Fluorogenic Detection of Sulfite in Water by Using Copper(II) Azacyclam Complexes

Copper(II) azacyclam complexes (azacyclam = 1,3,5,8,12-pentaazacyclotetradecane) containing naphthyl or dansyl subunits can be prepared by template synthesis involving proper sulfonamide derivatives as locking fragments. The macrocyclic complexes are very poorly emissive due to the fluorescence-quenching behavior displayed by Cu2+ ions. However, the fluorescence can be recovered as a result of the decomposition of the complexes, which induces the release of free light-emitting subunits to the solution. This reaction takes place very slowly in neutral water but its rate is increased by the presence of sulfite. Therefore, [Cu(azacyclam)]2+ derivatives have been investigated as simple chemical probes for the fluorogenic detection of sulfite both on laboratory and real samples. Preliminary tests performed on samples of white wine provided sulfite concentration values that are in agreement with those obtained by a standard analytical method.


Introduction
Sulfite and related sulfiting agents (i.e., bisulfite and metabisulfite) are well-known as preservatives and antioxidants and are widely used as food and drink additives [1][2][3]. They may sometimes occur spontaneously (i.e., without external addition) in some alimentary products [4,5]. Moreover, sulfite finds application in commercial products other than food and beverages (e.g., cosmetics, drugs), as well as in several industrial processes (e.g., leather, textile, pulp) [3,6]. In addition, sulfur dioxide (SO 2 ) is a common air pollutant and can be dissolved in aqueous media with consequent formation of sulfite (SO 3 2− ) and hydrogensulfite or bisulfite (HSO 3 − ) at neutral pH. Many studies showed that frequent exposure to SO 2 , as well as to its anionic derivatives, could induce harmful effects on human health, including cardiovascular diseases, neurological disorders, and cancer. Moreover, some individuals exhibit extremely high sensitivity even to very low levels of these compounds [6], resulting in a wide variety of adverse clinical effects that can range from mild-to-life-threatening reactions [3,6,7]. Due to these potential health concerns, in many countries the content of sulfite and/or its related compounds has been strictly limited in recent decades, particularly in foodstuffs and drinks (e.g., wine and beer) [8][9][10][11].

Synthesis and Crystal Structure of the Azacyclam Complexes
The copper complexes of azacyclam ligands 1-3 were prepared according to the previously reported metal-template route, which is depicted in Scheme 1 [65]. In particular, an EtOH solution of [Cu(2.3.2-tet)] 2+ complex (prepared in situ; 2,3,2-tet = N,N -bis(2aminoethyl)propane-1,3-diamine) was moderately heated and stirred in a common roundbottom flask while the other reagents (locking fragment, base, and formaldehyde) were consecutively added. The macrocyclic copper(II) complexes were isolated as pink-violet solids, usually formed at the end of the reaction (3-7 days) or after addition of excess concentrated perchloric acid to the deep blue-violet reaction mixtures. The addition of HClO 4 induces the decomposition of possible open-chain by-products and favors the precipitation of the azacyclam complexes as perchlorate or nitrate salts.
When dissolved in water (concentration lower than 10 −3 M), copper(II) complexes of ligands 1-3 display strong absorptions in the UV region (ε = 1500-75000), which are due to the aromatic residues belonging to the locking fragments, and a distinctly less intense band (ε = 76-83 M −1 cm −1 ) centered between 500 and 550 nm. Solutions with different concentrations (about 10 −5 and 10 −3 M) of the complexes were examined in order to accurately detect both the bands, which display very different intensities. The absorption spectra of the investigated azacyclam complexes taken in the 200-400 nm (about 10 −5 M) as well as in the 400-800 nm range (about 10 −3 M) are reported in Figures S1-S3. The weak band observed in the visible region results from an envelope of the three possible d-d transitions (xz, yz→x 2 -y 2 , z 2 →x 2 -y 2 , and xy→x 2 -y 2 ) in the square-planar copper(II)tetramine chromophore [85,86]. Spectral data of the investigated complexes as well as of [Cu(2.3.2-tet)] 2+ , taken as a reference, are resumed in Table 1. The macrocyclic complexes display a very similar absorption in the visible range both concerning the maximum wavelength and the intensity, indicating that the square-planar coordination environment is poorly affected by the different structures of the LF moieties. On the contrary, the d-d band energy of [Cu(1)] 2+ , [Cu (2)] 2+ , and [Cu(3)] 2+ is distinctly higher than [Cu(2.3.2-tet)] 2+ , due to the macrocyclic nature of azacyclam ligands, which provide a stronger ligand field than the acyclic tetra-amine.
Crystals containing the [Cu(1)] 2+ , [Cu (2)] 2+ , and [Cu(3)] 2+ molecular cations suitable for X-ray diffraction study were obtained by slow vapor exchange between ethanol and saturated aqueous solutions of the complexes (filtered immediately before starting the crystallization). In particular, [Cu (1)] 2+ molecular cation occurs as a nitrate salt, whose crystal structure is constituted by three similar but not symmetrically equivalent azacyclam ligands. In addition, the [Cu (2)] 2+ molecular cation forms as a nitrate salt with additional water-solvent molecules and, as [Cu (1)] 2+ , the crystal structure contains three independent azacyclam molecular complexes. On the contrary, the [Cu (3)] 2+ molecular cation forms as a perchlorate salt, and its crystal structure is made by a single independent azacyclam molecular complex. Plots showing thermal ellipsoids for one of the three independent [Cu(1)](NO 3 ) 2 molecular complexes, for one of the three independent [Cu (2)](NO 3 ) 2 molecular complexes, and for the [Cu (3)](ClO 4 ) 2 molecular complex are reported in Figure 1 (plots of the remaining independent molecular complexes are reported in Figure S4).  Figure 1 (plots of the remaining independent molecular complexes reported in Figure S4).  All azacyclam ligands are arranged accordingly to a trans-III (RRSS) configuration, with the copper(II) centers bonded to the four secondary amines according to a squareplanar coordination geometry. Cu-N bond distances are in the range 2.00(1)-2.04(1) Å for the  [87].
The tertiary amine of each azacyclam ligands is far from the metal center, with Cu-N tertiary distances in the range 3.21(1)-3.22(1) Å for the [Cu(1)] 2+ molecular cations, 3.22(1)-3.24(1) Å for the [Cu(2)] 2+ molecular cations, and 3.24(1) Å for the [Cu(3)] 2+ molecular cation. Such distances preclude any significant Cu(II)-N tertiary interaction in the azacyclam ligand. However, the observed distances are shorter than the corresponding values of 3.34 Å observed in cyclam-like compounds [88] where a CH 2 group takes the place of the tertiary amine. Such behavior was already observed and described in the nickel(II) complexes of ligands 1 and 3 [55,78] for which Ni-N tertiary distances are in the range 3.20(1)-3.28(1) Å and it characterizes azacyclam Cu II and Ni II complexes having the tertiary amine connected to electron-withdrawing functional groups.
In all the investigated metal complexes, the Cu II metal center remains in the best plane of the equatorial coordinated amines (deviation from the N 4 best plane are in the range 0.03(1)-0.07(1) Å) and the copper(II) coordination sphere is completed by two oxygen atoms belonging to the counterions placed in two axial positions. The Cu-O axial distances are longer than the Cu-N equatorial ones, being in the range 2.44 (1) O] crystals exhibit face-to-face π-stacking interactions involving the aromatic moieties of the LF: the centroid-centroid separations are in the range 3.58-3.99 Å and the dihedral angles between adjacent aromatic groups are in the range 3.3-11.6 • . These π-stacking interactions originate supramolecular chains extending along the direction of the b crystallographic axis ( Figure S5). In both these crystals, further intermolecular connections are provided by the nitrate ions, which bridge adjacent molecular compounds by means of N-H···O interactions having the secondary amines of the azacyclam ligands as H-donor species and the O atoms of the nitrate counterions as H-acceptors. These interactions also induce the formation of chains extending along b ( Figure S6). The geometrical features of such H-bond interactions are reported in Table S1.
On the contrary, in the [Cu  Figure S7 and Table S1, respectively.

Decomposition of Azacyclam Complexes in Aqueous Solution
All the investigated compounds are expected to display a strong stability in water, as previously observed for other azacyclam complexes, and more in general, for metal complexes of 14-membered macrocyclic ligands displaying a 5-6-5-6 sequence of chelate rings [86,89,90].
The inertness toward demetallation of [Cu(1)] 2+ , [Cu(2)] 2+ , and [Cu(3)] 2+ is indeed confirmed by the persistence of the d-d band in aqueous solutions within a wide range of pH. For instance, visible spectra measured at room temperature on aqueous solutions of the corresponding perchlorate salts (6-8 × 10 −4 M) do not undergo significant changes after 24 h at neutral pH (buffered at 7.2) and even in the presence of HClO 4 (0.1 M). This suggests that, in these conditions, the macrocyclic-complex species do not decompose or decompose to an undetectable extent. Anyway, we performed further investigations in order to check if the stability of the azacyclam complexes in water can be affected by increasing temperature. Therefore, the aqueous solutions of [Cu(1)] 2+ , [Cu(2)] 2+ , and [Cu(3)] 2+ (6-8 × 10 -4 M) were buffered at pH 7.2 (to avoid any possible effect of uncontrolled pH), heated for 1 h at 70 • C, and their visible spectra were measured before and after the prolonged heating. This treatment induced in all the samples a small but significant redshift (up to 8 nm) of the d-d absorption band (Figure 2). It should be noted that such a batochromic shift may be related to a ligand field weakening in the N 4 square-planar coordination environment and could be explained on the basis of a replacement of the macrocyclic ligand by an acyclic polyamine species. In fact, the d-d bands in the spectra obtained from the heated solutions of azacyclam complexes match quite well with the visible spectrum of [Cu(2.3.2-tet)] 2+ . This is in agreement with the hypothesis that the macrocyclic compounds decompose, producing the complex with the acyclic tetra-amino ligand. In particular, the d- The inertness toward demetallation of [Cu(1)] 2+ , [Cu(2)] 2+ , and [Cu(3)] 2+ is confirmed by the persistence of the d-d band in aqueous solutions within a wide ra pH. For instance, visible spectra measured at room temperature on aqueous solut the corresponding perchlorate salts (6-8 × 10 −4 M) do not undergo significant change 24 h at neutral pH (buffered at 7.2) and even in the presence of HClO4 (0.1 M suggests that, in these conditions, the macrocyclic-complex species do not decomp decompose to an undetectable extent. Anyway, we performed further investigat order to check if the stability of the azacyclam complexes in water can be affec increasing temperature. Therefore, the aqueous solutions of [Cu(1)] 2+ , [Cu(2)] [Cu(3)] 2+ (6-8 × 10 -4 M) were buffered at pH 7.2 (to avoid any possible ef uncontrolled pH), heated for 1 h at 70 °C, and their visible spectra were measured and after the prolonged heating. This treatment induced in all the samples a sm significant redshift (up to 8 nm) of the d-d absorption band (Figure 2). It should be that such a batochromic shift may be related to a ligand field weakening in the N4 s planar coordination environment and could be explained on the basis of a replacem the macrocyclic ligand by an acyclic polyamine species. In fact, the d-d bands spectra obtained from the heated solutions of azacyclam complexes match quite we the visible spectrum of [Cu(2.3.2-tet)] 2+ . This is in agreement with the hypothesis t macrocyclic compounds decompose, producing the complex with the acyclic tetraligand. In particular, the d-d band of [Cu   Moreover, the absorption spectrum of a [Cu(3)] 2+ aqueous solution (2 × 10 -5 M) registered after heating (1 h, 70 • C, pH 7.2) in the 200-400 nm range clearly shows a variation of the UV absorption bands when compared with the spectrum before decomposition (see Figure S8). In particular, the spectrum of the decomposed complex is superimposable to that of dansyamide, supporting the hypothesis of the macrocyclic ring opening with consequent release of the LF subunit.
ESI-MS experiments were performed to investigate the identity of the supposed decomposition products. At room temperature, the characteristic peaks ascribed to the macrocyclic complexes (e.g., M 2+ , {M-H} + , and {M + anion} + signals) can be observed for The decomposition of [Cu(2)] 2+ and [Cu (3)] 2+ was also investigated by performing light-emission measurements. As already discussed, these two complexes are very poorly emissive due to the fluorescence quenching induced by copper(II), but after heating, their aqueous solutions (2 × 10 −5 M) display very intense fluorescence bands at 345 and 565 nm, which correspond to the emission of the locking fragments released in solution, i.e., 2-naphthalenesulfonamide and dansylamide, respectively ( Figure S11). The decomposition of [Cu(3)] 2+ has been also monitored by collecting the emission spectra at different times. As an example, a family of dansylamide spectra collected during a monitoring experiment (about 15 min) and the corresponding time-dependent profile of fluorescence intensity, I F , are reported in Figure 3.
decomposition (see Figure S8). In particular, the spectrum of the decomposed comp superimposable to that of dansyamide, supporting the hypothesis of the macrocycl opening with consequent release of the LF subunit.
ESI-MS experiments were performed to investigate the identity of the sup decomposition products. At room temperature, the characteristic peaks ascribed macrocyclic complexes (e,g., M 2+ , {M -H} + , and {M + anion} + signals) can be observ [Cu (1) The decomposition of [Cu(2)] 2+ and [Cu (3)] 2+ was also investigated by perfo light-emission measurements. As already discussed, these two complexes are very p emissive due to the fluorescence quenching induced by copper(II), but after heating aqueous solutions (2 × 10 −5 M) display very intense fluorescence bands at 345 and 56 which correspond to the emission of the locking fragments released in solution, naphthalenesulfonamide and dansylamide, respectively ( Figure S11). The decompo of [Cu(3)] 2+ has been also monitored by collecting the emission spectra at different As an example, a family of dansylamide spectra collected during a monitoring exper (about 15 min) and the corresponding time-dependent profile of fluorescence intens are reported in Figure 3. The above-described experiments clearly indicate that copper(II) azac complexes, which seem to be extremely stable at room temperature, can un decomposition at neutral pH by heating, providing the starting compounds used in template synthesis, i.e., [Cu(2.3.2-tet)] 2+ and the locking fragments.
Other similar experiments have been performed to better assess the eff temperature on the decomposition process of the azacyclam complexes. In part aqueous solutions of [Cu(2)] 2+ and [Cu(3)] 2+ , buffered at pH 7.2, were thermosta temperatures between 40 and 70 °C, while their emission intensity was monitored. A spectra were collected by using the same instrumental conditions, in order to cor compare the data obtained in the different monitoring experiments. The IF vs. time p obtained at different temperatures are reported in Figure 4. As the time required f The above-described experiments clearly indicate that copper(II) azacyclam complexes, which seem to be extremely stable at room temperature, can undergo decomposition at neutral pH by heating, providing the starting compounds used in their template synthesis, i.e., [Cu(2.3.2-tet)] 2+ and the locking fragments.
Other similar experiments have been performed to better assess the effect of temperature on the decomposition process of the azacyclam complexes. In particular, aqueous solutions of [Cu(2)] 2+ and [Cu(3)] 2+ , buffered at pH 7.2, were thermostated at temperatures between 40 and 70 • C, while their emission intensity was monitored. All the spectra were collected by using the same instrumental conditions, in order to correctly compare the data obtained in the different monitoring experiments. The I F vs. time profiles obtained at different temperatures are reported in Figure 4. As the time required for the complete decomposition varies a lot depending on the temperature, the time-dependent profiles up to 20 min are reported.
The I F vs. time plots reported in Figure 4 corresponds to the initial straight line of the complete time-dependent I F profiles; therefore, their slopes, dI F /dt, are directly related to the rate of the slowest step. As expected, the rate of the decomposition process exhibits an exponential increase with temperature ( Figure S12).
On the basis of the above-mentioned results, a plausible mechanism for the decomposition reaction (Scheme 2) can be hypothesized. The first step may consist of the deprotonation of an amine adjacent to the sulfonamide group, also favored by the double-positive charge of the coordinated metal ion. The deprotonated species can then undergo a ring opening step, forming an imine group on an extremity of the chain and the deprotonated sulfonamide group on the other. Afterwards, the decomposition reaction may proceed following the opposite direction of the template synthesis pathway [74], providing again [Cu(2.3.2-tet)] 2+ , formaldehyde, and the original LFs, as demonstrated by MS and emission spectroscopy measurements.  The IF vs. time plots reported in Figure 4 corresponds to the initial straight line complete time-dependent IF profiles; therefore, their slopes, dIF/dt, are directly rela the rate of the slowest step. As expected, the rate of the decomposition process exhi exponential increase with temperature ( Figure S12).
On the basis of the above-mentioned results, a plausible mechanism f decomposition reaction (Scheme 2) can be hypothesized. The first step may consist deprotonation of an amine adjacent to the sulfonamide group, also favored by the d positive charge of the coordinated metal ion. The deprotonated species can then un a ring opening step, forming an imine group on an extremity of the chain an deprotonated sulfonamide group on the other. Afterwards, the decomposition re may proceed following the opposite direction of the template synthesis pathwa providing again [Cu(2.3.2-tet)] 2+ , formaldehyde, and the original LFs, as demonstra MS and emission spectroscopy measurements. The same final result can be expected considering, as an alternative decompo route, the direct nucleophilic attack of OH − to the diazamethylene group, as depic Scheme S1.
Experiments performed at different pH values (ranging between 6 and 8) and °C showed that the decomposition rate evaluated by dIF/dt, as described above, dis increases with pH ( Figure S13), supporting both the hypothesized mechanis decomposition, which are strongly favored by increasing OH − concentration anyw  The IF vs. time plots reported in Figure 4 corresponds to the initial straight line of the complete time-dependent IF profiles; therefore, their slopes, dIF/dt, are directly related to the rate of the slowest step. As expected, the rate of the decomposition process exhibits an exponential increase with temperature ( Figure S12).
On the basis of the above-mentioned results, a plausible mechanism for the decomposition reaction (Scheme 2) can be hypothesized. The first step may consist of the deprotonation of an amine adjacent to the sulfonamide group, also favored by the doublepositive charge of the coordinated metal ion. The deprotonated species can then undergo a ring opening step, forming an imine group on an extremity of the chain and the deprotonated sulfonamide group on the other. Afterwards, the decomposition reaction may proceed following the opposite direction of the template synthesis pathway [74], providing again [Cu(2.3.2-tet)] 2+ , formaldehyde, and the original LFs, as demonstrated by MS and emission spectroscopy measurements. The same final result can be expected considering, as an alternative decomposition route, the direct nucleophilic attack of OH − to the diazamethylene group, as depicted in Scheme S1.
Experiments performed at different pH values (ranging between 6 and 8) and at 40 °C showed that the decomposition rate evaluated by dIF/dt, as described above, distinctly increases with pH ( Figure S13), supporting both the hypothesized mechanisms of decomposition, which are strongly favored by increasing OH − concentration anyway. The same final result can be expected considering, as an alternative decomposition route, the direct nucleophilic attack of OH − to the diazamethylene group, as depicted in Scheme S1.
Experiments performed at different pH values (ranging between 6 and 8) and at 40 • C showed that the decomposition rate evaluated by dI F /dt, as described above, distinctly increases with pH ( Figure S13), supporting both the hypothesized mechanisms of decomposition, which are strongly favored by increasing OH − concentration anyway.

Decomposition in the Presence of Sulfite
The following experiment was preliminarily carried out in order to check the possible effect of sulfite on the decomposition of azacyclam complexes. As the reaction is affected by pH and T, the experiment was performed by carefully controlling the experimental conditions (pH = 7.2, phosphate buffer; T = 40 • C, thermostatic bath). An aqueous solution of [Cu(3)] 2+ (2 × 10 −5 M) was treated with excess Na 2 SO 3 (5 × 10 −4 M) and its emission was monitored over time. It should be noted that at neutral pH, both SO 3 − and HSO 3 − are present in relevant concentrations. For simplicity, the term "sulfite" will be used in the following, bearing in mind the two species in equilibrium. For comparison, a similar experiment was simultaneously performed on another portion of the same complex solution without adding sulfite. The corresponding profiles I F vs. time obtained from the fluorescence monitoring are reported in Figure 5. In the absence of sulfite, dansylamide emission linearly increased with time during the experiment (up to 72 h), although the fluorescence enhancement was almost undetectable after the first hour, coherently with the slow decomposition of complex [Cu(3)] 2+ at 40 • C and neutral pH. On the contrary, in the presence of excess sulfite, the I F enhancement was distinctly larger even at short time, suggesting that the decomposition rate had considerably increased (see inset in Figure 5). (3)] 2+ (2 × 10 −5 M) was treated with excess Na2SO3 (5 × 10 −4 M) and its emissio monitored over time. It should be noted that at neutral pH, both SO3 − and HSO present in relevant concentrations. For simplicity, the term "sulfite" will be used following, bearing in mind the two species in equilibrium. For comparison, a s experiment was simultaneously performed on another portion of the same com solution without adding sulfite. The corresponding profiles IF vs. time obtained fro fluorescence monitoring are reported in Figure 5. In the absence of sulfite, dansyla emission linearly increased with time during the experiment (up to 72 h), althoug fluorescence enhancement was almost undetectable after the first hour, coherently the slow decomposition of complex [Cu(3)] 2+ at 40 °C and neutral pH. On the contra the presence of excess sulfite, the IF enhancement was distinctly larger even at short suggesting that the decomposition rate had considerably increased (see inset in Figu were treated with a variety of anions (fluoride, chloride, bromide, iodide, n nitrate, azide, cyanate, thiocyanate, sulfate, thiosulfate, sulfide, hydrogencarb acetate, perchlorate) and their fluorescence was monitored. In all the considered solu the emission intensity increased very slowly and the corresponding time-depe profiles were very similar to that obtained in the absence of any anion ( Figure S14 the contrary, as mentioned above, the presence of sulfite induced a distinctly h fluorescence increase even after a short time (e,g., 20 min), suggesting that it is the anionic species that considerably affects the decomposition of azacyclam complexe unique behavior of sulfite can also be clearly inferred from the comparison o decomposition rates, i.e., dIF/dt values obtained from the slope of time-depe fluorescence profiles, as described in the previous section. A graphical representat dIF/dt values corresponding to the decomposition of [Cu(2)] 2+ and [Cu(3)] 2+ in the pre of the envisaged anions is reported in Figure 6. When sulfite is added to the com solution, the decomposition rate underwent a ten-fold increase, while most of the anions did not substantially modify the reaction rate of the complexes at neutral pH 2, T = 40 • C) were treated with a variety of anions (fluoride, chloride, bromide, iodide, nitrite, nitrate, azide, cyanate, thiocyanate, sulfate, thiosulfate, sulfide, hydrogencarbonate, acetate, perchlorate) and their fluorescence was monitored. In all the considered solutions the emission intensity increased very slowly and the corresponding time-dependent profiles were very similar to that obtained in the absence of any anion ( Figure S14). On the contrary, as mentioned above, the presence of sulfite induced a distinctly higher fluorescence increase even after a short time (e.g., 20 min), suggesting that it is the only anionic species that considerably affects the decomposition of azacyclam complexes. The unique behavior of sulfite can also be clearly inferred from the comparison of the decomposition rates, i.e., dI F /dt values obtained from the slope of time-dependent fluorescence profiles, as described in the previous section. A graphical representation of dI F /dt values corresponding to the decomposition of [Cu(2)] 2+ and [Cu(3)] 2+ in the presence of the envisaged anions is reported in Figure 6. When sulfite is added to the complex solution, the decomposition rate underwent a ten-fold increase, while most of the other anions did not substantially modify the reaction rate of the complexes at neutral pH.

of [Cu
A somewhat different behavior was observed in the case of HS − : the demetalation of macrocyclic Cu 2+ complexes induced by HS − was previously reported and even exploited for the detection of this anion [82][83][84]; therefore, its effect on the decomposition of [Cu(2)] 2+ and [Cu(3)] 2+ could be expected. To limit the possible interference of HS − , its concentration level was kept the same as sulfite in the corresponding competition test. In this situation the effect of HS − is however minimal when compared to HSO 3 − /SO 3 2− . On the basis of the experimental data, two different mechanisms could be hypothesized in order to explain the role of sulfite in the decomposition reaction of macrocyclic complexes: i. coordination of HSO 3 − /SO 3 2− to Cu 2+ , which could favor the reduction of the coordinated metal center and the formation of poorly stable copper(I) intermediates. The macrocyclic [Cu(N 4 )] + species easily undergoes demetalation, as the squareplanar coordination environment does not sufficiently stabilize that oxidation state. As a result of this mechanism, the unstable free ligand can be hydrolyzed generating the free sulfonamide LF. Although it could seem plausible, this hypothesis is not completely convincing because sulfite is not effective in the decomposition of macrocyclic copper(II) complexes other than azacyclam ones (e.g., derivatives of [Cu(cyclam)] 2+ ) [83]; ii.
nucleophilic reaction of sulfite and methylenediamine groups of the azacyclam framework, with consequent formation of aminomethanesulfonate derivatives, and release of sulfonamide locking fragments (Scheme 3).
Molecules 2022, 27, x FOR PEER REVIEW 10 of 21 A somewhat different behavior was observed in the case of HS − : the demetalation of macrocyclic Cu 2+ complexes induced by HS − was previously reported and even exploited for the detection of this anion [82][83][84]; therefore, its effect on the decomposition of [Cu(2)] 2+ and [Cu(3)] 2+ could be expected. To limit the possible interference of HS − , its concentration level was kept the same as sulfite in the corresponding competition test. In this situation the effect of HS − is however minimal when compared to HSO3 − /SO3 2− . On the basis of the experimental data, two different mechanisms could be hypothesized in order to explain the role of sulfite in the decomposition reaction of macrocyclic complexes: i.
coordination of HSO3 − /SO3 2− to Cu 2+ , which could favor the reduction of the coordinated metal center and the formation of poorly stable copper(I) intermediates. The macrocyclic [Cu(N4)] + species easily undergoes demetalation, as the squareplanar coordination environment does not sufficiently stabilize that oxidation state. As a result of this mechanism, the unstable free ligand can be hydrolyzed generating the free sulfonamide LF. Although it could seem plausible, this hypothesis is not completely convincing because sulfite is not effective in the decomposition of macrocyclic copper(II) complexes other than azacyclam ones (e,g., derivatives of [Cu(cyclam)] 2+ ) [83]; ii.
nucleophilic reaction of sulfite and methylenediamine groups of the azacyclam framework, with consequent formation of aminomethanesulfonate derivatives, and release of sulfonamide locking fragments (Scheme 3).
In order to check the second hypothesis of the mechanism, the sulfite-induced decomposition was studied in more detail. In particular, a buffered (pH = 7.2) aqueous solution of [Cu(1)] 2+ (2.6 × 10 −3 M) was treated with excess Na2SO3 (2.6 × 10 −2 M) at 40 °C and the resulting solution was analyzed after 2 h by mass spectroscopy. The ESI-MS spectrum exhibits a peak at m/z 194, corresponding to {Toluenesulfonamide + Na} + and a base peak at m/z 432, which corresponds to the {[Cu(4)] 0 + Na} + species, i.e., to the neutral bis-aminomethanesulfonate acyclic complex hypothesized as decomposition product in Scheme 3. The same peak at m/z 432 can be also observed in the ESI-MS spectrum obtained after carrying out the same experiment on [Cu  Moreover, after treatment with sulfite, the absorption spectrum of the resulting solution in the visible range showed a broad band centered at 568 nm (ε = 117 M −1 cm −1 , Figure S16). Notably, the redshift of the d-d band is considerably higher than that observed after the decomposition in neutral water or in the presence of OH − , suggesting again that a copper complex different from [Cu(2,3,2-tet)] 2+ is obtained after decomposition induced by HSO3 − /SO3 2− . The bis-aminomethanesulfonate acyclic ligand 4, i.e., the hypothesized final product of sulfite-induced azacyclam decomposition (Scheme 3), should provide a weaker ligand field than 2,3,2-tet due to the electron-withdrawing character of sulfonate, thus it could induce a larger shift of the d-d band towards lower energy. Moreover, we cannot completely rule out the formation of different coordination species (i.e., penta-coordinated or distorted octahedral hexa-coordinated) in which oxygen atoms of sulfonate groups are involved in the coordination to copper(II). The presence of different coordination species may result in the overlapping of several d-d absorptions, with consequent broadening and significant redshift of the resulting band. The coordination of aminomethylsulfonate group to copper(II) ion according to the chelate mode in hexacoordinated complexes has been already reported in literature [91].

Quantitative Detection of Sulfite
The experiments described above demonstrated that sulfite significantly increases the decomposition rate of azacyclam complexes. When considering complexes [Cu(2)] 2+ Scheme 3. Proposed mechanism of the sulfite-induced decomposition of copper(II) azacyclam complexes. The possible coordination of sulfonate groups in ligand 4 to the copper center is not represented in the scheme.
In order to check the second hypothesis of the mechanism, the sulfite-induced decomposition was studied in more detail. In particular, a buffered (pH = 7.2) aqueous solution of [Cu(1)] 2+ (2.6 × 10 −3 M) was treated with excess Na 2 SO 3 (2.6 × 10 −2 M) at 40 • C and the resulting solution was analyzed after 2 h by mass spectroscopy. The ESI-MS spectrum exhibits a peak at m/z 194, corresponding to {Toluenesulfonamide + Na} + and a base peak at m/z 432, which corresponds to the {[Cu(4)] 0 + Na} + species, i.e., to the neutral bis-aminomethanesulfonate acyclic complex hypothesized as decomposition product in Scheme 3. The same peak at m/z 432 can be also observed in the ESI-MS spectrum obtained after carrying out the same experiment on [Cu  Figure S15.
Moreover, after treatment with sulfite, the absorption spectrum of the resulting solution in the visible range showed a broad band centered at 568 nm (ε = 117 M −1 cm −1 , Figure S16). Notably, the redshift of the d-d band is considerably higher than that observed after the decomposition in neutral water or in the presence of OH − , suggesting again that a copper complex different from [Cu(2,3,2-tet)] 2+ is obtained after decomposition induced by HSO 3 − /SO 3 2− . The bis-aminomethanesulfonate acyclic ligand 4, i.e., the hypothesized final product of sulfite-induced azacyclam decomposition (Scheme 3), should provide a weaker ligand field than 2,3,2-tet due to the electron-withdrawing character of sulfonate, thus it could induce a larger shift of the d-d band towards lower energy. Moreover, we cannot completely rule out the formation of different coordination species (i.e., penta-coordinated or distorted octahedral hexa-coordinated) in which oxygen atoms of sulfonate groups are involved in the coordination to copper(II). The presence of different coordination species may result in the overlapping of several d-d absorptions, with consequent broadening and significant redshift of the resulting band. The coordination of aminomethylsulfonate group to copper(II) ion according to the chelate mode in hexacoordinated complexes has been already reported in literature [91].

Quantitative Detection of Sulfite
The experiments described above demonstrated that sulfite significantly increases the decomposition rate of azacyclam complexes. When considering complexes [Cu(2)] 2+ and [Cu(3)] 2+ , the fluorescence enhancement due to the release of the fluorophore can be in principle exploited to signal the presence of sulfite in solution. However, the decomposition of azacyclam complexes, even in the presence of sulfite, is a quite slow process (see Figure 5) and the related variation of I F at short times (i.e., <1 h) cannot be reliably used for analytical application. On the other hand, the decomposition rate, dI F /dt, is strongly affected by sulfite and could be usefully related to the concentration of this species in solution. To demonstrate the relationship between dI F /dt and [SO 3 2− ], the decomposition of [Cu(2)] 2+ was investigated in the presence of an increasing amount of sodium sulfite under the same experimental conditions described above (pH = 7.2; T = 40 • C). The fluorescence intensity at 345 nm (λ exc = 275) was monitored at the initial stage of the reaction (20 min) in each experiment, and the dI F /dt value corresponding to each considered [Na 2 SO 3 ] value was determined from I F vs. time profile. The resulting graph obtained by plotting dI F /dt vs. sulfite concentration is reported in Figure 7. The decomposition rate increases with sulfite concentration until this is almost equivalent to the concentration of azacyclam complex; then, it reaches a "plateau" and does not undergo further significant changes. A satisfactory fit of the experimental data was obtained by using Equation (1): where v0 is the reaction rate in the absence of SO3 2− , a is the difference between the "plateau rate", and v0, b is a parameter used to describe the curvature and c(SO3 2− ) is the global concentration of sulfites ([HSO3 − ] + [SO3 2− ], expressed in mol/L in the following data). The A satisfactory fit of the experimental data was obtained by using Equation (1): where v 0 is the reaction rate in the absence of SO 3 2− , a is the difference between the "plateau rate", and v 0 , b is a parameter used to describe the curvature and c(SO 3 2− ) is the global concentration of sulfites ([HSO 3 − ] + [SO 3 2− ], expressed in mol/L in the following data). The resulting curve matches the experimental points in the investigated concentration range. The same behavior was also observed for [Cu(3)] 2+ (Figure 8). The fitting data for both compounds are resumed in Table S2.

inset.
A satisfactory fit of the experimental data was obtained by using Equation (1): where v0 is the reaction rate in the absence of SO3 2− , a is the difference between the "plateau rate", and v0, b is a parameter used to describe the curvature and c(SO3 2− ) is the global concentration of sulfites ([HSO3 − ] + [SO3 2− ], expressed in mol/L in the following data). The resulting curve matches the experimental points in the investigated concentration range. The same behavior was also observed for [Cu(3)] 2+ (Figure 8). The fitting data for both compounds are resumed in Table S2. Although the overall response of both probes is nonlinear, the dIF/dt vs. c(SO3 2-) profiles are almost linear at lower concentration values (see insets in Figures 7 and 8) and can be properly used as calibration lines for the quantitative determination of sulfite Although the overall response of both probes is nonlinear, the dI F /dt vs. c(SO 3 2-) profiles are almost linear at lower concentration values (see insets in Figures 7 and 8) and can be properly used as calibration lines for the quantitative determination of sulfite roughly in the 0.5-10 µM range. Therefore, possible sensitivity problems when analyzing samples with higher sulfite concentration can be easily circumvented by diluting the sample solution (e.g., with a pH buffer) before starting the analysis.
Limit of detection (LOD) and limit of quantification (LOQ) values were also estimated for both probes [92] and are reported in Table S2. Interestingly, a detection limit as low as 30 ppb (calculated as SO 2 ) was found.
In order to check any possible interference of other anions on the sulfite-induced decomposition rate, more experiments with a fixed concentration of SO 3 2− (1 × 10 −5 M) and the simultaneous presence of a second anion (5 × 10 −4 M) were performed. In general, the dI F /dt data obtained by these tests were very similar to the decomposition rate in the presence of sulfite alone (Figure 9). For [Cu(2)] 2+ a slight decrease was observed in the case of S 2 O 3 2− and NO 2 − , while in the case of [Cu(3)] 2+ acetate (decrease) and hydrogencarbonate (increase) also induced a small but detectable change with respect to the sulfite alone.
Further experiments were performed in order to test the applicability of the abovedescribed method to evaluate the concentration of sulfite in real samples.
Sulfite is generally present in three forms in food or beverage systems: free, reversibly bound, and irreversibly bound. Free sulfite, as its name suggests, is not bound to any other component and can be more easily quantified. Reversibly and irreversibly bound sulfites exist when adducts form between sulfite and various food or beverage components (i.e., acetaldehyde, sugar monomers, or sugar acids) [2].
Determination of bound sulfite is usually not straightforward because their quantification requires particular pretreatment of samples or is scarcely reliable in the presence of irreversibly bound sulfite derivatives, which are usually very stable. Therefore, the check of the present method was limited to the determination of free sulfite in a sample of a popular branded white wine.
In order to check any possible interference of other anions on the sulfite-indu decomposition rate, more experiments with a fixed concentration of SO3 2− (1 × 10 −5 M) the simultaneous presence of a second anion (5 × 10 −4 M) were performed. In general, dIF/dt data obtained by these tests were very similar to the decomposition rate in presence of sulfite alone (Figure 9). For [Cu(2)] 2+ a slight decrease was observed in the c of S2O3 2− and NO2 − , while in the case of [Cu(3)] 2+ acetate (decrease) and hydrogencarbon (increase) also induced a small but detectable change with respect to the sulfite alone. Further experiments were performed in order to test the applicability of the abo described method to evaluate the concentration of sulfite in real samples.
Sulfite is generally present in three forms in food or beverage systems: free, revers bound, and irreversibly bound. Free sulfite, as its name suggests, is not bound to any ot component and can be more easily quantified. Reversibly and irreversibly bound sulf exist when adducts form between sulfite and various food or beverage components ( acetaldehyde, sugar monomers, or sugar acids) [2].
Determination of bound sulfite is usually not straightforward because th quantification requires particular pretreatment of samples or is scarcely reliable in presence of irreversibly bound sulfite derivatives, which are usually very sta Therefore, the check of the present method was limited to the determination of free sul in a sample of a popular branded white wine.
At first, the content of sulfite in the selected wine was determined by the widely u Ripper standard method [23,25], and a concentration of 39 ± 5 mg/L (calculated as S corresponding to about 4.9 × 10 −4 M) was found. Then, different samples of wine w properly diluted in order to obtain a sulfite concentration in the range corresponding the "linear" portion of profiles described by Equation (1)  At first, the content of sulfite in the selected wine was determined by the widely used Ripper standard method [23,25], and a concentration of 39 ± 5 mg/L (calculated as SO 2 , corresponding to about 4.9 × 10 −4 M) was found. Then, different samples of wine were properly diluted in order to obtain a sulfite concentration in the range corresponding to the "linear" portion of profiles described by Equation (1)

Materials and General Procedures
Unless otherwise stated, all reagents and solvents were supplied by Sigma-Aldrich and used as received.
Concentrated solutions of the complexes were stored in a refrigerator to ensure longterm stability.
UV-vis absorption spectra were recorded on a Cary 50 (Varian Ltd., Mulgrave, Victoria, Australia) spectrophotometer using a 1 cm path-length optical-glass cuvette. Emission spectra were recorded on a Cary Eclipse (Varian Ltd., Mulgrave, Victoria, Australia) spectrofluorimeter using a 1 cm × 1 cm quartz cuvette; the spectra were smoothed using a 5 points Savitzky-Golay filter [93]. ESI-MS spectra were recorded on a Thermo-Finnigan LCQ Advantage Max (Thermo Electron, San Josè, CA, USA) equipped with an ESI source. IR spectra were recorded on a Spectrum 100 FT-IR (PerkinElmer, Waltham, MA, USA) spectrometer equipped with an ATR accessory.

Synthesis
[Cu(1)](NO 3 ) 2 and [Cu(2)](NO 3 ) 2 were prepared following the previously reported procedure [74]. (3)](ClO 4 ) 2 (3-dansyl-1,3,5,8,12-pentaazacyclotetradecane copper(II) perchlorate) was performed following a similar procedure. Cu(NO 3 ) 2 ·3H 2 O (1.0 mmol) was dissolved in EtOH (6 mL) in a round-bottom flask and 2,3,2-tet (1.0 mmol diluted in a small amount of EtOH) was added dropwise while stirring. Dansylamide (1.0 mmol) suspended in a EtOH:MeCN mixture was added, followed by Et 3 N (0.15 mL) and 37% aqueous CH 2 O (0.4 mL). The resulting mixture was kept at 50-60 • C for 7 days with continuous stirring. Further, 37% aqueous CH 2 O (0.2 mL) was added every day for the first 4 days. After a week, the purple solution was slightly concentrated with a rotary evaporator and cooled in an ice bath. 30% HClO 4 was slowly added while stirring until an abundant precipitate was obtained. The solid was collected using a Buchner funnel, washed with several portions of cold EtOH followed by cold diluted HClO 4 and finally with another portion of cold EtOH. The pink powder was further suspended in THF, filtered, and washed with the same solvent in order to ensure the complete removal of any unreacted dansylamide.   (3)](ClO 4 ) 2 crystals (pale orange, platy prismatic, 0.50 × 0.20 × 0.08 mm 3 ) were collected on a Bruker AXS CCD-based three-circle diffractometer (Bruker AXS Inc., Madison, Wisconsin, USA). Both instruments work at ambient temperature with graphite-monochromatized Mo Kα X-radiation (λ = 0.7107 Å). Crystal data for the studied molecular compounds are reported in Table 2.

Synthesis of [Cu
Data reduction (including intensity integration, background, Lorentz, and polarization corrections) for intensities collected with the conventional diffractometer was performed with the WinGX package [99]. Absorption effects were evaluated with the psi-scan method [100] and absorption correction was applied to the data. Frames collected by the CCD-based system were processed for data reduction with the SAINT software [101] and intensities were corrected for Lorentz and polarization effects. Absorption effects were empirically evaluated by SADABS software [102] and absorption corrections were applied to the data. All crystal structures were solved by direct methods (SIR 97) [103] and refined by full-matrix least-squares procedures on F 2 using all reflections (SHELXL 2018/3) [104]. Positions for hydrogens bonded to the secondary amines were identified in the final ∆F maps and refined, restraining the N-H distances to be 0.90 ± 0.01 Å Other hydrogen atoms were placed at calculated positions with the appropriate AFIX instructions and were refined using a riding model. Anisotropic displacement parameters were refined for all non-hydrogen atoms. For the 3[[Cu (2)](NO 3 ) 2 ]·2H 2 O] crystal, soft restraints on the molecular geometry (SAME) and enhanced rigid bond restraints (RIGU) were applied for a naphthalene aromatic ring, probably affected by unresolved positional disorder. Positions for protons belonging to water solvent molecules remained undetermined. CCDC 2150891, 2150892, and 2150893 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre.

Conclusions
Macrocyclic copper(II) complexes, i.e., [Cu(azacyclam)] 2+ , can be easily prepared by a template reaction involving a variety of locking fragments, including fluorescent ones. Complexes [Cu(2)] 2+ or [Cu(3)] 2+ , although functionalized with naphthyl and dansyl moiety, respectively, do not display light-emitting properties due to the fluorescence quenching process activated by the copper(II) ions. Nevertheless, the typical emission of 2-naphthalenesulfonamide and dansylamide used as locking fragments in the template synthesis of the macrocyclic compounds can be recovered as a result of complex decomposition. In fact, the ring-opening process takes place with the simultaneous release of the locking fragments and the consequent turn-on of their fluorescence. The decomposition is extremely slow in neutral water at room temperature but its rate significantly increases at basic pH and/or at high temperature, as demonstrated by different spectroscopic investigations. Interestingly, decomposition of the macrocyclic species was observed to be strongly increased in the presence of sulfite at neutral pH. The reaction with SO 3 2− was apparently not observed before in analogous Cu 2+ macrocyclic complexes (e.g., [Cu(cyclam)] 2+ derivatives) and may be related to the unique structure of azacyclam compounds. No other anion affected the complexes' decomposition, apart from sulfide, which induced a moderate increase in the reaction rate, albeit distinctly lower than sulfite. Therefore, the copper(II) azacyclam complexes were investigated as possible chemical probes displaying a fluorescence-turn-on behavior towards sulfite. In particular, in order to gain a faster response, the correlation between decomposition rate (dI F /dt) and sulfite concentration was exploited to perform quantitative determination of this anionic substrate on laboratory as well as real samples. Although the nonlinear response of these systems can induce a decrease in sensitivity with increasing SO 3 2− concentrations (ultimately leading to an upper concentration limit), in practice this limit may be easily circumvented by diluting the sample solution (e.g., with a pH buffer).
Preliminary tests on white-wine samples provided free sulfite-concentration values that were confirmed by independent analyses performed by the standard Ripper method.
In conclusion, this work has shown that [Cu(azacyclam)] 2+ complexes, which are extremely inert toward decomposition in very acidic solution, can decompose in the presence of sulfite and this reaction can be usefully exploited to develop a fluorogenic procedure for the selective detecting of SO 3 2− . This procedure is based on a novel approach and may represent an interesting addition to the existing sulfite-determination methods.

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Informed Consent Statement: Not applicable.

Data Availability Statement:
The data used to support the findings of this study are included within the article and in Supplementary Materials.