A novel profluorescent paramagnetic diaza-crown ether: synthesis, characterization and alkaline metal-ion complexation

Starting from Kryptofix 22 two different branches were covalently attached through the nitrogen atoms, one containing a fluorescent moiety and the other the stable free radical TEMPO. The novel derivative exhibits fluorescence and paramagnetic properties, while the diaza-crown part ensures the affinity for alkaline metal-ions.

Fluorescence 1 and electron paramagnetic resonance (EPR) 2 are two versatile techniques that found useful applications in various elds, especially for detection purposes, taking advantage of the two very different working principles, i.e. uorescence as an optical method, and EPR as a magnetic method. Combination of these two techniques is possible by coupling in the same molecule two different moieties, one uorescent and one paramagnetic, yielding a dual behaviour of the resulted compound, usually called prouorescent free radicals. 3 Such examples are very useful for the detection of numerous biological analytes with very interesting performance. 4 Stable free radicals of nitroxide type are oen encountered in the literature due to their great stability in an open atmosphere (they do not react with oxygen, nor dimerize) over a large range of temperatures. Usually, covalent attachment of a nitroxide moiety to a uorescent compound leads to intramolecular uorescence quenching. By switching off the paramagnetic centre (i.e. via reduction reactions), the uorescence can be restored, providing dual paramagnetic-uorogenic probes useful as detection tools.
Our previous work 5 in the eld of prouorescent nitroxides involved synthesis of novel compounds based on a new type of uorogenic core, 2,5-disubstituted-1,3,4-oxadiazoles and TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) stable free radical, as paramagnetic component that were demonstrated useful in the detection and quantication of some analytes, i.e. sodium ascorbate.
Continuing this topic, we aimed for preparation of new prouorescent nitroxides that contain a third functional unit. Aza-crown ether moieties 6 are widely encountered in supramolecular assemblies, for synthesis of host-guest systems, generally acting as receptors for metal-ions. 7 Combining all these structural motifs may result in unusual chemical, optical, or electronic properties, considering that aza-crown ethers covalently functionalized with uorophores were successfully used in analytical chemistry as (chemo)sensors, due to high sensitivity of the uorescence technique and the affinity of the azacrown ether moiety for specic cations. 8 Whereas uorescent (aza)crown ethers are widely encountered in literature, spinlabelled (aza)crown ethers were much less explored; very few available papers describe, besides the expected complexation, structural and dynamical information about the environmental recognition processes. 9 To the best of our knowledge, there is only one paper 10 describing a double crown ether sensor, containing a uorophore (acridine) and a paramagnetic core (nitroxide), covalently linked through a 18-crown-6 ether moiety. In this context, we describe the synthesis and characterisation of a new nitrobenzo-1,2,5-oxadiazole (NBD) and/or TEMPO functionalised compounds in which the uorophore and free radical are linked through a diaza-crown ether (Scheme 1). We investigated the possibility to switch between luminescence and paramagnetism as well as the ability to form complexes with alkaline metal-ions. The choice for NBD as uorogenic unit was based on its widely use for development of chemosensors for various analytes i.e. cysteine, homocysteine, glutathione, vitamin C. 11 NBD-chloride is a versatile reagent that is highly reactive in S N Ar with common N, O or S nucleophiles, and, therefore, it was one of the rst reagents used in non-specic protein labelling. 12 Synthesis of the novel uorescent diaza-crown ether 3 was accomplished by reaction of the furazane 1 and the aza-crown ether 2, well known as Kryptox 22, in a good yield (62%) by simply stirring the two reactants in dichloromethane (DCM), using triethylamine as base. Compound 3 has not been described up to now. However, the disubstituted aza-crown derivative was previously obtained through a slightly modied procedure. 8b,13 Further, compound 3 underwent an amide coupling reaction with 4-carboxy-TEMPO, using common amide coupling conditions: activating reagent PyBOP (benzotriazol-1yl-oxytripyrrolidinophosphonium hexauoro-phosphate) and DIPEA (N,N-diisopropylethylamine) as base in DCM. The reaction proceeded smoothly, in a very short reaction time, in 81% isolated yield.
The NMR study ( Fig. 1) of compound 3 was performed in DMSO-d 6 and CDCl 3 (see ESI † for full spectra). In the aromatic region of the spectrum registered in DMSO-d 6 , we could observe a downeld of the chemical shis corresponding to the signals of the aromatic protons (d H 5 ¼ 8.46/8.40 ppm and d H 6 ¼ 6.54/ 6.21 ppm for DMSO-d 6 /CDCl 3 ), whereas in the aliphatic region, the signals corresponding to the methylene protons of the ether residue are slightly shielded (i.e. d H 8/8' ¼ 4.23/4.30 ppm d H 9/9' ¼ 3.79/3.86 ppm for DMSO-d 6 /CDCl 3 ). The data obtained in DMSO-d 6 is consistent with previously reported data for the disubstituted derivative. 8b,13 However, the high shield of the signals corresponding to the aromatic protons, especially H 6 was intriguing. Structurally similar aza-crown ethers bearing only one nitrogen atom displayed a chemical shi corresponding to H 6 at d ¼ 6.35 ppm. 8b Previous studies showed that the conformation of the aza-crown ethers is highly dependent on the substituents of the nitrogen atoms, which undergo inversion when substituted, with the lone pair electrons oriented toward the interior of the cavity. 14 In this context, the high shield of the aromatic protons in the vicinity of the azacrown ether moiety could be caused by conformation changes in environments of different polarities.
Another interesting observation with respect to previous reported data is the value of the chemical shis corresponding to protons H 8/8' and H 9/9' which are signicantly downeld compared to similar compound bearing N-methyl groups (d ¼ 2.74 ppm and d ¼ 3.56 ppm). 14 A possible hindered rotation between the aza-crown ether and the p-nitrofurazane moieties may also be the cause of the signals broadening. This could be observed for the signal corresponding to H 8 (d ¼ 4.23 ppm, broad signal). The carbon spectrum also displays broad signals of the carbons labelled with C-8 and C-9 (d ¼ 53.6 ppm and d ¼ 68.6 ppm, respectively, see ESI †). Finally, the proton of the free nitrogen atom is visible in the spectrum registered in CDCl 3 , at d ¼ 2.67 ppm, in accordance to previously reported data for similar compounds, although slightly downeld shied. 15 The absorption spectrum of compound 3 registered in DMSO at 20 mM (Fig. 2) showed absorption maxima around 350 and 500 nm (Table 1), corresponding to the transitions of the NBD moiety, 13 indicating that attachment of the diaza-crown ether moiety did not inuence the absorption behaviour of the oxadiazole. Variation of absorption maxima according to the solvent used, as inferred from the literature data 8b conrm  Further measurement of the uorescence spectra also showed a variation of the luminescence intensity according to the solvent polarity. Thus, the spectra recorded in DMSO or mixture of DMSO/water (1% DMSO in PBS buffer or 10% DMSO in ultrapure water) displayed emission maxima at l em ¼ 550 nm (l ex ¼ 500 nm), corresponding to the NBD moiety emission. However, the intensity of the emission bands decreased upon polarity increase, suggesting uorescence quenching as a result of the polar solvent induced aggregation of the organic molecules.
Solid state uorescence of compound 3 (Fig. 2) indicated an emission maximum at l em ¼ 564 nm (l ex ¼ 490 nm), slightly red shied compared to the emission in solution (Table 1), with an appreciable Stokes shi (74 nm).
We further studied the behaviour of compound 3 in presence of alkaline metal ions (Li + , Na + , K + ) by NMR, in order to assess the ability of the new compound for complexation. As previously mentioned, the nitrogen lone pair electrons were demonstrated to afford a better binding when substituents are present. 14 However, most of the examples include aliphatic substituents 8b,14 while in our case conjugation occurs by coupling the aza-crown ether to the aromatic NBD moiety.
Thus, addition of excess amounts of KClO 4 in D 2 O to a solution of 3 in DMSO-d 6 (approx. 25 mM nal concentration) led to more visible changes in the 1 H NMR spectra proles, compared to addition of lithium or sodium ions (Fig. 3 and  ESI †). This could be correlated with the dimensional t between the size of the crown-ether cavity and the diameter of the cation, suggesting a higher affinity of the compound for potassium ions. 16 However, the chemical shis slightly changed also for the two other cations, indicating a non-selective behaviour of 3. Generally, the signals were well resolved and could be assigned to most protons in the structure of compound 3. The signal multiplicities preserved upon addition of the aqueous metal ion solution and we could notice a shield of the aromatic doublets with approximately Dd ¼ 0.13 ppm for the proton in the vicinity of the nitro group (H 5 , d ¼ 8.46 ppm, d' ¼ 8.33 ppm) and Dd ¼ 0.21 ppm for the proton in the vicinity of the crown-ether moiety (H 6 , d ¼ 6.53 ppm, d' ¼ 6.32 ppm) upon addition of potassium ions solution (Fig. 3). Signicant changes were also visible in the aliphatic region: all signals of the mixtures were shielded. For example, the protons next to the conjugated nitrogen atom (H 8 , d ¼ 4.23 ppm, d' ¼ 4.14 ppm) yield a broad signal, which shis with approximately Dd ¼ 0.08 ppm. Spectra containing only deuterated water and no metal ion were also recorded to conrm that the changes in the spectra were the effect of the alkaline metal ions. The unusual shielding effect could be explained by changes in the conformation of the compound upon complexation, as also previously observed by others in studies regarding behaviour of diazacrown ethers toward metal ions like barium. 17 We have also performed titration experiments with potassium ions in DMSO-d 6 and conrmed 1 : 1 stoichiometry (see ESI †). However, the association constant was found to be rather low (<10 2 M À1 , see ESI †), suggesting that the nitrogen atom contributes less, due to involvement in conjugation with the aromatic system. 18 Complexation studies were also performed by ESI(+)-MS experiments. The ability of compound 3 to host alkaline metal ions was assessed by running experiments with alkaline metal ions (LiClO 4 , NaClO 4 , KClO 4 and CsClO 3 ), indicating formation of all supramolecular complexes between compound 3 and each of the tested alkaline metal ions (see ESI † for full spectra).
Once compound 3 characterized, we turned our attention to the triple functional compound 4 and investigated the paramagnetic properties, optical behaviour and complexation properties. Thus, the EPR spectrum showed the expected triplet of a nitroxide free radical (Fig. 4), with a hyperne coupling constant of 1.575 mT. The intensity of the high eld line is slightly smaller, conrming the successful attachment of the free radical moiety to the diaza-crown ether central unit. The HRMS spectrum also conrms the formation of the target structure (see ESI †).
Recently, 19 TEMPO-derived compounds were investigated by 1 H and 13 C NMR experiments, indicating that carbon spectra can be very informative for the atoms located far enough from the free radical site. This was also conrmed in our case (see  This journal is © The Royal Society of Chemistry 2019 ESI † for full spectra and Fig. 4). While the proton spectrum displays broad, unresolved signals, the 13 C NMR spectrum of compound 4 indicated signals that could be assigned to the furazan and diaza-crown ether cores as well as the carbonyl carbon from the amide moiety at d ¼ 170.2 ppm. In addition, the spectrum showed signals that seem to correspond to carbons of the TEMPO skeleton. However, precise assignment could not be performed, due to lower solubility of our compound (unlike previous results, which were possible using very high concentrations) and low accuracy of the 2D NMR experiments. In addition, the stability of the compound in DMSO-d 6 seems to be affected in time, making the analysis of lengthy experiments to be less reliable.
In a recent paper 20 Lucarini et al. demonstrated the use of a novel spin-labelled crown-ether in sensing of metal and organic cations, in host-guest complexation processes. Measurements were based on the change that occurs on EPR hyperne splitting constants of benzylic hydrogen and/or of nitrogen atom from nitroxide moiety. In our case, only nitrogen hyperne splitting constants might be affected, but test experiments showed no change outside the experimental errors.
Investigation of compound 4 by ESI(+)-MS revealed a prole of the mass spectrum that is coherent with previous studies of nitroxide based free radicals. 21 Various redox processes may occur under the ionisation conditions leading to fragmentation of the free radical moiety (see ESI † for full spectrum). Thus, the resulted base peak (m/z ¼ 593.3257) should correspond to species [M + H 2 -O] + , previously observed 21 for TEMPO-based radicals. In addition, a peak at m/z ¼ 609.3207 (14%) corresponding to [M + H 2 ] + ion could have resulted from the protonated hydroxylamine via a reduction process. The spectrum also shows the [M + Na] + ion peak (630.2946, 27%). Complexation studies with alkaline metal ions showed the expected host guest complexes for all cations (LiClO 4 , NaClO 4 , KClO 4 and CsClO 3 ). The competitive experiments using solutions containing equimolar amounts of the three metal ion salts, in ve fold excess, yielded peaks with different relative abundances: base peak for [M + Li] + (614.3284), 68% for [M + Cs] + peak (740.2188), 23% for [M + Na] + (630.3020) and 18% for [M + K] + peak (646.2761). All these data suggest that compound 4 acts as a non-selective host for the alkaline metal-ions.
We further set to study the potent prouorescent behaviour. The absorption spectra registered in solution in the same conditions as for compound 3 indicated a similar prole, caused by the uorogenic NBD moiety (Fig. 2). The uorescence spectra also behaved similarly in terms of proles in organic solvent and water, namely we noticed a decrease in the luminescence intensity by increase in the solvent polarity (Fig. 2). However, a much more interesting observation was the decreased uorescence intensity of approximately 20% with respect to precursor 3 which suggested that the uorophore did not strongly interact with the free radical system, in order to cause complete luminescence quench, as in the case of our previously described compounds. 5 This could be mainly attributed to the lack of a conjugated system which usually affords complete switching cycles between the paramagnetic and uorescent states, upon applying a redox trigger. In addition, in solid state (Fig. 2) we could observe a signicant quench of the uorescence, along with a blue shi of the emission maximum (l em ¼ 552 nm, l ex ¼ 490 nm, Table 1), suggesting stronger interactions between uorophore and free radical and the prouorescent character of 4.
In uorescence studies (Fig. 2), we observed that addition of vefold excess of alkaline metal ions (LiClO 4 , NaClO 4 , KClO 4 and CsClO 3 ) to compound 4 led to decrease in the uorescence intensity for all metal ions. The experiments were preformed in triplicates and were reproducible. The effect of water over the uorescence quench was negligible (we used aprox. 2% water in DMSO, which has limited effect on the uorescence) The uorescence quench was more signicant for potassium ions, especially with respect to the same experiments performed for the precursor 3 (see ESI †). This might be an indication of some interactions that occur between the uorophore unit and the free radical that are mediated by metal ions. However, the EPR spectra of the mixtures between compound 4 and metal-ions do not show any major change. Use of heavy metals or other solvents could bring other interesting observations and we are currently expanding our studies.

Conclusions
In conclusion, we described the synthesis of new uorophoreaza-crown-ether and uorophore-aza-crown-ether-free radical conjugates and their structural investigation, photophysical properties and behaviour in presence of alkaline metal ions. The collected data suggested a weak interaction between the uorophore and the free radical (visible from preservation of the luminescence when the free radical is active) in the uorophore-crown-ether-free radical conjugate. On the other hand, complexation experiments with the alkaline metal ions (Li + , Na + , K + , Cs + ) was studied by NMR and ESI(+)-MS, indicating the ability of the compounds to form supramolecular complexes, with a slight preference for potassium ions. Partial quench of uorescence in solid state or by addition of metal ions may be further exploited and such multifunctional systems containing: (i) an aza-crown ether residue, (ii) a paramagnetic component and (iii) a uorophore could nd useful applications as sensors for monitoring many processes that occur in (bio)chemical systems (i.e. spin-probes, markers, imaging).

Conflicts of interest
There are no conicts to declare.