2-Aza-1,3-butadiene ligands for the selective detection of Hg 2+ and Cu 2+ ions

We report a set of novel receptors with the structural feature of having an naphthyl-, pyrenyl-or ferrocenyl subunits, directly linked to a 2-aza-1,3-butadiene moiety which, in general, can be used for the rapid and selective detection of Hg 2+ . After coordination with this metal cation, the derivatives having two different signaling units, such as a fluorophore and a redox moiety, undergo significant changes not only in their oxidation potentials but also in their UV-vis and emission spectra. Interestingly, in such cases addition of Cu 2+ cation promotes the oxidation of the free ligands. By contrast, pyrenyl derivative 10 selectively senses both Hg 2+ and Cu 2+ through two different channels: colorimetric and fluorescent. Furthermore, the association constants for the 1:1 complexes formed as well as the detection limits for the analysis of Hg 2+ and Cu 2+ are also reported. These findings are not only a supplement to the detecting methods for these pollutant metal ions, but also adds new merits to the chemistry of the 2-aza-1,3-butadiene derivatives.


Introduction
The design and synthesis of sensors for heavy and transition-metal (HTM) ions, such as Hg 2+ and Cu 2+ , is currently a task of prime importance due to their fundamental role in biological, environmental and chemical processes. 1Mercury is considered as a highly toxic element and its contamination is a global problem.A major source of human exposure stems from a variety of

Synthesis
The synthetic route used for the preparation of these ligands is depicted in Scheme 1. Specifically, the preparation of the ferrocene derivatives 6,7 and 10 bearing a fluorophore unit linked to the 1 position of the 2-azadiene bridge were prepared starting from the appropriate Nsubstituted diethylaminophosphonate 2 or 3 8b which were obtained in almost quantitative yield, by condensation of diethylaminophosphonate 1 9 with the adequate aldehyde R 1 -CHO.Treatment of those N-substituted diethylaminophosphonates n-BuLi at -78ºC and subsequent reaction of the resulting metalloylide with a new carbonyl derivative R 2 -CHO provided 6, 7 and 10 in good yields (Scheme 1).
Starting from the N-substituted diethylaminophosphonates 4 10 or 5, derived from formylferrocene and 4'-formylbenzo-15-crown-5, respectively, and following the above mentioned Horner-Wadsworth-Emmons (HWE) methodology, the isomeric derivatives 8 and 9 as well as compound 11 were also prepared.These new ligands present the structural feature of having the fluorophore unit linked to the 1 position of the 2-azadiene bridge (Scheme 1).
The novel 2-aza-1,3-butadiene derivatives 6-11 were fully characterized by using 1 H and 13 C NMR spectroscopies and EI mass spectrometry.In general, the protons present in the 2-aza-1,3butadiene bridge appeared in their 1 H NMR spectra as one singlet (-CH=N-) and two doublets (-CH= CH-).Assignment of the configuration of the double bonds present in the 2-aza-1,3butadiene bridge was achieved by inspection of the corresponding 1 H NMR spectroscopic data.It is generally accepted that the stereoselectivity in HWE olefination reactions is a result of both kinetic and thermodynamic control upon the reversible formation of the erytro and threo adducts and their decomposition to olefins.That is, the stereochemistry is determined by a combination of the stereoselectivity in the initial carbon-carbon bond forming step and the reversibility of the intermediate adducts.However, in general, this reaction preferentially gives the more stable Edisubstituted olefins, as a consequence of the predominant formation of the thermodynamically more stable threo adducts. 11Thus, the E-configuration of the carbon-carbon double bond in compounds 6-11, as is expected in the olefination process, was confirmed by the value of the vicinal coupling constants (J = 13.1-14.0Hz).In addition, NOE and two-dimensional NOESY experiments carried out on CDCl3 solutions, confirmed the (E,E)-configuration of the double bonds present in the aza-bridge of these derivatives.
Interestingly, 1 H NMR spectra of those receptors bearing the ferrocene subunit (6-9) also showed two pseudotriplets, integrating two protons each, assigned to the four protons within the monosubstituted ciclopentadienenyl (Cp) ring, and one singlet corresponding to the unsubstituted Cp ring.

Redox and optical properties
Electrochemical studies were carried out by using cyclic (CV) and differential pulse (DPV) voltammetry of acetonitrile solutions of the receptors (c = 10 -3 M) containing 0.1 M [(n-Bu)4N]ClO4 (TBAP).Compounds 6-9 show one reversible 12 oxidation wave in the range 0.0-1.0V vs decamethylferrocene (DMFc) assigned to the oxidation of the ferrocene subunits (Table 1).The oxidation potentials of the ferrocenyl units (Fc) are dependent on the position of the 2-azadiene bridge to which they are attached: E1/2 = 0.440 and 0.543 V, in 6 and 7, with the Fc at the 4 position, and E1/2 = 0.576 and 0.672, V in 8 and 9, in which the Fc unit is linked to the 1 position of the bridge.Moreover, when the CV were carried out in the range 0-1.8 V, compounds 7 and 9 also shown two additional irreversible waves due to the oxidation of both the azadiene bridge (Ep = 1.09V for 7 and Ep = 1.16 V for 9) and the pyrenyl subunit (Ep = 1.61 V for 7 and Ep = 1.57V for 9).Spectrophotometric studies of these compounds were also carried out in acetonitrile solutions (c = 10 -4 or 2.5x10 -5 M).The electronic absorption spectra show the typical absorption bands corresponding to the naphthalene or pyrene 13 chromophore subunits in the region 265-347 nm for 6 and 8 and 235-391 nm for 7 and 9. Additionally, the ferrocenyl derivatives 6-9 also exhibit another weaker low-energy (LE) absorption band in the region 484-501 nm which is assigned to a ferrocenyl-based metal-to-ligand charge transfer process (MLCT) 14 (Table 1).By contrast, the UV-vis spectra of 10 and 11 showed a LE band, centered at 409 nm attributed to the aza-bridge, along with the typical pyrene absorption bands.
As expected, receptors 6-11 showed a very weak fluorescence ( = 0.00013-0.002)(Table 2).The emission spectrum of 7, 9, 10 and 11 in acetonitrile (exc = 350 nm), displays typical emission bands at 388 and 409 nm, which are attributed to the pyrene monomeric emission. 15On the other hand, the corresponding spectrum of the receptors 6 and 8 in the same solvent (exc = 310 nm) exhibit a structureless band centered at 387 and 408 nm, respectively, due to the naphthalene monomer emission.
The photophysical properties of the pyrene family receptors, 7, 9, 10 and 11, have also been investigated in CH3CN/H2O (7/3) solution (exc = 350 nm).Under these conditions, the pyrenetype emission is also quenched although in a less extent than in the pure organic solvent. 16oreover, their emission spectrum shows together with the above mentioned pyrene monomeric emission bands ( = 388 and 409 nm) a red-shifted structureless and broad fluorescence band with a maximum around  = 450 nm, typical of pyrene excimer fluorescence. 15
The ability of these receptors to offer an electrochemical response upon addition of such set of metal cations was investigated by using the DPV 19 technique.Nevertheless, due to the oxidizing character of the Cu 2+ metal cation, preliminary linear sweep voltammetry (LSV) studies were carried out by addition of this cation to a CH3CN solution of these ligands.In all cases, the results obtained demonstrate that an oxidation of the free ligand was taking place during this process, because a significant shift of the sigmoidal voltammetric wave toward cathodic currents was observed (Figure 1).On the other hand, ligands 6-9 undergo the same response when Hg 2+ cation was added: the appearance of a new redox peak, anodically shifted from the original ferrocene potential in the free ligand (Figure 2).However, the magnitude of these redox shifts were different depending on the position of the ring to which the ferrocene unit is bonded (Table 1).In contrast, addition of Li + , Na + , K + , Mg 2+ , Ca 2+ , Zn 2+ , Cd 2+ to these receptors do not promote any change in the corresponding electrochemical responses, with the only exception of ligand 8 which undergoes a significant redox shift upon addition of Zn 2+ cation (see Supplementary Information) In order to discriminate whether an oxidation or recognition process was taking place upon addition of Hg 2+ to these ligands, further experiments were carried out.Thus, a LSV study showed a different behaviour to that mentioned for the Cu 2+ cation.Then, addition of Hg 2+ revealed a shift of the linear sweep vomtammogram toward more positive pontentials, which is in agreement with the complexation process previously observed by DPV (Figure 1).Similar results were also obtained when the recognition behaviour was studied through spectrophotometric titrations by addition of the above-mentioned set of metal cations (c = 2.5 10 - 5 M in CH3CN), with the only exception of Cu 2+ , to receptors 6-9.The results obtained clearly demonstrate that only the addition of increasing amounts of Hg 2+ ions promotes some changes in their absorption spectra until 1 equiv was added (Figure 3 and Supplementary Information) (Table 2).Moreover, these changes also result in a variation of the colour in their solutions from orange, in the free ligands, to purple, which can be used for the naked eye detection of this metal cation.The appearance of well defined isosbestic points indicates the presence of a unique complex in equilibrium with the corresponding free ligand.Analysis of the absorption spectral data 21 confirmed a 1:1 L•Hg 2+ stoichiometry for the complexes formed, which calculated association constants are given in  The recognition process was also monitored by changes in the emission spectrum of these naphthyl and pyrenyl derivatives, both in CH3CN and CH3CN/H2O (7/3).In general, the emission of the weakly fluorescent ligands 6 and 8 only undergoes significant changes upon addition of Hg 2+ metal cation.Thus, when increasing amounts of this cation was added to a solution of these ligands, the appearance of a new emission band was observed at = 471 nm and = 428 nm for 6 and 8, respectively.The intensity of this emission band gradually increased until 1 equiv of the cation was added.In both cases, the calculated fluorescence enhancement factor (FEF) was 189 for 6 and 64 for 8.It is worth mentioning that only ligand 8 also emits a strong fluorescence (= 428 nm) when it complexes Zn 2+ metal cation (FEF = 39) (Supplementary Information).Similar results were obtained when the fluoroionophoric properties of ligands 7 and 9 were tested upon addition of the same set of metal cations.Remarkably, the fluorescence of a CH3CN solution of these free ligands was only influenced upon addition of Hg 2+ ion.Thus, the increase of the concentration of this cation in such solutions results in the gradual appearance of an emission pyrene-like spectrum, with three maxima at 388, 409 and 428 nm.The emission intensity of these bands continuously increase until 1 equiv of Hg 2+ ion was added, the calculated FEF being 27 and 5, for 7 and 9, respectively (Figure 4).Similar results were obtained in CH3CN/H2O (7/3), although a more intense pyrene excimer emission was observed at 453 nm (Figure 5).These data suggest that the coordination of the metal ion with the N atom in the aza-bridge is taking place so that the responsible mechanism for fluorescence quenching in the free ligand is minimized in its metal-bound state.Moreover, both ligands 7 and 9 were found to have a detection limit (Dlim) 20 of 3.8 x 10 -6 M in CH3CN, and 4.4x10 -6 M in CH3CN/H2O (7/3), as fluorogenic sensor for the analysis of Hg 2+ in these solvents (Supplementary Information).Analogous recognition studies by using the above mentioned set of metal cations were also carried out with the receptors 10 and 11, in which no redox subunit is linked to the azadiene bridge.Instead, two different potential ionophores are present in these molecules: the azadiene binding unit and a macrocyclic subunit, bearing binding sites for alkali metal cations.The Uv-vis and fluorescence experiments carried out demonstrate that only significant changes were observed upon addition of Cu 2+ and Hg 2+ cations to the ligand 10, in both CH3CN (Supplementary Information) and CH3CN/H2O (7/3) (Figure 6), while 11 do not show either optical or fluorescence selectivity for any of the metal ions tested.Thus, addition of increasing amounts of Cu 2+ and Hg 2+ to 10 promotes in its absorption spectrum remarkable responses, although the latter in a considerable less extent.These changes can be summarized as follows: (i) the bands at λ = 235 and 288 nm increase in intensity along with a blue shift (Table 2), reaching a maximum when 1 equiv of these metal cations was added; (ii) the band at λ = 409 nm progressively disappears, and at the same time, new bands at λ = 343, 361 and 393 nm continuously increase in intensity, reaching a maximum when 1 equiv of Cu 2+ was added; (iii) three well-defined isosbestic points at λ = 290, 325 and 365 nm were found, indicating the presence of a unique complex in equilibrium with the neutral ligand.The Job's plot clearly indicates a 1:1 binding model, for the complexation process (Supplementary Information).Remarkably, the titration experiments carried out in CH3CN/H2O (7/3) showed an analogous behaviour.
The evolution of the emission spectrum of 10 in CH3CN (Ф = 0.004) (Figure 7) or CH3CN/H2O (7/3) (Ф = 0.022) (see Supplementary Information) upon addition of the metal cations demonstrates that, after addition of 1 equiv of Cu 2+ , the fluorescence quantum yield increased by a factor of 15 (Ф = 0.062) and 5.5 (Ф = 0.122), respectively.Similar results were also obtained when Hg 2+ was added, although the observed fluorescence enhancement was considerably poorer (Ф = 0,020 in CH3CN and Ф = 0,075 in CH3CN/H2O (7/3)) (Figure 7).These data suggest that the coordination of the metal ion with the N atom in the aza-bridge is taking place so that the responsible mechanism for fluorescence quenching in the free ligand is minimized in its metal-bound state.Moreover, ligand 10 was found to have a detection limit 20 of 4.9 x 10 -6 and 3.8 x 10 -6 M as fluorogenic sensor for the analysis of Cu 2+ in CH3CN and CH3CN/H2O (7/3), respectively (Supplementary Information).As the general principle in designing fluorogenic and chromogenic chemosensors is based on analyte coordination events, therefore, both the interaction with the analyte and the change in colour or fluorescence should be reversible.Extraction experiments with EDTA confirmed the high degree of reversibility of the complexation/decomplexation processes (see Supplementary Information).
Due to the difficulties in obtaining suitable crystals for X-ray analysis of the resulting complexes, theoretical calculations at the DFT level have been carried out concerning the simplest example --naphthyl substitution -of the performance-enhanced 4-ferrocenyl azadiene derivatives in order to give an approximation to the binding mode taking place during the above mentioned recognition processes.In this regard some specific DFT methods have proved quite useful for studying systems with noncovalent interactions, offering an electron correlation correction frequently comparable, or in certain cases and for certain purposes even superior to MP2, but at considerably lower computational cost 21 We have used the Truhlar's hybrid meta functional mPW1B95 22 that has been recommended for general purpose applications and was developed in order to produce a better performance where weak interactions are involved such as those between ligands and heavy metals. 23At the working level of theory we have localized a global minimum 6 among several possible conformers.This compound forms a moderately stable complex with Hg(OTf)2 in acetonitrile (EMeCN = -6.44Kcal•mol -1 ) (Figure 8) despite the relatively high energetic cost to be paid for adaptation of the ligand (Lstrain = 5.14 Kcal•mol -1 ).Although in solution the triflate counteranions could be displaced to a second coordination sphere by the coordinating solvent (acetonitrile) molecules, we have used a [6•Hg(OTf)2] species as model for the complexation product, provided that it is expected to constitute an acceptable approximation to the binding of the Hg 2+ cation to receptor 6.The ligand 6 provides only one strong linkage to the Hg 2+ ion through the azadiene N-2 atom (dHg-N = 2.274 Å, WBI 0.254).We have also used the Bader's AIM (Atoms-In-Molecules) methodology 24 to perform a topological analysis of the electronic charge density (r), in order to search for significant bond critical points (BCPs) around the spatial region where the host-guest interactions are taking place, which are of diagnostic relevance for chemical bonding between two neighboring atoms.The electron density of the BCP for the above mentioned Hg 2+ -azadiene interaction agrees with the proposed high bond strength ((rc[Hg-N]) = 7.34•10 -2 au).In addition, four coordination positions are occupied by O atoms belonging to two triflate units. 25  According to our calculations the complex resulting from the reaction of 6 with Cu(OTf)2 had a geometry corresponding to the oxidized receptor 6 •+ interacting with the reduced Cu(I) cation, as evidenced by the electronic and structural features collected in Table 3, which nicely agree with the above mentioned experimental results.Specially relevant is the total natural charge within the ferrocenyl unit, very much higher than the residual values obtained for 6 or its complex with Hg(OTf)2.Furthermore we have recently found 10 that the distance between the metal atom and the centroid of the cyclopentadienyl rings has diagnostic relevance for characterizing the oxidation degree in ferrocene or ruthenocene derivatives, as far as it results almost insensitive to the nature of electron donating or withdrawing substituents but is considerably increased in radical-cation metallocinium species, probably because of the removal of an electron which is slightly bonding with respect to the metal-ring interaction.In addition, it has been previously reported 27 that neutral ferrocenyl units can participate in alleviating an electron deficiency at neighboring positions by folding the Cp a -Fe-Cp b axis.Thus, high values for in-plane angles 28 account for a tilting of the Fe dz2-type orbital which is involved in the  interaction with both the a1g MO of the unsubstituted Cp b unit and the LUMO (*) of the fulvene-like structure of Cp a (including the exocyclic bond).Oxidized ferrocenil units lack this possibility, therefore featuring low in-plane values (Table 3).a Distance between Fe and Cp centroid for the unsubstituted ring (Cp b ), referenced to that calculated for parent ferrocene, 1.635 Å (in Å•10 -2 ).b In degrees.c Total natural charge (in au) along the Fe atom or the ferrocenyl unit.

Conclusions
In summary, we have demonstrated that by using a structurally simple motif, whereby a fluorophore unit and a ferrocenyl moiety are linked by a 2-aza-1,3-butadiene bridge, highly selective sensing of Hg 2+ can be achieved in acetonitrile solution.Additionally, we have also developed a colorimetric and fluorescent ligand 10 which shows selectivity for Hg 2+ and Cu 2+ over other common metal ions, both in CH3CN and CH3CN/H2O (7/3).

Experimental Section
General Procedures.All reactions were carried out under N2 and using solvents which were dried by routine procedures.Melting points were determined on a Kofler hot-plate melting point apparatus and are uncorrected. 1H, 13 C and 31 P NMR spectra were recorded on a Bruker AC300, and 400.The following abbreviations for stating the multiplicity of the signals have been used; s (singlet), d (doublet), dd (double doublet), t (triplet), st (pseudotriplet), Cq (quaternary carbon).Chemical shifts refer to signals of tetramethylsilane in the case of 1 H and 13 C spectra.The mass spectra were recorded on a Fisons AUTOSPEC 500 VG spectrometer.Microanalyses were performed on a Carlo Erba 1108 instrument in the Department of Organic Chemistry (University of Murcia).CV and DPV techniques were performed with a conventional three-electrode configuration consisting of platinum working and auxiliary electrodes and a SCE reference electrode.The experiments were carried out with a 10 -3 M solution of sample in CH3CN containing 0.1 M (n-C4H9)4ClO4 (TBAP) (WARNING: CAUTION: potential formation of highly explosive perchlorate salts or organic derivatives) as supporting electrolyte.All the potential values reported are relative to the decamethylferrocene (DMFc) couple at room temperature.Deoxygenation of the solutions was achieved by bubbling nitrogen for at least 10 min and the working electrode was cleaned after each run.The cyclic voltammograms were recorded with a scan rate increasing from 0.05 to 1.00 V s -1 , while the DPV were recorded at a scan rate of 100 mV s -1 with a pulse height of 10 mV and a step time of 50ms.Typically, receptor (1 x 10 -3 M) was dissolved in CH3CN (5 mL) and TBAP (base electrolyte) (0.170 g) added.The guest under investigation was then added as a 0.1 M solution in appropriate solvent using a microsyringe whilst the cyclic voltammetric properties of the solution were monitored.DMFc was used as an external reference both for potential calibration and for reversibility criteria.Under similar conditions the DMFc has E = -0.07V vs SCE and the anodic peak-cathodic peak separation is 67 mV.

Computational details
Calculated geometries were fully optimized in the gas-phase with tight convergence criteria at the DFT level with the Gaussian 03 package 29 , using the hybrid meta functional mPW1B95.The 6-311G** basis set was used for all atoms, adding diffuse functions on donor atoms (N, O and F) (denoted as aug6-311G**) as well as the Stuttgart Relativistic Small Core basis set with effective core potential (StRSC-ecp) for Hg and Cu.Ultrafine grids (99 radial shells and 590 angular points per shell) were employed for numerical integrations.From these gas-phase optimized geometries all reported data were obtained by means of single-point (SP) calculations.Energy values were computed at the same level and considering solvent (acetonitrile) effects by using the Cossi and Barone's CPCM (conductor-like polarizable continuum model) modification 30 of the Tomasi's PCM formalism 31 and correcting the basis set superposition error (BSSE) by means of the Bq-approach.All energies are uncorrected for the zero-point vibrational energy.The same level of theory was used to perform the Natural Bond Orbital (NBO) population analysis.Bond orders were characterized by the Wiberg's bond index (WBI) 32 and calculated with the NBO method as the sum of squares of the off-diagonal density matrix elements between atoms.Timedependent DFT (TD-DFT) calculations were performed at the current level of theory onto gasphase optimized geometries and taking into account solvent effects.The topological analysis of the electronic charge density was conducted by means of the Bader's AIM (Atoms-In-Molecules) 24 methodology using the AIM2000 software. 33neral procedure for the preparation of N-substituted diethyl aminomethylphosphonates

General procedure for the preparation of 1,4-disubstituted 2-aza-1,3-butadienes 6-11
To a solution of the appropriate diethyl phosphonate (4.64 mmol) in dry THF (20 ml), at -78 ºC and under nitrogen atmosphere, was dropped the adequate amount of n-BuLi (1.6 M in hexane).Then, a solution of the appropriate aldehyde (4.64 mmol) in dry THF (10 ml) was added dropwise and the solution was stirred for 1.5 h.The reaction mixture was allowed to reach the room temperature and, afterward, it was heated under reflux temperature overnight.After the solution was cooled to room temperature, the solvent was evaporated under reduced pressure and the resulting solid was slurried with diethyl ether (25 ml) to give a crude product which was recrystallized from dichloromethane/diethyl ether (1/10).

Figure 1 .
Figure 1.Changes in the linear sweep voltammogram of 8 ( 1 x 10 -3 M ) in CH3CN with TBAP ( 0.1 M ) as supporting electrolyte, obtained using a rotating disk electrode at 100 mV s -1 and 1000 rpm, when metal cations are added: (a) upon addition of increasing amounts of Hg 2+ cations and (b) upon addition of increasing amount of Cu 2+ cations.

Figure 3 .
Figure 3. Changes in the absorption spectra of 8 a) and 9 b) (1 x 10 -4 M) in CH3CN upon addition of Hg 2+ (2.5 x 10 -2 M) in CH3CN, from 0 to 1 equiv.Arrows indicate the absorption that increase or decrease during the experiment.
Finally the 6-coordination sphere around the Hg 2+ metal cation is completed by an additional weak interaction with the naphthalene C-1 atom (dHg-C = 2.984 Å, WBI 0.035, (rc[Hg-C]) = 1.95•10 -2 au) whose -bonding nature is evidenced by the moderately high bond ellipticity value (ε = 0.963).All six interactions with Hg display negative values of the Laplacian of the electron density at their respective BCPs,  2 (rc), revealing that the charge is depleted, as in closed shell electrostatic interactions.26

Table 2 .
UV-Vis and fluorescente data of compounds 6-11 and of their corresponding metal compexes in CH3CN  in dm 3 mol -1 cm -1 ; b max in nm;cThe fluorescence quantum yields were measured with respect to anthracene as standard (Ф = 0.27).Dawson, W. R.; Windsor, M. W. J Phys.

Table 3 .
Selected electronic, structural and thermodynamic parameters for the calculated structures of receptor 6 and its complexes with Hg(OTf)2 and Cu(OTf)2 dFe-Cp b a in-plane b QFe c , an equimolar amount of the appropriate aldehyde was added dropwise.The resulting solution was stirred at room temperature for 2 h and then filtered.From the filtrate the solvent was removed under vacuum to give the corresponding aminomethylphosphonate in almost quantitative yield, as colored oils which were used, without further purification, in the next step.