Binding Properties of a Dinuclear Zinc(II) Salen-Type Molecular Tweezer with a Flexible Spacer in the Formation of Lewis Acid-Base Adducts with Diamines

: In this paper we report the binding properties, by combined 1 H NMR, optical absorption, and ﬂuorescence studies, of a molecular tweezer composed of two Zn(salen)-type Schiff-base units connected by a ﬂexible spacer, towards a series of ditopic diamines having a strong Lewis basicity, with different chain length and rigidity. Except for the 1,2-diaminoethane, in all other cases the formation of stable 1:1 Lewis acid-base adducts with large binding constants is demonstrated. For α , ω -aliphatic diamines, binding constants progressively increase with the increasing length of the alkyl chain, thanks to the ﬂexible nature of the spacer and the parallel decreased conformational strain upon binding. Stable adducts are also found even for short diamines with rigid molecular structures. Given their preorganized structure, these latter species are not subjected to loss of degrees of freedom. The binding characteristics of the tweezer have been exploited for the colorimetric and ﬂuorometric selective and sensitive detection of piperazine. binding interactions between the aggregate molecular tweezer 1 , having a flexible spacer, and the structure of the ditopic Lewis bases.


Introduction
"Molecular tweezers" refer to bifunctional molecular receptors characterized by the presence of two binding sites connected with a more or less rigid spacer [1,2]. They have the ability to form complexes with a substrate molecule, resembling a tweezer holding an object. Depending on the nature of the binding sites and on the conformational rigidity of the spacer, they find various applications such as in molecular recognition [1,3], including biomolecules [4][5][6], or fullerenes [7], enzyme inhibition or prevention of protein aggregation [8][9][10], catalysis [11], switchable molecular tweezers [12,13], electrochemical switches [14,15], and as building blocks for supramolecular nanostructures [16,17].
The aim of this work is to study, through 1 H NMR, UV/vis, and fluorescence spectroscopies, the binding interactions of the tweezer 1 with diamines of different chain length and rigidity, to better understand their Lewis acid-base interactions.

Results and Discussion
To study the binding of various diamines to the molecular tweezer 1, either aliphatic, alicyclic, and aromatic diamines were considered (Scheme 2). In particular, the flexible primary α,ω-aliphatic diamines, NH2(CH2)nNH2 (n = 2-12), were studied, and the results compared with those related to the rigid diamines, piperazine (PZ), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 4,4′-bipyridine (BPY), and the semi-rigid 1,2-bis(4-pyridyl)ethane (DPE). As the dinuclear aggregate complex 1 acts as a "molecular tweezer" upon deaggregation, this is an unusual feature compared to conventional tweezers characterized by binding sites kept separate by a spacer. Thus, starting from the defined aggregate 1, in the formation of Lewis acid-base adducts, it will not be subjected to binding as a consequence of a specific conformation of the Lewis base. Rather, the ability of the Lewis base to bind the aggregate could be related to its basicity and to the stability of the adduct. It is thus interesting to investigate the features affecting the binding interactions between the aggregate molecular tweezer 1, having a flexible spacer, and the structure of the ditopic Lewis bases.
The aim of this work is to study, through 1 H NMR, UV/vis, and fluorescence spectroscopies, the binding interactions of the tweezer 1 with diamines of different chain length and rigidity, to better understand their Lewis acid-base interactions.

Results and Discussion
To study the binding of various diamines to the molecular tweezer 1, either aliphatic, alicyclic, and aromatic diamines were considered (Scheme 2). In particular, the flexible primary α,ω-aliphatic diamines, NH 2 (CH 2 ) n NH 2 (n = 2-12), were studied, and the results compared with those related to the rigid diamines, piperazine (PZ), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 4,4 -bipyridine (BPY), and the semi-rigid 1,2-bis(4-pyridyl)ethane (DPE). Recently, a dinuclear Zn(salmal) Schiff-base complex (1, Scheme 1)whose units are connected with a non-conjugated, flexible spacer, has been synthesized and characterized [45]. It has been found that in non-coordinating solvents 1 is stabilized by the formation of intramolecular aggregates, which hardly deaggregate by addition of monotopic Lewis bases. However, in the presence of strong ditopic Lewis bases, such as diamines, the complex easily deaggregates with formation of 1:1 adducts, thus acting as a "molecular tweezer". Deaggregation is accompanied by relevant optical absorption changes and a substantial enhancement of the fluorescence. Therefore, 1 has been investigated for the selective and sensitive detection of some biogenic diamines [46]. Scheme 1. Structure of the dinuclear complex 1.As the dinuclear aggregate complex 1 acts as a "molecular tweezer" upon deaggregation, this is an unusual feature compared Table 1. in the formation of Lewis acid-base adducts, it will not be subjected to binding as a consequence of a specific conformation of the Lewis base. Rather, the ability of the Lewis base to bind the aggregate could be related to its basicity and to the stability of the adduct. It is thus interesting to investigate the features affecting the binding interactions between the aggregate molecular tweezer 1, having a flexible spacer, and the structure of the ditopic Lewis bases.
The aim of this work is to study, through 1 H NMR, UV/vis, and fluorescence spectroscopies, the binding interactions of the tweezer 1 with diamines of different chain length and rigidity, to better understand their Lewis acid-base interactions.
In all instances, the binding of diamines to 1 involves an increase of the optical absorption band centered at λ max = 580 nm and an enhancement of fluorescence intensity at λ max ∼ = 614 nm (e.g., the fluorescence quantum yield, φ, increases from 0.03 to 0.29 on switching from 1 to the adduct with 1,10-diaminodecane). Moreover, with the exception of diaminoethane, optical absorption spectra show the presence of multiple isosbestic points, indicative of the formation of species with a defined stoichiometry. Job's plot analyses clearly indicate the formation of 1:1 adducts.  2 ; λ max = 579 nm (absorption); λ max = 613 nm (emission); log K 1 = 2.35; log K 2 = 3.58; from ref. [45]. b from ref. [46]. UV/vis optical absorption and fluorescence spectral data and binding constants for the formation of (1:1) 1·diamine adducts (equilibrium (1)) are reported in Table 1. Spectrophotometric and spectrofluorometric titrations for the representative 1,10-diaminodecane are reported in Figures 1 and 2.
In all instances, the binding of diamines to 1 involves an increase of the optical absorption band centered at λmax = 580 nm and an enhancement of fluorescence intensity at λmax  614 nm (e.g., the fluorescence quantum yield, ϕ, increases from 0.03 to 0.29 on switching from 1 to the adduct with 1,10-diaminodecane). Moreover, with the exception of diaminoethane, optical absorption spectra show the presence of multiple isosbestic points, indicative of the formation of species with a defined stoichiometry. Job's plot analyses clearly indicate the formation of 1:1 adducts.  [45]. b from ref. [46].

α,ω-Aliphatic Diamines
As is shown in Table 1, a substantial variation of the binding constants with the chain length is observed. Despite the analogous Lewis basicity [47] along this investigated series of aliphatic, linear primary diamines, binding constants span over more than three orders of magnitude. While shorter diamines are characterized by relatively low binding constants, the increasing of the chain length parallels an increase of binding constant values. Largest binding constants are reached with the 1,8-diaminooctane, and then remain almost unchanged, even if slightly smaller, on further increase of the chain length. 1 H NMR titration studies further support the formation of 1:1 adducts. The titration for the representative 1,10-diaminodecane is reported in Figure 3. In particular, after the addition of half stoichiometric amount of 1,10-diaminodecane to a CDCl3 solution of 1, the 1 H NMR spectrum shows some changes with the appearance of new broad signals. A complete variation of the 1 H NMR spectrum, with the presence of broad signals, is observed after the addition of a stoichiometric amount of 1,10-diaminodecane. Finally, upon the addition of 4-fold molar excess of 1,10-diaminodecane the spectrum evolves towards a set of sharp signals, except for the down-field shifted H3 and H3′ protons which remain slightly broad, indicative of the formation of a defined 1:1: adduct. Moreover, the doublet of doublet benzylic proton signals, H5, of the aggregate complex 1 becomes a sharp singlet, indicating that the restricted rotation around the benzylic bonds is no longer operating in the adduct.
1,2-Diaminoethane behaves quite differently. In fact, starting from 10 μM solutions of 1 a detectable variation of optical absorption or fluorescence spectra occurs after the addition of ca. 2-fold molar excess of diaminoethane, while the saturation point is reached by the addition of ca. 800-fold molar excess, unlike longer diamines which form 1:1 adducts with 1 by addition of stoichiometric amounts. Moreover, in contrast with longer diamines, optical absorption spectra do not show any isosbestic point. These observations suggest the formation of multiple, instead of single, adducts (e.g., 1:2 adducts), likely favored by the presence of the large stoichiometric excess of diamine.
As binding constants for the formation of 1·diamine adducts reflect the relative stability of the adducts with respect to the aggregate [48], given the analogous Lewis basic-

α,ω-Aliphatic Diamines
As is shown in Table 1, a substantial variation of the binding constants with the chain length is observed. Despite the analogous Lewis basicity [47] along this investigated series of aliphatic, linear primary diamines, binding constants span over more than three orders of magnitude. While shorter diamines are characterized by relatively low binding constants, the increasing of the chain length parallels an increase of binding constant values. Largest binding constants are reached with the 1,8-diaminooctane, and then remain almost unchanged, even if slightly smaller, on further increase of the chain length. 1 H NMR titration studies further support the formation of 1:1 adducts. The titration for the representative 1,10-diaminodecane is reported in Figure 3. In particular, after the addition of half stoichiometric amount of 1,10-diaminodecane to a CDCl 3 solution of 1, the 1 H NMR spectrum shows some changes with the appearance of new broad signals. A complete variation of the 1 H NMR spectrum, with the presence of broad signals, is observed after the addition of a stoichiometric amount of 1,10-diaminodecane. Finally, upon the addition of 4-fold molar excess of 1,10-diaminodecane the spectrum evolves towards a set of sharp signals, except for the down-field shifted H 3 and H 3 protons which remain slightly broad, indicative of the formation of a defined 1:1: adduct. Moreover, the doublet of doublet benzylic proton signals, H 5 , of the aggregate complex 1 becomes a sharp singlet, indicating that the restricted rotation around the benzylic bonds is no longer operating in the adduct.
1,2-Diaminoethane behaves quite differently. In fact, starting from 10 µM solutions of 1 a detectable variation of optical absorption or fluorescence spectra occurs after the addition of ca. 2-fold molar excess of diaminoethane, while the saturation point is reached by the addition of ca. 800-fold molar excess, unlike longer diamines which form 1:1 adducts with 1 by addition of stoichiometric amounts. Moreover, in contrast with longer diamines, optical absorption spectra do not show any isosbestic point. These observations suggest the formation of multiple, instead of single, adducts (e.g., 1:2 adducts), likely favored by the presence of the large stoichiometric excess of diamine.
increased stability of the intramolecular cyclic adducts. In view of the flexibility of the spacer in the tweezer 1, which in principle can accommodate almost any ditopic Lewis base, the different binding constants along the series could be attributed to a larger entropic cost of the loss of degrees of freedom for diamines with a shorter alkyl chain, while the involved longer α,ω-diamines are not subjected to conformational strain upon binding, resulting in larger intramolecular cyclic adducts, also with gain of degrees of freedom of the flexible spacer of the tweezer. In comparison to host-guest studies involving glycoluril-or porphyrin-based tweezers with ditopic guests of different length, 1 behaves quite differently, being characterized by increasing binding constants with increasing chain length of diamines. In fact, it has been found that most of these investigated tweezers, having rigid or semi-flexible spacers, show a preference for a particular guest, rather than for shorter or longer ones. This has been associated with the guest best matching the distance between the binding sites of the tweezer [27][28][29][30][49][50][51].

Rigid Diamines
The binding interaction for the formation of 1·diamine adducts is also affected by the rigidity of the diamine. PZ is a cyclic secondary diamine with a N-N distance comparable to that of 1,2-diaminoethane. In spite of this, PZ forms stable 1:1 adducts with 1, with a large binding constant (Table 1), especially if compared to that of aliphatic diamines with shorter chain length (n ≤ 4). DABCO is a bicyclic tertiary diamine whose N-N distance is comparable to that of PZ. It has been widely used to study the binding characteristics of porphyrin-based tweezers [22,30,[52][53][54]. Again, DABCO binds easily with 1 with a binding constant slightly higher than that of PZ. This is consistent with the greater Lewis basicity of the tertiary alicyclic DABCO with respect to the secondary alicyclic PZ [47].
It therefore turns out that the preorganized structure of PZ and DABCO favors the formation of stable adducts because, except for the entropic cost upon binding the dia-  As binding constants for the formation of 1·diamine adducts reflect the relative stability of the adducts with respect to the aggregate [48], given the analogous Lewis basicity along the series of diamines and the entropic cost upon binding the diamine to 1, the increasing binding constants with the increased length of diamines can be related to an increased stability of the intramolecular cyclic adducts. In view of the flexibility of the spacer in the tweezer 1, which in principle can accommodate almost any ditopic Lewis base, the different binding constants along the series could be attributed to a larger entropic cost of the loss of degrees of freedom for diamines with a shorter alkyl chain, while the involved longer α,ω-diamines are not subjected to conformational strain upon binding, resulting in larger intramolecular cyclic adducts, also with gain of degrees of freedom of the flexible spacer of the tweezer.
In comparison to host-guest studies involving glycoluril-or porphyrin-based tweezers with ditopic guests of different length, 1 behaves quite differently, being characterized by increasing binding constants with increasing chain length of diamines. In fact, it has been found that most of these investigated tweezers, having rigid or semi-flexible spacers, show a preference for a particular guest, rather than for shorter or longer ones. This has been associated with the guest best matching the distance between the binding sites of the tweezer [27][28][29][30][49][50][51].

Rigid Diamines
The binding interaction for the formation of 1·diamine adducts is also affected by the rigidity of the diamine. PZ is a cyclic secondary diamine with a N-N distance comparable to that of 1,2-diaminoethane. In spite of this, PZ forms stable 1:1 adducts with 1, with a large binding constant (Table 1), especially if compared to that of aliphatic diamines with shorter chain length (n ≤ 4). DABCO is a bicyclic tertiary diamine whose N-N distance is comparable to that of PZ. It has been widely used to study the binding characteristics of porphyrin-based tweezers [22,30,[52][53][54]. Again, DABCO binds easily with 1 with a binding constant slightly higher than that of PZ. This is consistent with the greater Lewis basicity of the tertiary alicyclic DABCO with respect to the secondary alicyclic PZ [47].
It therefore turns out that the preorganized structure of PZ and DABCO favors the formation of stable adducts because, except for the entropic cost upon binding the diamine to 1, these species are not subjected to loss of degrees of freedom, contrary to aliphatic diamines with short alkyl chain. 1 H NMR titrations using DABCO as titrant suggest the formation of stable 1:1 adducts even for alicyclic diamines (Figure 4). In this case, however, when half stoichiometric amount of DABCO is added to CDCl 3 solution of 1, two sets of signals are evident in the spectrum. This indicates the presence in solution of the aggregate complex 1 and its adduct with DABCO, in a slow equilibrium on the NMR time scale. Moreover, from the comparison of the signal of H 5 protons, a sharper singlet is observed for the adduct with 1,10-diaminodecane, with respect to the broad signal for that with DABCO. This suggests a greater mobility of benzyl hydrogens in the larger intramolecular cyclic 1·diaminodecane adduct compared to those of the 1·DABCO adduct ( Figure 5).
Inorganics 2021, 9,49 6 of 13 mine to 1, these species are not subjected to loss of degrees of freedom, contrary to aliphatic diamines with short alkyl chain. 1 H NMR titrations using DABCO as titrant suggest the formation of stable 1:1 adducts even for alicyclic diamines (Figure 4). In this case, however, when half stoichiometric amount of DABCO is added to CDCl3 solution of 1, two sets of signals are evident in the spectrum. This indicates the presence in solution of the aggregate complex 1 and its adduct with DABCO, in a slow equilibrium on the NMR time scale. Moreover, from the comparison of the signal of H5 protons, a sharper singlet is observed for the adduct with 1,10-diaminodecane, with respect to the broad signal for that with DABCO. This suggests a greater mobility of benzyl hydrogens in the larger intramolecular cyclic 1·diaminodecane adduct compared to those of the 1·DABCO adduct ( Figure 5).  The structure of DPE is more rigid than that of α,ω-aliphatic diamines because of the presence of two heterocyclic aromatic rings linked by an ethyl group. In the anti-conformation, a N-N distance of 9.3 Å can be estimated, slightly longer than that of 1,6-diaminohexane (8.7 Å ). However, the binding constant of DPE results are one order of magnitude lower than that of 1,6-diaminohexane. This can be attributed to a higher    mine to 1, these species are not subjected to loss of degrees of freedom, contrary to aliphatic diamines with short alkyl chain. 1 H NMR titrations using DABCO as titrant suggest the formation of stable 1:1 adducts even for alicyclic diamines (Figure 4). In this case, however, when half stoichiometric amount of DABCO is added to CDCl3 solution of 1, two sets of signals are evident in the spectrum. This indicates the presence in solution of the aggregate complex 1 and its adduct with DABCO, in a slow equilibrium on the NMR time scale. Moreover, from the comparison of the signal of H5 protons, a sharper singlet is observed for the adduct with 1,10-diaminodecane, with respect to the broad signal for that with DABCO. This suggests a greater mobility of benzyl hydrogens in the larger intramolecular cyclic 1·diaminodecane adduct compared to those of the 1·DABCO adduct ( Figure 5).  The structure of DPE is more rigid than that of α,ω-aliphatic diamines because of the presence of two heterocyclic aromatic rings linked by an ethyl group. In the anti-conformation, a N-N distance of 9.3 Å can be estimated, slightly longer than that of 1,6-diaminohexane (8.7 Å ). However, the binding constant of DPE results are one order of magnitude lower than that of 1,6-diaminohexane. This can be attributed to a higher  The structure of DPE is more rigid than that of α,ω-aliphatic diamines because of the presence of two heterocyclic aromatic rings linked by an ethyl group. In the anti-conformation, a N-N distance of 9.3 Å can be estimated, slightly longer than that Inorganics 2021, 9, 49 7 of 13 of 1,6-diaminohexane (8.7 Å). However, the binding constant of DPE results are one order of magnitude lower than that of 1,6-diaminohexane. This can be attributed to a higher conformational strain upon binding DPE to 1, in comparison with the flexible diaminohexane.
BPY was also investigated as rigid diamine. Spectrophotometric titrations again indicate the formation of a defined species; by the presence of multiple isosbestic points, however, the saturation point is reached with ca. a 500-fold molar excess of BPY. In this case the binding isotherm is fitted with a model involving a 1:2 adduct (equilibria (2)), instead of a 1:1 adduct (equilibrium (1)).

Sensing Piperazine
The molecular tweezer 1 can be used as a suitable chemodosimeter for the detection of piperazine. PZ possesses important pharmacological properties and is used, together with its salts, as an anthelmintic [55], in industrial gas treatments such as CO 2 capture system [56,57], and also as the precursor for a class of psychogenic drugs [58,59]. Piperazine may cause allergic dermatitis [60], and it has been demonstrated that, although not extremely toxic, it has a low biodegradability [61]. Detection of PZ is thus relevant for environment monitoring and protection [62].
The tweezer 1 allows both the colorimetric and fluorometric selective and sensitive detection of PZ. A calculated limit of detection (LOD) down to 0.76 µM and 0.33 µM is obtained from the spectrophotometric and spectrofluorometric data, respectively, with a linear dynamic range up to 10 µM (Figures 6 and 7). These values are better than those reported in the literature using spectrophotometric methods [63][64][65][66]. Various other techniques have been developed for the detection and quantitation of piperazine, such as HPLC [67], voltammetry [68], or capillary electrophoresis [69]; most of them, however, require time-consuming procedures. Therefore, the development of simple and direct methods for sensing piperazine is highly desirable. In this regard, only a few optical chemosensors are reported in the literature for the selective detection of PZ [70][71][72]. conformational strain upon binding DPE to 1, in comparison with the flexible diaminohexane. BPY was also investigated as rigid diamine. Spectrophotometric titrations again indicate the formation of a defined species; by the presence of multiple isosbestic points, however, the saturation point is reached with ca. a 500-fold molar excess of BPY. In this case the binding isotherm is fitted with a model involving a 1:2 adduct (equilibria (2)), instead of a 1:1 adduct (equilibrium (1)).

Sensing Piperazine
The molecular tweezer 1 can be used as a suitable chemodosimeter for the detection of piperazine. PZ possesses important pharmacological properties and is used, together with its salts, as an anthelmintic [55], in industrial gas treatments such as CO2 capture system [56,57], and also as the precursor for a class of psychogenic drugs [58,59]. Piperazine may cause allergic dermatitis [60], and it has been demonstrated that, although not extremely toxic, it has a low biodegradability [61]. Detection of PZ is thus relevant for environment monitoring and protection [62].
The tweezer 1 allows both the colorimetric and fluorometric selective and sensitive detection of PZ. A calculated limit of detection (LOD) down to 0.76 µ M and 0.33 µ M is obtained from the spectrophotometric and spectrofluorometric data, respectively, with a linear dynamic range up to 10 μM (Figures 6 and 7). These values are better than those reported in the literature using spectrophotometric methods [63][64][65][66]. Various other techniques have been developed for the detection and quantitation of piperazine, such as HPLC [67], voltammetry [68], or capillary electrophoresis [69]; most of them, however, require time-consuming procedures. Therefore, the development of simple and direct methods for sensing piperazine is highly desirable. In this regard, only a few optical chemosensors are reported in the literature for the selective detection of PZ [70][71][72].   The selectivity of 1 towards PZ was proven by performing competitive experiments. These were conducted by mixing a solution of 1 with PZ (1:1 molar ratio) and a 10-fold molar excess of interferent ( Figure 8). These results were then compared with those obtained by adding to 1 either PZ in an equimolar amount, or the interferent in a 10-fold molar excess. As potential interferents, some monotopic species with a strong Lewis basicity were considered. Pyridine was chosen as the heterocyclic aromatic amine, while isopropylamine, diethylamine, and triethylamine were chosen as prototype compounds of primary, secondary, and tertiary amines. Moreover, the heteroditopic 4-amino-1-butanol, bearing two different coordinating sites with a different Lewis basicity, was also considered. As shown in Figure 8, very small or no changes of the absorbance at λmax = 580 nm are observed after the addition of each potential interferent, especially for diethylamine and triethylamine. Therefore, these data suggest that 1 can be considered a selective receptor towards PZ, even in the presence of common aliphatic or aromatic monotopic or heteroditopic amines. The selectivity of 1 towards PZ was proven by performing competitive experiments. These were conducted by mixing a solution of 1 with PZ (1:1 molar ratio) and a 10-fold molar excess of interferent ( Figure 8). These results were then compared with those obtained by adding to 1 either PZ in an equimolar amount, or the interferent in a 10-fold molar excess. As potential interferents, some monotopic species with a strong Lewis basicity were considered. Pyridine was chosen as the heterocyclic aromatic amine, while isopropylamine, diethylamine, and triethylamine were chosen as prototype compounds of primary, secondary, and tertiary amines. Moreover, the heteroditopic 4-amino-1-butanol, bearing two different coordinating sites with a different Lewis basicity, was also considered. As shown in Figure 8, very small or no changes of the absorbance at λ max = 580 nm are observed after the addition of each potential interferent, especially for diethylamine and triethylamine. Therefore, these data suggest that 1 can be considered a selective receptor towards PZ, even in the presence of common aliphatic or aromatic monotopic or heteroditopic amines. The selectivity of 1 towards PZ was proven by performing competitive experiments. These were conducted by mixing a solution of 1 with PZ (1:1 molar ratio) and a 10-fold molar excess of interferent ( Figure 8). These results were then compared with those obtained by adding to 1 either PZ in an equimolar amount, or the interferent in a 10-fold molar excess. As potential interferents, some monotopic species with a strong Lewis basicity were considered. Pyridine was chosen as the heterocyclic aromatic amine, while isopropylamine, diethylamine, and triethylamine were chosen as prototype compounds of primary, secondary, and tertiary amines. Moreover, the heteroditopic 4-amino-1-butanol, bearing two different coordinating sites with a different Lewis basicity, was also considered. As shown in Figure 8, very small or no changes of the absorbance at λmax = 580 nm are observed after the addition of each potential interferent, especially for diethylamine and triethylamine. Therefore, these data suggest that 1 can be considered a selective receptor towards PZ, even in the presence of common aliphatic or aromatic monotopic or heteroditopic amines.

Materials and General Procedures
All the chemicals used were purchased from Sigma-Aldrich (Darmstadt, Germany) and used as received. Complex 1 was synthesized and purified as previously reported [45]. Chloroform stabilized with amylene was used for optical absorption and fluorescence titrations. Before being used, it was purified as follows: dried on anhydrous K 2 CO 3 for 2 h, filtered and stored over molecular sieves (3 Å) in the dark under argon atmosphere. Chloroform solutions of 1 were prepared by dissolving the compound in chloroform and filtering it through a 0.2 µm Teflon membrane filter. CDCl 3 was stored over molecular sieves (3 Å).

Physical Measurements
1 H NMR measurements were run at 27 • C on a Varian Unity S 500 (499.88 MHz for 1 H) spectrometer. Tetramethylsilane was used as internal reference for all NMR experiments. Optical absorption spectra were recorded at room temperature with an Agilent Cary 60 spectrophotometer. Fluorescence spectra were recorded at room temperature using a JASCO FP-8200 spectrofluorometer (JASCO Europe). Spectrophotometric and fluorometric titrations were performed with a 1 cm path cell using 15 µM chloroform solutions of 1. Chloroform solutions of involved Lewis bases were added to the solution of 1 using Rainin (METTLER TOLEDO, Columbus, OH, USA) positive displacements pipettes. At least three replicate titrations were performed for each diamine. In fluorometric titrations, the wavelength of excitation was chosen in an isosbestic point. The fluorescence quantum yield was obtained using fluorescein (φ F = 0.925) in 0.1 M NaOH as a standard. The absorbance value of the samples at and above the excitation wavelength was lower than 0.1 for 1 cm path length cuvettes.

Calculation of Binding Constants and Limit of Detection
Binding constants, K, for the formation of 1 diamine adducts (equilibrium 1) were calculated by fitting the binding isotherms, obtained from the plot of A vs. c A from spectrophotometric titration data, with Equation (1) [73,74].
where A 0 is the initial absorbance of the solution having a concentration c 0 , A is the absorbance intensity after addition of a given amount of diamine (DA) at a concentration c DA , and A lim is the limiting absorbance reached in the presence of an excess of DA. Further details are reported elsewhere [46,48]. These calculated binding constants are comparable to those previously obtained by a multivariate analysis from spectrophotometric titrations of 1 with PZ, DPE, and 1,4-diaminobutane [45]. In the case of BPY, binding constants K 1 and K 2 for a 1:2 adduct (equilibria (2)) were calculated by fitting the binding isotherm using Equation (35) of Ref. [73]. The limit of detection (LOD) was estimated, both from optical absorption or fluorescence data, according to IUPAC recommendations (Equation (2)) [75,76].
where K = 3, S b is the standard deviation of the blank solution, i.e., the absorbance or fluorescence signal of 1, and S is the slope of the calibration curve obtained from the plot of the absorbance or fluorescence intensity of 1 vs. the concentration of the DA added. Each point is related to the mean value obtained from at least three replicate measurements. Twenty blank replicates were considered.

Conclusions
The binding properties of a molecular tweezer, composed of two Zn(salmal) units connected by a flexible spacer, towards a series of ditopic diamines having strong Lewis basicity have been explored by means of combined 1 H NMR, optical absorption, and fluorescence studies. The formation of stable 1:1 Lewis acid-base adducts with large binding constants is demonstrated. For α,ω-aliphatic diamines, binding constants progressively increase with the increasing length of the alkyl chain, thanks to the flexible nature of the spacer and there is a parallel decrease of the conformational strain upon binding for longer diamines, reaching the largest value for the 1,8-diaminooctane. Stable adducts are also found even for short diamines with rigid molecular structures. The preorganized structure of these ditopic species which, except for the entropic cost upon binding the diamine to 1, are not subjected to loss of degrees of freedom, accounts for the large binding constants.
These binding characteristics can be exploited for the detection of ditopic strong Lewis bases. The colorimetric and fluorometric selective and sensitive detection has been demonstrated for piperazine.