Multistimuli-Responsive Fluorescent Switches Based on Spirocyclic Meisenheimer Compounds: Smart Molecules for the Design of Optical Probes and Electrochromic Materials

Fluorescent switches based on spirocyclic zwitterionic Meisenheimer (SZMC) complexes are stimuli-responsive organic molecules with application in a variety of areas. To expand their functionality, novel switching mechanisms are herein reported for these systems: (a) acidand redox-triggered formation of an additional protonation state with distinct optical properties, and (b) solvent-induced fluorescence modulation. We demonstrate that these new features, which enable both multistimuli and multistate operation of SZMC switches, can be exploited in the preparation of smart organic materials: wide-range pH optical probes, electrochromic and electrofluorochromic films, and polymer-based fluorescent detectors of organic liquids.


INTRODUCTION
In the last decades fluorescent switches have emerged as smart functional systems with application in a variety of areas, ranging from (bio)chemical sensing [1][2][3][4] and imaging 5-8 to information storage, 9,10 processing 11,12 and protection. 13 As such, the development of new molecules and materials enabling stimulus-induced modulation of their emission properties has become an active area of research. Of special interest is the preparation of fluorescent switches capable to respond to two or more different inputs, a multifunctional behavior that can be exploited in complex logic-gate 7,8,14 and analog operations, 15 multimode information storage, 16 7 ppm) and isopropyl (δ= 4.54-3.77 and 1.33-1.12 ppm) protons of 1d (Figure 1a), we were particularly intrigued by the broad resonance at δ= 7.25 ppm with an integral of 2H, which resulted from the coalescence of the two other broad downfield-shifted singlets registered at intermediate additions of TFA. Actually, when cooling the protonated sample down to 248 K, the signal at δ= 7.25 ppm split into two separated doublets of equal intensity (Figure 1b), which showed correlation with distinct CH isopropyl protons in the corresponding 2D COSY spectrum ( Figure 1c). Therefore, these resonances should correspond to two different NH groups that are directly attached to those isopropyl substituents. Based on these findings, the structure shown in Scheme 2 is proposed for 1d, which assumes acid-mediated ring-opening of the spirocyclic backbone of 1b and protonation of the resulting guanidine group to yield a cationic compound. For such a structure, fast conformational flexibility of the pending chain of the aromatic moiety at room temperature should not only account for the coalescence of the two different NH signals at   7.4 ppm, but also of the CH and CH3 resonances of the two terminal isopropyl substituents of the compound, which explains the lower number of signals registered at 298 K for these groups at δ ~ 4.5 -3.75 ppm and δ ~ 1.5 -1.0 ppm (Figure 1a). This process was reverted upon cooling and, as a consequence, splitting into separated resonances was also observed for the broad signals of those CH and CH3 protons when lowering the temperature (Figure 1b). In fact, molecular rotation was sufficiently hindered at 248 K as to also make the two aromatic protons of 1d become anisochronous, as already described for related non spirocyclic compound 2c. 15 1 R 1 = -NO 2 R 2 = isopropyl 3d' 2 R 1 = -CF 3  and 3d, as well as the structure 3d' reported by Das and co-workers 39 .
Similar results were obtained when investigating the protonation of a tautomeric mixture of 2b and 2c with TFA in acetonitrile. Despite the higher complexity of the initial 1 H NMR spectrum of this mixture, disappearance of the signals for both neutral 2b and 2c compounds and growth of a simpler set of resonances were again observed ( Figure S1a). The latter were assigned to protonated state 2d, the two broad NH signals of which at δ ~ 7.0 ppm did not fully coalesce at room temperature in this case. However, they clearly separated into narrower resonances upon cooling and also showed crossed peaks with CH isopropyl protons in the 2D COSY spectrum (Figure S1b-c). Therefore, these evidences further support our conclusion that the protonation of the neutral state of SZMC switches occurs at their guanidinium group and induces the aperture of their spirocyclic structure (Scheme 2). As such, protonation causes loss of the cyclohexadienyl anion chromophore of this type of switches, which explains the color bleaching observed upon acid addition for both the acetonitrile solutions of 1b and of the tautomeric mixture 2b+2c in our NMR experiments.
Recently, SZMC switch 3 bearing a 2,4,6-trinitrocyclohexadienyl anion and cyclohexyl substituents was also reported to undergo an interconversion process to a non colored product when its zwitterionic state 3b was treated with TFA. 39 In this case, however, Das and co-workers ascribed this observation to the formation of the cationic spirocyclic compound 3d' shown in Scheme 2. Because of the discrepancies between this structure and those proposed in our work for the cationic state of SZMC switches, we also investigated the protonation of 3b by 1 H NMR.
A detailed description of the results obtained in these experiments are given in the Supporting Information ( Figures S2 and S3), where crossed peaks between two distinct NH resonances and cyclohexyl signals were observed for 3b. This finding allows disambiguation of the actual structure of the cationic state of the system: the new proton introduced cannot be attached to the unsaturated ring of the compound, but it must lie next to one of the pending alkyl chains (3d), as proposed in our work for other SZMC switches and in contrast to the assignment of Das and coworkers 39 (3d', Scheme 2).

SZMC switches as wide range pH probes
Once uncovered the nature of the cationic state of SZMCs, we investigated the pH-induced optical switching behavior expected for compounds 1 and 2 upon protonation of their neutral form. Figure 2a-b shows the variation in the absorption and emission spectra of 1b and of the tautomeric mixture 2b+2c in acetonitrile upon acid addition. Although HClO4 was preferably used in these experiments because it is a strong acid in acetonitrile, 32 identical results were achieved when employing a non oxidizing acid such as TFA. In both cases, continuous decrease of the visible absorption and emission signals of both compounds was observed by protonation, as previously described for 3b 39 and in agreement with the loss of the chromophoric cyclohexadienyl anion core in 1d and 2d. It must be noted that acid-induced conversion of the neutral state of 1 into its cationic counterpart yielded defined isosbestic points in the UV-vis spectra. In accordance to 1 H NMR data, this suggests that protonation of 1b and of the mixture Based on the capacity of SZMCs to reversibly interconvert between three different protonation states with distinct optical properties, they could be envisaged as wide range acidity probes 28,43,44 responding to both low and high pH values. As a proof of concept of this behavior, we focused our attention on 1, since it only presents a colored, fluorescent zwitterionic form in the neutral state of the system, thus increasing the contrast in optical properties upon switching.
For this compound, the variation in visible light absorbance and emission was measured as a function of pH in acetonitrile (Figure 3a), which was taken as the solvent for these studies because of: (a) the low solubility of 1 in aqueous media, which resulted in additional, more intricate optical changes due to dye aggregation; (b) the practical interest of pH determination in organic solvents for a wide range of applications. [45][46][47] From our experiments, the pKa constants for the acid-base equilibria of 1 in acetonitrile could be determined: pKa = 15.8 ± 0.1 and 21.2 ± 0.1 for the 1d→1b and 1b→1a interconversion processes, respectively. Since the autoprotolysis constant of acetonitrile is pK ACN ≥ 33, the pH scale in acetonitrile spreads over a much larger range than in water (typically, pH ~ 1-33). 48 As such, the pKa values determined for 1d and 1b indicate that both compounds behave as weak acids in acetonitrile and undergo deprotonation at rather different pH windows. Therefore, the changes in optical properties that concomitantly occur could be used for pH sensing over a large range. plot showing the correspondence between pH and the absorption and fluorescence signals measured at 423 nm and 575 nm, respectively, for 1 in acetonitrile.
In the case of the pH-dependent fluorescence response of 1, it follows a symmetric "off-onoff" behavior ( Figure 3a), since the neutral state 1b is the single emissive species of the system and it is only predominant for intermediate values of pH (in the case of acetonitrile solutions, for pH ~ [16][17][18][19][20][21]. On the other hand, by properly selecting the detection wavelength (e.g. λ = 423 nm), a stepwise "off-on1-on2" profile can be obtained for the variation of the absorption signal owing to the different absorption spectra of 1a and 1b in the visible region (Figure 3a), which resembles the behavior described for other compounds proposed for ternary molecular logic. 49,50 Because of the complementarity of these "off-on-off" and "off-on1-on2" optical responses, a univocal value of pH can be retrieved from each pair of absorption and fluorescence values measured upon acid or base addition over acetonitrile solutions of 1, as shown by the 3D representation in Figure 3b. Therefore, this demonstrates that 1 operates as a pH optical probe over a large interval, which in the case of acetonitrile media covers about 10 pH units (pH ~ 14-24). Importantly, this behavior could not only be extended to other SZMCs with three-state switching capabilities, but also modulated by tuning the pKa constants of their pH-active guanidine group upon variation of the appended substituents.
Wide range pH optical detection using SZMC switch 1 could be transferred from solution to the solid state by simply dispersing this halochromic molecule in a polymer matrix. In particular, a polymethyl methacrylate (PMMA) thin film was prepared and loaded with 1b, and then

Extended redox switching of SZMCs: preparation of electrofluorochromic materials
An added advantage of SZMCs over other pH-responsive fluorescent switches is their capacity to undergo redox-induced interconversion between their anionic and neutral states in organic solvents such as acetonitrile or DMF. 15,32-34 Inspired by this behavior, we explored herein the reversible transformation between the neutral and cationic forms of these compounds by means of electrochemical stimuli. Because of its simpler switching scheme owing to the thermal stability of its zwitterionic state, we took compound 1 as a benchmark case for these studies.  well-known hydrogen atom donor properties in electrochemical reactions. 51 As already reported, zwitterion 1b shows two characteristic redox waves: a one-electron reversible oxidation wave at E 0 = +1.52 V (vs SCE), and a one-electron pseudoreversible reduction wave at Ep,c = -0.85 V (vs SCE), which was proven to lead to the formation of anion 1a after hydrogen transfer from the solvent to the radical anion 1b •generated electrochemically (Scheme 3a). 32,33 Under the same experimental conditions, no oxidation wave was registered for 1d, whereas two different oneelectron reduction waves were observed at Ep,c = -0.50 and -0.85 V (vs SCE). Two aspects must be particularly noted about these waves. On one hand, the second pseudoreversible wave at -0.85 V (vs SCE) resembles that measured for 1b, which suggests that the first irreversible reduction process of 1d at lower potentials (-0.50 V (vs SCE)) couples to a hydrogen atom loss chemical reaction that would result in product deprotonation and formation of the zwitterionic state of the switch. On the other hand, a very low intensity irreversible reduction wave at -0.50 V (vs SCE) could also be detected in the cyclic voltammogram of 1b. In view of the electrochemical data registered for 1d, this could be an indication that the previously scanned oxidation wave of 1b at +1.52 V (vs SCE) was not fully reversible at this cyclic voltammogramm scale time and it led to the generation of a small amount of the cationic state of the system.
Prompted by these results, we assayed the redox interconversion between the neutral and cationic forms of 1 using oxidative (to induce 1b → 1d transformation) and reductive (to induce 1d → 1b transformation) controlled potential electrolysis, which were monitored by means of spectroelectrochemical and cyclic voltammetry measurements. Figure 5b shows the variation in absorption spectra measured for 1b in acetonitrile when subjecting this sample to consecutive oxidative (Eapplied= +1.7 V (vs SCE) for 40 s) and reductive (Eapplied= -0.6 V (vs SCE) for 80 s) potentials, while the corresponding cyclic voltammograms at t = 0, 40 and 120 s are shown in Figure 5c. Clearly, oxidation of 1b resulted in decoloration of the sample and formation of the same type of cyclic voltammogram previously registered for 1d. Hence, this reveals that, upon one-electron oxidation of 1b, its radical cation abstracts a hydrogen atom from the solvent and undergoes ring opening of its spirocyclic motif, thus generating 1d (Scheme 3b). In addition, subsequent reduction of 1d allowed the absorption spectrum and the cyclic voltammogram of 1b to be recovered, which suggests that 1d • releases a hydrogen atom that may eventually evolve into molecular hydrogen 52 and is then capable to retake the spirocyclic structure of SZMC systems (Scheme 3b). In combination with previous reported data 32,33 and the results shown in the previous sections, this demonstrates that 1 operates as a multistate and a multiresponsive fluorescent switch, since it interconverts between its anionic, neutral and cationic forms through both acid-base and redox stimuli, a behavior also expected for other SZMCs. It must be noted that such diverse types of stimuli produce the same molecular effects (i.e. protonation and deprotonation of the switches) and, as a consequence, identical variation of their optical acid-base and redox stimuli are used and, indeed, combined chemical-electrochemical operation of these compounds could be achieved. The capability of 1 to switch electrochemically between states with different absorption and fluorescence properties could be exploited in the preparation of electrochromic and electrofluorochromic devices. To illustrate this behavior, we loaded 1b into ion gels prepared by mixing an ionic liquid (1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [BMIM][TFSI]) and a polymer (poly(vinylidene fluoride-co-hexafluoropropylene, P(VDF-co-HFP)). 53 As shown in Figure 6a-b, this resulted in the formation of a slightly conductive elastic gel (conductivity = 0.686 mS cm -1 at room temperature) that conserved the color and emissive behavior of the embedded spirocyclic zwitterionic molecules. Later, thin films of this gel (~ 1 mm in thickness) were placed between two ITO-coated glass slides and different electric potentials were applied to induce the conversion between 1b and 1a as well as between 1b and 1d. Although large potentials were required due to the poor conductivity of the ion gels obtained, changes in color and fluorescence emission were registered that revealed successful redoxtriggered transformation between the different states of 1. Thus, by applying a reduction potential at -4.5 V and a subsequent oxidation potential at +2.5 V, we achieved reversible interconversion between the fluorescent 1b and non fluorescent 1a forms of the switch ( Figure   6c). In addition, transformation between 1b and 1d could also be accomplished within the ion gel applying an oxidation potential at +4.0 V and a reduction potential at -3.0 V, thus resulting in reversible decoloration and loss of fluorescence of the material (Figure 6d-e). In this case, however, these effects could not be fully achieved in certain regions of the gel, which we associated to those with lower conductivities and/or higher thicknesses. In spite of this, these exploratory experiments demonstrate the capacity to construct electrochromic and electrofluorochromic devices based on compound 1 and other SZMC switches.  All these results can be rationalized on the basis of different factors. On one hand, polarity increases the relative concentration of tautomer 2b in aprotic media, as expected due to the zwitterionic nature of this product. Accordingly, relatively large 2b values were measured in acetonitrile and DMSO even when heating. In addition, the capacity of the solvent molecules to stabilize the guanidinium group of isomer 2b via hydrogen bond interactions does play a role in aprotic media, thus accounting for the lower thermal decrement of 2b observed in THF, acetone, acetonitrile and DMSO with respect to toluene, CH2Cl2 and CHCl3. On the other hand, dissolution in polar protic solvents (e.g. methanol) significantly promotes the formation of the non zwitterionic species, thus leading to relatively low 2b values regardless of the temperature.

Solvent-induced switching of SZMCs
Although the actual mechanism underlying this behavior is unclear to us, we hypothesize that hydrogen bonding interactions of the nitrogen atoms next to the spiro carbon atom with solvent molecules reduces the stability of the cyclic structure of 2b and favors ring opening to yield 2c. Similarly, large solvatofluorochromic effects were measured for 2b, the emission spectra of the mixture 2b+2c bathochromically shifting with solvent polarity (fl,max=581 and 605 nm for toluene and DMSO, respectively; Figure 7c). In spite of this, minimal variations of the fluorescence quantum yield (fl) of 2b were mainly registered, and high fl,2b values (>0.65) were determined for all the apolar and polar aprotic solvents considered (Table S1). As a result, the fluorescence intensities measured for the equilibrium mixture 2b+2c in those cases essentially depended on the actual concentration of the emissive state 2b (Figure 7c). For protic solvents such as methanol and water, however, a clear decrease in fl, 2b was observed (< 0.40, Table S1), which in combination with the low  2b also measured in these media resulted in rather weak emission intensities (Figure 7c).
Owing to the solvent variation of 2b and fl,2b, the equilibrium mixture 2b+2c could be employed as a fluorescent probe of organic liquids and vapors. As a proof of concept, we explored its application to the fluorescence detection of acetonitrile, since maximal emission intensity was registered in this solvent for the neutral state of 2 at room temperature due to the large stability and fluorescence quantum yield of tautomer 2b. As a consequence, if this compound is dissolved in organic media where 2b presents low stability (e.g. toluene) and/or low fluorescence quantum yield (e.g. water), the addition of increasing amounts of acetonitrile should result in a large increase in emission intensity, as experimentally demonstrated in Figure   8a-b. Aiming at reproducing this behavior in solid materials that could be applied in functional devices, the tautomerix mixture 2b+2c was embedded in thin films of polystyrene (PS) and polyvinyl alcohol (PVA). In both cases, dimly colored and fluorescent polymer films were obtained due to the low stability of 2b in apolar (PS) and protic (PVA) media as well as to the reduced fl of this compound in protic environments (PVA, Figure 8c

Materials
Organic solvents used in the synthesis were distilled over CaH2 and stored over activated molecular sieves (3 Å). Spectroscopic grade solvents were used as received. All chemicals used for the synthesis were of reagent grade with > 98% purity and they were used without further purification. Flash column chromatography was performed on silica gel 60 Å with average particle size 35-70 m.

Optical characterization
UV-vis absorption spectra were recorded using a HP 8452A spectrophotometer (Agilent) with Chemstation software. Fluorescence spectra were recorded by means of a custom-made spectrofluorometer using cw lasers (BeamQ, exc = 473 nm; Z-laser, exc = 532 nm) as excitation sources. Emitted photons were detected using an Andor ICCD camera coupled to a spectrograph.
All the emission spectra registered were corrected by the wavelength dependence of the spectral response of the detection system. In all the cases spectroscopic quality solvents and 1-cm quartz cuvettes were used. Temperature was controlled using a refrigerated circulator bath (Huber MPC-K6) connected to the sample holder. Fluorescence quantum yields were determined using the standard method 55 for highly diluted solutions of the compounds of interest to prevent selfabsorption processes (absorption < 0.05 at the excitation wavelength) and relative to N,N'-bis(1hexylheptyl)perylene-3,4,9,10 tetracarboxybismide in acetonitrile (Φfl=1 56 ) or N,N'-bis(butyl)-1,6,7,12-tetra-(4-tert-butylphenoxy)perylene-3,4:9,10-tetracarboxylic diimide in CH2Cl2 (Φfl values are given relative to the acetonitrile solvent ( s spH scale). 58 To calibrate the electrode system we used reference buffers in acetonitrile (pyridine-pyridinium bromide and phenolsodium phenolate), whose s spH can be derived from the Henderson-Hasselbach equation using the pKa values in acetonitrile reported for these systems. 58

Fabrication of electrofluorochromic ion gels
Electrofluorochromic ion gels were prepared following the procedure described in ref. ºC for a few minutes. The solution was cast onto a glass slide and, after solvent evaporation, a flexible ion gel was obtained. The "cut-and-stick" strategy 53 was then followed to measure the electrochromic and electrofluorochromic behavior of the ion gels prepared, for which they were cut and the thin films obtained were sandwiched between two ITO-coated glass substrates that were fixed using double-sided tape.

Preparation of polymer thin films
Halochromic polymer films were prepared by dissolving 143 mg of PMMA (Mw=120000) in 5 mL of chloroform. Afterwards, 2 mg of 1b were added to the mixture and the solution was

SUPPORTING INFORMATION
Additional spectroscopic data about SZMC switches 1-3 is provided in the Supporting Information (Figures S1-S5 and Table S1).

Protonation of SZMC switch 3
To establish the structure of the cation obtained upon protonation of zwitterion 3b, we monitored the titration of 3b with trifluoroacetic acid (TFA) by 1 H NMR. In this case, we used deuterated dimethylformamide as a solvent since: (a) it allowed direct monitorization of the new proton introduced into the system along the process, and (b) 3b was found to be poorly soluble in deuterated acetonitrile, in contrast to 1b and 2b. As shown in Figure S2a As previously observed for 1b and 2b, this resulted in sample discoloration, which revealed that the 2,4,6-trinitrocyclohexadienyl anion chromophore of 3b had been disrupted upon TFA addition and, therefore, was not longer present in the protonated product.
The loss of the chromophore unit of 3b could be explained on the basis of two different processes: (a) protonation-induced ring-opening reaction of 3b, which would lead to an aromatic structure by analogy to the results obtained for 1b and 2b (structure 3d in Figure S3); (b) C4-protonation of the cyclohexadienyl anion, which would preserve the spirocyclic structure of the system, as proposed by Das and co-workers when conducting the experiment in CDCl3 (structure 3d' in Figure S3). 1 To discriminate between structures 3d and 3d', we performed two additional 1 H NMR experiments: (a) temperature variable 1 H NMR spectroscopy of the cationic state of 3 within the 318-258 K range ( Figure S2b); (b) 2D COSY spectroscopy of this compound at 288 K ( Figure S2c). When analyzing these new spectra, we particularly focused our attention on the four 1 H NMR signals of equal intensity detected between   9.6-9.0 ppm. Since cyclohexyl groups cannot provide 1 H NMR resonances at such S6 low fields, these signals must correspond to H3, H5, NH and the new proton introduced upon TFA addition for any of the two structures proposed for the cationic state of 3.
The most significant effect observed at low fields in temperature variable measurements was the broadening and coalescence of the 1 H NMR signals at   9.5 and 9.3 ppm when heating up. More importantly, correlation was observed between those two resonances in the 2D COSY spectrum at 288 K, and they did not show cross peaks with any other nucleus in the molecule ( Figure S2c). Based on these features, we assigned the 1 H NMR signals at   9.5 and 9.3 ppm to the two aromatic protons H3 and H5 in structure 3d, which should become anisochronous if the rotation around the bulky urea substituent sufficiently slows down when cooling, as also described for analogue compound 2c. 2 Noticeably, if those two signals had arisen from structure 3d', additional cross-peaks would have been found in the 2D COSY spectrum with the new proton H4 introduced in this compound.
On the other hand, clear doublet multiplicities were observed at 298-288 K for resonances at   9.2 and 9.0 ppm, which are in agreement with those expected for the two NH groups of structure 3d. This assignment is further corroborated by the 2D COSY spectrum registered at 288 K, where crossed peaks were observed between each of those doublets and two different CH protons of the cyclohexyl groups at   3.7 and 3.5 ppm ( Figure S2c). These results would not have been possible for structure 3d', where correlation with cyclohexyl CH protons would have not been observed for H4, but only for NH (i.e. only for one of the resonances at  9.6-9.0 ppm).
In conclusion, our 1 H NMR analysis of 3b protonation allows clear disambiguation of the actual structure of the cationic state of the system: the new proton introduced cannot be attached to the unsaturated ring of the compound, but it must lie next to a cyclohexyl substituent, as proposed in our work (3d) and in contrast to the assignment of Das and co-workers (3d'). 1 S7 Figure S2. (a) Variation of the 1 H NMR (400 MHz, DMF-d7, 298 K) spectrum of 3b upon addition of 1.2 equivalents of TFA, which leads to the quantitative formation of a new protonated state that we assigned to structure 3d (see Figure S3). Black stars are used to indicate the formation of two new signals corresponding to the two NH groups of 3d. (b) Variation of the 1 H NMR (400 MHz, DMF-d7) S8 spectrum of 3d with temperature. c) 2D COSY spectrum (400 MHz, DMF-d7, 288 K) of 3d, where cross-peaks are observed between each of the NH resonances at   9.2 and 9.0 ppm and the cyclohexyl CH multiplets at   3.7 and 3.5 ppm (CH Chyl ). Figure S3. Two possible structures proposed for the cationic state of 3: structure 3d (this work) and structure 3d' (Das and co-workers). 1 For each structure, the main 2D COSY spectral features expected are indicated, which allow discrimination between 3d and 3d'.

Solvent dependence on the tautomeric equilibrium between 2b and 2c
As already established in a previous work, 2 the relative concentration between the spirocyclic zwitterion 2b and the aromatic neutral tautomer 2c can be estimated from the integrals of the 1 H NMR signals arising from their cyclohexadienyl (for 2b) and aromatic (for 2c) protons, which appear in the low field region of the spectrum. To illustrate this procedure, Figure S4 plots these 1 H NMR signals for two of the solvents considered (acetone-d6 and methanol-d4) at 273, 298 and 313 K.