nitro group of PETN was only 2.3 Å (Figure 4, Table 3). Table 3. Change of the distance between NO 2 -and methylene moiety for the sample 3a (using the oxygen atom and hydrogen atom as a reference, d1 H-O ), between NO 2 -and triazole (using the oxygen atom and hydrogen atom as a reference, d2 H-O ), and between NO 2 -and phenyl moiety for 3b (using the oxygen atom and hydrogen atom as a reference, d3 H-O ). Gibbs electronic energies were calculated to evaluate the optimized model structures ( Table 4). Interactions of samples with explosives were considered as hypothetical supramolecular association processes. The results of DFT calculations showed that the "3b-TNT" and "3b-PETN" adducts were the most stable, with a lowest energy of −3672.38 and −3541.75 a.u., respectively. This demonstrates the most energetically favorable combination between the sample 3b with aromatic (TNT) and nitroaliphatic (PETN) analytes, which correlates well with both the K SV values and DFT-optimized adduct configurations.

Probes
The process of photoinduced electron transfer (PET) occurs from the LUMO of the excited sample to the LUMO of NAC, followed by the reverse nonradiative electron transfer to the HOMO of the sample, which leads to fluorescence quenching [28]. Therefore, higher fluorescence quenching efficiency of samples 3a-b was observed for DNT/TNT nitroaromatic compounds with a marginal electron deficit due to their relatively lower LUMO energy levels (LUMO (DNT) 3.00 eV, LUMO (TNT) 3.39 eV) ( Figure 5). Because there was no overlap between the emission spectra of chemosensors 3a-b and the absorption spectrum of the PETN, no energy transfer between them had occurred [35]. Therefore, due to the high LUMO energy level of chemosensors 3a-b (2.35 and 2.36 eV, respectively), a more pronounced driving force of the PET process from the LUMO level of the chemosensor 3b to the LUMO level of PETN takes place (Table 5, Figure 6).  Gibbs electronic energies were calculated to evaluate the optimized model structures ( Table 4). Interactions of samples with explosives were considered as hypothetical supramolecular association processes. The results of DFT calculations showed that the "3b-TNT" and "3b-PETN" adducts were the most stable, with a lowest energy of −3672.38 and −3541.75 a.u., respectively. This demonstrates the most energetically favorable combination between the sample 3b with aromatic (TNT) and nitroaliphatic (PETN) analytes, which correlates well with both the KSV values and DFT-optimized adduct configurations. The process of photoinduced electron transfer (PET) occurs from the LUMO of the excited sample to the LUMO of NAC, followed by the reverse nonradiative electron transfer to the HOMO of the sample, which leads to fluorescence quenching [28]. Therefore, higher fluorescence quenching efficiency of samples 3a-b was observed for DNT/TNT nitroaromatic compounds with a marginal electron deficit due to their relatively lower LUMO energy levels (LUMO (DNT) 3.00 eV, LUMO (TNT) 3.39 eV) ( Figure 5). Because  there was no overlap between the emission spectra of chemosensors 3a-b and the absorption spectrum of the PETN, no energy transfer between them had occurred [35]. Therefore, due to the high LUMO energy level of chemosensors 3a-b (2.35 and 2.36 eV, respectively), a more pronounced driving force of the PET process from the LUMO level of the chemosensor 3b to the LUMO level of PETN takes place (Table 5, Figure 6).

Recognition of Hg 2+ via Fluorescence "Turn Off" Process
Visual detection of highly toxic Hg 2+ cation is important for food safety and environmental monitoring. Hg 2+ is known to form stable complexes with azaheterocyclic ligands [36][37][38] and crown ethers [39][40][41]. Therefore, as a final step, we studied the fluorescence response in solution of the chemosensors 3a-b to the Hg 2+ cation and other metal cations. Thus

Recognition of Hg 2+ via Fluorescence "Turn Off" Process
Visual detection of highly toxic Hg 2+ cation is important for food safety and environmental monitoring. Hg 2+ is known to form stable complexes with azaheterocyclic ligands [36][37][38] and crown ethers [39][40][41]. Therefore, as a final step, we studied the fluorescence response in solution of the chemosensors 3a-b to the Hg 2+ cation and other metal cations.
Thus, in MeCN:H 2 O [90:10 (vol.%)] solutions of chemosensors 3a-b (2 × 10 −4 M), upon UV light excitation (λ max = 365 nm), a dramatic fluorescence quenching was observed when an aqueous solution of mercury acetate was added (2 × 10 −2 M), while for other metal cations, no fluorescence quenching was observed ( Figure S34). To obtain more evidence, we carried out fluorescence titration of the solution of sensor 3a (10 −6 M) by the solution of mercury acetate (2 × 10 −4 M), and a dramatic fluorescence quenching was observed. Similar to 3a, for the sample 3b, a noticeable fluorescence quenching at a very low concentration of Hg 2+ was observed. This quenching, calculated by the 3σ limit of detection (LOD) method, was measured as~2.59 × 10 -6 M ( Table 6, Figure S26). The binding constant for the complex "Hg 2+ :3a" was calculated as a tangent of the slope of the SV plot and amounted to~1.0 × 10 4 M −1 , and the quenching of the fluorescence of 3a by Hg 2+ was as high as 73%. Therefore, chemosensor 3a can be considered as a highly sensitive fluorescent "turn-off" probe for Hg 2+ . Selective recognition of cation Hg 2+ by the sample 3a was also demonstrated by additional experiments in the presence of other metal cations ( Figure S34). Thus, after the addition of other metal cations, such as Cu 2+ , Co 2+ , Cd 2+ , Hg 2+ , Sn 2+ , Zn 2+ , Ni 2+ , and Mg 2+ , only in the case of Hg 2+ was a very clear quenching of the fluorescence of the chemosensor 3a observed. Thus, a very high selectivity of 3a towards Hg 2+ was confirmed. Again, static and/or dynamic quenching was suggested. In order to understand the nature of the quenching mechanism, fluorescence lifetime measurements for the sample 3a in the presence of various concentrations of Hg 2+ were carried out. As a result, no lifetime decrease upon increasing concentrations of Hg 2+ was observed, which rules out a dynamic quenching mechanism (Table S4, Figures S31 and S32) and suggests the static quenching mechanism. However, upon UV-Vis spectrophotometric titration studies, only subtle changes in the UV spectra of sample 3a in the presence of various concentrations of Hg 2+ was observed; thus, no formation of stable molecular complex "3a*Hg 2+ " was confirmed ( Figure S30). These results suggest the false static fluorescence quenching mechanism to be predominant for 3a in the presence of Hg 2+ . Therefore, in the presence of both the nitro-analytes and Hg 2+ cation the fluorescence quenching of chemosensor 3a was observed and, in all the cases, the false static mechanism was suggested to be the predominant one.

Chemical Experiment
Unless otherwise indicated, all common reagents and solvents used were obtained from commercial suppliers without further purification. Melting points were determined on Boetius combined heating stages. TLC and column chromatography were carried out on SiO 2 . Spectra of 1 H NMR and 13 C NMR were recorded at room temperature at 400 and 100 MHz, respectively, on a Bruker DRX-400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) using CDCl 3 as the solvent. Peaks were labeled as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), doublet of doublets of doublets (ddd), and multiplet (m). The mass spectra (electron impact) were measured on a Shimadzu GCMS-QP2010 Ultra instrument (Shimadzu, Kyoto, Japan). Elemental analyses were performed on a PerkinElmer 2400 Series II CHN Analyzer (PerkinElmer, Waltham, MA, USA). UV-Vis absorption spectra were recorded on the Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan), and emission spectra were measured on a HORIBA FluoroMax-4 (HORIBA Jobin Yvon S.A.S., Longjumeau, France) by using quartz cells with 1 cm path length at room temperature. Absolute quantum yields of luminescence of target compounds in solution were measured by using the Quanta-ϕ Integrating Sphere of the HORIBA FluoroMax-4 fluorometer (HORIBA Jobin Yvon S.A.S., Longjumeau, France) at room temperature.

Conclusions
The main goal of the research reported herein was to develop new bola-type azole derivatives by using click reaction methodology in order to investigate their photophysical properties and their sensory response to metal cations and nitro-explosives.